Activity-dependent regulation of NR2B translation contributes to metaplasticity in mouse visual cortex

Visual experience and deprivation bidirectionally modify the NR2A and NR2B subunit composition of NMDARs, and these changes in turn modify the properties of synaptic plasticity in the visual cortex. Deprivation-induced lowering of the NR2A/2B ratio can occur by altering either NR2A or NR2B protein levels, but how a reduction in synaptic activity regulates these changes in a subunit-specific manner is poorly understood. Here, we find that visual deprivation in juvenile mice by dark-rearing or monocular lid suture reduces the NR2A/2B ratio in the deprived cortex in temporally distinct phases--initially by increasing NR2B protein levels, and later by decreasing NR2A protein levels. Brief dark-exposure of juvenile rats likewise produces an increase in NR2B expression. Furthermore, we are able to model the early increase in NR2B by blocking NMDARs in vitro, and we find that translation of NR2B is likely a major point of regulation. Translation of NR2A is not regulated in this manner. Therefore, the differential translational regulation of NR2A and NR2B may contribute to experience-dependent modification of NMDAR subunit composition.     

Differential roles of NR2A and NR2B subtypes in NMDA receptor-dependent protein synthesis in dendrites

Protein synthesis in dendrites is critical for long-term synaptic plasticity. Previous studies have identified an essential role of NMDA receptors in control of activity-dependent dendritic protein synthesis, but the contribution of NR2A- and NR2B-containing NMDA receptors, the two predominant subtypes of NMDA receptors in the forebrain, has not been determined. Using a pharmacological approach, we investigated the role of NR2A and NR2B subtypes in the regulation of NMDA-induced dendritic translation of a GFP reporter mRNA controlled by CaMKII untranslated regions (UTRs). We found that ifenprodil and Ro25-6981, two specific inhibitors of NR2B-containing NMDA receptors, did not affect dendritic GFP synthesis induced by NMDA. In contrast, NVP-AAM077, an antagonist that preferentially blocks the NR2A subtype, completely abolished NMDA-induced GFP synthesis in dendrites. Our results together suggest that NR2A but not NR2B subtypes are indispensable for NMDA receptor-dependent dendritic protein synthesis.     

Experience-dependent changes in NMDA receptor composition at mature central synapses

N-methyl-D-aspartate receptor (NMDAR) activation is obligatory for the induction of diverse forms of synaptic plasticity. The molecular composition and the function of NMDARs are themselves modified by synaptic activity, which, in turn, alters the ability of synapses to undergo subsequent plastic modification. This homeostatic control of synaptic plasticity is well-known for the experience-dependent development of sensory cortices. However, it is now becoming clear that NMDAR properties may not only be altered at juvenile, but also at mature synapses. Diverse types of behavioral manipulation, such as sensory experience, learning and sleep deprivation alter the NR2A/NR2B ratio of hippocampal or cortical NMDARs. As an additional facet to the dynamics of NMDAR function, NMDAR trafficking is regulated by G-protein-coupled neurotransmitter receptors implicated in learning and arousal, such as orexin and dopamine. These findings suggest that mature glutamatergic synapses may be modified by recent activity via alterations in synaptic NMDAR function. Rapid forms of NMDAR trafficking, perhaps controlled by the neurochemical environment featuring specific states of arousal and learning, may regulate plasticity and modulate cognitive abilities in adulthood.     

Fear memory impairing effects of systemic treatment with the NMDA NR2B subunit antagonist, Ro 25-6981, in mice: attenuation with ageing

N-methyl-D-aspartate receptors (NMDARs) are mediators of synaptic plasticity and learning and are implicated in the pathophysiology of neuropsychiatric disease and age-related cognitive dysfunction. NMDARs are heteromers, but the relative contribution of specific subunits to NMDAR-mediated learning is not fully understood. We characterized pre-conditioning systemic treatment of the NR2B subunit-selective antagonist Ro 25-6981 for effects on multi-trial, one-trial and low-shock Pavlovian fear conditioning in C57BL/6J mice. Ro 25-6981 was also profiled for effects on novel open field exploration, elevated plus-maze anxiety-like behavior, startle reactivity, prepulse inhibition of startle, and nociception. Three-month (adult) and 12-month old C57BL/6Tac mice were compared for Ro 25-6981 effects on multi-trial fear conditioning, and corticolimbic NR2B protein levels. Ro 25-6981 moderately impaired fear learning in the multi-trial and one-trial (but not low-shock) conditioning paradigms, but did not affect exploratory or anxiety-related behaviors or sensory functions. Memory impairing effects of Ro 25-6981 were absent in 12-month old mice, although NR2B protein levels were not significantly altered. Present data provide further evidence of the memory impairing effects of selective blockade of NR2B-containing NMDARs, and show loss of these effects with ageing. This work could ultimately have implications for elucidating the pathophysiology of learning dysfunction in neuropsychiatric disorders and ageing.     

Allosteric modulators of NR2B-containing NMDA receptors: molecular mechanisms and therapeutic potential

N-methyl-D-aspartate receptors (NMDARs) are ion channels gated by glutamate, the major excitatory neurotransmitter in the mammalian central nervous system (CNS). They are widespread in the CNS and are involved in numerous physiological and pathological processes including synaptic plasticity, chronic pain and psychosis. Aberrant NMDAR activity also plays an important role in the neuronal loss associated with ischaemic insults and major degenerative disorders including Parkinson''s and Alzheimer''s disease. Agents that target and alter NMDAR function may, thus, have therapeutic benefit. Interestingly, NMDARs are endowed with multiple extracellular regulatory sites that recognize ions or small molecule ligands, some of which are likely to regulate receptor function in vivo. These allosteric sites, which differ from agonist-binding and channel-permeation sites, provide means to modulate, either positively or negatively, NMDAR activity. The present review focuses on allosteric modulation of NMDARs containing the NR2B subunit. Indeed, the NR2B subunit confers a particularly rich pharmacology with distinct recognition sites for exogenous and endogenous allosteric ligands. Moreover, NR2B-containing receptors, compared with other NMDAR subtypes, appear to contribute preferentially to pathological processes linked to overexcitation of glutamatergic pathways. The actions of extracellular H⁺, Mg²⁺, Zn²⁺, of polyamines and neurosteroids, and of the synthetic compounds ifenprodil and derivatives (‘prodils’) are presented. Particular emphasis is put upon the structural determinants and molecular mechanisms that underlie the effects exerted by these agents. A better understanding of how NR2B-containing NMDARs (and NMDARs in general) operate and how they can be modulated should help define new strategies to counteract the deleterious effects of dysregulated NMDAR activity.     

Effect of transgenic overexpression of NR2B on NMDA receptor function and synaptic plasticity in visual cortex.

The NMDA receptor (NMDAR) is a heteromer comprised of NR1 and NR2 subunits. Mice that overexpress the NR2B subunit exhibit enhanced hippocampal LTP, prolonged NMDAR currents, and improved memory ( Tang et al., 1999). In the current study, we explored visual cortex plasticity and NMDAR function in NR2B overexpressing transgenic mice. Unlike the hippocampus, in vitro synaptic plasticity of the visual cortex was unaltered by NR2B overexpression. Consistent with the plasticity findings, NMDAR excitatory postsynaptic current (EPSC) durations from layer 2/3 pyramidal cells were similar in wild-type (wt) and transgenic (tg) mice. Furthermore, temporal summation of NMDAR EPSCs to 10, 20, and 40 Hz stimulation did not differ between cells from wt and tg mice. Finally, although in situ studies clearly demonstrate overexpression of NR2B mRNA in visual cortex, we failed to observe a significant elevation in the synaptic expression of NR2B protein. We conclude that the synaptic ratio of NR2B over NR2A in the NMDA receptor complex in the visual cortex is not significantly influenced by the transgene overexpression. These data suggest that mRNA availability is not a limiting factor for the synthesis of NR2B protein in the visual cortex, and support the hypothesis that levels of NR2A, rather than NR2B, normally determine the subunit composition of NMDARs in visual cortex.     

The role of NMDA receptors in regulating group II metabotropic glutamate receptor-mediated long-term depression in rat medial prefrontal cortex

Previous work has shown that brief application of group II metabotropic glutamate receptor (mGluR) agonist (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl) glycine (DCG-IV) can induce long-term depression (LTD) of excitatory transmission on layer V pyramidal neurons of rat medial prefrontal cortex (mPFC). An unusual feature of this LTD is that it relies on activation of both group II mGluRs and N-methyl-d-aspartate receptors (NMDARs). However, it is not known whether other specific group II mGluR agonists also induce LTD and whether they depend on the conjoint activation of group II mGluRs and NMDARs. We show here that the ability of DCG-IV to induce LTD was mimicked by a more selective group II mGluR agonist, LY379268. The induction of LTD by a lower concentration of DCG-IV (0.2 μM) or LY379268 (0.03 μM) was blocked by the NMDAR antagonist APV or the interruption of synaptic stimulation during drug application. In contrast, application of a higher concentration of DCG-IV (1 μM) or LY379268 (0.1 μM) can induce LTD that was independent of synaptic NMDAR activation. These results suggest that although molecular cooperation between group II mGluRs and synaptic NMDARs may facilitate the induction of group II mGluR-mediated LTD at excitatory synapses onto mPFC layer V pyramidal neurons, enhancing group II mGluR activation may remove NMDAR involvement in this form of synaptic plasticity.     

Developmental switch in requirement for PKA RIIbeta in NMDA-receptor-dependent synaptic plasticity at Schaffer collateral to CA1 pyramidal cell synapses

The cAMP/protein kinase A (PKA) signaling cascade is crucial for synaptic plasticity in a wide variety of species. PKA regulates Ca²⁺ permeation through NMDA receptors (NMDARs) and induction of NMDAR-dependent synaptic plasticity at the Schaffer collateral to CA1 pyramidal cell synapse. Whereas the role of PKA in induction of NMDAR-dependent LTP at CA1 synapses is established, the identity of PKA isoforms involved in this phenomenon is less clear. Here we report that protein synthesis-independent NMDAR-dependent LTP at the Schaffer collateral-CA1 synapse in the hippocampus is deficient, but NMDAR-dependent LTD is normal, in young (postnatal day 10 (P10)-P14) mice lacking PKA RIIβ, the PKA regulatory protein that links PKA to NMDARs at synaptic sites. In contrast, in young adult (P21-P28) mice lacking PKA RIIβ, LTP is normal and LTD is abolished. These findings indicate that distinct PKA isoforms may subserve distinct forms of synaptic plasticity and are consistent with a developmental switch in the signaling cascades required for LTP induction.     

急性阿片耐受大鼠脊髓NMDA受体NR2A及NR2B亚基表达的改变

目的：了解大鼠短期多次应用芬太尼能否发生急性阿片耐受以及急性阿片耐受大鼠脊髓NMDA受体NR2A和NR2B亚基表达的改变。方法：24只体重为200-220g的雄性SD大鼠随机分为3组（n=8）：对照组（C）,生理盐水组（S）及芬太尼组（F）。F组大鼠给予皮下注射芬太尼30μg/kg,共4次,每两次注射之间间隔15min,S组大鼠以生理盐水代替芬太尼,C组大鼠未给药。给药前及给药结束后每30min以Von-Frey仪测定各组大鼠的机械刺激缩足阈值（paw withdrawal threshold,PWT）。当F组大鼠的PWT恢复到给药前基础水平时,各组大鼠均给予腹腔注射吗啡5mg/kg。随后仍每30min测定各组大鼠的PWT,直至F组大鼠的PWT再次回到基础水平。对各组大鼠不同时间点的PWT进行组内和组间比较。另24只体重为2000-220g的雄性SD大鼠分组及给药方法同前（分为C^＊、S^＊及F^＊组）,当F＊组大鼠的PWT首次恢复到基础值时,不给予吗啡,处死各组大鼠,取脊髓,以Western blotting方法测定NMDA受体NR2A及NR2B亚基的蛋白表达水平。结果：连续4次皮下注射芬太尼（F组）后大鼠首先表现为PWT较基础值显著升高,随后PWT降低到基础值以下,然后逐渐恢复至基础值水平,此时皮下注射吗啡后,吗啡的镇痛效果显著低于其他两组大鼠（S组,C组）。F^＊组大鼠脊髓的NR2B亚基表达水平显著高于C^＊组及S^＊组,各组大鼠脊髓的NR2A亚基表达水平的差异无统计学意义。结论：短期应用芬太尼可导致大鼠发生急性阿片耐受。急性阿片耐受大鼠的脊髓NMDA受体NR2B亚基表达水平显著升高,NR2A亚基表达水平无明显变化。     

Influence of the intracellular GluN2 C-terminal domain on NMDA receptor function

Excitatory neurotransmission mediated by N-methyl-d-aspartate receptors (NMDARs) is fundamental to learning and memory and, when impaired, causes certain neurological disorders. NMDARs are heterotetrameric complexes composed of two GluN1 [NR1] and two GluN2(A-D) [NR2(A-D)] subunits. The GluN2 subunit is responsible for subunit-specific channel activity and gating kinetics including activation (rise time), peak open probability (peak Po) and deactivation (decay time). The peak Po of recombinant NMDARs was recently described to be controlled by the extracellular GluN2 N-terminal domain (NTD). The cytoplasmic GluN2 C-terminal domain (CTD) could also be involved, because the Po of synaptic NMDARs is reduced in mice expressing C-terminally truncated GluN2 subunits. Here, we examined the role of the GluN2 cytoplasmic tail for NMDAR channel activity and gating in HEK-293 cells. C-terminal truncation of GluN2A, GluN2B or GluN2C did not change the subunit-specific rise time but accelerated the decay time of glutamate-activated currents. Furthermore, the peak Po was reduced by about 50% for GluN2A and GluN2B but not for GluN2C. These results indicated that the CTD of GluN2 has a modulating role in NMDAR gating even in the absence of interacting synaptic proteins. Reduction of peak Po and deactivation kinetics following GluN2 C-terminal truncation were reversed by re-introducing a CTD from a different GluN2 subunit. Thus, the CTDs of GluN2 subunits behave as constitutive structural elements required for normal functioning of NMDARs but are not involved in determining the subunit-specific gating properties of NMDARs.     

Site within N-Methyl-D-aspartate receptor pore modulates channel gating

N-methyl-d-aspartate-type glutamate receptors (NMDARs) are ligand-gated ion channels activated by coagonists glutamate and glycine. NMDARs play a critical role in synaptic plasticity and excitotoxicity, largely because of their high calcium permeability and slow deactivation and desensitization kinetics. NR1 is an obligate subunit in all NMDAR complexes, where it combines with NR2A, 2B, 2C, and/or 2D. NR1 binds glycine, and residue Asn598 in the re-entrant membrane loop M2 largely determines NMDAR calcium permeability. In contrast, NR2 subunits bind glutamate and contain regions that regulate receptor desensitization and deactivation. Here, we report that mutations of NR1(Asn598) in combination with wild-type NR2A, expressed in human embryonic kidney 293 cells, exhibit altered glycine-independent desensitization. In the absence of extracellular calcium, substitution of Arg for Asn598 (NR1R) slowed desensitization by 2- to 3-fold compared with wild-type NR1/NR2A, and glutamate-evoked peak current EC50 and deactivation rate were also affected. Replacement of Asn by Gln (NR1Q) produced two distinct rates of calcium- and glycine-independent desensitization. Moreover, in the presence of extracellular calcium, the voltage-dependent pore block by calcium for the NR1Q mutant mimicked the effects of the positively charged Arg at this site in NR1R on slowing desensitization and deactivation. A kinetic model of the NMDA receptor-channel suggests that these results can be explained by altered gating and not ligand binding. Our data increase understanding of the role that amino acids within the NMDAR pore play in channel gating.     

Studies on the subtype selectivity of CP-101,606: evidence for two classes of NR2B-selective NMDA receptor antagonists

The subtype-selectivity of racemic [(3)H]CP-101,606, a novel high-affinity NMDA receptor radioligand was determined using defined recombinant NMDA receptor subunits expressed in HEK 293 cells. [(3)H]CP-101,606 binds to adult rodent forebrain and NR1/NR2B receptors expressed in HEK 293 cells with K(D)=4.2 nM and 6.0 nM, respectively. In contrast, no high affinity specific binding was detected to NR1, NR2A, NR2B subunits expressed alone or NR1/NR2A receptors. HEK 293 cells were transfected with NR1, NR2A and NR2B receptor subunits and complexes comprising all three subunits were isolated by anti-NR2A immunoaffinity chromatography. Based on immunoblotting with subunit-selective antibodies, the immunopurified material contained all three NMDA receptor subunit polypeptides. However, in contrast to parallel studies in which high affinity [(3)H]Ro-25,6981 binding activity was observed, no high affinity [(3)H]CP-101,606 binding sites were detected to the immunopurified material. This study provides further evidence for two distinct classes of NR2B-directed NMDA receptor antagonists, one which binds with high affinity irrespective whether another NR2 subunit type is present (Ro-25,6981) and a second class which is affected significantly by the presence of another NR2 subunit type within the receptor complex, exemplified by CP-101,606.     

Acquisition of fear extinction requires activation of NR2B-containing NMDA receptors in the lateral amygdala.

N-methyl-D-aspartate receptors (NMDARs) contribute to synaptic plasticity underlying learning in a variety of brain systems. Fear extinction, which involves learning to suppress the expression of previously learned fear, appears to require NMDAR activation in the amygdala. However, it is unclear whether amygdala NMDARs are required for the acquisition of extinction learning, and it is unknown whether NR2B-containing NMDARs are required in fear extinction. Here, we assessed the effects of selective NR2B blockade with ifenprodil on fear extinction learning, and found that both systemic and intra-amygdala ifenprodil treatment, given before extinction training, impaired the initial acquisition, and subsequent retrieval of fear extinction. These results confirm previous evidence showing that NMDARs in the amygdala are involved in fear extinction, and additionally show that NR2B-containing NMDARs are required. Contrary to the conclusion of previous studies, our findings demonstrate NMDARs are required for the initial acquisition, rather than only the retention, of fear extinction learning. Thus, our results support a previously not known role for NMDA-dependent plasticity in the lateral amygdala during the acquisition of fear extinction.     

Postsynaptic density-95 (PSD-95) and calcineurin control the sensitivity of N-methyl-D-aspartate receptors to calpain cleavage in cortical neurons

The N-methyl-D-aspartate receptor (NMDAR) is a Ca(2+)-permeable glutamate receptor mediating many neuronal functions under normal and pathological conditions. Ca(2+) influx via NMDARs activates diverse intracellular targets, including Ca(2+)-dependent protease calpain. Biochemical studies suggest that NR2A and NR2B subunits of NMDARs are substrates of calpain. Our physiological data showed that calpain, activated by prolonged NMDA treatment (100 microM, 5 min) of cultured cortical neurons, irreversibly decreased the whole-cell currents mediated by extrasynaptic NMDARs. Animals exposed to transient forebrain ischemia, a condition that activates calpain, exhibited the reduced NMDAR current density and the lower full-length NR2A/B level in a calpain-dependent manner. Disruption of the association between NMDARs and the scaffolding protein postsynaptic density (PSD)-95 facilitated the calpain regulation of synaptic NMDAR responses and NR2 cleavage in cortical slices, whereas inhibition of calcineurin activity blocked the calpain effect on NMDAR currents and NR2 cleavage. Calpain-cleaved NR2B subunits were removed from the cell surface. Moreover, cell viability assays showed that calpain, by targeting NMDARs, provided a negative feedback to dampen neuronal excitability in excitotoxic conditions. These data suggest that calpain activation suppresses NMDAR function via proteolytic cleavage of NR2 subunits in vitro and in vivo, and the susceptibility of NMDARs to calpain cleavage is controlled by PSD-95 and calcineurin.     

Differential roles of NR2A and NR2B-containing NMDA receptors in LTP and LTD in the CA1 region of two-week old rat hippocampus

The role of NMDA receptors in the induction of long-term potentiation (LTP) and long-term depression (LTD) is well established but which particular NR2 subunits are involved in these plasticity processes is still a matter of controversy. We have studied the effects of subtype selective NMDA receptor antagonists on LTP induced by high frequency stimulation (100 Hz for 1s) and LTD induced by low frequency stimulation (1 Hz for 15 min) in the CA1 region of hippocampal slices from 14 day old Wistar rats. Against recombinant receptors in HEK293 cells NVP-AAM077 (NVP) was approximately 14-fold selective for NR2A vs NR2B receptors, whilst Ro 25-6981 (Ro) was highly selective for NR2B receptors. On NMDA receptor-mediated EPSCs from Schaffer collaterals in CA1 neurones, NVP and Ro both reduced the amplitude but differentially affected the time constant of decay. The data are compatible with the selective effect of NVP (0.1 microM) and Ro (4 microM) on native NR2A and NBR2B receptors, respectively. NVP reduced both LTP and LTD whereas Ro reduced only LTP. Thus, LTP was reduced by 63% at 0.1 microM NVP and almost completely at 0.4 microM whereas 5 microM Ro reduced LTP by 45%. These data are consistent with a role for both NR2A and NR2B in the induction of LTP, under our experimental conditions. In comparison, LTD was unaffected by Ro (5 microM) even in the presence of a glutamate uptake inhibitor threo-beta-benzylaspartic acid (TBOA) to increase the concentration of glutamate at NR2B containing receptors. NVP (0.2-0.4 microM), however, produced a concentration dependent inhibition of LTD which was complete at 0.4 microM. The lack of effect of 0.1 microM NVP on LTD contrasts with its marked effect on LTP and raises the possibility that different NVP-sensitive NR2 subunit-containing NMDA receptors are required for LTP and LTD in this preparation.     

Activation of NR2B-containing NMDA receptors is not required for NMDA receptor-dependent long-term depression

The triggering of both NMDA receptor-dependent long-term potentiation (LTP) and long-term depression (LTD) in the CA1 region of the hippocampus requires a rise in postsynaptic calcium. A prominent hypothesis has been that the detailed properties of this postsynaptic calcium signal dictate whether LTP or LTD is generated by a given pattern of synaptic activity. Recently, however, evidence has been presented that the subunit composition of the NMDA receptor (NMDAR) determines whether a synapse undergoes LTP or LTD with NR2A-containing NMDARs triggering LTP and NR2B-containing NMDARs triggering LTD. In the present study, the role of NR2B-containing synaptic NMDARs in the induction of LTD in CA1 pyramidal cells has been studied using the selective NR2B antagonists, ifenprodil and Ro25-6981. While both antagonists reduced NMDAR-mediated synaptic currents, neither prevented induction of LTD. These results demonstrate that activation of NR2B-containing NMDARs is not an absolute requirement for the induction of LTD in the hippocampus.     

Subunit-specific roles of glycine-binding domains in activation of NR1/NR3 N-methyl-D-aspartate receptors

N-Methyl-D-aspartate receptors (NMDARs) composed of NR1 and NR3 subunits differ from other NMDAR subtypes in that they require glycine alone for activation. However, little else is known about the activation mechanism of these receptors. Using NMDAR glycine-site agonists/antagonists in conjunction with functional mutagenesis of the NR1 and NR3 ligand-binding cores, we demonstrate quite surprisingly that agonist binding to NR3 alone is sufficient to activate a significant component of NR1/NR3 receptor currents. Thus, the apo conformation of NR1 in NR1/NR3 receptors is permissive for receptor activation. Agonist-bound NR1 may also contribute to peak NR1/NR3 receptor currents but specifically enables significant NR1/NR3 receptor current decay under the conditions studied here, pre-sumably via a slow component of desensitization. Ligand studies of NR1/NR3 receptors also suggest differential agonist selectivity between NR3 and NR1, as some high-affinity NR1 agonists only minimally activate NR1/NR3 receptors, whereas other NR1 agonists are as potent as glycine. Furthermore, liganded NR3 subunits seem necessary for effective engagement of NR1 in NR1/NR3 receptor activation, suggesting significant interactivity between the two subunits. NR3 subunits thus induce plasticity in NR1 with respect to subunit assembly and ligand binding/channel coupling that is unique among ligand-gated ion channel subunits.     

GABAergic disinhibition induced pain hypersensitivity by upregulating NMDA receptor functions in spinal dorsal horn

Intense noxious stimuli impair GABAergic inhibition in spinal dorsal horn, which has been proposed as a critical contributor to pathological pain. However, how the reduced inhibition exacerbates the transfer of nociceptive information at excitatory glutamatergic synapses is still poorly understood. The present study demonstrated that one of the striking consequences of GABAergic disinhibition was to enhance the function of N-methyl-D-aspartate subtype glutamate receptors (NMDARs), a well-characterized player in central sensitization. We found that intrathecal application of bicuculline, a GABA_A receptor antagonist, to remove the inhibition readily elicited mechanical allodynia in naive mice, which could be dose-dependently attenuated by NMDARs antagonist D-APV. Biochemical analysis demonstrated that bicuculline did not affect the total expression levels of the obligatory NMDARs subunit NR1 and the regulatory subunit NR2A and NR2B. However, bicuculline promoted NR1 phosphorylation at Serine 897 (NR1-S897) by cAMP-dependent protein kinase (PKA). This PKA-mediated phosphorylation incorporated NR1 along with NR2B into synapses. When PKA inhibitor H-89 was intrathecally applied, it totally eliminated bicuculline-induced NMDARs phosphorylation, synaptic redistribution as well as pain sensitization. Importantly, the reduced inhibition also operated to enhance NMDARs functions after peripheral inflammation, because spinal injection of diazepam to rescue the inhibition in inflamed mice greatly depressed PKA phosphorylation of NR1-S897, reduced the synaptic concentration of NR1/NR2B and meanwhile, alleviated the inflammatory pain. These data suggested that removal of GABAergic inhibition allowed for PKA-mediated NMDARs phosphorylation and synaptic accumulation, thus exaggerating NMDARs-dependent nociceptive transmission and behavioral sensitization.     

The role of NMDAR subtypes and charge transfer during hippocampal LTP induction

Activation of NMDA receptors (NMDARs) is a requirement for persistent synaptic alterations, such as long-term potentiation of synaptic transmission (LTP). NMDARs are composed of NR1 and NR2 subunits, and NR2 subunit-dependent gating properties of NMDAR subtypes cause dramatic differences in the timing of charge transfer. These postsynaptic temporal profiles are further influenced by the frequency of synaptic activation. Here, we investigated in the CA1 region of hippocampal slices from P28 mice, whether particular NMDAR subtypes are recruited based on NR2 subunit-specific gating following different induction protocols. For high frequency afferent stimulation (HFS), we found that genetic impairment of NR2A or pharmacological block of NR2A- or NR2B-type NMDARs can reduce field LTP. In contrast, when pairing low frequency synaptic stimulation with postsynaptic depolarization (LFS pairing) in single CA1 neurons, pharmacological antagonism of either subtype modestly reduced the charge transfer during LFS pairing without reducing the LTP magnitude. These results indicate that HFS-triggered LTP is induced by more than one NMDAR subtype, whereas a single subtype is sufficient during LFS pairing. Analysis of charge transfer during LFS pairing in 13 different conditions revealed a threshold for LTP induction, which was independent of the NR2 antagonist tested. Thus, at least for LFS pairing, the amount of charge transfer, and thus Ca2+ influx, during LTP induction is a factor more critical than the participation of a particular NMDAR subtype.     

Functional evidence for a twisted conformation of the NMDA receptor GluN2A subunit N-terminal domain

Ionotropic glutamate receptors (iGluRs) possess in their extracellular region a large N-terminal domain (NTD) that precedes the agonist-binding domain and displays a clamshell-like architecture similar to the bacterial leucine/isoleucine/valine-binding protein (LIVBP). In addition to their role in receptor assembly, in NMDA receptors (NMDARs), the NTDs of GluN2A and GluN2B subunits form a major site for subunit-specific regulation of ion channel activity, in particular through binding of allosteric modulators such as the synaptically-enriched zinc ion. A recent crystallographic study of the isolated GluN2B NTD has revealed an unexpected twisted closed-cleft conformation caused by a rotation of ∼50° in the interlobe orientation compared with all other known LIVBP-like structures (Karakas et al., 2009). By measuring currents carried by recombinant NMDARs, we now provide functional evidence, through disulfide cross-linking and the identification of a new zinc-binding residue (D283), that the GluN2A NTD of intact GluN1/GluN2A receptors adopts a similar twisted conformation in its closed-cleft state. We propose that the twisted NTD conformation is a distinct structural feature of NMDARs (at least for GluN2A and GluN2B subunits), arguing for interactions between the NTDs in the tetrameric complex that are likely to differ between NMDA and AMPA/kainate receptors.