Subunit-containing N-methyl-D-aspartate Receptors Research Articles

Ketamine inside neurons?

Clinically used antidepressants, such as selective serotonin reuptake inhibitors (SSRIs), aid only a fraction of patients. Furthermore, even successful use of SSRIs takes 2 to 6 weeks of maintained medication. Depressed patients need faster help. Since 2000, several clinical studies report that depressed patients given subanesthetic doses of ketamine showed improvement within 2 hours. Trials continue for various dosing regimens, formulations, and populations. It is not understood what causes the therapeutic action of the SSRIs, and it is also not clear how ketamine exerts its effects. The best-known behavioral effect of ketamine is dissociative anesthesia. The drug retains Food and Drug Administration (FDA) approval for anesthesia in special populations as well as for veterinary use. The dissociative effects presumably arise from ketamine’s action to block N-methyl-D-aspartate (NMDA) receptor channels that have been opened by glutamate. The kinetics, equilibrium, and voltage sensitivity of open-channel blockers is a well-studied topic, and recent work shows how ketamine becomes trapped within the channel pore of NMDA receptors at local concentrations of ∼1 µM, which are expected to occur at the clinically effective antidepressant human doses. How might blockade of NMDA receptors lead to the antidepressant effects? Most studies emphasize signal transduction pathways that could be modulated by the locally decreased Ca^(2+) flux through NMDA receptors, especially extrasynaptic GluN2B subunit-containing NMDA receptors. In one series of experiments, the decreased Ca^(2+) flux led to decreased activity of eukaryotic elongation factor 2 kinase, which in turn desuppressed eukaryotic elongation factor 2. This ribosome-binding protein then increased translation of brain-derived neurotrophic factor (BDNF). Many other experiments show that BDNF is released during antidepressant action.

Caffeine potentiates the release of GABA mediated by NMDA receptor activation: Involvement of A1 adenosine receptors

Caffeine, a methylated derivative of xanthine and widely consumed psychoactive substance, acts in several targets in the nervous system. We investigated its role in retinal explants of chick embryo analyzing the role of purinergic receptors in [3H]-GABA release induced by d-aspartate (d-asp). d-Asp increases GABA-release 4.5-fold when compared to basal levels from 13-day-old chick embryo retinal explants. Caffeine 500μM elevated d-asp-induced GABA release in 60%. The release was inhibited in the presence of NNC-711, a GABA transporter-1 (GAT-1) blocker or by MK-801, an N-methyl-d-aspartate receptor (NMDAR) antagonist. Caffeine did not modify [3H]-GABA uptake carried out for 5, 10, 30 and 60min and did not increase the release of d-asp or glutamate at basal or stimulated conditions. The caffeine effect was mimicked by the adenosine A1 receptor antagonist DPCPX and by the adenylyl cyclase (AC) activator forskolin. It was also blocked by the protein kinase A (PKA) inhibitor H-89, tyrosine kinase inhibitor genistein or by the src family kinase (SFK) inhibitor PP1. Forskolin-stimulated cyclic adenosine monophosphate (cAMP) levels were reduced in the presence of the A1 receptor agonist CHA. Western blot analysis revealed that 500μM caffeine increased phosphoGluN2B expression levels in approximately 60% when compared to total GluN2B levels in embryonic E13 retina. The GluN2B subunit-containing NMDAR antagonist ifenprodil inhibited the caffeine effect. Our results suggest that caffeine potentiates d-asp-induced GABA release, which is mediated by GAT-1, via inhibition of adenosine A1 receptor and activation of the PKA pathway. Regulation of NMDAR by phosphorylation of GluN2B subunit by a SFK may also be involved in the effect promoted by caffeine.

Synthesis and preclinical evaluation of carbon-11 labelled N-((5-(4-fluoro-2-[(11)C]methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine as a PET tracer for NR2B subunit-containing NMDA receptors.

The N-methyl-D-Aspartate (NMDA) receptor plays an important role in learning and memory. Overactivation is thought to play an important role in neurodegenerative disorders such as Alzheimer's disease. Currently, it is not possible to assess N-methyl-D-aspartate receptor (NMDAr) bio-availability in vivo. The purpose of this study was to develop a positron emission tomography (PET) ligand for the NR2B binding site of the NMDA receptor. N-((5-(4-fluoro-2-methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine was radiolabelled with carbon-11 in the phenyl moiety. Biodistribution and blocking studies were carried out in anaesthetized mice and in non-anaesthetized rats. N-((5-(4-fluoro-2-[(11)C]methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine was prepared in 49±3% (decay-corrected) yield, affording 4.1±0.3 GBq of formulated product at the end of synthesis with a radiochemical purity of >99% and with a specific activity of 78±10 GBq/μmol. A new NR2B PET ligand was developed in high yield. [(11)C]4 readily enters the brain and binds to the NR2B subunit-containing NMDAr in the rodent brain. High sigma-1 receptor binding may, however, limit its future application as a PET probe for imaging the NR2B subunit-containing NMDAr. Anaesthesia has an effect on NMDAr function and therefore can complicate interpretation of preclinical in vivo results. In addition, effects of endogenous compounds cannot be excluded. Despite these potential limitations, further studies are warranted to investigate the values of [(11)C]4 as an NR2B PET ligand.

Dopamine D3 receptor-regulated NR2B subunits of N-methyl-d-aspartate receptors in the nucleus accumbens involves in morphine-induced locomotor activity.

Dopamine and glutamate receptors are densely expressed in the nucleus accumbens (NAc). Active interactions between these receptors contribute to the development of neuropsychiatric diseases, such as drug addiction and relapse. However, the molecular mechanisms underlying these interactions remain unclear. This study established a mouse model of intermittent morphine-induced mouse behavioral sensitization model. Western blot and electrophysiological recording methods were performed to directly identify the affective components of morphine behavioral sensitization. Interval morphine administration could cause significant locomotor sensitization. Hyperlocomotion and behavioral locomotor sensitization were significantly suppressed when ifenprodil (5mg/kg), a selective NR2B subunit-containing N-methyl-d-aspartate (NMDA) receptor antagonist, or nafadotride (25μg/kg), a dopamine D3 receptor (D3R)-preferring antagonist, was coadministered with morphine. Western blot analysis showed that morphine behavioral sensitization induced a region-specific increase in phosphorylation of NR2B (pNR2B) and total levels of NR2B (NR2B) expression in the NAc. Systemically administered nafadotride attenuated behavioral locomotor sensitization induced by morphine and significantly reversed the overexpression of pNR2B and NR2B subunit-containing NMDA receptor in the NAc. NMDA receptor-mediated excitatory postsynaptic currents in the NAc were also significantly reduced by nafadotride. These findings suggest that D3Rs are involved in morphine-induced behavioral locomotor sensitization in mice by regulating the NR2B subunits of NMDA receptors in the NAc.

GluN2B‐Selective N‐Methyl‐d‐aspartate (NMDA) Receptor Antagonists Derived from 3‐Benzazepines: Synthesis and Pharmacological Evaluation of Benzo[7]annulen‐7‐amines

Given their high neuroprotective potential, ligands that block GluN2B-containing N-methyl-D-aspartate (NMDA) receptors by interacting with the ifenprodil binding site located on the GluN2B subunit are of great interest for the treatment of various neuronal disorders. In this study, a novel class of GluN2B-selective NMDA receptor antagonists with the benzo[7]annulene scaffold was prepared and pharmacologically evaluated. The key intermediate, N-(2-methoxy-5-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-yl)acetamide (11), was obtained by cyclization of 3-acetamido-5-(3-methoxyphenyl)pentanoic acid (10 b). The final reaction steps comprise hydrolysis of the amide, reduction of the ketone, and reductive alkylation, leading to cis- and trans-configured 7-(ω-phenylalkylamino)benzo[7]annulen-5-ols. High GluN2B affinity was observed with cis-configured γ-amino alcohols substituted with a 3-phenylpropyl moiety at the amino group. Removal of the benzylic hydroxy moiety led to the most potent GluN2B antagonists of this series: 2-methoxy-N-(3-phenylpropyl)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-amine (20 a, Ki =10 nM) and 2-methoxy-N-methyl-N-(3-phenylpropyl)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-amine (23 a, Ki =7.9 nM). The selectivity over related receptors (phencyclidine binding site of the NMDA receptor, σ1 and σ2 receptors) was recorded. In a functional assay measuring the cytoprotective activity of the benzo[7]annulenamines, all tested compounds showed potent NMDA receptor antagonistic activity. Cytotoxicity induced via GluN2A subunit-containing NMDA receptors was not inhibited by the new ligands.

Effect of the N‐methyl‐D‐aspartate NR2B subunit antagonist ifenprodil on precursor cell proliferation in the hippocampus

The N-methyl-D-aspartate (NMDA) receptor, one of the ionotropic glutamate receptor, plays important physiological and pathological roles in learning and memory, neuronal development, acute and chronic neurological diseases, and neurogenesis. This work examines the contribution of the NR2B NMDA receptor subunit to adult neurogenesis/cell proliferation under physiological conditions and following an excitotoxic insult. We have previously shown in vitro that a discrete NMDA-induced, excitotoxic injury to the hippocampus results in an increase in neurogenesis within the dentate gyrus. Here we have characterized adult neurogenesis or proliferation, using BrdU, in an in vivo model of excitotoxic injury to the CA1 subfield of the hippocampus. We demonstrate a peak in neural stem cell proliferation/neurogenesis between 6 and 9 days after the excitotoxic insult. Treatment with ifenprodil, an NR2B subunit-specific NMDA receptor antagonist, without prior injury induction, also increased the number of BrdU-positive cells within the DG and posterior periventricle, indicating that ifenprodil itself could modulate the rate of proliferation. Interestingly, though, the increased level of cell proliferation did not change significantly when ifenprodil was administered following an excitotoxic insult. In conclusion, our results suggest and add to the growing evidence that NR2B subunit-containing NMDA receptors play a role in neural stem cell proliferation.

GluN2A and GluN2B subunit-containing NMDA receptors in hippocampal plasticity.

N-Methyl-d-aspartate receptor (NMDAR)-dependent synaptic plasticity is a strong candidate to mediate learning and memory processes that require the hippocampus. This plasticity is bidirectional, and how the same receptor can mediate opposite changes in synaptic weights remains a conundrum. It has been suggested that the NMDAR subunit composition could be involved. Specifically, one subunit composition of NMDARs would be responsible for the induction of long-term potentiation (LTP), whereas NMDARs with a different subunit composition would be engaged in the induction of long-term depression (LTD). Unfortunately, the results from studies that have investigated this hypothesis are contradictory, particularly in relation to LTD. Nevertheless, current evidence does suggest that the GluN2B subunit might be particularly important for plasticity and may make a synapse bidirectionally malleable. In particular, we conclude that the presence of GluN2B subunit-containing NMDARs at the postsynaptic density might be a necessary, though not a sufficient, condition for the strengthening of individual synapses. This is owing to the interaction of GluN2B with calcium/calmodulin-dependent protein kinase II (CaMKII) and is distinct from its contribution as an ion channel.

Inhibition of protein tyrosine phosphatases in spinal dorsal horn attenuated inflammatory pain by repressing Src signaling

Tyrosine phosphorylation of N-methyl-d-aspartate (NMDA) subtype glutamate receptors by Src-family protein tyrosine kinases (SFKs) plays a critical role in spinal sensitization. Besides SFKs, the tyrosine phosphorylation levels of proteins are also determined by protein tyrosine phosphatases (PTPs). However, whether PTPs are involved in spinal nociceptive processing is largely unknown. The present study found that intrathecal application of broad-spectrum PTPs inhibitors orthovanadate or Bpv (phen) generated little effects on the paw withdrawal thresholds of intact rats to Von Frey filament stimuli. Although the basal nociceptive responses didn't require the involvement of PTPs, the mechanical allodynia evoked by intrathecal injection of NMDA was greatly attenuated by orthovanadate and Bpv (phen), suggesting that PTPs activity, once stimulated by NMDA receptors, became essential for spinal sensitization. Biochemical analysis demonstrated that PTPs functioned to activate SFKs member Src and promote Src interaction with NR2B subunit-containing NMDA receptors (NR2B receptors). As a result, PTPs inhibition largely suppressed Src-mediated NR2B phosphorylation at Tyr1472 and reduced the synaptic concentration of NR2B receptors in spinal dorsal horn of NMDA-treated rats. Importantly, intraplantar injection of Complete Freund's Adjuvant (CFA) naturally activated spinal PTPs to initiate Src signaling, because PTPs inhibition significantly repressed Src activity, reduced Src phosphorylation of NR2B, decreased NR2B synaptic accumulation and eventually ameliorated inflammatory pain. These data indicated an important role played by spinal PTPs in inducing Src-dependent NR2B receptor hyperfunction and suggested that PTPs inhibition might represent an effective strategy for the treatment of inflammatory pain.

Open-channel blockade is less effective on GluN3B than GluN3A subunit-containing NMDA receptors

The GluN3 subunits of the N-methyl-d-aspartate (NMDA) receptor are known to reduce its Ca2+ permeability and Mg2+ sensitivity, however, little is known about their effects on other channel blockers. cRNAs for rat NMDA receptor subunits were injected into Xenopus oocytes and responses to NMDA and glycine were recorded using two electrode voltage clamp. Channel block of receptors containing GluN1-1a/2A, GluN1-1a/2A/3A or GluN1-1a/2A/3B subunits was characterised using Mg2+, memantine, MK-801, philanthotoxin-343 and methoctramine. IC50 values for Mg2+ and memantine increased when receptors contained GluN3A subunits and were further increased when they contained GluN3B, e.g. IC50s at −75mV for block of GluN1-1a/2A, GluN1-1a/2A/3A and GluN1-1a/2A/3B receptors respectively were 4.2, 22.4 and 40.1μM for Mg2+, and 2.5, 7.5 and 17.5μM for memantine. Blocking activity was found to be fully or partially restored when G or R (at the N and N+1 sites respectively) were mutated to N in GluN3A. Thus, the changes cannot be attributed to the loss of the N or N+1 sites alone, but rather involve both sites or residues elsewhere. Block by MK-801 and philanthotoxin-343 was also reduced by GluN3A, most strongly at −100mV but not at −50mV, and by GluN3B at all Vh. Methoctramine was the least sensitive to introduction of GluN3 subunits suggesting a minimal interaction with the N and N+1 sites. We conclude that GluN3B-containing receptors provide increased resistance to channel block compared to GluN3A-containing receptors and this must be due to differences outside the deep pore region (N site and deeper).

β-Estradiol unmasks metabotropic receptor-mediated metaplasticity of NMDA receptor transmission in the female rat dentate gyrus

Loss of estrogen in women following menopause is associated with increased risk for cognitive decline, dementia and depression, all of which can be prevented by estradiol replacement. The dentate gyrus plays an important role in cognition, learning and memory. The gatekeeping function of the dentate gyrus to filter incoming activity into the hippocampus is modulated by estradiol in a frequency-dependent manner and involves activation of metabotropic glutamate receptors (mGluR). In the present study, we investigated whether estradiol (EB) modulates the metaplastic effect of inducing synaptic long-term potentiation (LTP) on subsequent propensity for expression of LTP in the dentate gyrus. At medial perforant path-dentate granule cell synapses in hippocampal slices of ovariectomized female rats, EB replacement was critical for an initial induction of LTP to enhance the magnitude of subsequent LTP elicited by a second high-frequency stimulation, metaplasticity, which was not present in slices from oil-treated control animals. EB enhanced expression of group I mGluRs, and the metaplastic effect of EB on LTP required activation of group I mGluRs that led to Src-family tyrosine kinase-mediated phosphorylation of NR2B subunits of N-methyl-d-aspartate receptors (NMDAR) that enhanced the magnitude of NMDAR-dependent LTP. Our data show that EB effects on LTP in the hippocampal dentate gyrus require activation of group I mGluRs, which in turn leads to functional metaplastic regulation of NR2B subunit-containing NMDARs, as opposed to direct effects of EB on NMDARs.

Involvement of NMDA receptors in ryanodine receptor expression in dopaminergic neurons in the ventral tegmental area of mice with intermittent methamphetamine treatment

Excitatory synapses on dopaminergic neurons of the ventral tegmental area (VTA) represent an important role in psychostimulant-induced rewarding effect. This study investigated the regulation of ryanodine receptor (RyR) and N-methyl-D-aspartate (NMDA) receptor expression in mice under intermittent methamphetamine (METH) treatment using a place preference procedure. RyR-1 and -2 significantly increased in the VTA of mice with METH-induced place preference, whereas RyR-3 showed no changes. In addition, the levels of NR1, NR2A, and NR2B subunits were increased in the VTA. The METH-induced place preference was inhibited by intracerebroventricular pretreatment with MK-801, a noncompetitive NMDA receptor antagonist, and ifenprodil, a selective NR2B subunit-containing NMDA receptor antagonist, in a dose-dependent manner. Under these conditions, the increase of RyR-1 and -2 in the VTA was significantly blocked by ifenprodil. The immunohistochemical analysis revealed the colocalization of RyR-1 and -2 with NR2B subunits in dopaminergic neurons in the mouse VTA. These findings suggest that RyRs could be involved in the development of METH-induced place preference and that NR2B subunit-containing NMDA receptors in mice showing METH-induced place preference play an important role in expression of RyRs.

Partial involvement of NMDA receptors and glial cells in the nociceptive behaviors induced by intrathecally administered histamine

The involvement of spinal glial cells in the nociceptive behaviors induced by 800 pmol of histamine was determined in mice. Histamine at 800 pmol injected intrathecally (i.t.) produced nociceptive behaviors, consisting of scratching, biting and licking. The nociceptive behaviors induced by histamine were significantly suppressed by i.t. co-administration with tachykinin NK 1 receptor antagonist CP99,994 or competitive antagonist for N-methyl- d-aspartate (NMDA) receptor d-(−)-2-amino-5-phosphonovaleric acid ( d-APV). The i.t. pretreatment with the glial cell inhibitor dl-fluorocitric acid or minocycline failed to affect the nociceptive behaviors induced by histamine. However, in mice pretreated i.t. with dl-fluorocitric acid or minocycline, the nociceptive behaviors induced by histamine were significantly suppressed by i.t. co-administration with CP99,994 but not d-APV. In Western blot analysis using lumbar spinal cords, i.t. treatment with 800 pmol of histamine increased the phosphorylation of the NR1 subunit of NMDA receptors. The increased phosphorylation of the NR1 subunit of NMDA receptors by histamine was abolished by i.t. pretreatment with dl-fluorocitric acid or minocycline. The present results suggest that histamine at 800 pmol elicits nociceptive behaviors through activation of the neuronal NK 1 receptor and the NR1 subunit-containing NMDA receptors on glial cells in the spinal cord.

Activation of N-Methyl-d-aspartate (NMDA) Receptors in the Dorsal Vagal Complex Lowers Glucose Production

Diabetes is characterized by hyperglycemia due partly to increased hepatic glucose production. The hypothalamus regulates hepatic glucose production in rodents. However, it is currently unknown whether other regions of the brain are sufficient in glucose production regulation. The N-methyl-D-aspartate (NMDA) receptor is composed of NR1 and NR2 subunits, which are activated by co-agonist glycine and glutamate or aspartate, respectively. Here we report that direct administration of either co-agonist glycine or NMDA into the dorsal vagal complex (DVC), targeting the nucleus of the solitary tract, lowered glucose production in vivo. Direct infusion of the NMDA receptor blocker MK-801 into the DVC negated the metabolic effect of glycine. To evaluate whether NR1 subunit of the NMDA receptor mediates the effect of glycine, NR1 in the DVC was inhibited by DVC NR1 antagonist 7-chlorokynurenic acid or DVC shRNA-NR1. Pharmacological and molecular inhibition of DVC NR1 negated the metabolic effect of glycine. To evaluate whether the NMDA receptors mediate the effects of NR2 agonist NMDA, DVC NMDA receptors were inhibited by antagonist D-2-amino-5-phosphonovaleric acid (D-APV). DVC D-APV fully negated the ability of DVC NMDA to lower glucose production. Finally, hepatic vagotomy negated the DVC glycine ability to lower glucose production. These findings demonstrate that activation of NR1 and NR2 subunits of the NMDA receptors in the DVC is sufficient to trigger a brain-liver axis to lower glucose production, and suggest that DVC NMDA receptors serve as a therapeutic target for diabetes and obesity.

Polyaminergic agents modulate contextual fear extinction in rats

Polyamines, such as spermidine and spermine, have been reported to improve memory retention through the activation of N-methyl- d-aspartate receptors (NMDAr). However whether polyamine agonists and antagonists alter extinction remains unclear. In the current study, we investigated whether spermidine and polyamine antagonists that selectively block the NR2B subunit at the NMDAr alter the extinction of contextual conditioned fear in male Wistar rats. The bilateral intra-hippocampal administration of exogenous spermidine (2 nmol/site) immediately after, but not 6 h after extinction training, facilitated the extinction of fear conditioning. The injection of the NMDAr antagonists arcaine (0.2 nmol/site), ifenprodil (20 nmol/site) and traxoprodil (0.2 nmol/site), disrupted fear extinction and, at doses that had no effect per se, reversed the facilitatory effect of spermidine on fear extinction. These results suggest that exogenous and endogenous polyamines facilitate the extinction of contextual conditioned fear through activation of NR2B subunit-containing NMDAr in the hippocampus. Since extinction-based exposure therapy is widely used as treatment for a number of anxiety-related disorders, including phobias and post-traumatic stress, the currently reported facilitation of extinction by polyaminergic agents suggest these compounds as putative candidates for drug development.

Double Dissociation of Spike Timing–Dependent Potentiation and Depression by Subunit-Preferring NMDA Receptor Antagonists in Mouse Barrel Cortex

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.

Induction and expression of GluA1 (GluR‐A)‐independent LTP in the hippocampus

Long-term potentiation (LTP) at hippocampal CA3–CA1 synapses is thought to be mediated, at least in part, by an increase in the postsynaptic surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptors induced by N-methyl-d-aspartate (NMDA) receptor activation. While this process was originally attributed to the regulated synaptic insertion of GluA1 (GluR-A) subunit-containing AMPA receptors, recent evidence suggests that regulated synaptic trafficking of GluA2 subunits might also contribute to one or several phases of potentiation. However, it has so far been difficult to separate these two mechanisms experimentally. Here we used genetically modified mice lacking the GluA1 subunit (Gria1−/− mice) to investigate GluA1-independent mechanisms of LTP at CA3–CA1 synapses in transverse hippocampal slices. An extracellular, paired theta-burst stimulation paradigm induced a robust GluA1-independent form of LTP lacking the early, rapidly decaying component characteristic of LTP in wild-type mice. This GluA1-independent form of LTP was attenuated by inhibitors of neuronal nitric oxide synthase and protein kinase C (PKC), two enzymes known to regulate GluA2 surface expression. Furthermore, the induction of GluA1-independent potentiation required the activation of GluN2B (NR2B) subunit-containing NMDA receptors. Our findings support and extend the evidence that LTP at hippocampal CA3–CA1 synapses comprises a rapidly decaying, GluA1-dependent component and a more sustained, GluA1-independent component, induced and expressed via a separate mechanism involving GluN2B-containing NMDA receptors, neuronal nitric oxide synthase and PKC.

Plasticity of NMDA receptor NR2B subunit in memory and chronic pain

Glutamatergic synapses play critical roles in brain functions and diseases. Long-term potentiation (LTP) is a most effective cellular model for investigating the synaptic changes that underlie learning as well as brain disease – although different molecular mechanisms are likely involved in LTP in physiological and pathological conditions. In the case of learning, N-methyl-D-aspartate (NMDA) receptor is known to be important for triggering learning-related plasticity; alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptors are thought to be important for the expression of synaptic changes. In this review, I will examine recent evidence on the novel roles of NMDA receptors, in particular NR2B subunit-containing NMDA receptors in learning and chronic pain. A positive feedback control of NR2B receptor subunit is proposed to explain cortical sensitization involved in chronic pain, but not learning and memory.

Polyamine deficient diet to relieve pain hypersensitivity

There is a compelling body of evidence that N-methyl-d-aspartate receptors (NMDA-R) play a critical role in the development and maintenance of pain hypersensitivity. However, long-term treatments with NMDA-R antagonists are limited by unacceptable side effects. Since polyamines modulate the functioning of NMDA-R and mainly originate from normal dietary intake and bacterial metabolism in the gut, we developed a nutritional therapy based on dietary polyamine deficiency. Here, we reported that a polyamine deficient diet (PD diet) for 7 days prevented the enhancement of tyrosine phosphorylation of the spinal NR2B subunit-containing NMDA-R associated with inflammation in rats. Based on these data, we studied the ability of PD diet to prevent long-lasting pain hypersensitivity associated with tissue injury on one hind paw by evaluating long-lasting changes in both mechanical nociceptive threshold and weight bearing. A PD diet strongly reduced long-lasting hyperalgesia induced by inflammation or incision, especially in fentanyl-treated rats. Moreover a PD diet also prevented the exaggerated hyperalgesia induced by a second inflammation performed 7 days after the first one. A PD diet also opposed paradoxical hyperalgesia induced by non-nociceptive environmental stress in rats with pain and opioid experiences. A PD diet reversed pain hypersensitivity associated with monoarthritis or neuropathy and restored the analgesic effect of morphine. Since PD diet was devoid of any noticeable side effects, this nutritional therapy could be part of an effective and safe strategy for pre-emptive analgesia and for reducing the transition from acute to chronic pain and its outcomes in various pain syndromes.

Neural Cell Adhesion Molecule-associated Polysialic Acid Inhibits NR2B-containing N-Methyl-d-aspartate Receptors and Prevents Glutamate-induced Cell Death

The neural cell adhesion molecule (NCAM) and its associated glycan polysialic acid play important roles in the development of the nervous system and N-methyl-D-aspartate(NMDA)receptor-dependent synaptic plasticity in the adult. Here, we investigated the influence of polysialic acid on NMDA receptor activity. We found that glutamate-elicited NMDA receptor currents in cultured hippocampal neurons were reduced by approximately 30% with the application of polysialic acid or polysialylated NCAM but not by the sialic acid monomer, chondroitin sulfate, or non-polysialylated NCAM. Polysialic acid inhibited NMDA receptor currents elicited by 3 microm glutamate but not by 30 microm glutamate, suggesting that polysialic acid acts as a competitive antagonist, possibly at the glutamate binding site. The polysialic acid induced effects were mimicked and fully occluded by the NR2B subunit specific antagonist, ifenprodil. Recordings from single synaptosomal NMDA receptors reconstituted in lipid bilayers revealed that polysialic acid reduced open probability but not the conductance of NR2B-containing NMDA receptors in a polysialic acid and glutamate concentration-dependent manner. The activity of single NR2B-lacking synaptosomal NMDA receptors was not affected by polysialic acid. Application of polysialic acid to hippocampal cultures reduced excitotoxic cell death induced by low micromolar concentration of glutamate via activation of NR2B-containing NMDA receptors, whereas enzymatic removal of polysialic acid resulted in increased cell death that occluded glutamate-induced excitotoxicity. These observations indicate that the cell adhesion molecule-associated glycan polysialic acid is able to prevent excitotoxicity via inhibition of NR2B subunit-containing NMDA receptors.

NR2B antagonists restrict spatiotemporal spread of activity in a rat model of cortical dysplasia

Freeze-lesion-induced focal cortical dysplasia in rats closely resembles human microgyria, a neuronal migration disorder associated with drug-resistant epilepsy. Alterations in expression of N-methyl- d-aspartate receptors (NMDARs) containing NR2B subunits have been suggested to play a role in the hyperexcitability seen in this model. We examined the effect of NMDAR antagonists selective for NR2B subunits (Ro 25-6981 and ifenprodil) on activity evoked by intracortical stimulation in brain slices from freeze-lesioned rat neocortex. Whole-cell voltage-clamp recordings showed that Ro 25-6981 (1 μM) significantly reduced the response area of evoked postsynaptic currents in pyramidal cells from the paramicrogyral area whereas responses were unaffected in slices from control (sham operated) animals. Voltage-sensitive dye imaging was used to examine spatiotemporal spread of evoked activity in lesioned and control cortices. The imaging experiments revealed that peak amplitude, duration, and lateral spread of evoked activity in the paramicrogyral area was reduced by bath application of Ro 25-6981 (1 μM) and ifenprodil (10 μM). Ro 25-6981 had no effect on evoked activity in neocortical slices from control animals. The non-selective NMDAR antagonist d-2-amino-5-phosphonvaleric acid (APV, 20 μM) reduced activity evoked in presence of 50 μM 4-aminopyridine (known to increase excitability by enhancing neurotransmitter release) in neocortical slices from control animals whereas Ro 25-6981 (1 μM) did not. These results suggest that NR2B subunit-containing NMDARs contribute significantly to the enhanced spatiotemporal spread of paroxysmal activity observed in vitro in the rat freeze-lesion model of focal cortical dysplasia.