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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.  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
4.  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(Pt 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
5.  Distinct functional and pharmacological properties of triheteromeric GluN1/GluN2A/GluN2B NMDA receptors 
Neuron  2014;81(5):1084-1096.
Summary
NMDA receptors are tetrameric ligand-gated ion channels comprised of GluN1, GluN2, and GluN3 subunits. Two different GluN2 subunits have been identified in most NMDA receptor-expressing cells, and the majority of native receptors are triheteromers containing two GluN1 and two different GluN2. In contrast to diheteromeric NMDA receptors, little is known about the function of triheteromers. We developed a method to provide selective cell-surface expression of recombinant GluN1/GluN2A/GluN2B triheteromers, and compared properties of these receptors with those of GluN1/GluN2A and GluN1/GluN2B diheteromers. We show that glutamate deactivation of triheteromers is distinct from those of GluN1/GluN2A and GluN1/GluN2B, and reveal modulation of triheteromers by subunit-selective antagonists ifenprodil, CP-101,606, TCN-201, and extracellular Zn2+. Furthermore, kinetic measurements suggest variation in the ifenprodil binding site of triheteromers compared to GluN1/GluN2B diheteromers. This work provides new insight to the distinct properties of GluN1/GluN2A/GluN2B triheteromers, which are presumably the most abundant NMDA receptors in the adult forebrain.
doi:10.1016/j.neuron.2014.01.035
PMCID: PMC3957490  PMID: 24607230
6.  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
7.  Structure-based discovery of antagonists for GluN3-containing N-methyl-D-aspartate receptors 
Neuropharmacology  2013;0:10.1016/j.neuropharm.2013.08.003.
NMDA receptors are ligand-gated ion channels that assemble into tetrameric receptor complexes composed of glycine-binding GluN1 and GluN3 subunits (GluN3A-B) and glutamate-binding GluN2 subunits (GluN2A-D). NMDA receptors can assemble as GluN1/N2 receptors and as GluN3-containing NMDA receptors, which are either glutamate/glycine-activated triheteromeric GluN1/N2/N3 receptors or glycine-activated diheteromeric GluN1/N3 receptors. The glycine-binding GluN1 and GluN3 subunits display strikingly different pharmacological selectivity profiles. However, the pharmacological characterization of GluN3-containing receptors has been hampered by the lack of methods and pharmacological tools to study GluN3 subunit pharmacology in isolation. Here, we have developed a method to study the pharmacology of GluN3 subunits in recombinant diheteromeric GluN1/N3 receptors by mutating the orthosteric ligand-binding pocket in GluN1. This method is suitable for performing compound screening and characterization of structure-activity relationship studies on GluN3 ligands. We have performed a virtual screen of the orthosteric binding site of GluN3A in the search for antagonists with selectivity for GluN3 subunits. In the subsequent pharmacological evaluation of 99 selected compounds, we identified 6-hydroxy-[1,2,5]oxadiazolo[3,4-b]pyrazin-5(4H)-one (TK80) a novel competitive antagonist with preference for the GluN3B subunit. Serendipitously, we also identified [2-hydroxy-5-((4-(pyridin-3-yl)thiazol-2-yl)amino]benzoic acid (TK13) and 4-(2,4-dichlorobenzoyl)-1H-pyrrole-2-carboxylic acid (TK30), two novel non-competitive GluN3 antagonists. These findings demonstrate that structural differences between the orthosteric binding site of GluN3 and GluN1 can be exploited to generate selective ligands.
doi:10.1016/j.neuropharm.2013.08.003
PMCID: PMC3865070  PMID: 23973313
NMDA receptor; GluN3 subunit; antagonist; selectivity; virtual screening; Xenopus oocyte electrophysiology
8.  Distinct pharmacological and functional properties of NMDA receptors in mouse cortical astrocytes 
British Journal of Pharmacology  2011;163(8):1755-1766.
BACKGROUND AND PURPOSE
Astrocytes of the mouse neocortex express functional NMDA receptors, which are not blocked by Mg2+ ions. However, the pharmacological profile of glial NMDA receptors and their subunit composition is far from complete.
EXPERIMENTAL APPROACH
We tested the sensitivity of NMDA receptor-mediated currents to the novel GluN2C/D subunit-selective antagonist UBP141 in mouse cortical astrocytes and neurons. We also examined the effect of memantine, an antagonist that has substantially different affinities for GluN2A/B and GluN2C/d-containing receptors in physiological concentrations of extracellular Mg2+.
KEY RESULTS
UBP141 had a strong inhibitory action on NMDA receptor-mediated transmembrane currents in the cortical layer II/III astrocytes with an IC50 of 2.29 µM and a modest inhibitory action on NMDA-responses in the pyramidal neurons with IC50 of 19.8 µM. Astroglial and neuronal NMDA receptors exhibited different sensitivities to memantine with IC50 values of 2.19 and 10.8 µM, respectively. Consistent with pharmacological differences between astroglial and neuronal NMDA receptors, NMDA receptors in astrocytes showed lower Ca2+ permeability than neuronal receptors with PCa/PNa ratio of 3.4.
CONCLUSIONS AND IMPLICATIONS
The biophysical and pharmacological properties of the astrocytic NMDA receptors strongly suggest that they have a tri-heteromeric structure composed of GluN1, GluN2C/D and GluN3 subunits. The substantial difference between astroglial and neuronal NMDA receptors in their sensitivity to UBP141 and memantine may enable selective modulation of astrocytic signalling that could be very helpful for elucidating the mechanisms of neuron-glia communications. Our results may also provide the basis for the development of novel therapeutic agents specifically targeting glial signalling.
doi:10.1111/j.1476-5381.2011.01374.x
PMCID: PMC3166701  PMID: 21449975
Memantine; UBP141; NMDA receptor; GluN3A subunit; calcium permeability; neuron-glia communication; GluN3B; mouse cortex
9.  Synaptic GluN2A and GluN2B Containing NMDA Receptors within the Superficial Dorsal Horn Activated following Primary Afferent Stimulation 
The Journal of Neuroscience  2014;34(33):10808-10820.
NMDA receptors are important elements in pain signaling in the spinal cord dorsal horn. They are heterotetramers, typically composed of two GluN1 and two of four GluN2 subunits: GluN2A-2D. Mice lacking some of the GluN2 subunits show deficits in pain transmission yet functional synaptic localization of these receptor subtypes in the dorsal horn has not been fully resolved. In this study, we have investigated the composition of synaptic NMDA receptors expressed in monosynaptic and polysynaptic pathways from peripheral sensory fibers to lamina I neurons in rats. We focused on substance P receptor-expressing (NK1R+) projection neurons, critical for expression of hyperalgesia and allodynia. EAB-318 and (R)-CPP, GluN2A/B antagonists, blocked both monosynaptic and polysynaptic NMDA EPSCs initiated by primary afferent activation by ∼90%. Physiological measurements exploiting the voltage dependence of monosynaptic EPSCs similarly indicated dominant expression of GluN2A/B types of synaptic NMDA receptors. In addition, at synapses between C fibers and NK1R+ neurons, NMDA receptor activation initiated a secondary, depolarizing current. Ifenprodil, a GluN2B antagonist, caused modest suppression of monosynaptic NMDA EPSC amplitudes, but had a widely variable, sometimes powerful, effect on polysynaptic responses following primary afferent stimulation when inhibitory inputs were blocked to mimic neuropathic pain. We conclude that GluN2B subunits are moderately expressed at primary afferent synapses on lamina I NK1R+ neurons, but play more important roles for polysynaptic NMDA EPSCs driven by primary afferents following disinhibition, supporting the view that the analgesic effect of the GluN2B antagonist on neuropathic pain is at least in part, within the spinal cord.
doi:10.1523/JNEUROSCI.0145-14.2014
PMCID: PMC4131007  PMID: 25122884
disinhibition; ifenprodil; NK1 receptor; polysynaptic; spinal cord
10.  Mapping the high-affinity binding domain of 5-substituted benzimidazoles to the proximal N-terminus of the GluN2B subunit of the NMDA receptor 
British Journal of Pharmacology  2010;159(2):449-461.
Background and purpose:
N-methyl-D-aspartate (NMDA) receptors represent an attractive drug target for the treatment of neurological and neurodegenerative disorders associated with glutamate-induced excitotoxicity. The aim of this study was to map the binding domain of high affinity 5-substituted benzimidazole derivatives [N-{2-[(4-benzylpiperidin-1-yl)methyl]benzimidazol-5-yl}methanesulphonamide (XK1) and N-[2-(4-phenoxybenzyl)benzimidazol-5-yl]methanesulphonamide (XK2)] on the GluN2B subunit of the NMDA receptor.
Experimental approach:
The pharmacological antagonistic profiles of XK1 and XK2 were assessed using in vitro rat primary cerebrocortical neurones and two-electrode voltage clamp on Xenopus oocytes expressing heterologous GluN1/GluN2B receptors. Direct ligand binding was determined using the recombinant amino-terminal domain (ATD) of GluN2B.
Key results:
XK1 and XK2 effectively protected against NMDA-induced excitotoxicity in rat primary cortical neurones. Low concentrations of XK1 (10 nM) and XK2 (1 nM) significantly reversed neuronal death. Both compounds failed to inhibit currents measured from oocytes heterologously expressing GluN1-1a subunit co-assembled with the ATD-deleted GluN2B subunit. XK1 and XK2 showed specific binding to recombinant protein of GluN2B ATD with low nanomolar affinities. Several residues in the recombinant ATD of GluN2B were identified to be critical for conferring XK1 and XK2 sensitivity. The inhibitory effects of XK1 and XK2 were pH-sensitive, being increased at acidic pH.
Conclusions and implications:
These results demonstrate that XK1 and XK2 are effective neuroprotective agents in vitro and indicate that 5-substituted benzimidazole derivatives inhibit GluN1/GluN2B receptors via direct binding to the ATD of the GluN2B subunit. These compounds represent valuable alternatives to the classical antagonist ifenprodil as pharmacological tools for studying GluN2B-containing NMDA receptors.
doi:10.1111/j.1476-5381.2009.00549.x
PMCID: PMC2825366  PMID: 20082612
NMDA receptors; benzimidazole; recombinant protein; amino-terminal domain; GluN2B subunit; circular dichroism; ifenprodil
11.  Deletion of the N-terminal Domain (NTD) Alters the Ethanol Inhibition of NMDA Receptors in a Subunit-Dependent Manner 
Alcoholism, clinical and experimental research  2013;37(11):10.1111/acer.12168.
Background
Ethanol inhibition of NMDA receptors is poorly understood due in part to the organizational complexity of the receptor that provides ample locations for sites of action. Among these the N-terminal domain of NMDA receptor subunits contains binding sites for a variety of modulatory agents including zinc, protons and GluN2B selective antagonists such as ifenprodil or Ro-25–6981. Ethanol inhibition of neuronal NMDA receptors expressed in some brain areas has been reported to be occluded by the presence of ifenprodil or similar compounds suggesting that the N-terminal domain may be important in regulating the ethanol sensitivity of NMDA receptors.
Methods
Wild-type GluN1 and GluN2 subunits and those in which the coding sequence for the N-terminal domain was deleted were expressed in HEK293 cells. Whole-cell voltage-clamp recording was used to assess ethanol inhibition of wild-type and mutant receptors lacking the N-terminal domain.
Results
As compared to wild-type GluN1/GluN2A receptors, ethanol inhibition was slightly greater in cells expressing GluN2A subunits lacking the N-terminal domain. In contrast, GluN2B N-terminal deletion mutants showed normal ethanol inhibition while those lacking the N-terminal domain in both GluN1 and GluN2B subunits had decreased ethanol inhibition as compared to wild-type receptors. N-terminal domain lacking GluN2B receptors were insensitive to ifenprodil but retained normal sensitivity to ethanol.
Conclusions
These findings indicate that the N-terminal domain modestly influences the ethanol sensitivity of NMDA receptors in a subunit-dependent manner. They also show that ifenprodil’s actions on GluN2B containing receptors can be dissociated from those of ethanol. These results suggest that while the N-terminal domain is not a primary site of action for ethanol on NMDA receptors, it likely affects sensitivity via actions on intrinsic channel properties.
doi:10.1111/acer.12168
PMCID: PMC3812356  PMID: 23905549
Ethanol; NMDA receptors; N-terminal domain; glutamate
12.  GluN2B-containing NMDA receptors regulate depression-like behavior and are critical for the rapid antidepressant actions of ketamine 
eLife  null;3:e03581.
A single, low dose of the NMDA receptor antagonist ketamine produces rapid antidepressant actions in treatment-resistant depressed patients. Understanding the cellular mechanisms underlying this will lead to new therapies for treating major depression. NMDARs are heteromultimeric complexes formed through association of two GluN1 and two GluN2 subunits. We show that in vivo deletion of GluN2B, only from principal cortical neurons, mimics and occludes ketamine's actions on depression-like behavior and excitatory synaptic transmission. Furthermore, ketamine-induced increases in mTOR activation and synaptic protein synthesis were mimicked and occluded in 2BΔCtx mice. We show here that cortical GluN2B-containing NMDARs are uniquely activated by ambient glutamate to regulate levels of excitatory synaptic transmission. Together these data predict a novel cellular mechanism that explains ketamine's rapid antidepressant actions. In this model, basal glutamatergic neurotransmission sensed by cortical GluN2B-containing NMDARs regulates excitatory synaptic strength in PFC determining basal levels of depression-like behavior.
DOI: http://dx.doi.org/10.7554/eLife.03581.001
eLife digest
Depression is the leading cause of disability worldwide, with hundreds of millions of people living with the condition. The ‘gold standard’ for depression treatment involves a combination of psychotherapy and medication. Unfortunately, current antidepressant medications do not help everyone, waiting lists for psychotherapy are often long, and both normally take a number of weeks of regular treatment before they begin to have an effect. As patients are often at a high risk of suicide, it is crucial that treatments that act more quickly, and that are safe and effective, are developed.
One substance that may fulfill these requirements is a drug called ketamine. Studies have shown that depression symptoms can be reduced within hours by a single low dose of ketamine, and this effect on mood can last for more than a week. However, progress has been hindered by a lack of knowledge about what ketamine actually does inside the brain.
Neurons communicate with one another by releasing chemicals known as neurotransmitters, which transfer information by binding to receptor proteins on the surface of other neurons. Drugs such as ketamine also bind to these receptors. Ketamine works by blocking a specific receptor called the n-methyl d-aspartate (NMDA) receptor, but how this produces antidepressant effects is not fully understood.
The NMDA receptor is actually formed from a combination of individual protein subunits, including one called GluN2B. Now Miller, Yang et al. have created mice that lack receptors containing these GluN2B subunits in neurons in their neocortex, including the prefrontal cortex, a brain region involved in complex mental processes such as decision-making. This allowed Miller, Yang et al. to discover that when the neurotransmitter glutamate binds to GluN2B-containing NMDA receptors, it limits the production of certain proteins that make it easier for signals to be transmitted between neurons. Suppressing the synthesis of these proteins too much may cause depressive effects by reducing communication between the neurons in the prefrontal cortex.
Both mice lacking GluN2B-containing receptors in their cortical neurons and normal mice treated with ketamine showed a reduced amount of depressive-like behavior. This evidence supports Miller, Yang et al.'s theory that by blocking these NMDA receptors, ketamine restricts their activation. This restores normal levels of protein synthesis, improves communication between neurons in the cortex, and reduces depression.
Understanding how ketamine works to alleviate depression is an important step towards developing it into a safe and effective treatment. Further research is also required to determine the conditions that cause overactivation of the GluN2B-containing NMDA receptors.
DOI: http://dx.doi.org/10.7554/eLife.03581.002
doi:10.7554/eLife.03581
PMCID: PMC4270067  PMID: 25340958
depression; cortex; synapse; ketamine; electrophysiology; protein synthesis; mouse; rat
13.  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
14.  Structure-activity relationships for allosteric NMDA receptor inhibitors based on 2-naphthoic acid 
Neuropharmacology  2011;62(4):1730-1736.
Over-activation of N-methyl-D-aspartate (NMDA) receptors is critically involved in many neurological conditions, thus there has been considerable interest in developing NMDA receptor antagonists. We have recently identified a series of naphthoic and phenanthroic acid compounds that allosterically modulate NMDA receptors through a novel mechanism of action. In the present study, we have determined the structure-activity relationships of 18 naphthoic acid derivatives for the ability to inhibit the four GluN1/GluN2(A-D) NMDA receptor subtypes. 2-Naphthoic acid has low activity at GluN2A-containing receptors and yet lower activity at other NMDA receptors. 3-Amino addition, and especially 3-hydroxy addition, to 2-naphthoic acid increased inhibitory activity at GluN1/GluN2C and GluN1/GluN2D receptors. Further halogen and phenyl substitutions to 2-hydroxy-3-naphthoic acid leads to several relatively potent inhibitors, the most potent of which is UBP618 (1-bromo-2-hydroxy-6-phenylnaphthalene-3-carboxylic acid) with an IC50 ~ 2 μM at each of the NMDA receptor subtypes. While UBP618 is non-selective, elimination of the hydroxyl group in UBP618, as in UBP628 and UBP608, leads to an increase in GluN1/GluN2A selectivity. Of the compounds evaluated, specifically those with a 6-phenyl substitution were less able to fully inhibit GluN1/GluN2A, GluN1/GluN2B and GluN1/GluN2C responses (maximal % inhibition of 60 – 90%). Such antagonists may potentially have reduced adverse effects by not excessively blocking NMDA receptor signaling. Together, these studies reveal discrete structure-activity relationships for the allosteric antagonism of NMDA receptors that may facilitate the development of NMDA receptor modulator agents for a variety of neuropsychiatric and neurological conditions.
doi:10.1016/j.neuropharm.2011.11.019
PMCID: PMC3269548  PMID: 22155206
15.  The Effects of NMDA Subunit Composition on Calcium Influx and Spike Timing-Dependent Plasticity in Striatal Medium Spiny Neurons 
PLoS Computational Biology  2012;8(4):e1002493.
Calcium through NMDA receptors (NMDARs) is necessary for the long-term potentiation (LTP) of synaptic strength; however, NMDARs differ in several properties that can influence the amount of calcium influx into the spine. These properties, such as sensitivity to magnesium block and conductance decay kinetics, change the receptor's response to spike timing dependent plasticity (STDP) protocols, and thereby shape synaptic integration and information processing. This study investigates the role of GluN2 subunit differences on spine calcium concentration during several STDP protocols in a model of a striatal medium spiny projection neuron (MSPN). The multi-compartment, multi-channel model exhibits firing frequency, spike width, and latency to first spike similar to current clamp data from mouse dorsal striatum MSPN. We find that NMDAR-mediated calcium is dependent on GluN2 subunit type, action potential timing, duration of somatic depolarization, and number of action potentials. Furthermore, the model demonstrates that in MSPNs, GluN2A and GluN2B control which STDP intervals allow for substantial calcium elevation in spines. The model predicts that blocking GluN2B subunits would modulate the range of intervals that cause long term potentiation. We confirmed this prediction experimentally, demonstrating that blocking GluN2B in the striatum, narrows the range of STDP intervals that cause long term potentiation. This ability of the GluN2 subunit to modulate the shape of the STDP curve could underlie the role that GluN2 subunits play in learning and development.
Author Summary
The striatum of the basal ganglia plays a key role in fluent motor control; pathology in this structure causes the motor symptoms of Parkinson's Disease and Huntington's Chorea. A putative cellular mechanism underlying learning of motor control is synaptic plasticity, which is an activity dependent change in synaptic strength. A known mediator of synaptic potentiation is calcium influx through the NMDA-type glutamate receptor. The NMDA receptor is sensitive to the timing of neuronal activity, allowing calcium influx only when glutamate release and a post-synaptic depolarization coincide temporally. The NMDA receptor is comprised of specific subunits that modify its sensitivity to neuronal activity and these subunits are altered in animal models of Parkinson's disease. Here we use a multi-compartmental model of a striatal neuron to investigate the effect of different NMDA subunits on calcium influx through the NMDA receptor. Simulations show that the subunit composition changes the temporal intervals that allow coincidence detection and strong calcium influx. Our experiments manipulating the dominate subunit in brain slices show that the subunit effect on calcium influx predicted by our computational model is mirrored by a change in the amount of potentiation that occurs in our experimental preparation.
doi:10.1371/journal.pcbi.1002493
PMCID: PMC3334887  PMID: 22536151
16.  Pharmacological Modulation of NMDA Receptor Activity and the Advent of Negative and Positive Allosteric Modulators 
Neurochemistry international  2012;61(4):581-592.
The NMDA receptor (NMDAR) family of L-glutamate receptors are well known to have diverse roles in CNS function as well as in various neuropathological and psychiatric conditions. Until recently, the types of agents available to pharmacologically regulate NMDAR function have been quite limited in terms of mechanism of action and subtype selectivity. This has changed significantly in the past two years. The purpose of this review is to summarize the many drug classes now available for modulating NMDAR activity. Previously, this included competitive antagonists at the L-glutamate and glycine binding sites, high and low affinity channel blockers, and GluN2B-selective N-terminal domain binding site antagonists. More recently, we and others have identifed new classes of NMDAR agents that are either positive or negative allosteric modulators (PAMs and NAMs, respectively). These compounds include the pan potentiator UBP646, the GluN2A-selective potentiator/GluN2C & GluN2D inhibitor UBP512, the GluN2D-selective potentiator UBP551, the GluN2C/GluN2D-selective potentiator CIQ as well as the new NMDAR-NAMs such as the pan-inhibitor UBP618, the GluN2C/GluN2D-selective inhibitor QZN46 and the GluN2A inhibitors UBP608 and TCN201. These new agents do not bind within the L-glutamate or glycine binding sites, the ion channel pore or the N-terminal regulatory domain. Collectively, these new allosteric modulators appear to be acting at multiple novel sites on the NMDAR complex. Importantly, these agents display improved subtype-selectivity and as NMDAR PAMs and NAMs, they represent a new generation of potential NMDAR therapeutics.
doi:10.1016/j.neuint.2012.01.004
PMCID: PMC3360989  PMID: 22269804
NMDA receptors; allosteric modulators; glycine; potentiators; competitive inhibitors; channel blockers; antagonists
17.  Subtype selective NMDA receptor antagonists induce recovery of synapses lost following exposure to HIV-1 Tat 
British Journal of Pharmacology  2012;166(3):1002-1017.
BACKGROUND AND PURPOSE
Neurocognitive disorders afflict approximately 20% of HIV-infected patients. HIV-1-infected cells in the brain shed viral proteins such as transactivator of transcription (Tat). Tat elicits cell death and synapse loss via processes initiated by NMDA receptor activation but mediated by separate downstream signalling pathways. Subunit selective NMDA receptor antagonists may differentially modulate survival relative to synaptic changes.
EXPERIMENTAL APPROACH
Tat-evoked cell death was quantified by measuring propidium iodide uptake into rat hippocampal neurons in culture. The effects of Tat on synaptic changes were measured using an imaging-based assay that quantified clusters of the scaffolding protein postsynaptic density 95 fused to green fluorescent protein.
KEY RESULTS
Dizocilpine, a non-competitive NMDA receptor antagonist, inhibited Tat-induced synapse loss, subsequent synapse recovery and Tat-induced cell death with comparable potencies. Memantine (10 µM) and ifenprodil (10 µM), which preferentially inhibit GluN2B-containing NMDA receptors, protected from Tat-induced cell death with no effect on synapse loss. Surprisingly, memantine and ifenprodil induced synapse recovery in the presence of Tat. In contrast, the GluN2A-prefering antagonist TCN201 prevented synapse loss and recovery with no effect on cell death.
CONCLUSIONS AND IMPLICATIONS
Synapse loss is a protective mechanism that enables the cell to cope with excess excitatory input. Thus, memantine and ifenprodil are promising neuroprotective drugs because they spare synaptic changes and promote survival. These GluN2B-preferring drugs induced recovery from Tat-evoked synapse loss, suggesting that synaptic pharmacology changed during the neurotoxic process. NMDA receptor subtypes differentially participate in the adaptation and death induced by excitotoxic insult.
doi:10.1111/j.1476-5381.2011.01805.x
PMCID: PMC3417425  PMID: 22142193
HIV Tat; neurotoxicity; dizocilpine; memantine; ifenprodil; TCN201; synapse loss; NMDA receptor; synapse recovery; HIV-associated neurocognitive disorders (HAND)
18.  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
19.  Effects of NMDA receptor antagonists with different subtype selectivities on retinal spreading depression 
British Journal of Pharmacology  2012;165(1):235-244.
BACKGROUND AND PURPOSE
Spreading depression (SD) is a local, temporary disruption of cellular ionic homeostasis that propagates slowly across the cerebral cortex and other neural tissues such as the retina. Spreading depolarization associated with SD occurs in different types of stroke, and this phenomenon correlates also with the initiation of classical migraine aura. The aim of this study was to investigate how NMDA receptor antagonists with different subtype selectivity alter SD.
EXPERIMENTAL APPROACH
Immunoblotting was applied to the chick retina for NMDA receptor subunit protein analysis, and an efficient in vitro chick retinal model used with SD imaging for NMDA receptor pharmacology.
KEY RESULTS
The prominent NMDA receptor subtypes GluN1, GluN2A and GluN2B were found highly expressed in the chick retina. Nanomolar concentrations of NVP-AAM077 (GluN2A-preferring receptor antagonist) markedly suppressed high K+-induced SD; that is, ∼30 times more effectively than MK801. At sub-micromolar concentrations, Ro 25-6981 (GluN2B-preferring receptor antagonist) produced a moderate SD inhibition, whereas CP-101,606 (also GluN2B-preferring receptor antagonist) and UBP141 (GluN2C/2D-preferring receptor antagonist) had no effect.
CONCLUSIONS AND IMPLICATIONS
The expression of major NMDA receptor subtypes, GluN1, GluN2A and GluN2B in the chick retina makes them pertinent targets for pharmacological inhibition of SD. The high efficacy of NVP-AAM077 on SD inhibition suggests a critical role of GluN2A-containing receptors in SD genesis. Such high anti-SD potency suggests that NVP-AAM077, and other GluN2A-selective drug-like candidates, could be potential anti-migraine agents.
doi:10.1111/j.1476-5381.2011.01553.x
PMCID: PMC3252980  PMID: 21699507
spreading depression; NMDA receptor subtypes; NVP-AAM077; GluN2A; GluN2A/2B; immunoblotting; chick retina; GluN2B; CP-101; 606; GluN2D; UBP141
20.  Selective inhibition of GluN2D-containing N-methyl-D-aspartate receptors prevents tissue plasminogen activator-promoted neurotoxicity both in vitro and in vivo 
Background
Tissue plasminogen activator (tPA) exerts multiple functions in the central nervous system, depending on the partner with which it interacts. In particular, tPA acts as a positive neuromodulator of N-methyl-D-aspartate glutamatergic receptors (NMDAR). At the molecular level, it has been proposed that the pro-neurotoxicity mediated by tPA might occur through extrasynaptic NMDAR containing the GluN2D subunit. Thus, selective antagonists targeting tPA/GluN2D-containing NMDAR signaling would be of interest to prevent noxious effects of tPA.
Results
Here, we compared three putative antagonists of GluN2D-containing NMDAR and we showed that the new compound UBP145 ((2R*,3S*)-1-(9-bromophenan-threne-3-carbonyl)piperazine-2,3-dicarboxylic acid) is far more selective for GluN2D subunits than memantine and PPDA (phenanthrene derivative (2S*, 3R*)-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid). Indeed, in vitro, in contrast to the two other compounds, UBP145 prevented NMDA toxicity only in neurons expressing GluN2D (ie, in cortical but not hippocampal neurons). Furthermore, in cultured cortical neurons, UBP145 fully prevented the pro-excitotoxic effect of tPA. In vivo, we showed that UBP145 potently prevented the noxious action of exogenous tPA on excitotoxic damages. Moreover, in a thrombotic stroke model in mice, administration of UBP145 prevented the deleterious effect of late thrombolysis by tPA.
Conclusions
In conclusion, tPA exerts noxious effects on neurons by acting on GluN2D-containing NMDAR and pharmacological antagonists of GluN2D-containing NMDAR could be used to prevent the ability of tPA to promote neurotoxicity.
doi:10.1186/1750-1326-6-68
PMCID: PMC3204249  PMID: 21975018
Tissue plasminogen activator; GluN2D; UBP145; excitotoxicity; stroke; glutamatergic neurotransmission
21.  Long-Term Ethanol and Corticosterone Co-Exposure Sensitize the Hippocampal CA1 Region Pyramidal Cells to Insult During Ethanol Withdrawal in an NMDA GluN2B Subunit-Dependent Manner 
Background
Chronic ethanol (EtOH) exposure produces neuroadaptations in NMDA receptor function and/or abundance and alterations in hypothalamic–pituitary–adrenal (HPA) axis functioning that contribute to neuronal excitation and neurotoxicity during ethanol withdrawal (EWD). Both EtOH and corticosterone (CORT) promote synthesis of polyamines, which allosterically potentiate NMDA receptor function at the GluN2B subunit. The current studies investigated the effect of 10-day EtOH and CORT co-exposure on toxicity during EWD in rat hippocampal explants and hypothesized that alterations in function and/or density of GluN2B subunits contribute to the toxicity.
Methods
Organotypic hippocampal slice cultures were exposed to CORT (0.01–1.0 μM) during 10-day EtOH exposure (50 mM) and 1 day of EWD. EtOH-naïve cultures were exposed to CORT for 11 days. Additional cultures were exposed to a membrane impermeable form of CORT (BSA-CORT) with and without 10-day EtOH exposure and EWD. Cytotoxicity (uptake of propidium iodide) was assessed in the pyramidal cell layer of the CA1 region. Western blot analysis was employed to assess the density of GluN2B subunits following EtOH and CORT exposure.
Results
EWD did not produce overt neurotoxicity. However, co-exposure to EtOH/EWD and CORT produced significant neurotoxicity in the CA1 region pyramidal cell layer. Ifenprodil, a GluN2B polyamine site antagonist, significantly reduced toxicity from EtOH and CORT (0.1 μM) co-exposure during EWD. However, Western blots did not reveal differences in GluN2B subunit density among groups. Exposure to BSA-CORT did not produce toxicity, suggesting that membrane-bound CORT receptors did not significantly contribute to the observed toxicity.
Conclusions
These data suggest that CORT and EtOH co-exposure result in increased function of polyamine-sensitive GluN2B subunits, but this toxicity does not appear dependent on the abundance of hippocampal NMDA GluN2B subunits or membrane-bound CORT receptor function.
doi:10.1111/acer.12195
PMCID: PMC4166417  PMID: 23889203
Alcohol Dependence; HPA Axis; Ifenprodil; NMDA Receptor
22.  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
23.  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
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.  Probing NMDA receptor GluN2A and GluN2B subunit expression and distribution in cortical neurons 
Neuropharmacology  2014;79:542-549.
The spatial distribution of N-methyl-D-aspartate receptor (NMDAR) subunits in layer 5 (L5) neurons of the medial prefrontal cortex (mPFC) is important for integrating input-output signals involved in cognitive functions and motor behavior. In this study, focal laser scanning photostimulation of caged glutamate, slice electrophysiology, and small peptide pharmacology, were used to map the distribution of functional GluN2A and GluN2B subunits of the NMDAR from L5 neurons of wild-type (WT) and GluN2A−/− mice. Focal uncaging of glutamate evoked spatially-restricted glutamatergic responses on various dendritic locations of pyramidal neurons in the mPFC. Analyses of the spatial arrangements of the GluN2A and GluN2B subunits were performed by comparing inhibition of glutamatergic responses in the presence of the GluN2A-selective pharmacological antagonist, NVP-AAM077 (NVP), and the GluN2B-selective peptidic antagonist, conantokin-G (con-G). We found that apical and basal expression and distribution of GluN2A and GluN2B were similar in L5 mPFC neurons of WT mice. However, the inhibition of glutamatergic responses by NVP in brain slices of GluN2A−/− mice were dramatically decreased, while con-G inhibition remained similar to that observed in WT brain slices. The data obtained show that expression and spatial arrangement of GluN2B subunits is independent of GluN2A in L5 neurons of the mPFC. These findings have important ramifications for NMDAR organization and function in L5 pyramidal neurons of the mPFC, and show that specific populations of NMDARs can be antagonized, while sparing other subgroups of NMDARs, thus preserving selective NMDAR functions, an important therapeutic advantage.
doi:10.1016/j.neuropharm.2014.01.005
PMCID: PMC3951114  PMID: 24440368
NMDARs; pyramidal L5 neurons; conantokins; slice electrophysiology; medial prefrontal cortex; laser-scanning photostimulation

Results 1-25 (816744)