The magnitude of hippocampal long term depression depends on the synaptic location of activated NR2-containing N-methyl-D-aspartate receptors

Activation of N-methyl-d-aspartate receptors (NMDARs) is the first step in the induction of certain forms of synaptic plasticity in the hippocampus. In the adult rat hippocampus, NMDARs are composed almost exclusively of NR1 and NR2 subunits with NR1 subunits being mainly associated with either NR2A and/or NR2B subunits. The role played by the different subunits in synaptic plasticity is still controversial. In the present study, we used two different long term depression (LTD) -inducing protocols (electrical and chemical stimulation) to show that activation of NR2A-containing NMDAR subunits leads to the induction of LTD. We also demonstrated that extrasynaptic NR2B-containing NMDARs regulate the magnitude of LTD by exerting a control over the function of synaptic NR2A-containing NMDARs while having no effect on plasticity in the absence of synaptic receptor activation. Taken as a whole, these experiments demonstrate that NMDAR subunits play different roles according to their nature (NR2A or NR2B) and location (synaptic versus extrasynaptic). This sheds new light on the functional role of extrasynaptic NR2B containing-NMDARs. These results are particularly important for a better understanding of certain pathological disorders associated with glutamatergic overactivity.     

NMDA receptor desensitization regulated by direct binding to PDZ1-2 domains of PSD-95

Regulation of N-methyl-d-aspartate receptor (NMDAR) activity by desensitization is important in physiological and pathological states; NMDAR desensitization contributes in shaping synaptic responses and may be protective by limiting calcium influx during sustained glutamate insults. We previously reported that glycine-independent desensitization decreases during hippocampal neuronal development, correlating with NMDAR synaptic localization and association with postsynaptic density 95 (PSD-95). PSD-95/Discs large/zona occludens (PDZ)-1,2 domains of PSD-95 bind to the C-terminus of NMDAR NR2 subunits. The role of PSD-95 in anchoring signaling proteins near NMDARs is well documented. To determine if PSD-95-induced changes in NMDAR desensitization occur because of direct binding to NR2 or due to recruitment of regulatory proteins, we tested the effects of various PSD-95 constructs on NMDAR currents in human embryonic kidney 293 (HEK293) cells and neurons. In HEK cells, wild-type PSD-95 significantly reduced wild-type NMDAR desensitization without altering currents of NMDARs containing NR2A-S1462A, a mutation that abolishes PSD-95 binding. The PSD-95 N-terminus truncated after the PDZ1-2 domains was sufficient for this effect in neurons with low endogenous PSD-95 levels; in NMDAR-expressing HEK cells, the effect persisted when PSD-95 multimerization was eliminated. Moreover other PSD-95 family members with highly homologous PDZ1-2 domains significantly reduced NMDAR desensitization. In mature neurons, disruption of PSD-95/NMDAR interaction through protein kinase C (PKC) activation increased desensitization to levels found in immature neurons, and this effect was not due to PKC direct regulation of NMDAR activity. We conclude that direct binding of PSD-95 increases stability of NMDAR responses to agonist exposure in neuronal and nonneuronal cells.     

NR2A/B-containing NMDA receptors mediate cocaine-induced synaptic plasticity in the VTA and cocaine psychomotor sensitization

Cocaine-induced modifications of glutamatergic synaptic transmission in the mesolimbic system play a key role in adaptations that promote addictive behaviors. In particular, the activation of ionotropic glutamate N-methyl-d-aspartate receptor (NMDAR) in the ventral tegmental area (VTA) is critical for both cocaine-induced synaptic plasticity induced by a single cocaine injection and for the initiation of cocaine psychomotor sensitization. In this study, we set to determine whether the NR2 subunits of the NMDAR play a specific role in triggering cocaine-induced alterations in synaptic plasticity and the development of psychomotor sensitization. We found that inhibition of NR2A-containing NMDARs by NVP-AAM077, or NR2B-containing receptors by ifenprodil, blocked cocaine-induced increase in the AMPAR/NMDAR currents ratio, a measure of long-term potentiation (LTP) in vivo, in VTA neurons 24 h following a single cocaine injection. Furthermore, inhibition of the NR2A subunit during the development of psychomotor sensitization attenuated the enhanced locomotor activity following repeated cocaine injections. Together, these results suggest that NR2-containing NMDA receptors play an important role in the machinery that triggers synaptic and behavioral adaptations to drugs of abuse such as cocaine.     

Hippocampal long-term synaptic plasticity and signal amplification of NMDA receptors

The direction of plasticity at CA3-CA1 hippocampal synapses is determined by the strength of afferent stimulation. Weak stimuli lead to long-term depression (LTD) and strong stimuli to long-term potentiation (LTP), but both require activation of synaptic N-methyl-D-aspartate receptors (NMDARs). These receptors are therefore necessary and required for the induction of plasticity at CA3-CA1 synapses even though they carry little of the current responsible for the basal excitatory post-synaptic potential (EPSP). The influx of Ca(2+) via NMDARs triggers the subsequent and persistent changes in the expression of alpha-amino-3-hydroxy-5 methylisoxazole-4-proprionic acid receptors (AMPARs) and these receptors are responsible for the major part of the basal EPSP. The degree of activity of NMDARs is determined in part by extracellular Mg(2+) and by the co-agonists for this receptor, glycine and D-serine. During strong stimulation, a relief of the voltage-dependent block of NMDARs by Mg(2+) provides a positive feedback for NMDAR Ca(2+) influx into postsynaptic CA1 spines. In this review, we discuss how the induction of LTP at CA3-CA1 synapses requires further signal amplification of NMDAR activity. We discuss how the regulation of NMDARs by protein kinases and phosphatases is brought into play. Evidence is presented that Src family kinases (SFKs) play a "core" role in the induction of LTP by enhancing the function and expression of NMDARs. At CA3-CA1 synapses, NMDARs are largely composed of NR1 (NMDA receptor subunit 1)-NR2A or NR1-NR2B containing subunits. Recent, but controversial, evidence has correlated NR1-NR2A receptors with the induction of LTP and NR1-NR2B receptors with LTD. However, LTP can be induced by activation of either subtype of NMDAR and the ratio of NR2A:NR2B receptors has been proposed as an alternative determinant of the direction of synaptic plasticity. Many transmitters and signal pathways can modify NMDAR function and expression and, for a given stimulus strength, they can potentially lead to a change in the balance between LTP and LTD. As opposed to the "core" mechanisms of LTP and LTD, the resulting alterations in this balance underlie "meta-plasticity." Thus, in addition to their contribution to core mechanisms, we will also discuss how Src-family kinases could preferentially target NR1-NR2A or NR1-NR2B receptors to alter the relative contribution of these receptor subtypes to synaptic plasticity.     

Immunolocalization of NR1, NR2A, and PSD-95 in rat hippocampal subregions during postnatal development

Although the expression of NMDARs and synaptic-associated proteins has been widely studied, the temporospatial distribution of NMDAR subunits and synaptic proteins in different hippocampal subregions during postnatal development still lacks detailed information, and the relationship between NR1 or NR2 subunits and PSD-95 family proteins is controversial. In this study, we used immunofluorescent staining to assess NR1 or NR2A and PSD-95 expressions and the relationship between them in CA1, CA3, and DG of rat hippocampus on postnatal (P) days: P0, P4, P7, P10, P14, P21, P28, P56. The results showed that from P0 to P56, NR1, NR2A, and PSD-95 expressions increased gradually, and the time points of their expression peak differed in CA1, CA3, and DG during postnatal development. Interestingly, although the expression of PSD-95 was positively correlated to both NR1 and NR2A, the NR1 and PSD-95 coexpressed puncta were greatest in CA3, while NR2A and PSD-95 coexpressed puncta were greatest in CA1, compared to other subregions. Surprisingly, at P21, among different strata of CA1, the area of highest expression of NR2A was dramatically changed from stratum pyramidale to stratum polymorphum and stratum moleculare, and returned to stratum pyramidale gradually on the later observed days again, indicating that P21 may be one critical timepoint during postnatal development in CA1. The specific temporospatial distribution pattern of NR1, NR2A, and PSD-95 might be related to the different physiological functions during postnatal development. Discovering the alteration of the relationship between PSD-95 and NMDAR subunits expression may be helpful for understanding mechanisms and therapy of neurodegenerative diseases.     

NMDA receptor antagonists reveal age-dependent differences in the properties of visual cortical plasticity

The suggestion that NMDA receptor (NMDAR)-dependent plasticity is subunit specific, with NR2B-types required for long-term depression (LTD) and NR2A-types critical for the induction of long-term potentiation (LTP), has generated much attention and considerable debate. By investigating the suggested subunit-specific roles of NMDARs in the mouse primary visual cortex over development, we report several important findings that clarify the roles of NMDAR subtypes in synaptic plasticity. We observed that LTD was not attenuated by application of ifenprodil, an NR2B-type antagonist, or NVP-AAM007, a less selective NR2A-type antagonist. However, we were surprised that NVP-AAM007 completely blocked adult LTP (postnatal day (P) 45–90), while only modestly affecting juvenile LTP (P21-28). To assess whether this developmental transition reflected an increasing role for NR2A-type receptors with maturity, we characterized the specificity of NVP-AAM007. We found not only that NVP-AAM007 lacks discernable subunit specificity but also that the effects of NVP-AAM077 on LTP could be mimicked using subsaturating concentrations of APV, a global NMDAR antagonist. These results indicate that the effects of NVP-AAM077 on synaptic plasticity are largely explained by nonspecific blockade of NMDARs. Moreover our findings are the first to reveal a developmental increase in the sensitivity of LTP to NMDAR antagonism. We suggest that discrepant reports describing the effect of NVP-AAM077 on LTP may be partially explained by this developmental shift in the properties of LTP. These results indicate that the degree of NMDAR activation required for LTP increases with development, providing insight into a novel underlying mechanism governing the properties of synaptic plasticity.     

Postsynaptic density-93 deficiency protects cultured cortical neurons from N-methyl-d-aspartate receptor-triggered neurotoxicity

It has been reported that N-methyl-d-aspartate receptor (NMDAR)-triggered neurotoxicity is related to excessive Ca²⁺ loading and an increase in nitric oxide (NO) concentration. However, the molecular mechanisms that underlie these events are not completely understood. NMDARs and neuronal NO synthase each binds to the scaffolding protein postsynaptic density (PSD)-93 through its PDZ domains. In this study, we determined whether PSD-93 plays a critical role in NMDAR/Ca²⁺/NO-mediated neurotoxicity. We found that the targeted disruption of the PSD-93 gene attenuated the neurotoxicity triggered by NMDAR activation, but not by non-NMDAR activation, in cultured mouse cortical neurons. PSD-93 deficiency reduced the amount of NMDAR subunits NR2A and NR2B in synaptosomal fractions from the cortical neurons and significantly prevented NMDA-stimulated increases in cyclic guanosine 3′,5′-monophosphate and Ca²⁺ loading in the cortical neurons. These findings indicate that PSD-93 deficiency could block NMDAR-triggered neurotoxicity by disrupting the NMDAR-Ca²⁺-NO signaling pathway and reducing expression of synaptic NR2A and NR2B. Since NMDARs, Ca²⁺, and NO play a critical role during the development of brain trauma, seizures, and ischemia, the present work suggests that PSD-93 might contribute to molecular mechanisms of neuronal damage in these brain disorders.     

Role of NR2B-type NMDA receptors in selective neurodegeneration in Huntington disease

N-Methyl-D-aspartate receptor (NMDAR)-mediated excitotoxicity has been proposed to play a role in Huntington disease (HD), caused by expansion of a polyglutamine tract in the protein huntingtin. HD is characterized by selective neurodegeneration most severely affecting striatal medium-sized spiny projection neurons (MSNs), where expression of the NMDAR subunit NR2B is increased relative to other NR2 subunits. Here, we review our data that NR2B-type NMDAR currents are selectively potentiated by mutant huntingtin in transfected non-neuronal cells and acutely dissociated striatal MSNs from the YAC72 transgenic mouse model of HD. As well, we report increased striatal MSN NMDAR-mediated synaptic currents in corticostriatal slice recordings from YAC72 compared with wild-type mice. This effect was associated with a larger NMDAR- to AMPAR-mediated current ratio, suggesting specific potentiation of postsynaptic NMDARs. Enhanced NMDAR current likely involves increased surface receptor numbers or activity, since we observed no differences between genotypes in striatal NR2B expression. Potentiation of NR2B-containing NMDAR current in striatal MSNs expressing mutant huntingtin may help explain the exquisite vulnerability of these neurons to degeneration in HD.     

Prenatal morphine alters the synaptic complex of postsynaptic density 95 with N-methyl-d-aspartate receptor subunit in hippocampal CA1 subregion of rat offspring leading to long-term cognitive deficits

Infants who are passively exposed to morphine or heroin through their addicted mothers usually develop neurobiological changes. The postsynaptic density 95 (PSD-95) protein, a submembranous cytoskeletal specialization, is dynamically linked with N-methyl-d-aspartate receptors (NMDARs) to form a synaptic complex in postsynaptic neurons. This complex serves important neurobiological functions, including mammalian learning and memory. However, the effects of prenatal morphine exposure on this synaptic complex are not well understood. In this study, we determined whether prenatal morphine exposure altered the synaptic complex association between PSD-95 and three major NMDAR subunits (NR1, NR2A, and NR2B), at the mRNA and protein levels, within the hippocampal CA1 subregion (an important integration area for mammalian learning and memory) of rat offspring along with the performance of long-term cognitive functions. Sprague–Dawley rat offspring from morphine-addicted mothers were studied at a younger age (postnatal day 14; P14) and at an older age (P45). Subsequently, an eight-arm radial maze task was applied to analyze the working and cued reference memory in such offspring (P45). The real-time polymerase chain reaction results showed that prenatal morphine exposure caused significant decreases in mRNA levels of the PSD-95 and three NMDAR subunits (NR1, NR2A, and NR2B) in offspring (P14 and P45). Similarly, at the protein level, immunoblotting showed that decreased whole levels of PSD-95 and NMDAR subunits were seen in offspring subjected with prenatal morphine. Furthermore, the protein interaction of the synaptic complex between the PSD-95 and NMDAR subunit, as indicated by coimmunoprecipitation, was less in prenatal morphine samples than in vehicle controls (P14 and P45). The prenatal morphine group also showed poorer performance for an eight-arm radial maze task than the vehicle-control group. These results are particularly important for a better understanding of certain opioid-mediated neurobehavioral cognitive changes in offspring associated with altered protein interaction between PSD-95 and NMDAR subunits within the developing brain.     

Modulation of NMDA receptor properties and synaptic transmission by the NR3A subunit in mouse hippocampal and cerebrocortical neurons

Expression of the NR3A subunit with NR1/NR2 in Xenopus oocytes or mammalian cell lines leads to a reduction in N-methyl-d-aspartate (NMDA)-induced currents and decreased Mg(2+) sensitivity and Ca(2+) permeability compared with NR1/NR2 receptors. Consistent with these findings, neurons from NR3A knockout (KO) mice exhibit enhanced NMDA-induced currents. Recombinant NR3A can also form excitatory glycine receptors with NR1 in the absence of NR2. However, the effects of NR3A on channel properties in neurons and synaptic transmission have not been fully elucidated. To study physiological roles of NR3A subunits, we generated NR3A transgenic (Tg) mice. Cultured NR3A Tg neurons exhibited two populations of NMDA receptor (NMDAR) channels, reduced Mg(2+) sensitivity, and decreased Ca(2+) permeability in response to NMDA/glycine, but glycine alone did not elicit excitatory currents. In addition, NMDAR-mediated excitatory postsynaptic currents (EPSCs) in NR3A Tg hippocampal slices showed reduced Mg(2+) sensitivity, consistent with the notion that NR3A subunits incorporated into synaptic NMDARs. To study the function of endogenous NR3A subunits, we compared NMDAR-mediated EPSCs in NR3A KO and WT control mice. In NR3A KO mice, the ratio of the amplitudes of the NMDAR-mediated component to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated component of the EPSC was significantly larger than that seen in WT littermates. This result suggests that NR3A subunits contributed to the NMDAR-mediated component of the EPSC in WT mice. Taken together, these results show that NR3A subunits contribute to NMDAR responses from both synaptic and extrasynaptic receptors, likely composed of NR1, NR2, and NR3 subunits.     

PSD-95 regulates NMDA receptors in developing cerebellar granule neurons of the rat

We transfected a green fluorescent protein-tagged PSD-95 (PSD-95gfp) into cultured rat cerebellar granule cells (CGCs) to investigate the role of PSD-95 in excitatory synapse maturation. Cells were grown in low potassium to favour functional synapse formation in vitro . Transfected cells displayed clear clusters of PSD-95gfp, often at the extremities of the short dendritic trees. We recorded NMDA and AMPA miniature excitatory postsynaptic currents (NMDA- and AMPA-mESPCs) in the presence of TTX and bicuculline. At days in vitro ( DIV) 7–8 PSD-95gfp-transfected cells had NMDA-mEPSCs with faster decay and smaller amplitudes than matching controls. In contrast, AMPA-mEPSC frequencies and amplitudes were increased. Whole-cell current density and ifenprodil sensitivity were reduced in PSD-95gfp cells, indicating a reduction of NR2B subunits containing NMDA receptors. No changes were observed compared to control when cells were transfected with cDNA for PSD-95gfp with palmitoylation site mutations that prevent targeting to the synapse. Overexpression of the NMDA receptor NR2A subunit, but not the NR2B subunit, prevented NMDA-mEPSC amplitude reduction when cotransfected with PSD-95gfp. PSD-95gfp overexpression produced faster NMDA-mEPSC decay when transfected alone or with either NR2 subunit. Surface staining of the epitope-tagged NR2 subunits revealed that colocalization with PSD-95gfp was higher for flag-tagged NR2A subunit clusters than for flag-tagged NR2B subunit clusters. These data suggest that PSD-95 overexpression in CGCs favours synaptic maturation by allowing synaptic insertion of NR2A and depressing expression of NR2B subunits.     

Endocytosis and synaptic removal of NR3A-containing NMDA receptors by PACSIN1/syndapin1

A key step in glutamatergic synapse maturation is the replacement of developmentally expressed N-methyl-D-aspartate receptors (NMDARs) with mature forms that differ in subunit composition, electrophysiological properties and propensity to elicit synaptic plasticity. However, the mechanisms underlying the removal and replacement of synaptic NMDARs are poorly understood. Here we demonstrate that NMDARs containing the developmentally regulated NR3A subunit undergo rapid endocytosis from the dendritic plasma membrane in cultured rat hippocampal neurons. This endocytic removal is regulated by PACSIN1/syndapin1, which directly and selectively binds the carboxy-terminal domain of NR3A through its NPF motifs and assembles a complex of proteins including dynamin and clathrin. Endocytosis of NR3A by PACSIN1 is activity dependent, and disruption of PACSIN1 function causes NR3A accumulation at synaptic sites. Our results reveal a new activity-dependent mechanism involved in the regulation of NMDAR expression at synapses during development, and identify a brain-specific endocytic adaptor that confers spatiotemporal and subunit specificity to NMDAR endocytosis.     

Lesion-induced enhancement of LTP in rat visual cortex is mediated by NMDA receptors containing the NR2B subunit

There is emerging evidence that injury of the cerebral cortex is followed by processes of enhanced neuroplasticity. In the present study, we investigate the functional properties of NMDA receptors (NMDARs) in the surround of focal lesions with recordings of extracellular field potentials (FPs) in acute slices of rat visual cortex at survival times of 2–6 days. FPs were recorded in cortical layer III lateral to the lesion, while long-term potentiation (LTP) was induced by theta-burst stimulation (TBS) in layer IV. The predominantly AMPA receptor-mediated FPs displayed a significantly enhanced LTP in the surround of the lesion at distances of 2–3.2 mm. The LTP was completely blocked by the NMDAR antagonist D-AP5. Ifenprodil, an antagonist of NMDARs containing the NR2B subunit, only slightly affected the LTP in slices from sham-operated animals, but significantly reduced the LTP in slices from lesioned rats. We quantitatively analysed the proportion of NMDARs containing the NR2B subunit after lesions by applying ifenprodil to pharmacologically isolated NMDAR-FPs. The NR2B antagonist reduced the NMDAR-FPs significantly more strongly at distances of 2.0–3.2 mm from the border of the lesion. This indicates that the early phase of increased synaptic long-term plasticity in the surround of cortical lesions is accompanied by an up-regulation of NMDARs containing the NR2B subunit.     

Functional contributions of synaptically localized NR2B subunits of the NMDA receptor to synaptic transmission and long-term potentiation in the adult mouse CNS

The NMDA-type glutamate receptor is a heteromeric complex composed of the NR1 and at least one of the NR2 subunits. Switching from the NR2B to the NR2A subunit is thought to underlie functional alteration of the NMDA receptor during synaptic maturation, and it is generally believed that it results in preferential localization of NR2A subunits on the synaptic site and that of NR2B subunits on the extracellular site in the mature brain. It has also been proposed that activation of the NR2A and NR2B subunits results in long-term potentiation (LTP) and long-term depression (LTD), respectively. Furthermore, recent reports suggest that synaptic and extrasynaptic receptors may have distinct roles in synaptic plasticity as well as in gene expression associated with neuronal death. Here, we have investigated whether NR2B subunit-containing receptors are present and functional at mature synapses in the lateral nucleus of the amygdala (LA) and the CA1 region of the hippocampus, comparing their properties between the two brain regions. We have found, in contrast to the above hypotheses, that the NR2B subunit significantly contributes to synaptic transmission as well as LTP induction. Furthermore, its contribution is greater in the LA than in the CA1 region, and biophysical properties of NMDA receptors and the NR2B/NR2A ratio are different between the two brain regions. These results indicate that NR2B subunit-containing NMDA receptors accumulate on the synaptic site and are responsible for the unique properties of synaptic function and plasticity in the amygdala.     

The NR1-4 C-terminus interferes with N-methyl-D-aspartate receptor-mediated excitotoxicity: evidence against a typical T/SXV-PDZ interaction

The N-methyl-D-aspartate receptor (NMDAR) plays a key role in the neural plasticity that underlies learning and memory in vivo. The plasticity exhibited by NMDARs may also contribute to disease pathogenesis, as a number of disorders are caused or exacerbated by exaggerated NMDAR activity. The NMDAR is composed of two obligatory types of subunits, NR1 and NR2. These transmembrane proteins include large intracellular C-termini that have yet to be fully characterized. We have developed a three-color fluorescence system in order to visualize NMDAR expression in living cells. Using excitotoxicity as a proxy for exaggerated NMDAR activity, we analyzed the effect of over-expressing NR1-4 and NR2A C-terminal domains on exaggerated NMDAR function. We demonstrate that a determinant within the C-terminal domain of NR1-4 (C02') is important for NMDAR excitotoxicity, whereas no novel determinants were identified in the NR2A C-terminus. Through the use of heterologous cells, and by examining the interaction between the prototypical NMDAR-binding partner postsynaptic density-95 (PSD-95), we show that this effect is unlikely to be mediated through a classical interaction with PSD-95.     

Deficits in the expression of the NR2B subunit in the hippocampus of aged Fisher 344 rats

The NMDA receptor (NMDAR) has been implicated in the induction of LTP at hippocampal synapses, and has been proposed to play a significant role in the involvement of the hippocampus with learning and memory. Aged rats are known to have deficits in LTP, learning and memory. We tested the hypothesis that aged rats might have deficits in expression of NMDAR subunits. Aged rats have significantly lower levels of NR2B mRNA and protein compared to young animals. This complements a recent report which showed improved learning and memory in mice which overexpress NR2B. No changes were seen in either the mRNA or the protein levels of the NMDAR subunit NR2A, nor in the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate receptor (AMPAR) subunit GluR2. Our data support the hypothesis that age related alterations in the expression of the NMDAR NR2B subunit might underlie deficits in LTP and learning and memory in aged animals.     

Modulation of glycine potency in rat recombinant NMDA receptors containing chimeric NR2A/2D subunits expressed in Xenopus laevis oocytes

Heteromeric NMDARs are composed of coagonist glycine-binding NR1 subunits and glutamate-binding NR2 subunits. The majority of functional NMDARs in the mammalian central nervous system (CNS) contain two NR1 subunits and two NR2 subunits of which there are four types (A-D). We show that the potency of a variety of endogenous and synthetic glycine-site coagonists varies between recombinant NMDARs such that the highest potency is seen at NR2D-containing and the lowest at NR2A-containing NMDARs. This heterogeneity is specified by the particular NR2 subunit within the NMDAR complex since the glycine-binding NR1 subunit is common to all NMDARs investigated. To identify the molecular determinants responsible for this heterogeneity, we generated chimeric NR2A/2D subunits where we exchanged the S1 and S2 regions that form the ligand-binding domains and coexpressed these with NR1 subunits in Xenopus laevis oocytes. Glycine concentration-response curves for NMDARs containing NR2A subunits including the NR2D S1 region gave mean glycine EC(50) values similar to NR2A(WT)-containing NMDARs. However, receptors containing NR2A subunits including the NR2D S2 region or both NR2D S1 and S2 regions gave glycine potencies similar to those seen in NR2D(WT)-containing NMDARs. In particular, two residues in the S2 region of the NR2A subunit (Lys719 and Tyr735) when mutated to the corresponding residues found in the NR2D subunit influence glycine potency. We conclude that the variation in glycine potency is caused by interactions between the NR1 and NR2 ligand-binding domains that occur following agonist binding and which may be involved in the initial conformation changes that determine channel gating.     

Influence of the NR3A subunit on NMDA receptor functions

Various combinations of subunits assemble to form the NMDA-type glutamate receptor (NMDAR), generating diversity in its functions. Here we review roles of the unique NMDAR subunit, NR3A, which acts in a dominant-negative manner to suppress receptor activity. NR3A-containing NMDARs display striking regional and temporal expression specificity, and, unlike most other NMDAR subtypes, they have a low conductance, are only modestly permeable to Ca²⁺, and pass current at hyperpolarized potentials in the presence of magnesium. While glutamate activates triheteromeric NMDARs composed of NR1/NR2/NR3A subunits, glycine is sufficient to activate diheteromeric NR1/NR3A-containing receptors. NR3A dysfunction may contribute to neurological disorders involving NMDARs, and the subunit offers an attractive therapeutic target given its distinct pharmacological and structural properties.     

Enhanced striatal NR2B-containing N-methyl-D-aspartate receptor-mediated synaptic currents in a mouse model of Huntington disease

Huntington disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine tract near the N terminus of the protein huntingtin, leading to dramatic loss of striatal medium-sized spiny GABAergic projection neurons (MSNs). Evidence suggests overactivation of N-methyl-D-aspartate (NMDA)-type glutamate receptors (NMDARs) contributes to selective degeneration of MSNs in HD. Striatal MSNs are enriched in NR2B, and whole cell current and excitotoxicity mediated predominantly by the NR2B subtype of NMDARs is increased with expression of mutant huntingtin in transfected cell lines and striatal MSNs from mice models. To test whether synaptic NMDAR current is altered by mutant huntingtin expression, we recorded striatal MSN excitatory postsynaptic currents (EPSCs) evoked by stimulation of cortical afferents in corticostriatal slices from YAC72 mice and their wild-type (WT) littermates at age 21-31 days. The ratio of NMDAR- to AMPAR-mediated EPSC amplitude was significantly increased in YAC72 compared to WT mice. Furthermore, using a paired-pulse stimulation protocol as a measure of presynaptic glutamate release probability, we found no significant differences between YAC72 and WT striatal MSN responses. These data suggest selective potentiation of postsynaptic NMDAR activity at corticostriatal synapses in YAC72 mice. Measurements of EPSC decay kinetics, as well as the effects of NR2B-subtype selective antagonists and glycine concentration on EPSC amplitude, are consistent with the majority of postsynaptic NMDARs being triheteromers of NR1/NR2A/NR2B in both WT and YAC72 mice. Together with previous results, our data suggest that enhanced activity of NR2B-containing NMDARs is one of the earliest changes leading to neuronal degeneration in HD.