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1.  Direct pharmacological monitoring of the developmental switch in NMDA receptor subunit composition using TCN 213, a GluN2A-selective, glycine-dependent antagonist 
British Journal of Pharmacology  2012;166(3):924-937.
BACKGROUND AND PURPOSE
Developmental switches in NMDA receptor subunit expression have been inferred from studies of GluN2 expression levels, changes in kinetics of glutamatergic synaptic currents and sensitivity of NMDA receptor-mediated currents to selective GluN2B antagonists. Here we use TCN 213, a novel GluN2A-selective antagonist to identify the presence of this subunit in functional NMDA receptors in developing cortical neurones.
EXPERIMENTAL APPROACH
Two-electrode voltage-clamp (TEVC) recordings were made from Xenopus laevis oocytes to determine the pharmacological activity of TCN 213 at recombinant NMDA receptors. TCN 213 antagonism was studied in cultures of primary cortical neurones, assessing the NMDA receptor dependency of NMDA-induced excitotoxicity and monitoring developmental switches in NMDA receptor subunit composition.
KEY RESULTS
TCN 213 antagonism of GluN1/GluN2A NMDA receptors was dependent on glycine but independent of glutamate concentrations in external recording solutions. Antagonism by TCN 213 was surmountable and gave a Schild plot with unity slope. TCN 213 block of GluN1/GluN2B NMDA receptor-mediated currents was negligible. In cortical neurones, at a early developmental stage predominantly expressing GluN2B-containing NMDA receptors, TCN 213 failed to antagonize NMDA receptor-mediated currents or to prevent GluN2B-dependent, NMDA-induced excitoxicity. In older cultures (DIV 14) or in neurones transfected with GluN2A subunits, TCN 213 antagonized NMDA-evoked currents. Block by TCN 213 of NMDA currents inversely correlated with block by ifenprodil, a selective GluN2B antagonist.
CONCLUSIONS AND IMPLICATIONS
TCN 213 selectively blocked GluN1/GluN2A over GluN1/GluN2B NMDA receptors allowing direct dissection of functional NMDA receptors and pharmacological profiling of developmental changes in native NMDA receptor subunit composition.
doi:10.1111/j.1476-5381.2011.01748.x
PMCID: PMC3417419  PMID: 22022974
NMDA; glutamate; glycine; antagonism; oocyte; two-electrode voltage clamp; electrophysiology; neurotoxicity; development
2.  TCN 201 selectively blocks GluN2A-containing NMDARs in a GluN1 co-agonist dependent but non-competitive manner 
Neuropharmacology  2012;63-540(3-7):441-449.
Antagonists that are sufficiently selective to preferentially block GluN2A-containing N-methyl-d-aspartate receptors (NMDARs) over GluN2B-containing NMDARs are few in number. In this study we describe a pharmacological characterization of 3-chloro-4-fluoro-N-[4-[[2-(phenylcarbonyl)hydrazino]carbonyl]benzyl]benzenesulphonamide (TCN 201), a sulphonamide derivative, that was recently identified from a high-throughput screen as a potential GluN2A-selective antagonist. Using two-electrode voltage-clamp (TEVC) recordings of NMDAR currents from Xenopus laevis oocytes expressing either GluN1/GluN2A or GluN1/GluN2B NMDARs we demonstrate the selective antagonism by TCN 201 of GluN2A-containing NMDARs. The degree of inhibition produced by TCN 201 is dependent on the concentration of the GluN1-site co-agonist, glycine (or d-serine), and is independent of the glutamate concentration. This GluN1 agonist-dependency is similar to that observed for a related GluN2A-selective antagonist, N-(cyclohexylmethyl)-2-[{5-[(phenylmethyl)amino]-1,3,4-thiadiazol-2-yl}thio]acetamide (TCN 213). Schild analysis of TCN 201 antagonism indicates that it acts in a non-competitive manner but its equilibrium constant at GluN1/GluN2A NMDARs indicates TCN 201 is around 30-times more potent than TCN 213. In cortical neurones TCN 201 shows only modest antagonism of NMDAR-mediated currents recorded from young (DIV 9–10) neurones where GluN2B expression predominates. In older cultures (DIV 15–18) or in cultures where GluN2A subunits have been over-expressed TCN 201 gives a strong block that is negatively correlated with the degree of block produced by the GluN2B-selective antagonist, ifenprodil. Nevertheless, while TCN 201 is a potent antagonist it must be borne in mind that its ability to block GluN2A-containing NMDARs is dependent on the GluN1-agonist concentration and is limited by its low solubility.
Highlights
► TCN 201 is a potent and selective GluN1/GluN2A NMDAR antagonist. ► TCN 201 antagonism is dependent on the GluN1-agonist concentration. ► TCN 201 antagonism is independent on the GluN2-agonist concentration. ► TCN 201 blocks GluN2A-containing NMDARs in a non-competitive manner. ► TCN 201 allows pharmacological identification of native GluN2 A-containing NMDAR populations.
doi:10.1016/j.neuropharm.2012.04.027
PMCID: PMC3384000  PMID: 22579927
TCN 201; TCN 213; NMDA receptor; GluN2A-selective; Glycine; d-serine; Schild analysis
3.  Subunit-selective allosteric inhibition of glycine binding to NMDA receptors 
The Journal of Neuroscience  2012;32(18):6197-6208.
NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain, and are involved in numerous neuropathological conditions. NMDA receptors are activated upon simultaneous binding of co-agonists glycine and glutamate to the GluN1 and GluN2 subunits, respectively. Subunit-selective modulation of NMDA receptor function by ligand binding to modulatory sites distinct from the agonist binding sites could allow pharmacological intervention with therapeutically beneficial mechanisms. Here, we show the mechanism of action for TCN-201, a new GluN1/GluN2A-selective NMDA receptor antagonist whose inhibition can be surmounted by glycine. Electrophysiological recordings from chimeric and mutant rat NMDA receptors suggest that TCN-201 binds to a novel allosteric site located at the dimer interface between the GluN1 and GluN2 agonist binding domains. Furthermore, we demonstrate that occupancy of this site by TCN-201 inhibits NMDA receptor function by reducing glycine potency. TCN-201 is therefore a negative allosteric modulator of glycine binding.
doi:10.1523/JNEUROSCI.5757-11.2012
PMCID: PMC3355950  PMID: 22553026
4.  Distinct modes of AMPA receptor suppression at developing synapses by GluN2A and GluN2B: analysis of single-cell GluN2 subunit deletion in vivo 
Neuron  2011;71(6):1085-1101.
Summary
During development there is an activity-dependent switch in synaptic 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 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.
doi:10.1016/j.neuron.2011.08.007
PMCID: PMC3183990  PMID: 21943605
5.  The Subtype of GluN2 C-terminal Domain Determines the Response to Excitotoxic Insults 
Neuron  2012;74(3):543-556.
Summary
It is currently unclear whether the GluN2 subtype influences NMDA receptor (NMDAR) excitotoxicity. We report that the toxicity of NMDAR-mediated Ca2+ influx is differentially controlled by the cytoplasmic C-terminal domains of GluN2B (CTD2B) and GluN2A (CTD2A). Studying the effects of acute expression of GluN2A/2B-based chimeric subunits with reciprocal exchanges of their CTDs revealed that CTD2B enhances NMDAR toxicity, compared to CTD2A. Furthermore, the vulnerability of forebrain neurons in vitro and in vivo to NMDAR-dependent Ca2+ influx is lowered by replacing the CTD of GluN2B with that of GluN2A by targeted exon exchange in a mouse knockin model. Mechanistically, CTD2B exhibits stronger physical/functional coupling to the PSD-95-nNOS pathway, which suppresses protective CREB activation. Dependence of NMDAR excitotoxicity on the GluN2 CTD subtype can be overcome by inducing high levels of NMDAR activity. Thus, the identity (2A versus 2B) of the GluN2 CTD controls the toxicity dose-response to episodes of NMDAR activity.
Highlights
► The CTD of GluN2B promotes excitotoxicity better than that of GluN2A ► GluN2 CTD subtype differences are seen in both WT and chimeric 2A/2B subunits ► The GluN2B CTD couples to a prodeath PSD-95/nNOS-dependent CREB shut-off pathway
Martel et al. find that the two subtypes (2A versus 2B) of the GluN2 C-terminal domain differentially couple to the CREB shut-off pathway, causing distinct effects on NMDA receptor-mediated neuronal death both in vitro and in vivo.
doi:10.1016/j.neuron.2012.03.021
PMCID: PMC3398391  PMID: 22578505
6.  New advances in NMDA receptor pharmacology 
Trends in pharmacological sciences  2011;32(12):726-733.
N-Methyl-D-aspartate (NMDA) receptors are tetrameric ion channels containing two of four possible GluN2 subunits. These receptors have been implicated for decades in neurological diseases such as stroke, traumatic brain injury, dementia, and schizophrenia. The GluN2 subunits contribute substantially to functional diversity of NMDA receptors and are distinctly expressed in development and among brain regions. Thus, subunit-selective antagonists and modulators that differentially target the GluN2 subunit might provide an opportunity to pharmacologically modify the function of select groups of neurons for therapeutic gain. A flurry of clinical, functional, and chemical studies have together reinvigorated efforts to identify subunit-selective modulators of NMDA receptor function, resulting in a handful of new compounds that appear to act at novel sites. Here we review the properties of new emerging classes of subunit-selective NMDA receptor modulators, which we predict will mark the beginning of a productive period of progress for NMDA receptor pharmacology.
doi:10.1016/j.tips.2011.08.003
PMCID: PMC3223280  PMID: 21996280
7.  Structural and mechanistic determinants of a novel site for non-competitive inhibition of GluN2D-containing NMDA receptors 
N-methyl-D-aspartate (NMDA) receptors are ionotropic glutamate receptors that mediate excitatory synaptic transmission and have been implicated in several neurological diseases. We have evaluated the mechanism of action of a class of novel subunit-selective NMDA receptor antagonists, typified by (E)-4-(6-methoxy-2-(3-nitrostyryl)-4-oxoquinazolin-3(4H)-yl)-benzoic acid (QNZ46). We found that QNZ46 inhibits NMDA receptor function in a non-competitive and voltage-independent manner by an unconventional mechanism that requires binding of glutamate to the GluN2 subunit, but not glycine binding to the GluN1 subunit. This dependency of antagonist association on glutamate binding to GluN2 renders these compounds nominally use-dependent, since inhibition will rely on synaptic release of glutamate. Evaluation of the structural determinants responsible for the subunit-selectivity of QNZ46 revealed that these compounds act at a new site that has not previously been described. Residues residing in the part of the agonist binding domain immediately adjacent to the transmembrane helices appear to control selectivity of QNZ46 for GluN2C- and GluN2D-containing receptors. These residues are well-positioned to sense glutamate binding to GluN2 and thus mediate glutamate-dependent actions. This new class of non-competitive antagonists could provide an opportunity for the development of pharmacological tools and therapeutic agents that target NMDA receptors at a new site and modulate function by a novel mechanism.
doi:10.1523/JNEUROSCI.5565-10.2011
PMCID: PMC3063124  PMID: 21389220
patch-clamp electrophysiology; Xenopus oocytes; pharmacology; allosteric modulation
8.  Open-channel blockade is less effective on GluN3B than GluN3A subunit-containing NMDA receptors 
European Journal of Pharmacology  2012;686(1-3):22-31.
The GluN3 subunits of the N-methyl-d-aspartate (NMDA) receptor are known to reduce its Ca2+ permeability and Mg2+ sensitivity, however, little is known about their effects on other channel blockers. cRNAs for rat NMDA receptor subunits were injected into Xenopus oocytes and responses to NMDA and glycine were recorded using two electrode voltage clamp. Channel block of receptors containing GluN1-1a/2A, GluN1-1a/2A/3A or GluN1-1a/2A/3B subunits was characterised using Mg2+, memantine, MK-801, philanthotoxin-343 and methoctramine. IC50 values for Mg2+ and memantine increased when receptors contained GluN3A subunits and were further increased when they contained GluN3B, e.g. IC50s at − 75 mV for block of GluN1-1a/2A, GluN1-1a/2A/3A and GluN1-1a/2A/3B receptors respectively were 4.2, 22.4 and 40.1 μM for Mg2+, and 2.5, 7.5 and 17.5 μM for memantine. Blocking activity was found to be fully or partially restored when G or R (at the N and N + 1 sites respectively) were mutated to N in GluN3A. Thus, the changes cannot be attributed to the loss of the N or N + 1 sites alone, but rather involve both sites or residues elsewhere. Block by MK-801 and philanthotoxin-343 was also reduced by GluN3A, most strongly at − 100 mV but not at − 50 mV, and by GluN3B at all Vh. Methoctramine was the least sensitive to introduction of GluN3 subunits suggesting a minimal interaction with the N and N + 1 sites. We conclude that GluN3B-containing receptors provide increased resistance to channel block compared to GluN3A-containing receptors and this must be due to differences outside the deep pore region (N site and deeper).
Graphical abstract
doi:10.1016/j.ejphar.2012.04.036
PMCID: PMC3657159  PMID: 22564863
N-methyl-d-aspartate receptors; GluN3 subunit; Channel block; Magnesium; Memantine; MK-801; Philanthotoxin-343; Methoctramine
9.  NMDA receptor subunits have different roles in NMDA-induced neurotoxicity in the retina 
Molecular Brain  2013;6:34.
Background
Loss of retinal ganglion cells (RGCs) is a hallmark of various retinal diseases including glaucoma, retinal ischemia, and diabetic retinopathy. N-methyl-D-aspartate (NMDA)-type glutamate receptor (NMDAR)-mediated excitotoxicity is thought to be an important contributor to RGC death in these diseases. Native NMDARs are heterotetramers that consist of GluN1 and GluN2 subunits, and GluN2 subunits (GluN2A–D) are major determinants of the pharmacological and biophysical properties of NMDARs. All NMDAR subunits are expressed in RGCs in the retina. However, the relative contribution of the different GluN2 subunits to RGC death by excitotoxicity remains unclear.
Results
GluN2B- and GluN2D-deficiency protected RGCs from NMDA-induced excitotoxic retinal cell death. Pharmacological inhibition of the GluN2B subunit attenuated RGC loss in glutamate aspartate transporter deficient mice.
Conclusions
Our data suggest that GluN2B- and GluN2D-containing NMDARs play a critical role in NMDA-induced excitotoxic retinal cell death and RGC degeneration in glutamate aspartate transporter deficient mice. Inhibition of GluN2B and GluN2D activity is a potential therapeutic strategy for the treatment of several retinal diseases.
doi:10.1186/1756-6606-6-34
PMCID: PMC3733768  PMID: 23902942
NMDA receptor; GluN2B; GluN2D; Excitotoxicity; Retina; Glaucoma; Glutamate transporter
10.  Contribution of NMDA Receptor Hypofunction in Prefrontal and Cortical Excitatory Neurons to Schizophrenia-Like Phenotypes 
PLoS ONE  2013;8(4):e61278.
Pharmacological and genetic studies support a role for NMDA receptor (NMDAR) hypofunction in the etiology of schizophrenia. We have previously demonstrated that NMDAR obligatory subunit 1 (GluN1) deletion in corticolimbic interneurons during early postnatal development is sufficient to confer schizophrenia-like phenotypes in mice. However, the consequence of NMDAR hypofunction in cortical excitatory neurons is not well delineated. Here, we characterize a conditional knockout mouse strain (CtxGluN1 KO mice), in which postnatal GluN1 deletion is largely confined to the excitatory neurons in layer II/III of the medial prefrontal cortex and sensory cortices, as evidenced by the lack of GluN1 mRNA expression in in situ hybridization immunocytochemistry as well as the lack of NMDA currents with in vitro recordings. Mutants were impaired in prepulse inhibition of the auditory startle reflex as well as object-based short-term memory. However, they did not exhibit impairments in additional hallmarks of schizophrenia-like phenotypes (e.g. spatial working memory, social behavior, saccharine preference, novelty and amphetamine-induced hyperlocomotion, and anxiety-related behavior). Furthermore, upon administration of the NMDA receptor antagonist, MK-801, there were no differences in locomotor activity versus controls. The mutant mice also showed negligible levels of reactive oxygen species production following chronic social isolation, and recording of miniature-EPSC/IPSCs from layer II/III excitatory neurons in medial prefrontal cortex suggested no alteration in GABAergic activity. All together, the mutant mice displayed cognitive deficits in the absence of additional behavioral or cellular phenotypes reflecting schizophrenia pathophysiology. Thus, NMDAR hypofunction in prefrontal and cortical excitatory neurons may recapitulate only a cognitive aspect of human schizophrenia symptoms.
doi:10.1371/journal.pone.0061278
PMCID: PMC3628715  PMID: 23613827
11.  Amino Terminal Domains of the NMDA Receptor Are Organized as Local Heterodimers 
PLoS ONE  2011;6(4):e19180.
The N-methyl-D-aspartate (NMDA) receptor, an obligate heterotetrameric assembly organized as a dimer of dimers, is typically composed of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. Despite the crucial role that the NMDA receptor plays in the nervous system, the specific arrangement of subunits within the dimer-of-dimer assemblage is not conclusively known. Here we studied the organization of the amino terminal domain (ATD) of the rat GluN1/GluN2A and GluN1/GluN2B NMDA receptors by cysteine-directed, disulfide bond-mediated cross-linking. We found that GluN1 ATDs and GluN2 ATDs spontaneously formed disulfide bond-mediated dimers after introducing cysteines into the L1 interface of GluN2A or GluN2B ATD. The formation of dimer could be prevented by knocking out endogenous cysteines located near the L1 interface of GluN1. These results indicate that GluN1 and GluN2 ATDs form local heterodimers through the interactions in the L1-L1 interface and further demonstrate a dimer-of-heterodimer arrangement in GluN1/GluN2A and GluN1/GluN2B NMDA receptors.
doi:10.1371/journal.pone.0019180
PMCID: PMC3081335  PMID: 21544205
12.  Pharmacological Activation/Inhibition of the Cannabinoid System Affects Alcohol Withdrawal-Induced Neuronal Hypersensitivity to Excitotoxic Insults 
PLoS ONE  2011;6(8):e23690.
Cessation of chronic ethanol consumption can increase the sensitivity of the brain to excitotoxic damages. Cannabinoids have been proposed as neuroprotectants in different models of neuronal injury, but their effect have never been investigated in a context of excitotoxicity after alcohol cessation. Here we examined the effects of the pharmacological activation/inhibition of the endocannabinoid system in an in vitro model of chronic ethanol exposure and withdrawal followed by an excitotoxic challenge. Ethanol withdrawal increased N-methyl-D-aspartate (NMDA)-evoked neuronal death, probably by altering the ratio between GluN2A and GluN2B NMDA receptor subunits. The stimulation of the endocannabinoid system with the cannabinoid agonist HU-210 decreased NMDA-induced neuronal death exclusively in ethanol-withdrawn neurons. This neuroprotection could be explained by a decrease in NMDA-stimulated calcium influx after the administration of HU-210, found exclusively in ethanol-withdrawn neurons. By contrast, the inhibition of the cannabinoid system with the CB1 receptor antagonist rimonabant (SR141716) during ethanol withdrawal increased death of ethanol-withdrawn neurons without any modification of NMDA-stimulated calcium influx. Moreover, chronic administration of rimonabant increased NMDA-stimulated toxicity not only in withdrawn neurons, but also in control neurons. In summary, we show for the first time that the stimulation of the endocannabinoid system is protective against the hyperexcitability developed during alcohol withdrawal. By contrast, the blockade of the endocannabinoid system is highly counterproductive during alcohol withdrawal.
doi:10.1371/journal.pone.0023690
PMCID: PMC3158793  PMID: 21886913
13.  Ligand-specific deactivation time course of GluN1/GluN2D NMDA receptors 
Nature Communications  2011;2:294.
N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors that mediate a majority of excitatory synaptic transmission. NMDA receptors are comprised of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits, of which there are four subtypes (GluN2A-D) that determine many functional properties of the receptors. One unique property of the GluN1/GluN2D NMDA receptors is an unusually prolonged deactivation time course that lasts several seconds following the removal of L-glutamate. Here, we show by a combination of x-ray crystallography and electrophysiology that the deactivation time course of the GluN1/GluN2D receptors is influenced both by the conformational variability of the ligand-binding domain as well as the chemical structure and stereochemistry of the activating ligand. Of all ligands tested, L-glutamate and L-CCG-IV induce a significantly slower deactivation time course on the GluN1/GluN2D receptors than other agonists. Furthermore, crystal structures of the isolated GluN2D ligand-binding domain monomer in complex with various ligands reveal that the binding of L-glutamate induces a unique conformation at the back side of the ligand-binding site in proximity to the region where the transmembrane domain would be located in the intact receptors. These data suggest that the activity of the GluN1/GluN2D NMDA receptor is controlled distinctively by the endogenous neurotransmitter L-glutamate.
doi:10.1038/ncomms1295
PMCID: PMC3302728  PMID: 21522138
ionotropic glutamate receptors; NMDA receptors; GluN1/GluN2D; x-ray crystallography; electrophysiology; deactivation; pharmacology
14.  Tyrosine phosphorylation regulates the endocytosis and surface expression of GluN3A-containing NMDA receptors 
Selective control of receptor trafficking provides a mechanism for remodeling the receptor composition of excitatory synapses, and thus supports synaptic transmission, plasticity and development. GluN3A (formerly NR3A) is a non-conventional member of the NMDA receptor (NMDAR) subunit family which endows NMDAR channels with low calcium permeability and reduced magnesium sensitivity compared to NMDARs comprising only GluN1 and GluN2 subunits. Because of these special properties, GluN3A subunits act as a molecular brake to limit the plasticity and maturation of excitatory synapses, pointing towards GluN3A removal as a critical step in the development of neuronal circuitry. However, the molecular signals mediating GluN3A endocytic removal remain unclear. Here we define a novel endocytic motif (YWL) that is located within the cytoplasmic carboxy-terminal tail of GluN3A and mediates its binding to the clathrin adaptor AP2. Alanine mutations within the GluN3A endocytic motif inhibited clathrin-dependent internalization and led to accumulation of GluN3A-containing NMDARs at the cell surface, whereas mimicking phosphorylation of the tyrosine residue promoted internalization and reduced cell-surface expression as shown by immunocytochemical and electrophysiological approaches in recombinant systems and rat neurons in primary culture. We further demonstrate that the tyrosine residue is phosphorylated by Src family kinases, and that Src-activation limits surface GluN3A expression in neurons. Together, our results identify a new molecular signal for GluN3A internalization that couples the functional surface expression of GluN3A-containing receptors to the phosphorylation state of GluN3A subunits, and provide a molecular framework for the regulation of NMDAR subunit composition with implications for synaptic plasticity and neurodevelopment.
doi:10.1523/JNEUROSCI.2721-12.2013
PMCID: PMC3682218  PMID: 23447623
15.  Triheteromeric NMDA Receptors at Hippocampal Synapses 
NMDA receptors are composed of two GluN1 (N1) and two GluN2 (N2) subunits. Constituent N2 subunits control the pharmacological and kinetic characteristics of the receptor. NMDA receptors in hippocampal or cortical neurons are often thought of as diheteromeric, i.e., containing only one type of N2 subunit. However, triheteromeric receptors with more than one type of N2 subunit also have been reported and the relative contribution of di- and triheteromeric NMDA receptors at synapses has been difficult to assess. Because wild-type hippocampal principal neurons express N1, N2A and N2B, we used cultured hippocampal principal neurons from N2A and N2B-knockout mice as templates for diheteromeric synaptic receptors. Summation of N1/N2B and N1/N2A excitatory postsynaptic currents could not account for the deactivation kinetics of wild-type excitatory postsynaptic currents (EPSCs) however. To make a quantitative estimate of NMDA receptor subtypes at wild-type synapses, we used the deactivation kinetics, as well as the effects of the competitive antagonist NVP-AAM077. Our results indicate that three types of NMDA receptors contribute to the wild-type EPSC, with at least two-thirds being triheteromeric receptors. Functional isolation of synaptic triheteromeric receptors revealed deactivation kinetics and pharmacology distinct from either diheteromeric receptor subtype. Because of differences in open probability, synaptic triheteromeric receptors outnumbered N1/N2A receptors by 5.8 to 1 and N1/N2B receptors by 3.2 to 1. Our results suggest that triheteromeric NMDA receptors must be either preferentially assembled or preferentially localized at synapses.
doi:10.1523/JNEUROSCI.0829-13.2013
PMCID: PMC3755730  PMID: 23699525
16.  Mg2+ block properties of triheteromeric GluN1–GluN2B–GluN2D NMDA receptors on neonatal rat substantia nigra pars compacta dopaminergic neurones 
The Journal of Physiology  2014;592(10):2059-2078.
Native NMDA receptors (NMDARs) are tetrameric channels formed by two GluN1 and two GluN2 subunits. So far, seven NMDARs subunits have been identified and they can form diheteromeric or triheteromeric NMDARs (more than one type of GluN2 subunit). Extracellular Mg2+ is an important regulator of NMDARs, and particularly the voltage dependence of Mg2+ block is crucial to the roles of NMDARs in synaptic plasticity and the integration of synaptic activity with neuronal activity. Although the Mg2+ block properties of diheteromeric NMDARs are fully investigated, properties of triheteromeric NMDARs are still not clear. Our previous data suggested that dopaminergic neurones expressed triheteromeric GluN1–GluN2B–GluN2D NMDARs. Here, using NMDARs in dopaminergic neurones from postnatal day 7 (P7) rats as a model system, we characterize the voltage-dependent Mg2+ block properties of triheteromeric NMDARs. In control conditions, external Mg2+ significantly inhibits the whole cell NMDA-evoked current in a voltage-dependent manner with IC50 values of 20.9 μm, 53.3 μm and 173 μm at −90 mV, −70 mV and −50 mV, respectively. When the GluN2B-selective antagonist ifenprodil was applied, the Mg2+ sensitivity of the residual NMDA-mediated currents (which is mainly carried by GluN1–GluN2B–GluN2D NMDARs) is reduced to IC50 values of 45.9 μm (−90 mV), 104 μm (−70 mV) and 276 μm (−50 mV), suggesting that triheteromeric GluN1–GluN2B–GluN2D NMDARs have less affinity for external Mg2+ than GluN1–GluN2B receptors. In addition, fitting INMDA–V curves with a trapping Mg2+ block model shows the triheteromeric GluN1–GluN2B–GluN2D NMDARs have weaker voltage-dependent Mg2+ block (δ = 0.56) than GluN1–GluN2B NMDARs. Finally, our concentration jump and single channel recordings suggest that GluN1–GluN2B–GluN2D rather than GluN1–GluN2D NMDARs are present. These data provide information relevant to Mg2+ block characteristics of triheteromeric NMDARs and may help to better understand synaptic plasticity, which is dependent on these triheteromeric NMDARs.
doi:10.1113/jphysiol.2013.267864
PMCID: PMC4027860  PMID: 24614743
17.  Mechanistic and structural determinants of NMDA receptor voltage-dependent gating and slow Mg2+ unblock 
NMDA receptor (NMDAR)-mediated currents depend on membrane depolarization to relieve powerful voltage-dependent NMDAR channel block by external magnesium (Mg2+o). Mg2+o unblock from native NMDARs exhibits a fast component consistent with rapid Mg2+o unbinding kinetics, and also a slower, ms time scale component (slow Mg2+o unblock). In recombinant NMDARs, slow Mg2+o unblock is prominent in GluN1/2A (NMDAR subtype composed of GluN1 and GluN2A subunits) and GluN1/2B receptors, with slower kinetics observed for GluN1/2B receptors, but absent from GluN1/2C and GluN1/2D receptors. Slow Mg2+o unblock from GluN1/2B receptors results from inherent voltage-dependent gating, which increases channel open probability with depolarization.
Here we examine the mechanisms responsible for NMDAR subtype dependence of slow Mg2+o unblock. We demonstrate that slow Mg2+o unblock from GluN1/2A receptors, like GluN1/2B receptors, results from inherent voltage-dependent gating. Surprisingly, GluN1/2A and GluN1/2B receptors exhibit equal inherent voltage dependence; faster Mg2+o unblock from GluN1/2A receptors can be explained by voltage-independent differences in gating kinetics. To investigate the absence of slow Mg2+o unblock in GluN1/2C and GluN1/2D receptors we examined the GluN2 S/L site, a site responsible for several NMDAR subtype-dependent channel properties. Mutating the GluN2 S/L site of GluN2A subunits from serine (found in GluN2A and GluN2B subunits) to leucine (found in GluN2C and GluN2D) greatly diminished both voltage-dependent gating and slow Mg2+o unblock. Thus, the residue at the GluN2 S/L site governs expression of both slow Mg2+o unblock and inherent voltage dependence.
doi:10.1523/JNEUROSCI.3712-12.2013
PMCID: PMC3629906  PMID: 23447622
18.  GluN2A and GluN2B NMDA Receptor Subunits Differentially Modulate Striatal Output Pathways and Contribute to Levodopa-Induced Abnormal Involuntary Movements in Dyskinetic Rats 
ACS Chemical Neuroscience  2013;4(5):808-816.
Dual probe microdialysis was used to investigate whether GluN2A and GluN2B NMDA receptor subunits regulate striatal output pathways under dyskinetic conditions. The preferential GluN2A antagonist NVP-AAM077 perfused in the dopamine-depleted striatum of 6-hydroxydopamine hemilesioned dyskinetic rats reduced GABA and glutamate levels in globus pallidus whereas the selective GluN2B antagonist Ro 25-6981 elevated glutamate without affecting pallidal GABA. Moreover, intrastriatal NVP-AAM077 did not affect GABA but elevated glutamate levels in substantia nigra reticulata whereas Ro 25-6981 elevated GABA and reduced nigral glutamate. To investigate whether GluN2A and GluN2B NMDA receptor subunits are involved in motor pathways underlying dyskinesia expression, systemic NVP-AAM077 and Ro 25-6981 were tested for their ability to attenuate levodopa-induced abnormal involuntary movements. NVP-AAM077 failed to prevent dyskinesia while Ro 25-6981 mildly attenuated it. We conclude that in the dyskinetic striatum, striatal GluN2A subunits tonically stimulate the striato-pallidal pathway whereas striatal GluN2B subunits tonically inhibit striato-nigral projections. Moreover, GluN2A subunits are not involved in dyskinesia expression whereas GluN2B subunits minimally contribute to it.
doi:10.1021/cn400016d
PMCID: PMC3656753  PMID: 23611155
GABA; microdialysis; NMDA receptor subunits; NVP-AAM077; 6-OHDA; Ro 25-6981
19.  Positive emotional learning is regulated in the medial prefrontal cortex by GluN2B-containing NMDA receptors 
Neuroscience  2011;192:515-523.
In rats, hedonic USVs is a validated model of positive affect and is best elicited by rough-and-tumble play. Here we report that modulation of GluN2B-containing NMDA receptors (NMDAR) in the medial prefrontal cortex (MPFC) is involved in positive emotional learning. Rough and tumble play increased both GluN1 and GluN2B NMDAR subunit mRNA and protein levels in the frontal cortex. GLYX-13, a GluN2B-preferring, NMDAR glycine-site partial agonist (1 mg / kg i.v.) significantly increased positive emotional learning whereas the GluN2B receptor-specific antagonist, ifenprodil (10 mg/kg i.p.), inhibited positive emotional learning. Animals selectively bred for low rates of hedonic USVs were returned to wild-type levels of positive emotional learning following GLYX-13 treatment. MPFC microinjections of GLYX-13 (0.1–10 μg / side) significantly increased rates of positive emotional learning. Thus GluN2B-containing NMDARs may be involved in positive emotional learning in the MPFC by similar mechanisms as spatial / temporal learning in the hippocampus.
doi:10.1016/j.neuroscience.2011.05.001
PMCID: PMC3166413  PMID: 21645591
GLYX-13; GluN2B; medial prefrontal cortex; positive emotion; learning; vocalizations
20.  Involvement of the N-methyl-d-aspartate receptor GluN2D subunit in phencyclidine-induced motor impairment, gene expression, and increased Fos immunoreactivity 
Molecular Brain  2013;6:56.
Background
Noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists evoke a behavioral and neurobiological syndrome in experimental animals. We previously reported that phencyclidine (PCP), an NMDA receptor antagonist, increased locomotor activity in wildtype (WT) mice but not GluN2D subunit knockout mice. Thus, the aim of the present study was to determine whether the GluN2D subunit is involved in PCP-induced motor impairment.
Results
PCP or UBP141 (a GluN2D antagonist) induced potent motor impairment in WT mice but not GluN2D KO mice. By contrast, CIQ, a GluN2C/2D potentiator, induced severe motor impairment in GluN2D KO mice but not WT mice, suggesting that the GluN2D subunit plays an essential role in the effects of PCP and UBP141, and an appropriate balance between GluN2C and GluN2D subunits might be needed for appropriate motor performance. The level of the GluN2D subunit in the mature mouse brain is very low and restricted. GluN2D subunits exist in brainstem structures, the globus pallidus, thalamus, and subthalamic nucleus. We found that the expression of the c-fos gene increased the most among PCP-dependent differentially expressed genes between WT and GluN2D KO mice, and the number of Fos-positive cells increased after PCP administration in the basal ganglia motor circuit in WT mice but not GluN2D KO mice.
Conclusion
These results suggest that the GluN2D subunit within the motor circuitry is a key subunit for PCP-induced motor impairment, which requires an intricate balance between GluN2C- and GluN2D-mediated excitatory outputs.
doi:10.1186/1756-6606-6-56
PMCID: PMC3878647  PMID: 24330819
GluN2C; GluN2D; Motor impairment; Motor loop; PCP
21.  Development of 2′-substituted (2S,1′R,2′S)-2-(carboxycyclopropyl)glycine analogues as potent N-methyl-d-aspartic acid receptor agonists 
Journal of medicinal chemistry  2013;56(10):4071-4081.
A series of 2′-substituted analogues of the selective NMDA receptor ligand (2S,1′R,2′S)-2-(carboxycyclopropyl)glycine ((S)-CCG-IV) have been designed, synthesized and pharmacologically characterized. The design was based on a docking study hypothesizing that substituents in the 2′-position would protrude into a region where differences among the NMDA receptor GluN2 subunits exist. Various synthetic routes were explored, and two different routes provided a series of alkyl-substituted analogues. Pharmacological characterization revealed that these compounds are NMDA receptor agonists and that potency decreases with increasing size of the alkyl groups. Variations in agonist activity are observed at the different recombinant NMDA receptor subtypes. This study demonstrates that it is possible to introduce substituents in the 2′-position of (S)-CCG-IV while maintaining agonist activity and that variation among NMDA receptor subtypes may be achieved by probing this region of the receptor.
doi:10.1021/jm400346a
PMCID: PMC3689883  PMID: 23614571
NMDA receptor; CCG; glutamate; molecular modeling; subtype selectivity; electrophysiology
22.  Identification of a single amino acid in GluN1 that is critical for glycine-primed internalization of NMDA receptors 
Molecular Brain  2013;6:36.
Background
NMDA receptors are ligand-gated ion channels with essential roles in glutamatergic synaptic transmission and plasticity in the CNS. As co-receptors for glutamate and glycine, gating of the NMDA receptor/channel pore requires agonist binding to the glycine sites, as well as to the glutamate sites, on the ligand-binding domains of the receptor. In addition to channel gating, glycine has been found to prime NMDA receptors for internalization upon subsequent stimulation of glutamate and glycine sites.
Results
Here we address the key issue of identifying molecular determinants in the glycine-binding subunit, GluN1, that are essential for priming of NMDA receptors. We found that glycine treatment of wild-type NMDA receptors led to recruitment of the adaptor protein 2 (AP-2), and subsequent internalization after activating the receptors by NMDA plus glycine. However, with a glycine-binding mutant of GluN1 – N710R/Y711R/E712A/A714L – we found that treating with glycine did not promote recruitment of AP-2 nor were glycine-treated receptors internalized when subsequently activated with NMDA plus glycine. Likewise, GluN1 carrying a single point mutation – A714L – did not prime upon glycine treatment. Importantly, both of the mutant receptors were functional, as stimulating with NMDA plus glycine evoked inward currents.
Conclusions
Thus, we have identified a single amino acid in GluN1 that is critical for priming of NMDA receptors by glycine. Moreover, we have demonstrated the principle that while NMDA receptor gating and priming share a common requirement for glycine binding, the molecular constraints in GluN1 for gating are distinct from those for priming.
doi:10.1186/1756-6606-6-36
PMCID: PMC3846451  PMID: 23941530
NMDA Receptors; Glycine; Internalization; Endocytosis; Dynamin; GluN1; GluN2
23.  NMDA Receptor Subunits in the Adult Rat Hippocampus Undergo Similar Changes after 5 Minutes in an Open Field and after LTP Induction 
PLoS ONE  2013;8(2):e55244.
NMDA receptor subunits change during development and their synaptic expression is modified rapidly after synaptic plasticity induction in hippocampal slices. However, there is scarce information on subunits expression after synaptic plasticity induction or memory acquisition, particularly in adults. GluN1, GluN2A and GluN2B NMDA receptor subunits were assessed by western blot in 1) adult rats that had explored an open field (OF) for 5 minutes, a time sufficient to induce habituation, 2) mature rat hippocampal neuron cultures depolarized by KCl and 3) hippocampal slices from adult rats where long term potentiation (LTP) was induced by theta-burst stimulation (TBS). GluN1 and GluN2A, though not GluN2B, were significantly higher 70 minutes –but not 30 minutes- after a 5 minutes session in an OF. GluN1 and GluN2A total immunofluorescence and puncta in neurites increased in cultures, as evaluated 70 minutes after KCl stimulation. Similar changes were found in hippocampal slices 70 minutes after LTP induction. To start to explore underlying mechanisms, hippocampal slices were treated either with cycloheximide (a translation inhibitor) or actinomycin D (a transcription inhibitor) during electrophysiological assays. It was corroborated that translation was necessary for LTP induction and expression. The rise in GluN1 depends on transcription and translation, while the increase in GluN2A appears to mainly depend on translation, though a contribution of some remaining transcriptional activity during actinomycin D treatment could not be rouled out. LTP effective induction was required for the subunits to increase. Although in the three models same subunits suffered modifications in the same direction, within an apparently similar temporal course, further investigation is required to reveal if they are related processes and to find out whether they are causally related with synaptic plasticity, learning and memory.
doi:10.1371/journal.pone.0055244
PMCID: PMC3562335  PMID: 23383317
24.  Double Dissociation of Spike Timing–Dependent Potentiation and Depression by Subunit-Preferring NMDA Receptor Antagonists in Mouse Barrel Cortex 
Cerebral Cortex (New York, NY)  2009;19(12):2959-2969.
Spike timing–dependent plasticity (STDP) is a strong candidate for an N-methyl-D-aspartate (NMDA) receptor-dependent form of synaptic plasticity that could underlie the development of receptive field properties in sensory neocortices. Whilst induction of timing-dependent long-term potentiation (t-LTP) requires postsynaptic NMDA receptors, timing-dependent long-term depression (t-LTD) requires the activation of presynaptic NMDA receptors at layer 4-to-layer 2/3 synapses in barrel cortex. Here we investigated the developmental profile of t-LTD at layer 4-to-layer 2/3 synapses of mouse barrel cortex and studied their NMDA receptor subunit dependence. Timing-dependent LTD emerged in the first postnatal week, was present during the second week and disappeared in the adult, whereas t-LTP persisted in adulthood. An antagonist at GluN2C/D subunit–containing NMDA receptors blocked t-LTD but not t-LTP. Conversely, a GluN2A subunit–preferring antagonist blocked t-LTP but not t-LTD. The GluN2C/D subunit requirement for t-LTD appears to be synapse specific, as GluN2C/D antagonists did not block t-LTD at horizontal cross-columnar layer 2/3-to-layer 2/3 synapses, which was blocked by a GluN2B antagonist instead. These data demonstrate an NMDA receptor subunit-dependent double dissociation of t-LTD and t-LTP mechanisms at layer 4-to-layer 2/3 synapses, and suggest that t-LTD is mediated by distinct molecular mechanisms at different synapses on the same postsynaptic neuron.
doi:10.1093/cercor/bhp067
PMCID: PMC2774397  PMID: 19363149
development; LTD; LTP; rodent; synaptic plasticity
25.  Immunogold electron microscopic evidence of differential regulation of GluN1, GluN2A and GluN2B, NMDA-type glutamate receptor subunits in rat hippocampal CA1 synapses during benzodiazepine withdrawal 
The Journal of comparative neurology  2010;518(21):4311-4328.
Benzodiazepine withdrawal-anxiety is associated with enhanced AMPA receptor (AMPAR)-mediated glutamatergic transmission in rat hippocampal CA1 synapses due to enhanced synaptic insertion and phosphorylation of GluA1 homomers. Interestingly, attenuation of withdrawal-anxiety is associated with a reduction in NMDA receptor (NMDAR)-mediated currents and subunit expression, secondary to AMPA receptor potentiation. Therefore, in this study ultrastructural evidence for possible reductions in NMDAR GluN1, GluN2A and GluN2B subunits was sought at CA1 stratum radiatum synapses in proximal dendrites using postembedding immunogold labeling of tissues from rats withdrawn for 2-days from 1-week daily oral administration of the benzodiazepine, flurazepam (FZP). GluN1-immunogold density and the percentage of immunopositive synapses were significantly decreased in tissues from FZP-withdrawn rats. Similar decreases were observed for GluN2B subunits, however the relative lateral distribution of GluN2B-immunolabeling within the postsynaptic density did not change after BZ withdrawal. In contrast to the GluN2B subunit, the percentage of synapses labeled with the GluN2A subunit antibody and the density of immunogold labeling for this subunit was unchanged. The spatial localization of immunogold particles asssociated with each NMDAR subunit was consistent with a predominantly postsynaptic localization. The data therefore provide direct evidence for reduced synaptic GluN1/GluN2B receptors and preservation of GluN1/GluN2A receptors in the CA1 stratum radiatum region during BZ withdrawal. Based on collective findings in this benzodiazepine withdrawal-anxiety model, we propose a functional model illustrating the changes in glutamate receptor populations at excitatory synapses during benzodiazepine withdrawal.
doi:10.1002/cne.22458
PMCID: PMC2943829  PMID: 20853509
Electron microscopy; Plasticity; Dependence; Glutamate; LTP; Anxiety

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