Glycine Binding Site Of NMDA Receptor Research Articles

NMDA Receptor Glycine Binding Site Modulators for Prevention and Treatment of Ketamine Use Disorder.

Ketamine offers a fast-acting approach to relieving treatment-resistant depression, but its abuse potential is an issue of concern. As ketamine is a noncompetitive N-methyl-D-aspartate receptor (NMDAR) ion channel blocker, modulation of NMDAR might be an effective strategy to counteract the abuse liability of ketamine and even to treat ketamine use disorder. This study evaluated whether NMDAR modulators that act on glycine binding sites can decrease motivation to obtain ketamine and reduce reinstatement to ketamine-seeking behavior. Two NMDAR modulators, D-serine and sarcosine were examined. Male Sprague-Dawley rats underwent training to acquire the ability to self-administer ketamine. The motivation to self-administer ketamine or sucrose pellets was examined under a progressive ratio (PR) schedule. The reinstatement of ketamine-seeking and sucrose pellet-seeking behaviors were assessed after extinction. The results showed that both D-serine and sarcosine significantly decreased the breakpoints for ketamine and prevented reinstatement of ketamine seeking. However, these modulators did not alter motivated behavior for sucrose pellets, the ability of the cue and sucrose pellets to reinstate sucrose-seeking behavior or spontaneous locomotor activity. These findings indicate that two NMDAR modulators can specifically reduce the measures of motivation and relapse for ketamine in rats, suggesting that targeting the glycine binding site of the NMDAR is a promising approach for preventing and treating ketamine use disorder.

PM432. Evaluation of the extrapyramidal side effects (EPS) liability of NMDA receptor glycine-binding site agonists

PM432. Evaluation of the extrapyramidal side effects (EPS) liability of NMDA receptor glycine-binding site agonists

Xenon-mediated neuroprotection in response to sustained, low-level excitotoxic stress.

Noble gases such as xenon and argon have been reported to provide neuroprotection against acute brain ischemic/anoxic injuries. Herein, we wished to evaluate the protective potential of these two gases under conditions relevant to the pathogenesis of chronic neurodegenerative disorders. For that, we established cultures of neurons typically affected in Alzheimer's disease (AD) pathology, that is, cortical neurons and basal forebrain cholinergic neurons and exposed them to L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) to generate sustained, low-level excitotoxic stress. Over a period of 4 days, PDC caused a progressive loss of cortical neurons which was prevented substantially when xenon replaced nitrogen in the cell culture atmosphere. Unlike xenon, argon remained inactive. Xenon acted downstream of the inhibitory and stimulatory effects elicited by PDC on glutamate uptake and efflux, respectively. Neuroprotection by xenon was mimicked by two noncompetitive antagonists of NMDA glutamate receptors, memantine and ketamine. Each of them potentiated xenon-mediated neuroprotection when used at concentrations providing suboptimal rescue to cortical neurons but most surprisingly, no rescue at all. The survival-promoting effects of xenon persisted when NMDA was used instead of PDC to trigger neuronal death, indicating that NMDA receptor antagonism was probably accountable for xenon’s effects. An excess of glycine failed to reverse xenon neuroprotection, thus excluding a competitive interaction of xenon with the glycine-binding site of NMDA receptors. Noticeably, antioxidants such as Trolox and N-acetylcysteine reduced PDC-induced neuronal death but xenon itself lacked free radical-scavenging activity. Cholinergic neurons were also rescued efficaciously by xenon in basal forebrain cultures. Unexpectedly, however, xenon stimulated cholinergic traits and promoted the morphological differentiation of cholinergic neurons in these cultures. Memantine reproduced some of these neurotrophic effects, albeit with less efficacy than xenon. In conclusion, we demonstrate for the first time that xenon may have a therapeutic potential in AD.

Dynamic Regulation of P-glycoprotein in Human Brain Capillaries

Considering its role as a major blood-brain barrier gatekeeper, the dynamic regulation of the efflux transporter P-glycoprotein is of considerable functional relevance. In particular, disease-associated alterations in transport function might affect central nervous system drug efficacy. Thus, targeting regulatory signaling cascades might render a basis for novel therapeutic approaches. Using capillaries freshly prepared from patient tissue resected during epilepsy surgery, we demonstrate dynamic regulation of P-glycoprotein in human brain capillaries. Glutamate proved to up-regulate P-glycoprotein efflux transport in a significant manner via endothelial NMDA receptors. Both inhibition of cyclooxygenase-2 and antagonism at the glycine-binding site of the NMDA receptor prevented the glutamate-mediated induction of P-glycoprotein transport function in human capillaries. In conclusion, the data argue against species differences in the signaling factors increasing endothelial P-glycoprotein transport function in response to glutamate exposure. Targeting of cyclooxygenase-2 and of the NMDA receptor glycine-binding site was confirmed as an efficacious approach to control P-glycoprotein function. The findings might render a basis for translational development of add-on approaches to improve brain penetration and efficacy of drugs.

Development of N-[ 11C]methylamino 4-hydroxy-2(1 H)-quinolone derivatives as PET radioligands for the glycine-binding site of NMDA receptors

In this study, we synthesized and evaluated several amino 4-hydroxy-2(1 H)-quinolone (4HQ) derivatives as new PET radioligand candidates for the glycine site of the NMDA receptors. Among these ligands, we discovered that 7-chloro-4-hydroxy-3-{3-(4-methylaminobenzyl) phenyl}-2-(1 H)-quinolone ( 12) and 5-ethyl-7-chloro-4-hydroxy-3-(3-methylaminophenyl)-2(1 H)-quinolone ( 32) have high affinity for the glycine site ( K i values; 11.7 nM for 12 and 11.8 nM for 32). In vitro autoradiography experiments indicated that [ 11C] 12 and [ 11C] 32 showed high specific binding in the brain slices, which were strongly inhibited by both glycine agonists and antagonists. In vivo brain uptake of these 11C-labeled 4HQs were examined in normal mice. Cerebellum to blood ratio of accumulation, of both [ 11C] 12 and [ 11C] 32 at 30 min were 0.058, which were slightly higher than those of cerebrum to blood ratio (0.043 and 0.042, respectively). These results indicated that [ 11C] 12 and [ 11C] 32 have poor blood brain barrier permeability. Although the plasma protein-binding ratio of [ 11C] 32 was much lower than methoxy analogs (71% vs 94–98%, respectively), [ 11C] 32 still binds with plasma protein strongly. It is conjectured that still acidic moiety and high affinity with plasma protein of [ 11C] 32 may prevent in vivo brain uptake. In conclusion, [ 11C] 12 and [ 11C] 32 are unsuitable for imaging cerebral NMDA receptors.

A plateau potential mediated by the activation of extrasynaptic NMDA receptors in rat hippocampal CA1 pyramidal neurons

Hippocampal pyramidal neurons express various extrasynaptic glutamate receptors. When glutamate spillover was facilitated by blocking glutamate uptake and fast synaptic transmission was blocked by antagonists of AMPA- and NMDA-type glutamate receptors and an ionotropic GABA receptor blocker, repetitive synaptic stimulation evoked a persistent membrane depolarization that consisted of an early Ca(2+)-independent component and a late Ca(2+)-dependent component. The early component, which we refer to as a plateau potential, had a half-width of 770 +/- 160 ms and a steady peak level of -9.54 +/- 3.50 mV. It was accompanied by an increase in membrane conductance, the I-V relationship of which showed a peak at -19.91 +/- 2.18 mV and reversal of the current at -4.32 +/- 2.13 mV, and was suppressed by high concentration of an NMDA receptor (NMDAR) antagonist d-APV, or an NMDAR glycine-binding site antagonist 5,7-dCK. After blocking synaptically located NMDARs using MK801, the potential was still evoked synaptically when spillover was facilitated. A sustained depolarization was evoked by iontophoretic application of glutamate in the presence or absence of a glutamate uptake blocker. This potential was not affected by Na(+) or Ca(2+) channel blockers, but was suppressed by 5,7-dCK, leaving an unspecified depolarizing potential. Iontophoresis of NMDA evoked a sustained depolarization that was blocked by a high concentration of d-APV or 5,7-dCK. The I-V relationship of the current during this potential was similar to that obtained during the synaptically induced plateau potentials. These results show that CA1 pyramidal neurons generate plateau potentials mediated most likely by activation of extrasynaptic NMDARs.

Activity-Dependent Shedding of the NMDA Receptor Glycine Binding Site by Matrix Metalloproteinase 3: A PUTATIVE Mechanism of Postsynaptic Plasticity

Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.

D-Cycloserine enhances conditioned taste aversion learning in rats

Conditioned taste aversion (CTA) is a form of associative learning in which the pairing of a taste with a toxin causes an animal to avoid the taste. NMDA receptor mediated neurotransmission has been implicated in CTA, but the role of the NMDA receptor glycine-binding site has not been examined. To examine the effects on CTA of the glycinergic NMDA receptor agonist d-cycloserine, rats received d-cycloserine (15 mg/kg, i.p.) or vehicle 15 min before 10-min access to 0.125% saccharin, followed by a low dose of LiCl (19 mg/kg, i.p.). CTA was measured with 24-h, 2-bottle preference tests between water and saccharin. Vehicle-treated rats formed a mild CTA that rapidly extinguished, while d-cycloserine-treated rats formed a stronger CTA that extinguished slowly. The effect of d-cycloserine was specific to the NMDA receptor glycine-binding site, because pretreatment with HA-966 (6 mg/kg), a partial glycinergic agonist, blocked enhancement by d-cycloserine. Three follow-up experiments suggest that the enhancement of CTA was not due to an aversive effect of d-cycloserine. First, saccharin paired with d-cycloserine (15 mg/kg) alone did not induce a CTA, although a higher dose (30 mg/kg) did significantly lower saccharin preference. Second, pretreatment with d-cycloserine did not increase the duration of “lying-on-belly” behavior induced by LiCl. Third, pretreatment with d-cycloserine did not increase c-Fos induction by either LiCl or vehicle injection in central visceral relays (the nucleus of the solitary tract, the parabrachial nucleus, the central nucleus of the amygdala, the supraoptic nucleus, and the paraventricular nucleus). These results confirm the participation of NMDA receptor, and specifically the glycine-binding site of NMDA receptor, in CTA learning.

Immunocytochemical analysis of D‐serine distribution in the mammalian brain reveals novel anatomical compartmentalizations in glia and neurons

D-Serine is a co-agonist at the NMDA receptor glycine-binding site. Early studies have emphasized a glial localization for D-serine. However the nature of the glial cells has not been fully resolved, because previous D-serine antibodies needed glutaraldehyde-fixation, precluding co-localization with fixation-sensitive antigens. We have raised a new D-serine antibody optimized for formaldehyde-fixation. Light and electron microscopic observations indicated that D-serine was concentrated into vesicle-like compartments in astrocytes and radial glial cells, rather than being distributed uniformly in the cytoplasm. In aged animals, patches of cortex and hippocampus were devoid of immunolabeling for D-serine, suggesting that impaired glial modulation of forebrain glutamatergic signaling might occur. Dual immunofluorescence labeling for glutamate and D-serine revealed D-serine in a subset of glutamatergic neurons, particularly in brainstem regions and in the olfactory bulbs. Microglia also contain D-serine. We suggest that some D-serine may be derived from the periphery. Collectively, our data suggest that the cellular compartmentation and distribution of D-serine may be more complex and extensive than previously thought and may have significant implications for our understanding of the role of D-serine in disease states including hypoxia and schizophrenia.

Ginsenoside Rg3 antagonizes NMDA receptors through a glycine modulatory site in rat cultured hippocampal neurons

We previously reported that ginseng, a well-known herbal medicine, inhibited NMDA receptors in cultured hippocampal neurons. Here, we further examined the detailed mechanism of ginseng-mediated inhibition using its main active ingredient, ginsenoside Rg3. Co-application of ginsenoside Rg3 with increasing concentrations of NMDA did not change the EC50 of NMDA to the receptor, suggesting that ginsenoside Rg3 inhibits NMDA receptors without competing with the NMDA-binding site. Ginsenoside Rg3-mediated inhibition also occurred in a distinctive manner from the well-characterized NMDA receptor open channel blocker, MK-801. However, ginsenoside Rg3 produced its effect in a glycine concentration-dependent manner and shifted the glycine concentration–response curve to the right without changing the maximal response, suggesting the role of ginsenoside Rg3 as a competitive NMDA receptor antagonist. We also demonstrated that ginsenoside Rg3 significantly protected neurons against NMDA insults. Therefore, these results suggest that ginsenoside Rg3 protects NMDA-induced neuronal death via a competitive interaction with the glycine-binding site of NMDA receptors in cultured hippocampal neurons.

Effects of endogenous agonists, glycine and D-serine, on in vivo specific binding of [11C]L-703,717, a PET radioligand for the glycine-binding site of NMDA receptors.

A positron-emitter (carbon-11) labeled antagonist for the glycine-binding site of NMDA receptors, [(11)C]L-703,717, has a unique in vivo binding characteristic, in which it preferentially binds to cerebellar-specific NMDA receptors consisting of a GluRepsilon3 subunit and eventually accumulates in rodent cerebellum under in vivo conditions, but not under in vitro conditions. In order to understand the in vivo-specific site and subunit localization of this radioligand, we examined the effect of the endogenous glycine site agonists, glycine and D-serine, on in vivo [(11)C]L-703,717 binding. An increase in extracellular glycine concentration by treatment with a glycine transporter 1 (GlyT1)-selective inhibitor, NFPS ethyl ester, significantly decreased the cerebellar localization of [(11)C]L-703,717 in rats. D-serine is known to be concentrated in mammalian forebrain regions. The lack of D-serine detection in the cerebellum may be due to the fact that it has the highest enzymatic activity of D-amino acid oxidase (DAO). It was found that the cerebellar localization of [(11)C]L-703,717 is greatly diminished in mutant mice lacking DAO, in which D-serine content in the cerebellum is drastically increased from a nondetectable level in normal mice. These studies indicate that [(11)C]L-703,717 is susceptible to inhibition by glycine site agonists in its in vivo binding, and suggest that regional differences in inhibitions by endogenous agonists may be a crucial factor in the site- and subunit-specific binding of this glycine-site antagonist.

SM-31900, a novel NMDA receptor glycine-binding site antagonist, reduces infarct volume induced by permanent middle cerebral artery occlusion in spontaneously hypertensive rats

The purpose of this study was to investigate the effect of (3 S)-7-chloro-3-[2-((1 R)-1-carboxyethoxy)-4-aminomethylphenyl]aminocarbonylmethyl-1,3,4,5-tetrahydrobenz[c,d]indole-2-carboxylic acid hydrochloride (SM-31900), an antagonist with high selectivity and affinity for the NMDA receptor glycine-binding site, on the cerebral infarct volume in a permanent middle cerebral artery occlusion (MCAo) model, which was constructed by electrocoagulation of a unilateral middle cerebral artery distal to the olfactory tract using spontaneously hypertensive rats (SHRs). To investigate the dose-response characteristics and the therapeutic time window of SM-31900 in this MCAo model, we conducted three experiments, in which the administration of SM-31900 was started 5 min (experiment I), 30 min (experiment II), or 60 min (experiment III) after MCAo, respectively. In all the studies, SM-31900 was administered by intravenous bolus injection followed by continuous intravenous infusion to obtain a steady-state level of this compound in blood immediately after its administration. The treatment with SM-31900 was continued until 24 h after MCAo, at which time the cerebral infarct volume was measured. In experiment I, SM-31900 significantly reduced the infarct volume by 37% at a dosage of 0.38 mg/kg bolus followed by 1.5 mg/kg/h continuous infusion (0.38 mg/kg+1.5 mg/kg/h). In experiment II, the neuroprotective effect of SM-31900 was also significant, with a 25% reduction in infarct volume at a dosage of 0.38 mg/kg+1.5 mg/kg/h, and a 40% reduction at 1.5 mg/kg+6.0 mg/kg/h. Furthermore, even in experiment III, SM-31900 exerted a significant neuroprotective effect, with a 20% reduction at 1.5 mg/kg+6.0 mg/kg/h. These studies revealed that SM-31900 can exert a neuroprotective effect when it is administered up to at least 60 min after the onset of ischemia in the MCAo model, an animal model of stroke, indicating that SM-31900 is a good candidate for treating acute brain ischemia.

Severe impairment of NMDA receptor function in mice carrying targeted point mutations in the glycine binding site results in drug-resistant nonhabituating hyperactivity.

NMDA receptor hypofunction has been implicated in the pathophysiology of schizophrenia, and pharmacological and genetic approaches have been used to model such dysfunction. We previously have described two mouse lines carrying point mutations in the NMDA receptor glycine binding site, Grin1(D481N) and Grin1(K483Q), which exhibit 5- and 86-fold reductions in receptor glycine affinity, respectively. Grin1(D481N) animals exhibit a relatively mild phenotype compatible with a moderate reduction in NMDA receptor function, whereas Grin1(K483Q) animals die shortly after birth. In this study we have characterized compound heterozygote Grin1(D481N/K483Q) mice, which are viable and exhibited biphasic NMDA receptor glycine affinities compatible with the presence of each of the two mutated alleles. Grin1(D481N/K483Q) mice exhibited a marked NMDA receptor hypofunction revealed by deficits in hippocampal long-term potentiation, which were rescued by the glycine site agonist d-serine, which also facilitated NMDA synaptic currents in mutant, but not in wild-type, mice. Analysis of striatal monoamine levels revealed an apparent dopaminergic and serotonergic hyperfunction. Behaviorally, Grin1(D481N/K483Q) mice were insensitive to acute dizocilpine pretreatment and exhibited increased startle response but normal prepulse inhibition. Most strikingly, mutant mice exhibited a sustained, nonhabituating hyperactivity and increased stereotyped behavior that were resistant to suppression by antipsychotics and the benzodiazepine site agonist Zolpidem. They also displayed a disruption of nest building behavior and were unable to perform a cued learning paradigm in the Morris water maze. We speculate that the severity of NMDA receptor hypofunction in these mice may account for their profound behavioral phenotype and insensitivity to antipsychotics.

Severe Impairment of NMDA Receptor Function in Mice Carrying Targeted Point Mutations in the Glycine Binding Site Results in Drug-Resistant Nonhabituating Hyperactivity

NMDA receptor hypofunction has been implicated in the pathophysiology of schizophrenia, and pharmacological and genetic approaches have been used to model such dysfunction. We previously have described two mouse lines carrying point mutations in the NMDA receptor glycine binding site, Grin1(D481N) and Grin1(K483Q), which exhibit 5- and 86-fold reductions in receptor glycine affinity, respectively. Grin1(D481N) animals exhibit a relatively mild phenotype compatible with a moderate reduction in NMDA receptor function, whereas Grin1(K483Q) animals die shortly after birth. In this study we have characterized compound heterozygote Grin1(D481N/K483Q) mice, which are viable and exhibited biphasic NMDA receptor glycine affinities compatible with the presence of each of the two mutated alleles. Grin1(D481N/K483Q) mice exhibited a marked NMDA receptor hypofunction revealed by deficits in hippocampal long-term potentiation, which were rescued by the glycine site agonist d-serine, which also facilitated NMDA synaptic currents in mutant, but not in wild-type, mice. Analysis of striatal monoamine levels revealed an apparent dopaminergic and serotonergic hyperfunction. Behaviorally, Grin1(D481N/K483Q) mice were insensitive to acute dizocilpine pretreatment and exhibited increased startle response but normal prepulse inhibition. Most strikingly, mutant mice exhibited a sustained, nonhabituating hyperactivity and increased stereotyped behavior that were resistant to suppression by antipsychotics and the benzodiazepine site agonist Zolpidem. They also displayed a disruption of nest building behavior and were unable to perform a cued learning paradigm in the Morris water maze. We speculate that the severity of NMDA receptor hypofunction in these mice may account for their profound behavioral phenotype and insensitivity to antipsychotics.

AFFINITY LABELING OF CYSTEINE-MUTANTS EVIDENCES CONTACT RESIDUES IN MODELED RECEPTOR BINDING SITES

ABSTRACTTo investigate the topology of binding sites in two ionotropic receptors, we have initiated a strategy combining affinity labeling with cysteine-scanning mutagenesis. For the GABAA receptor we have used reactive derivatives of non-competitive blockers (NCBs) to explore interacting positions in its channel. The polypeptide positions of the M2 segment of the α1 subunit which we mutated into cysteine were selected for their established accessibility, as determined by the substituted-cysteine accessibility method (SCAM). Using the Xenopus oocyte expression system, we show that receptors containing mutations V257C and S272C are inactivated by several reactive NCBs. These position-selective inactivations lead to an analysis of NCB binding in the channel. For the NMDA receptor glycine-binding site, the prototype antagonist L-701,324 was derivatized at different positions with different reactive groups. The receptor positions to mutate into cysteine were selected after a 3-D homology model. The observed receptor inactivations are mutant- and probe-selective, leading to an unambiguous chemical docking of the antagonist pharmacophore and supporting the model. The site-specificity of the inactivating reactions is assessed by protection experiments and by mutant to wild-type (WT) comparisons. The scope and limitations of the method are briefly discussed.

A positron‐emitter labeled glycineB site antagonist, [11C]L‐703,717, preferentially binds to a cerebellar NMDA receptor subtype consisting of GluR ϵ3 subunit in vivo, but not in vitro

In previous studies, we have found that [(11)C]L-703,717, a positron-emitter labeled antagonist for the glycine-binding site of NMDA receptors, only localizes in rodent cerebellum under in vivo conditions. In order to understand the unusual cerebellar localization, we have examined the binding of [(11)C]L-703,717 to a cerebellar-specific NMDA receptor subtype consisting of GLuRepsilon3 subunit, by comparing its autoradiographic distributions between GluRepsilon3-deficient and wild-type mice. Ex vivo [(11)C]L-703,717 binding to wild-type mice showed a highly specific localization of radioactivity in the cerebellum, whereas that to the GluRepsilon3-deficient mice showed no specific localization of radioactivity in any of the brain regions. In contrast to the ex vivo binding, in vitro [(11)C]L-703,717 binding displayed a similar binding characteristic between GluRepsilon3-deficient and wild-type mice with highly specific localizations in the hippocampus and cerebral cortex. Therefore, the present study clearly demonstrated that [(11)C]L-703,717 preferentially binds to a cerebellar NMDA receptor subtype consisting of GluRepsilon3 subunit in vivo, but not in vitro.

Synthesis of 7,8-dichloro-6-Nitro-1H-1,5-benzodiazepine-2,4-(3H, 5H)-dione as a potential NMDA receptor glycine site antagonist

An efficient procedure for the preparation of 7,8-dichloro-6-nitro-1H-1,5-benzodiazepine-2,4-(3H, 5H)-dione(7) as a potential lead compound for the NMDA receptor glycine binding site antagonist, starting from readily available 4,5-dichloro-2-nitroaniline(8), is described. The key step in the synthesis involves the cyclization of malonic ester amide 10 to compound 11.

O-271 - Pharmacological properties of SM-31900, a novel NMDA receptor glycine-binding site antagonist

O-271 - Pharmacological properties of SM-31900, a novel NMDA receptor glycine-binding site antagonist

5-Aminomethylquinoxaline-2,3-diones. Part II: N-aryl derivatives as novel NMDA/glycine and AMPA antagonists.

Potent antagonists at the glycine-binding site of NMDA receptors, as well as dual antagonists acting also at AMPA receptors have been identified in a series of 5-arylaminomethylquinoxaline-2,3-diones. A study of the structure-activity relationship of these compounds is reported here.