NR2B-containing Receptors Research Articles

6-Gingerol, a Major Constituent of Zingiber officinale Rhizoma, Exerts Anticonvulsant Activity in the Pentylenetetrazole-Induced Seizure Model in Larval Zebrafish.

Zingiber officinale is one of the most frequently used medicinal herbs in Asia. Using rodent seizure models, it was previously shown that Zingiber officinale hydroethanolic extract exerts antiseizure activity, but the active constituents responsible for this effect have not been determined. In this paper, we demonstrated that Zingiber officinale methanolic extract exerts anticonvulsant activity in the pentylenetetrazole (PTZ)-induced hyperlocomotion assay in larval zebrafish. Next, we isolated 6-gingerol (6-GIN)—a major constituent of Zingiber officinale rhizoma. We observed that 6-GIN exerted potent dose-dependent anticonvulsant activity in the PTZ-induced hyperlocomotion seizure assay in zebrafish, which was confirmed electroencephalographically. To obtain further insight into the molecular mechanisms of 6-GIN antiseizure activity, we assessed the concentration of two neurotransmitters in zebrafish, i.e., inhibitory γ-aminobutyric acid (GABA) and excitatory glutamic acid (GLU), and their ratio after exposure to acute PTZ dose. Here, 6-GIN decreased GLU level and reduced the GLU/GABA ratio in PTZ-treated fish compared with only PTZ-bathed fish. This activity was associated with the decrease in grin2b, but not gabra1a, grin1a, gria1a, gria2a, and gria3b expression in PTZ-treated fish. Molecular docking to the human NR2B-containing N-methyl-D-aspartate (NMDA) receptor suggests that 6-GIN might act as an inhibitor and interact with the amino terminal domain, the glutamate-binding site, as well as within the ion channel of the NR2B-containing NMDA receptor. In summary, our study reveals, for the first time, the anticonvulsant activity of 6-GIN. We suggest that this effect might at least be partially mediated by restoring the balance between GABA and GLU in the epileptic brain; however, more studies are needed to prove our hypothesis.

Leptin Controls Glutamatergic Synaptogenesis and NMDA-Receptor Trafficking via Fyn Kinase Regulation of NR2B.

Activation of the leptin receptor, LepRb, by the adipocytokine/neurotrophic factor leptin in the central nervous system has procognitive and antidepressive effects. Leptin has been shown to increase glutamatergic synaptogenesis in multiple brain regions. In contrast, mice that have a mutation in the LepRb gene show abnormal synapse development in the hippocampus as well as deficits in cognition and increased depressive-like symptoms. Leptin increases glutamatergic synaptogenesis, in part, through enhancement of N-methyl-D-aspartic acid (NMDA) receptor function; yet the underlying signaling pathway is not known. In this study, we examine how leptin regulates surface expression of NR2B-containing NMDA receptors in hippocampal neurons. Leptin stimulation increases NR2BY1472 phosphorylation, which is inhibited by the Src family kinase inhibitor, PP1. Moreover, we show that Fyn, a member of the Src family kinases, is required for leptin-stimulated NR2BY1472 phosphorylation. Furthermore, inhibiting Y1472 phosphorylation with either a dominant negative Fyn mutant or an NR2B mutant that lacks the phosphorylation site (NR2BY1472F) blocks leptin-stimulated synaptogenesis. Additionally, we show that LepRb forms a complex with NR2B and Fyn. Taken together, these findings expand our knowledge of the LepRb interactome and the mechanisms by which leptin stimulates glutamatergic synaptogenesis in the developing hippocampus. Comprehending these mechanisms is key for understanding dendritic spine development and synaptogenesis, alterations of which are associated with many neurological disorders.

NMDA receptors promote neurogenesis in the neonatal rat subventricular zone following hypoxic‑ischemic injury.

Evidence suggests the involvement of N-methyl-D-aspartate receptors (NMDAR) in the regulation of neurogenesis. Functional properties of NMDAR are strongly influenced by the type of NR2 subunits in the receptor complex. NR2A- and NR2B-containing receptors are expressed in neonatal fore-brain regions, such as the subventricular zone (SVZ). The aim of the present study was to examine the effect of the protein expression of hypoxic-ischemic injury NMDAR subunits 2A and 2B in the SVZ of neonatal rats. Expression of these and other proteins of interest was performed using immunohistochemistry. The results showed that NR2A expression was decreased at 6 h after hypoxic-ischemic injury. By contrast, a significant increase in NR2B expression was observed at 24 h after hypoxic-ischemic injury, induced by the clamping of the right common carotid artery. The functional effect of NMDAR subunits on neurogenesis was also examined by quantifying Nestin and doublecortin (DCX), the microtubule-associated protein expressed only in immature neurons. In addition, the effects of selective non-competitive NMDAR antagonist MK-801 (0.5 mg/kg), NR2B antagonist Ro25-6981 (5 mg/kg), and NR2A antagonist NVP-AAM077 (5 mg/kg) administered 30 min prior to the hypoxic-ischemic injury were examined. The number of Nestin- and DCX-positive cells increased significantly 48 h after hypoxic-ischemic injury, which was reverted by the MK-801 and Ro25-6981 antagonists. Notably, NVP-AAM077 had no significant effect on the expression of Nestin and DCX. In conclusion, the results of the present study demonstrate that hypoxia-ischemia inhibited the expression of NR2A, but promoted the expression of NR2B. Furthermore, NMDAR promoted neurogenesis in the SVZ of neonatal brains.

Effects of Chronic NMDA‐NR2b Inhibition in the Median Eminence of the Reproductive Senescent Female Rat

Gonadotrophin-releasing hormone (GnRH) neurones of the hypothalamic-pituitary-gonadal (HPG) axis drive reproductive function and undergo age-related decreases in activation during the transition to reproductive senescence. Decreased GnRH secretion from the median eminence (ME) partially arises from attenuated glutamatergic signalling via the NMDA receptor (NMDAR) and may be a result of changing NMDAR stoichiometry to favour NR2b over NR2a subunit expression with ageing. We have previously shown that the systemic inhibition of NR2b-containing receptors with ifenprodil, an NR2b-specific antagonist, stimulates parameters of luteinising hormone (used as a proxy for GnRH) release in both young and middle-aged females. In the present study, we chronically administered ifenprodil, an NR2b-specific antagonist, at the site of GnRH terminals in the ME or at GnRH perikarya in the preoptic area, in reproductively senescent middle-aged female rats, aiming to determine whether NR2b antagonism could restore aspects of reproductive functionality. Effects on oestrous cyclicity, serum hormones, and protein expression of GnRH, NR2b and phosphorylated NR2b (Tyr-1472) in the ME were measured. Chronic ifenprodil treatment in the ME (but not the preoptic area) altered oestrous cyclicity by increasing the percentage of days spent in pro-oestrus. This was accompanied by increased GnRH fluorescence intensity in the external ME zone and a greater proportion of GnRH terminals that co-labelled with pNR2b with treatment. We also observed changes in the relationships between protein immunofluorescence, serum hormone levels and other aspects of reproductive physiology in acyclic females, as revealed by bionetwork analysis. Together, these data support the hypothesis that NMDAR-NR2b expression and phosphorylation state play a role in reproductive senescence and highlight the ME as a major player in reproductive ageing.

Influences of NR2B-containing NMDA receptors knockdown on neural activity in hippocampal newborn neurons

Adult-born neurons undergo a transient period of plasticity during their integration into the neural circuit. This transient plasticity may involve NMDA receptors containing NR2B, the major subunit expressed at early developmental stages. The main objective of the present study was to investigate the effects of NR2B gene knockdown on the functional integration of the adult-born granule cells generated from the subgranule zone (SGZ) in the hippocampus. The small interfering RNA (siRNA) was used to knock down the NR2B gene in the adult-born hippocampal neurons. In the functional integration test, the mice were exposed to a novel environment (open field arena), and the expression of c-fos was immunohistochemically detected in the hippocampus. After exposure to the novel environment, siRNA-NR2B mice were significantly different from control mice in either the number of squares or the number of rears they crossed, showing decreased horizontal and vertical activity (P<0.05). Moreover, the c-fos expression was increased in both control and siRNA-NR2B mice after open field test. But, it was significantly lower in siRNA-NR2B neurons than in control neurons. It was concluded that the neural activity of newborn neurons is regulated by their own NR2B-containing NMDA glutamate receptors during a short, critical period after neuronal birth.

Dock3 interaction with a glutamate-receptor NR2D subunit protects neurons from excitotoxicity

BackgroundN-methyl-D-aspartate receptors (NMDARs) are critical for neuronal development and synaptic plasticity. Dysregulation of NMDARs is implicated in neuropsychiatric disorders. Native NMDARs are heteromultimeric protein complexes consisting of NR1 and NR2 subunits. NR2 subunits (NR2A–D) are the major determinants of the functional properties of NMDARs. Most research has focused on NR2A- and/or NR2B-containing receptors. A recent study demonstrated that NR2C- and/or NR2D-containing NMDARs are the primary targets of memantine, a drug that is widely prescribed to treat Alzheimer’s disease. Our laboratory demonstrated that memantine prevents the loss of retinal ganglion cells (RGCs) in GLAST glutamate transporter knockout mice, a model of normal tension glaucoma (NTG), suggesting that NR2D-containing receptors may be involved in RGC loss in NTG.ResultsHere we demonstrate that NR2D deficiency attenuates RGC loss in GLAST-deficient mice. Furthermore, Dock3, a guanine nucleotide exchange factor, binds to the NR2D C-terminal domain and reduces the surface expression of NR2D, thereby protecting RGCs from excitotoxicity.ConclusionsThese results suggest that NR2D is involved in the degeneration of RGCs induced by excitotoxicity, and that the interaction between NR2D and Dock3 may have a neuroprotective effect. These findings raise the possibility that NR2D and Dock3 might be potential therapeutic targets for treating neurodegenerative diseases such as Alzheimer’s disease and NTG.

Erratum: Phosphoinositide 3-kinase couples NMDA receptors to superoxide release in excitotoxic neuronal death

Sustained activation of neuronal N-methly D-aspartate (NMDA)-type glutamate receptors leads to excitotoxic cell death in stroke, trauma, and neurodegenerative disorders. Excitotoxic neuronal death results in part from superoxide produced by neuronal NADPH oxidase (NOX2), but how NMDA receptors are coupled to neuronal NOX2 activation is not well understood. Here, we identify a signaling pathway coupling NMDA receptor activation to NOX2 activation in primary neuron cultures. Calcium influx through the NR2B subunit of NMDA receptors leads to the activation of phosphoinositide 3-kinase (PI3K). Formation of phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P3) by PI3K activates the atypical protein kinase C, PKC zeta (PKCζ), which in turn phosphorylates the p47(phox) organizing subunit of neuronal NOX2. Calcium influx through NR2B-containing NMDA receptors triggered mitochondrial depolarization, NOX2 activation, superoxide formation, and cell death. However, equivalent magnitude calcium elevations induced by ionomycin did not induce NOX2 activation or neuronal death, despite causing mitochondrial depolarization. The PI3K inhibitor wortmannin prevented NMDA-induced NOX2 activation and cell death, without preventing cell swelling, calcium elevation, or mitochondrial depolarization. The effects of wortmannin were circumvented by exogenous supply of the PI3K product, PI(3,4,5)P3, and by transfection with protein kinase M, a constitutively active form of PKCζ. These findings demonstrate that superoxide formation and excitotoxic neuronal death can be dissociated from mitochondrial depolarization, and identify a novel role for PI3K in this cell death pathway. Perturbations in this pathway may either increase or decrease superoxide production in response to NMDA receptor activation, and may thereby impact neurological disorders, in which excitotoxicity is a contributing factor.

Phosphoinositide 3-kinase couples NMDA receptors to superoxide release in excitotoxic neuronal death

Sustained activation of neuronal N-methly D-aspartate (NMDA)-type glutamate receptors leads to excitotoxic cell death in stroke, trauma, and neurodegenerative disorders. Excitotoxic neuronal death results in part from superoxide produced by neuronal NADPH oxidase (NOX2), but how NMDA receptors are coupled to neuronal NOX2 activation is not well understood. Here, we identify a signaling pathway coupling NMDA receptor activation to NOX2 activation in primary neuron cultures. Calcium influx through the NR2B subunit of NMDA receptors leads to the activation of phosphoinositide 3-kinase (PI3K). Formation of phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P3) by PI3K activates the atypical protein kinase C, PKC zeta (PKCζ), which in turn phosphorylates the p47phox organizing subunit of neuronal NOX2. Calcium influx through NR2B-containing NMDA receptors triggered mitochondrial depolarization, NOX2 activation, superoxide formation, and cell death. However, equivalent magnitude calcium elevations induced by ionomycin did not induce NOX2 activation or neuronal death, despite causing mitochondrial depolarization. The PI3K inhibitor wortmannin prevented NMDA-induced NOX2 activation and cell death, without preventing cell swelling, calcium elevation, or mitochondrial depolarization. The effects of wortmannin were circumvented by exogenous supply of the PI3K product, PI(3,4,5)P3, and by transfection with protein kinase M, a constitutively active form of PKCζ. These findings demonstrate that superoxide formation and excitotoxic neuronal death can be dissociated from mitochondrial depolarization, and identify a novel role for PI3K in this cell death pathway. Perturbations in this pathway may either increase or decrease superoxide production in response to NMDA receptor activation, and may thereby impact neurological disorders, in which excitotoxicity is a contributing factor.

Group I mGluR antagonist rescues the deficit of D1-induced LTP in a mouse model of fragile X syndrome

BackgroundFragile X syndrome (FXS) is caused by the absence of the mRNA-binding protein Fragile X mental retardation protein (FMRP), encoded by the Fmr1 gene. Overactive signaling by group 1 metabotropic glutamate receptor (Grp1 mGluR) could contribute to slowed synaptic development and other symptoms of FXS. Our previous study has identified that facilitation of synaptic long-term potentiation (LTP) by D1 receptor is impaired in Fmr1 knockout (KO) mice. However, the contribution of Grp1 mGluR to the facilitation of synaptic plasticity by D1 receptor stimulation in the prefrontal cortex has been less extensively studied.ResultsHere we demonstrated that DL-AP3, a Grp1 mGluR antagonist, rescued LTP facilitation by D1 receptor agonist SKF81297 in Fmr1KO mice. Grp1 mGluR inhibition restored the GluR1-subtype AMPA receptors surface insertion by D1 activation in the cultured Fmr1KO neurons. Simultaneous treatment of Grp1 mGluR antagonist with D1 agonist recovered the D1 receptor signaling by reversing the subcellular redistribution of G protein-coupled receptor kinase 2 (GRK2) in the Fmr1KO neurons. Treatment of SKF81297 alone failed to increase the phosphorylation of NR2B-containing N-methyl D-aspartate receptors (NMDARs) at Tyr-1472 (p-NR2B-Tyr1472) in the cultures from KO mice. However, simultaneous treatment of DL-AP3 could rescue the level of p-NR2B-Tyr1472 by SKF81297 in the cultures from KO mice. Furthermore, behavioral tests indicated that simultaneous treatment of Grp1 mGluR antagonist with D1 agonist inhibited hyperactivity and improved the learning ability in the Fmr1KO mice.ConclusionThe findings demonstrate that mGluR1 inhibition is a useful strategy to recover D1 receptor signaling in the Fmr1KO mice, and combination of Grp1 mGluR antagonist and D1 agonist is a potential drug therapy for the FXS.

NR2A and NR2B subunits differentially mediate MAP kinase signaling and mitochondrial morphology following excitotoxic insult

NMDA receptors are essential for neurotransmission and key mediators of synaptic signaling, but they can also trigger deleterious degenerative processes that lead to cell death. Growing evidence suggests that selective blockade of the heterogeneous subunits that comprise the NMDA receptor may enable better control of pharmacotherapies for treating neurological diseases and injuries. We investigated the relationship between NMDAR activation, MAPK signaling, and mitochondrial shape following an excitotoxic insult. NR2A- and NR2B-containing NMDARs differentially mediated acute changes in cytosolic calcium, alterations in mitochondrial morphology, and phosphorylation of the MAPKs ERK and JNK. Activation of NR2A-containing NMDARs was associated with JNK phosphorylation that was neuroprotective in neuronal cultures subjected to excitotoxicity. In contrast, activation of NR2B-containing NMDARs triggered calcium accumulation in mitochondria that was strongly associated with mitochondrial swelling and neuronal cell death. Indeed, while blockade of NR2B-containing receptors was neuroprotective, this protection was lost when NR2A-initiated JNK phosphorylation was inhibited. Given the modest selectivity of the NR2A inhibitor, NVP-AAM077, the results highlight the significance of the relative, rather than absolute, activation of these two NMDA subtypes in modulating cell death pathways. Therefore, the balance between concurrent activation of NR2B-containing and NR2A-containing NMDARs dictates neuronal fate following excitotoxicity.

The Inhibition of Spinal Astrocytic JAK2-STAT3 Pathway Activation Correlates with the Analgesic Effects of Triptolide in the Rat Neuropathic Pain Model.

Neuropathic pain (NP) is an intractable clinical problem without satisfactory treatments. However, certain natural products have been revealed as effective therapeutic agents for the management of pain states. In this study, we used the spinal nerve ligation (SNL) pain model to investigate the antinociceptive effect of triptolide (T10), a major active component of the traditional Chinese herb Tripterygium wilfordii Hook F. Intrathecal T10 inhibited the mechanical nociceptive response induced by SNL without interfering with motor performance. Additionally, the anti-nociceptive effect of T10 was associated with the inhibition of the activation of spinal astrocytes. Furthermore, intrathecal administration of T10 attenuated SNL-induced janus kinase (JAK) signal transducers and activators of transcription 3 (STAT3) signalling pathway activation and inhibited the upregulation of proinflammatory cytokines, such as interleukin-6, interleukin-1 beta, and tumour necrosis factor-α, in dorsal horn astrocytes. Moreover, NR2B-containing spinal N-methyl D-aspartate receptor (NMDAR) was subsequently inhibited. Above all, T10 can alleviate SNL-induced NP via inhibiting the neuroinflammation in the spinal dorsal horn. The anti-inflammation effect of T10 may be related with the suppression of spinal astrocytic JAK-STAT3 activation. Our results suggest that T10 may be a promising drug for the treatment of NP.

A steroid modulatory domain in NR2A collaborates with NR1 exon‐5 to control NMDAR modulation by pregnenolone sulfate and protons

NMDA receptor (NMDAR)-mediated excitatory synaptic transmission plays a critical role in synaptic plasticity and memory formation, whereas its dysfunction may underlie neuropsychiatric and neurodegenerative diseases. The neuroactive steroid pregnenolone sulfate (PS) acts as a cognitive enhancer in impaired animals, augments LTP in hippocampal slices by enhancing NMDAR activity, and may participate in the reduction of schizophrenia's negative symptoms by systemic pregnenolone. We report that the effects of PS on NMDAR function are diverse, varying with subunit composition and NR1 splice variant. While PS potentiates NR1-1a/NR2B receptors through a critical steroid modulatory domain in NR2B that also modulates tonic proton inhibition, potentiation of the NMDA response is not dependent upon relief of such inhibition, a finding that distinguishes it from spermine. In contrast, the presence of an NR2A subunit confers enhanced PS-potentiation at reduced pH, suggesting that it may indeed act like spermine does at NR2B-containing receptors. Additional tuning of the NMDAR response by PS comes via the N-terminal exon-5 splicing insert of NR1-1b, which regulates the magnitude of proton-dependent PS potentiation. For NR2C- and NR2D-containing receptors, negative modulation at NR2C receptors is pH-independent (like NR2B) while negative modulation at NR2D receptors is pH-dependent (like NR2A). Taken together, PS displays a rich modulatory repertoire that takes advantage of the structural diversity of NMDARs in the CNS. The differential pH sensitivity of NMDAR isoforms to PS modulation may be especially important given the emerging role of proton sensors to both learning and memory, as well as brain injury.

NMDA Receptor Activation Suppresses Microtubule Growth and Spine Entry

Dynamic microtubules are important to maintain neuronal morphology and function, but whether neuronal activity affects the organization of dynamic microtubules is unknown. Here, we show that a protocol to induce NMDA-dependent long-term depression (LTD) rapidly attenuates microtubule dynamics in primary rat hippocampal neurons, removing the microtubule-binding protein EB3 from the growing microtubule plus-ends in dendrites. This effect requires the entry of calcium and is mediated by activation of NR2B-containing NMDA-type glutamate receptor. The rapid NMDA effect is followed by a second, more prolonged response, during which EB3 accumulates along MAP2-positive microtubule bundles in the dendritic shaft. MAP2 is both required and sufficient for this activity-dependent redistribution of EB3. Importantly, NMDA receptor activation suppresses microtubule entry in dendritic spines, whereas overexpression of EB3-GFP prevents NMDA-induced spine shrinkage. These results suggest that short-lasting and long-lasting changes in dendritic microtubule dynamics are important determinants for NMDA-induced LTD.

Soluble Aβ Oligomers Inhibit Long-Term Potentiation through a Mechanism Involving Excessive Activation of Extrasynaptic NR2B-Containing NMDA Receptors

In Alzheimer's disease (AD), dementia severity correlates strongly with decreased synapse density in hippocampus and cortex. Numerous studies report that hippocampal long-term potentiation (LTP) can be inhibited by soluble oligomers of amyloid β-protein (Aβ), but the synaptic elements that mediate this effect remain unclear. We examined field EPSPs and whole-cell recordings in wild-type mouse hippocampal slices. Soluble Aβ oligomers from three distinct sources (cultured cells, AD cortex, or synthetic peptide) inhibited LTP, and this was prevented by the selective NR2B inhibitors ifenprodil and Ro 25-6981. Soluble Aβ enhanced NR2B-mediated NMDA currents and extrasynaptic responses; these effects were mimicked by the glutamate reuptake inhibitor dl-threo-β-benzyloxyaspartic acid. Downstream, an Aβ-mediated rise in p38 mitogen-activated protein kinase (MAPK) activation was followed by downregulation of cAMP response element-binding protein, and LTP impairment was prevented by inhibitors of p38 MAPK or calpain. Thus, soluble Aβ oligomers at low nanomolar levels present in AD brain increase activation of extrasynaptic NR2B-containing receptors, thereby impairing synaptic plasticity.

Involvement of Spinal NR2B-Containing NMDA Receptors in Oxaliplatin-Induced Mechanical Allodynia in Rats

BackgroundOxaliplatin is a platinum-based chemotherapy drug characterized by the development of acute and chronic peripheral neuropathies. The chronic neuropathy is a dose-limiting toxicity. We previously reported that repeated administration of oxaliplatin induced cold hyperalgesia in the early phase and mechanical allodynia in the late phase in rats. In the present study, we investigated the involvement of NR2B-containing N-methyl-D-aspartate (NMDA) receptors in oxaliplatin-induced mechanical allodynia in rats.ResultsRepeated administration of oxaliplatin (4 mg/kg, i.p., twice a week) caused mechanical allodynia in the fourth week, which was reversed by intrathecal injection of MK-801 (10 nmol) and memantine (1 μmol), NMDA receptor antagonists. Similarly, selective NR2B antagonists Ro25-6981 (300 nmol, i.t.) and ifenprodil (50 mg/kg, p.o.) significantly attenuated the oxaliplatin-induced pain behavior. In addition, the expression of NR2B protein and mRNA in the rat spinal cord was increased by oxaliplatin on Day 25 (late phase) but not on Day 5 (early phase). Moreover, we examined the involvement of nitric oxide synthase (NOS) as a downstream target of NMDA receptor. L-NAME, a non-selective NOS inhibitor, and 7-nitroindazole, a neuronal NOS (nNOS) inhibitor, significantly suppressed the oxaliplatin-induced pain behavior. The intensity of NADPH diaphorase staining, a histochemical marker for NOS, in the superficial layer of spinal dorsal horn was obviously increased by oxaliplatin, and this increased intensity was reversed by intrathecal injection of Ro25-6981.ConclusionThese results indicated that spinal NR2B-containing NMDA receptors are involved in the oxaliplatin-induced mechanical allodynia.

Nickel modulates the electrical activity of cultured cortical neurons through a specific effect on N-methyl-d-aspartate receptor channels

Nickel (Ni2+) is a toxic metal that affects the function of several ionic channels. In the N-methyl-d-aspartate (NMDA) subtype of glutamate receptor (NR), it causes activity enhancement of the channels containing the NR2B subunit and voltage-independent inhibition of those containing NR2A. Thus, it may represent a functional marker for the identification of NR native channel subunits. We investigated the effect of Ni2+ on spontaneous NR currents in cortical neurons, dissociated from 18-day rat embryos and maintained in culture for up to ∼40 days. In whole-cell voltage-clamp at −60 mV, in a Mg2+-free bath containing the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) (10 μM), spontaneous currents were blocked by 10 μM D(–)-2-Amino-5-phosphonopentanoic acid (APV) (10 μM), and by NR2B antagonists, ifenprodil (10 μM) or Ro25-6981 (Ro25, 1 μM), indicating that they are due to NRs containing predominantly the NR2B subunit. In the presence of Ni2+ (30 μM) the amplitude and the frequency of spontaneous currents were increased and the decay time decreased. A higher dose (300 μM) blocked all electrical activity. In current-clamp, Ni2+ (30 μM) caused a ∼5 mV reversible depolarization. The effect of Ni2+, as well as that of NR2B antagonists, was almost independent of days in vitro (DIV) in the range from 18 to 33 DIV. The electrical activity of the neuronal networks measured by microelectrode arrays (MEAs) was also affected by Ni2+, which caused a decrease in firing rate, but an increase in burst duration, while Ro25 (1–10 μM) caused a decrease in both firing rate and burst duration. Finally, reverse transcription polymerase chain reaction (RT-PCR) revealed a predominant expression of NR2B, with no modification during DIV. These results demonstrate that, in these cultured cells, the NR spontaneous current is almost entirely due by NR2B-containing receptors and that Ni2+ affects the electrical activity through a specific effect on NR channels.

Kv4.2 block of long-term potentiation is partially dependent on synaptic NMDA receptor remodeling

Proper expression of synaptic NMDA receptors (NMDARs) is necessary to regulate synaptic Ca 2+ influx and the induction the long-term potentiation (LTP) in the mammalian hippocampus. Previously we reported that expressing the A-type K + channel subunit Kv4.2 in CA1 neurons of organotypic slice cultures reduced synaptic NR2B-containing NMDAR expression and completely blocked LTP induced by a pairing protocol. As pretreatment with an NMDAR antagonist (APV) overnight blocked the reduction of NR2B-containing receptors in neurons expressing EGFP-labeled Kv4.2 (Kv4.2g), we hypothesized that LTP would be rescued in Kv4.2g neurons by overnight treatment with APV. We report here that the overnight APV pretreatment in Kv4.2g-expressing neurons only partially restored potentiation. This partial potentiation was completely blocked by inhibition of the CAMKII kinase. These results indicate that A-type K + channels must regulate synaptic integration and plasticity through another mechanism in addition to their regulation of synaptic NR2 subunit composition. We suggest that dendritic excitability, which is regulated by Kv4.2 expression, also contributes to synaptic plasticity.

Identification and Characterization of Novel NMDA Receptor Antagonists Selective for NR2A- over NR2B-Containing Receptors

NR1/NR2A is a subtype of N-methyl-d-aspartate receptors (NMDARs), which are glutamate and glycine-gated Ca(2+)-permeable channels highly expressed in the central nervous system. A high-throughput screening (HTS) campaign using human osteosarcoma (U-2 OS) cells transiently transduced with NR1/NR2A NMDAR subunits, tested in a specifically designed fluorometric imaging plate reader (FLIPR)/Ca(2+) assay, identified sulfonamide derivative series, exemplified by 3-chloro-4-fluoro-N-[(4-{[2-(phenylcarbonyl)hydrazino]carbonyl}phenyl)methyl]benzenesulfonamide (compound 1) and thiodiazole derivative N-(cyclohexylmethyl)-2-({5-[(phenylmethyl)amino]-1,3,4-thiadiazol-2-yl}thio)acetamide (compound 13) as novel NR1/NR2A receptor antagonists. Compounds 1 and 13 displayed submicromolar and micromolar potency at NR1/NR2A receptor, respectively, although they did not show activity at NR2B-containing receptor up to 50 μM concentration. Addition of 1 mM glycine, but not 1 mM l-glutamate, was able to surmount compound 1 and 13 inhibitory effects in FLIPR NR1/NR2A assay. However, compounds 1 and 13 displaced a glutamate site antagonist [(3)H]d,l-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid ([(3)H]CGP 39653) to a greater extent than the glycine site antagonist [(3)H]3-[(E)-2-carboxy-2-phenylethenyl]-4,6-dichloro-1H-indole-2-carboxylic acid ([(3)H]MDL 105,519), in rat brain cortex binding assay. Results of FLIPR cell-based, electrophysiological, and biochemical binding assays suggest that compounds 1 and 13 are the prototypes of novel classes of NMDAR ligands, which to the best of our knowledge are the first selective antagonists at NR1/NR2A over NR1/NR2B receptor, and might constitute useful tools able to elucidate the relative role of the NR2A subunit in physiological and pathological conditions.

Long-Term Potentiation in the CA1 Hippocampus Induced by NR2A Subunit-Containing NMDA Glutamate Receptors Is Mediated by Ras-GRF2/Erk Map Kinase Signaling

BackgroundNMDA-type glutamate receptors (NMDARs) are major contributors to long-term potentiation (LTP), a form of synaptic plasticity implicated in the process of learning and memory. These receptors consist of calcium-permeating NR1 and multiple regulatory NR2 subunits. A majority of studies show that both NR2A and NR2B-containing NMDARs can contribute to LTP, but their unique contributions to this form of synaptic plasticity remain poorly understood.Methodology/Principal FindingsIn this study, we show that NR2A and NR2B-containing receptors promote LTP differently in the CA1 hippocampus of 1-month old mice, with the NR2A receptors functioning through Ras-GRF2 and its downstream effector, Erk Map kinase, and NR2B receptors functioning independently of these signaling molecules.Conclusions/SignificanceThis study demonstrates that NR2A-, but not NR2B, containing NMDA receptors induce LTP in pyramidal neurons of the CA1 hippocamus from 1 month old mice through Ras-GRF2 and Erk. This difference add new significance to the observation that the relative levels of these NMDAR subtypes is regulated in neurons, such that NR2A-containing receptors become more prominent late in postnatal development, after sensory experience and synaptic activity.

Contribution of the spinal cord BDNF to the development of neuropathic pain by activation of the NR2B-containing NMDA receptors in rats with spinal nerve ligation

The NMDA receptor and the brain-derived neurotrophic factor (BDNF) are involved in central sensitization and synaptic plasticity in the spinal cord. To determine whether the spinal cord BDNF contributes to the development and maintenance of neuropathic pain by activation of the dorsal horn NR2B-containing NMDA (NMDA-2B) receptors, this study was designed to investigate if alterations in BDNF and its TrkB receptor in the spinal dorsal horn would parallel the timeline of the development of neuropathic pain in lumbar 5 (L5) spinal nerve ligated (SNL) rats. The enzyme-linked immunosorbent assay (ELISA) showed that the BDNF concentration significantly increased during 24 h post-surgery, and the maximal enhancement lasted for 48 h. It declined as time progressed and returned to the level of pre-operation at 28 days after SNL. In parallel with the alteration of BDNF concentration in the spinal dorsal horn, the 50% paw withdrawal threshold (PWT) of the ipsilateral hind paw in SNL rats also showed a significant decrease during 24–48 h after SNL as compared with those in sham-operated rats. The correlation analysis revealed that the BDNF concentration had a negative correlation with 50% PWT in early stage (0–48 h) ( r = -0.974, p = 0.001), but not late stage (3–28 days) ( r = 0.3395, p = 0.6605), after SNL. Similarly, the immunohistochemical staining revealed that a significant up-regulation of BDNF expression in the spinal dorsal horn appeared as early as 12 h post-operation in SNL rats, peaked at 24–48 h, declined at 3 days and disappeared at 14 days after SNL. In contrast, an increase in NMDA-2B receptors expression in the spinal dorsal horn was delayed to 48 h after SNL. The increase reached peak at 3 days, lasted for 14 days, and returned to the control level of pre-operation at 28 days after SNL. The maximal enhancement of BDNF expression occurred in early stage (24–48 h) after nerve injury, while the peak of NMDA-2B receptors expression appeared in late stage (3–14 days) post-nerve ligation. As compared with the dynamic changes of 50% PWT in the timeline after nerve injury, the maximal enhancement of BDNF expression closely paralleled the maximal decline in the slope of 50% PWT, while the peak of NMDA-2B receptors expression corresponded with the plateau of the decreased 50% PWT. Therefore, the increased BDNF in the spinal dorsal horn was likely to be associated with the initiation of neuropathic pain in early stage (0–48 h), while the activation of NMDA-2B receptors was involved in the maintenance of persistent pain states in late stage (2–14 days) after nerve injury. Moreover, the present study also demonstrated that the BDNF/TrkB-mediated signaling pathway within the spinal cord might be involved in the induction of neuropathic pain in early stage after nerve injury, and the selective NMDA-2B receptors antagonist (Ro 25-6981) almost completely blocked the BDNF-induced mechanical allodynia in all of the tested rats. These data suggested that the BDNF/TrkB-mediated signaling pathway in the spinal cord was involved in the development of nerve injury-induced neuropathic pain through the activation of dorsal horn NMDA-2B receptors.