CGX-1007 prevents excitotoxic cell death via actions at multiple types of NMDA receptors

Glutamate induced excitotoxic injury through over-activation of N-methyl-D-aspartate receptors (NMDARs) plays a critical role in the development of many neurodegenerative diseases. The present study was undertaken to evaluate the role of CGX-1007 (Conantokin G) as a neuroprotective agent against NMDA-induced excitotoxicity. Conantokin G, a cone snail peptide isolated from Conus geographus is reported to selectively inhibit NR2B containing NMDARs with high specificity and is shown to have potent anticonvulsant and antinociceptive effects. CGX-1007 significantly reduced the excitotoxic cell death induced by NMDA in organotypic hippocampal brain slice cultures in a concentration-dependent manner. In contrast, ifenprodil, another NR2B specific antagonist failed to offer neuroprotection against NMDA-induced excitotoxicity. We further determined that the neuroprotection observed is likely due to the action of CGX-1007 at multiple NMDA receptor subtypes. In a series of electrophysiology experiments, CGX-1007 inhibited NMDA-gated currents in human embryonic kidney (HEK) 293 cells expressing NMDA receptors containing either NR1a/NR2B or NR1a/NR2A subunit combinations. CGX-1007 produced a weak inhibition at NR1a/NR2C receptors, whereas it had no effect on NR1a/NR2D receptors. Further, the inhibition of NMDA receptors by CGX-1007 was voltage-dependent with greater inhibition seen at hyperpolarized membrane potentials. The voltage-dependence of CGX-1007 activity was also observed in recordings of NMDA-gated currents evoked in native receptors expressed in cortical neurons in culture. Based on our results, we conclude that CGX-1007 is a potent neuroprotective agent that acts as an antagonist at both NR2A and NR2B containing receptors.     

Hippocampal synaptic metaplasticity requires the activation of NR2B-containing NMDA receptors

The potential to exhibit synaptic plasticity itself is modulated by previous synaptic activity, which has been termed as metaplasticity. In this paper, we demonstrated that the activation of N-methyl-d-aspartate (NMDA) receptor 2B (NR2B) subunit in NNDA receptors was required for hippocampal metaplasticity at Schaffer collateral-commissural fiber-CA1 synapses. Brief 5 Hz priming stimulation did not cause long-term synaptic plasticity; however, it could result in the inhibition of subsequently evoked long-term potentiation (LTP). Meanwhile, the application of selective antagonists for NR2B subunit of NMDA receptors after delivering priming stimulation could block the metaplasticity. In contrast, LTP induction was not affected by NR2B antagonists in slices without pre-treatment of priming stimulation. These results indicated that the activation of NR2B-containing NMDA receptors was required for metaplasticity.     

NMDA receptor 2B subunit-mediated synaptic transmission in the superficial dorsal horn of peripheral nerve-injured neuropathic mice

Previous research has shown that peripheral inflammation and peripheral nerve injury alter the properties of NMDA receptors in the spinal dorsal horn. However, there is no direct evidence that demonstrates the influence of peripheral nerve injury on NMDA receptor-mediated synaptic transmission in the spinal dorsal horn. Using whole cell tight-seal methods, NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) were recorded from superficial dorsal horn neurons in adult mouse spinal cord slices. Peripheral nerve injury-induced changes in the pharmacological and electrophysiological properties of synaptic NMDA receptors were studied. The ratio of the amplitude of NMDA EPSCs to that of non-NMDA EPSCs was larger in nerve-ligated neuropathic mice than in sham-operated control mice. The decay phase of the NMDA EPSCs was slower in nerve-ligated neuropathic mice. The NR2B subunit-specific NMDA receptor antagonist ifenprodil (10 microM) reduced the amplitude of the NMDA EPSCs and shortened their decay phase. The sensitivity of NMDA EPSCs to ifenprodil was significantly larger in nerve-ligated neuropathic mice than in sham-operated control mice. Single-cell RT-PCR analysis performed on superficial dorsal horn neurons showed that the incidence of NR2A mRNA-expressing neurons was reduced in nerve-ligated neuropathic mice. This result, together with the electrophysiological findings, suggests that the subunit composition of the subsynaptic NMDA receptors in the superficial dorsal horn was altered by peripheral nerve injury. Pharmacological and electrophysiological changes observed in the present experiments might be the underlying causes of the hyperalgesia and allodynia induced by peripheral nerve injury and inflammation.     

Felbamate block of recombinant N-methyl-D-aspartate receptors: selectivity for the NR2B subunit

The anticonvulsant felbamate blocks N-methyl-D-asparate (NMDA) receptors but fails to exhibit the neurobehavioral toxicity characteristic of other NMDA receptor antagonists. To investigate the possibility that felbamate's favorable toxicity profile could be related to NMDA receptor subtype selectivity, we examined the specificity of felbamate block of recombinant NMDA receptors composed of the NR1a subunit and various NR2 subunits. Felbamate produced a rapid, concentration-dependent block of currents evoked by 50 microM NMDA and 10 microM glycine in human embryonic kidney 293 cells expressing the rat NR1a subunit, and either the NR2A, NR2B or NR2C subunits; the IC50 values for block were 2.6, 0.52 and 2.4 mM, respectively (holding potential, - 60 mV). The Hill coefficient values were < 1 and, in kinetic analyses, onset and recovery from block were well fit by double exponential functions, indicating binding to more than one blocking site on the NMDA receptor channel complex. The higher affinity of felbamate block of NMDA receptors containing the NR2B subunit could be accounted for by more rapid association and slower dissociation from these sites. We conclude that felbamate exhibits modest selectivity for NMDA receptors composed of NR1a/NR2B subunits. This selectivity could, in part, account for the more favorable clinical profile of felbamate in comparison with NMDA receptor antagonists that do not show subunit selectivity.     

Lesion-induced changes in NMDA receptor subunit mRNA expression in rat visual cortex

Focal lesions in the visual cortex are well known to induce pronounced perilesional reorganization of the neuronal circuitry. Since NMDA receptors crucially control synaptic plasticity and reorganization, we studied lesion-induced changes in their subunit expression and biophysical properties. Between 8 and 10 days after focal thermolesioning, pyramidal neurones in the near surround of the lesion were studied in acute brain slices. We found a significant decrease in the ratio of NR2A and NR2B subunit mRNA as compared to neurones from sham operated animals. Interestingly, no significant differences in the properties of NMDA receptor-mediated postsynaptic currents (NMDA PSCs) were observed between lesioned and sham operated animals. Thus, the observed perilesional changes in the NR2A/NR2B mRNA ratio appear to be subthreshold to result in significant changes in the functional properties of NMDA receptors.     

Implication of NMDA Receptors in the Antidyskinetic Activity of Cabergoline,CI-1041, and Ro 61-8048 in MPTP Monkeys with Levodopa-induced Dyskinesias

This study assessed striatal N-methyl-D-aspartate (NMDA) glutamate receptors of 1-methyl 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys with levodopa (L-DOPA)-induced dyskinesias (LID). In a first experiment, four MPTP monkeys receiving L-DOPA/Benserazide alone developed dyskinesias. Four MPTP monkeys received L-DOPA/Benserazide plus CI-1041 an NMDA antagonist selective for NR1/NR2B and four were treated with L-DOPA/Benserazide plus a small dose of cabergoline; one monkey of each group developed mild dyskinesias at the end of treatment. In a second experiment, a kynurenine 3-hydroxylase inhibitor Ro 61-8048, combined with L-DOPA/Benserazide, reduced dyskinesias in MPTP monkeys. Drug-treated MPTP monkeys were compared to intact monkeys and saline-treated MPTP monkeys. Glutamate receptors were investigated by autoradiography using [³H]CGP-39653 (NR1/NR2A antagonist) and [³H]Ro25-6981 (NR1/NR2B antagonist). In general, striatal [³H]CGP-39653 specific binding was unaltered in all experimental groups. MPTP lesion decreased striatal [³H]Ro25-6981 specific binding; these levels were enhanced in the L-DOPA-alone-treated MPTP monkeys and decreased in antidyskinetic drugs treated monkeys. Maximal dyskinesias scores of the MPTP monkeys correlated significantly with [³H]Ro25-6981 specific binding in the rostral and caudal striatum. Hence, MPTP lesion, L-DOPA treatment and prevention of LID with CI-1041 and cabergoline, or reduction with Ro 61-8048 were associated with modulation of NR2B/NMDA glutamate receptors.     

Effect of a selective glutamate antagonist on L-dopa-induced dyskinesias in drug-naive parkinsonian monkeys

Alterations of striatal glutamate receptors are believed to be responsible, at least in part, for the pathogenesis of L-dopa-induced dyskinesias (LID). To evaluate whether co-administration of CI-1041, a novel NMDA receptor antagonist selective for the NR1A/NR2B subtype, with L-dopa might prevent the appearance of this side effect, eight de novo parkinsonian monkeys were treated chronically orally with either L-dopa alone or L-dopa plus CI-1041 (n= 4 for each group). After 4 weeks of treatment with L-dopa alone, all four animals developed moderate dyskinesias either choreic or dystonic in nature. CI-1041 co-treatment completely prevented the induction of dyskinesias in three animals and only one monkey developed mild dyskinesias at the end of the fourth week of treatment in the L-dopa + CI-1041 group. The magnitude and duration of the antiparkinsonian action of L-dopa was similar in both groups. These results suggest that selective NMDA receptor antagonism may be interesting for managing LID in Parkinson's disease patients.     

Studies on neuronal apoptoisis in primary forebrain cultures: Neuroprotective/anti-apoptotic action of NR2B NMDA antagonists

While the role of apoptosis in neuronal injury is continually being re-defined, approaches to intervene in the progression of apoptotic injury have been documented to provide neuroprotection against a variety of insults. The present studies were undertaken to systematically study the effects of certain neuroprotective agents against neuronal apoptosis mediated by staurosporine (ST). ST (0.01-5 micro M) produced a dose-related apoptotic injury (as characterized by cellular morphology, 'Comet' assay analysis [single cell gel electrophoresis] and caspase-3 activation) in primary cultures of forebrain neurons. ST significantly increased caspase-3 activity. The NMDA receptor subtype non-selective antagonist dizocilpine [(+) MK-801; 0.1-50 micro M] and a novel sodium channel blocker RS100642 (1.0-250 micro M) had no significant effects against ST-induced neurotoxicity. Conversely, NR2B-selective NMDA receptor antagonists CGX-1007 (0.01-50 micro M) and ifenprodil (0.01-50 micro M) provided dose-dependent neuroprotection against ST-induced neurotoxicity (as measured by neuronal viability and comet assay analysis). CGX-1007 had no significant effect on ST-induced caspase-3 activity; however, ifenprodil did block activation of caspase-3. These studies demonstrate that NR2B NMDA receptor antagonists are anti-apoptotic and may mediate their action via mechanism(s) that are dependent or independent of caspase-3 activation.     

Effects of exercise on NMDA receptor subunit contributions to bidirectional synaptic plasticity in the mouse dentate gyrus

We examined synaptic plasticity in the dentate gyrus (DG) of the hippocampus in vitro in juvenile C57Bl6 mice (28-40 days of age), housed in control conditions with minimal enrichment (Controls) or with access to an exercise wheel (Runners). LTP expression was significantly greater in slices from Runners than in those from Controls, but could be blocked by APV in both groups. LTP was significantly reduced by NR2B subunit antagonists in both groups. NVP-AAM077, an antagonist with a higher preference for NR2A subunits over NR2B subunits, blocked LTP in slices from Runners and produced a slight depression in Control animals. LTD in the DG was also blocked by APV, but not by either of the NR2B specific antagonists. Strikingly, NVP-AAM077 prevented LTD in Runners, but not in Control animals, suggesting an increased involvement of NR2A subunits in LTD in animals that exercise. NVP-AAM077 did not block LTD in NR2A Knock Out (KO) animals that exercised, as expected. In an attempt to discern whether NMDA receptors located at extrasynaptic sites could play a role in the induction of LTD, DL-TBOA was used to block excitatory amino acid transport and increase extracellular glutamate levels. Under these conditions, LTD was not blocked by the co-application of a specific NR2B subunit antagonist in either group, but NVP-AAM077 again blocked LTD selectively in Runners. These results indicate that NR2A and NR2B subunits play a significant role in LTP in the DG, and that exercise can significantly alter the contribution of NMDA NR2A subunits to LTD.     

The NR2B-selective NMDA receptor antagonist CP-101,606 exacerbates L-DOPA-induced dyskinesia and provides mild potentiation of anti-parkinsonian effects of L-DOPA in the MPTP-lesioned marmoset model of Parkinson's disease

In Parkinson's disease (PD), degeneration of the dopaminergic nigrostriatal pathway leads to enhanced transmission at NMDA receptors containing NR2B subunits. Previous studies have shown that some, but not all, NR2B-containing NMDA receptor antagonists alleviate parkinsonian symptoms in animal models of PD. Furthermore, enhanced NMDA receptor-mediated transmission underlies the generation of L-DOPA-induced dyskinesia (LID). The subunit content of NMDA receptors responsible for LID is not clear. Here, we assess the actions of the NMDA antagonist CP-101,606 in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned marmoset model of Parkinson's disease. CP-101,606 is selective for NMDA receptors containing NR2B subunits, with higher affinity for NR1/NR2B complexes compared to ternary NR1/NR2A/NR2B complexes. CP-101,606 had no significant effect on parkinsonian symptoms when administered as monotherapy over a range of doses (0.1-10 mg/kg). CP-101,606 provided a modest potentiation of the anti-parkinsonian actions of L-DOPA (8 mg/kg), although, at doses of 1 and 3 mg/kg, CP-101,606 exacerbated LID. Results of this study provide further evidence of differences in the anti-parkinsonian activity and effects on LID of the NR2B subunit selective NMDA receptor antagonists. These distinctions may reflect disparities in action on NR1/NR2B as opposed to NR1/NR2A/NR2B receptors.     

Activity dependent localization of synaptic NMDA receptors in spinal neurons

In cultured spinal neurons, NMDA receptors are absent from excitatory synapses under basal conditions, but can be made to appear at excitatory synapses following blockade of excitatory synaptic activity. The activity dependent synaptic localization of NMDA receptors is critically dependent on both the gradual, global accumulation of the NR2A and NR2B subunits and on a rapid, surface redistribution phase that is primed by the accumulation of NR2A and NR2B and inhibited by synaptic activity. Global changes in NR2A and NR2B accumulation and heterogeneous increases in synaptic NMDA receptor localization can also result from inhibitors of proteasomal processing, from manipulations of proteasomal subunit composition and from media conditioned by neurons undergoing synaptic scaling. While proteasomal processing is a mechanism shared with AMPA receptor scaling in cultured spinal neurons, diffusible factors, heterogeneity, and a rapid surface redistribution phase appear to be unique to activity dependent synaptic NMDA receptor localization.     

Augmentation by zinc of NMDA receptor-mediated synaptic responses in CA1 of rat hippocampal slices: mediation by Src family tyrosine kinases

Normal neuronal activity results in the release of zinc from the synaptic vesicles of glutamatergic terminals and subsequent entry into postsynaptic neurons. Although the exact physiological role of zinc translocation is currently unknown, it is very likely that intracellular zinc exerts long-term modulatory effects upon synaptic transmission since zinc affects various molecules involved in signaling pathways. In this study we used rat hippocampal slices to examine the effect of zinc on glutamatergic synaptic transmission in the Schaffer collateral-CA1 synapses. Following a 10-min exposure to 0.3-1 mM zinc, the magnitude of NMDA receptor-mediated field excitatory postsynaptic potentials (fEPSP) gradually increased over the subsequent 30-40 min. In contrast, the magnitude of AMPA/kainate receptor-mediated fEPSPs remained unchanged. The selective potentiation of NMDA receptor-mediated fEPSPs by zinc was unlikely to be a presynaptic event, since the degree of paired-pulse facilitation was unaltered. Interestingly, the specific Src family tyrosine kinase inhibitor PP2 completely blocked zinc-induced potentiation of NMDA receptor-mediated fEPSP while the inactive analog PP3 had no effect, thereby suggesting the involvement of Src family tyrosine kinases. Furthermore, zinc exposure increased levels of total and tyrosine-phosphorylated forms of NR2A and NR2B in a PP2-dependent manner in both hippocampal slices and cell cultures. In addition, zinc treatment of hippocampal cultures increased the levels of tyrosine phosphorylation at the two positive regulatory sites of Src family tyrosine kinases. Our results demonstrate that zinc increases NMDA receptor function via Src family tyrosine kinase-mediated increases of NR2A and 2B tyrosine phosphorylation. We speculate that intense release of endogenous synaptic zinc may potentiate NMDA receptor-mediated transmission in zinc-containing glutamatergic pathways by a similar mechanism.     

Expression of functional NR1/NR2B-type NMDA receptors in neuronally differentiated SK-N-SH human cell line

The present study demonstrates that human SK-N-SH neuroblastoma cells, differentiated by retinoic acid (RA), express functional NMDA receptors and become vulnerable to glutamate toxicity. During exposure to RA, SK-N-SH cells switched from non-neuronal to neuronal phenotype by showing antigenic changes typical of postmitotic neurons together with markers specific for cholinergic cells. Neuronally differentiated cells displayed positive immunoreactivity to the vesicular acetylcholine transporter and active acetylcholine release in response to depolarizing stimuli. The differentiation correlated with the expression of NMDA receptors. RT-PCR and immunoblotting analysis identified NMDA receptor subunits NR1 and NR2B, in RA-differentiated cultures. The NR1 protein immunolocalized to the neuronal cell population and assembled with the NR2B subunit to form functional N-methyl-D-aspartate (NMDA) receptors. Glutamate or NMDA application, concentration-dependently increased the intracellular Ca2+ levels and acetylcholine release in differentiated cultures, but not in undifferentiated SK-N-SH cells. Moreover, differentiated cultures became vulnerable to NMDA receptor-mediated excitotoxicity. The glutamate effects were enhanced by glycine application and were prevented by the NMDA receptor blocker MK 801, as well as by the NR2B selective antagonist ifenprodil. These data suggest that SK-N-SH cells differentiated by brief treatment with RA may represent an unlimited source of neuron-like cells suitable for studying molecular events associated with activation of human NR1/NR2B receptors.     

Long-term potentiation in the nucleus accumbens requires both NR2A- and NR2B-containing N-methyl-D-aspartate receptors

N-methyl-d-aspartate (NMDA) receptors play crucial roles in several forms of long-term changes in the efficacy of glutamatergic synaptic transmission. The suggestion that the NR2A subunit of the NMDA receptor may be selectively involved in the induction of long-term potentiation (LTP) in the hippocampus and cortex has been challenged. However, the contribution of NR2B in the induction of LTP is not always clearly established. The present study investigates the role of NR2A and NR2B in the induction of LTP in the nucleus accumbens (NAc), a brain region that expresses high levels of NR2B and an NMDA-dependent form of LTP. We recorded extracellular field excitatory postsynaptic potentials/population spikes in slices of mouse NAc. High-frequency stimulation of glutamatergic fibers consistently induced LTP of the field excitatory postsynaptic potential/population spike in the NAc. LTP was abolished in the presence of selective antagonists of either NR2B [R-(R*,S*)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenyl-methyl)-1-piperidine propanol and Ifenprodil] or NR2A ([(R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid) subunits. Recordings performed in a low concentration of Mg(2+) ions in the perfusion solution did not reveal a selective involvement of a particular NMDA receptor subunit because either NR2A or NR2B antagonists were able to block LTP. LTP was also abolished in the presence of a low concentration of the non-subunit-selective NMDA receptor antagonist dl-2-amino-5-phosphonopentanoic acid in normal Mg(2+) and low Mg(2+) in the perfusion solution. These results show that the degree of NMDA receptor activation, and not their subunit composition, determines whether LTP is induced in the NAc.     

NR2B receptors are involved in the mediation of spinal segmental reflex potentials but not in the cumulative motoneuronal depolarization in vitro

Windup, the frequency dependent build-up of spinal neuronal responses is an electrophysiological model of the development of the central sensitization in the chronic pain states. NR2B subunit containing NMDA-type glutamate receptors are implicated in the windup of dorsal horn neurons, while their role at the motoneuronal level is controversial. The cumulative motoneuronal depolarization in hemisected rat spinal cord preparation is an in vitro model of windup. The role of NR2B receptors in this process, and in the mediation of dorsal root stimulation evoked ventral root reflex potentials was elucidated. Three selective NR2B antagonists; CP-101,606; CI-1041 and Co-101244 (1 microM) were used. They had only weak, but statistically significant inhibitory effect on the early part of ventral root response, and did not influence the cumulative depolarization. On the contrary, non-selective NMDA antagonist APV (40 microM) decreased both responses markedly. We conclude that the pharmacological sensitivities of windup at the sensory and motor levels are different. NR2B containing NMDA receptors have major role in the mediation of the windup of dorsal horn neurons, but their contribution to this phenomenon at the motor level is negligible.     

Coactivation of NMDA receptors by glutamate and D-serine induces dilation of isolated middle cerebral arteries

N-methyl-D-aspartate (NMDA) receptors are glutamate-gated cation channels that mediate excitatory neurotransmission in the central nervous system. In addition to glutamate, NMDA receptors are also activated by coagonist binding of the gliotransmitter, D-serine. Neuronal NMDA receptors mediate activity-dependent blood flow regulation in the brain. Our objective was to determine whether NMDA receptors expressed by brain endothelial cells can induce vasodilation of isolated brain arteries. Adult mouse middle cerebral arteries (MCAs) were isolated, pressurized, and preconstricted with norepinephrine. N-methyl-D-aspartate receptor agonists, glutamate and NMDA, significantly dilated MCAs in a concentration-dependent manner in the presence of D-serine but not alone. Dilation was significantly inhibited by NMDA receptor antagonists, D-2-amino-5-phosphonopentanoate and 5,7-dichlorokynurenic acid, indicating a response dependent on NMDA receptor glutamate and D-serine binding sites, respectively. Vasodilation was inhibited by denuding the endothelium and by selective inhibition or genetic knockout of endothelial nitric oxide synthase (eNOS). We also found evidence for expression of the pan-NMDA receptor subunit, NR1, in mouse primary brain endothelial cells, and for the NMDA receptor subunit NR2C in cortical arteries in situ. Overall, we conclude that NMDA receptor coactivation by glutamate and D-serine increases lumen diameter in pressurized MCA in an endothelial and eNOS-dependent mechanism.     

BDNF enhancement of postsynaptic NMDA receptors is blocked by ethanol

The neurotrophin brain-derived neurotrophic factor (BDNF) modulates several distinct aspects of synaptic transmission. Physiological and biochemical evidence implicates the NMDA glutamate receptor as one of the targets for BDNF modulation. In the present studies, murine brain slices containing hippocampus and neocortex were used to study the effects of BDNF on excitatory neurotransmission. Acute exposure to BDNF rapidly and reversibly enhanced the magnitude of NMDA-mediated, but not AMPA receptor-mediated, synaptic currents, specifically enhancing the activity of NMDA receptors containing the NR2B subunit. This effect of BDNF was dependent on activation of trkB neurotrophin receptors because similar effects were not seen with the related neurotrophins NT-3 or NGF. Furthermore, activation of trkB receptors in the postsynaptic neuron was required, as BDNF-induced potentiation was blocked by postsynaptic injection of a trk tyrosine kinase inhibitor. Interestingly, the effect of BDNF was also completely blocked by pretreatment with ethanol, even at concentrations of ethanol that had minimal direct effects on NMDA-mediated responses. These results provide a potential mechanism for the proposed role for BDNF in activity-dependent synaptic plasticity and, potentially, learning and memory processes.     

Mapping the binding site of the neuroprotectant ifenprodil on NMDA receptors

Ifenprodil is a noncompetitive antagonist of NMDA receptors highly selective for the NMDA receptor 2B (NR2B) subunit. It is widely used as a pharmacological tool to discriminate subpopulations of NMDA receptors, and derivatives are currently being developed as candidate neuroprotectants. Despite numerous studies on the mechanism of action of ifenprodil on NMDA receptors, the structural determinants responsible for the subunit selectivity have not been identified. By combining functional studies on recombinant NMDA receptors and biochemical studies on isolated domains, we now show that ifenprodil binds to the N-terminal leucine/isoleucine/valine-binding protein (LIVBP)-like domain of NR2B. In this domain, several residues, both hydrophilic and hydrophobic, were found to control ifenprodil inhibition. Their location in a modeled three-dimensional structure suggests that ifenprodil binds in the cleft of the LIVBP-like domain of NR2B by a mechanism (Venus-flytrap) resembling that of the binding of Zn on the LIVBP-like domain of NR2A. These results reinforce the proposal that the LIVBP-like domains of NMDA receptors, and possibly of other ionotropic glutamate receptors, bind modulatory ligands. Moreover, they identify the LIVBP-like domain of the NR2B subunit as a promising therapeutic target and provide a framework for designing structurally novel NR2B-selective antagonists.     

Deletion of the C-terminal domain of the NR2B subunit alters channel properties and synaptic targeting of N-methyl-D-aspartate receptors in nascent neocortical synapses

Channel properties and synaptic targeting of N-methyl-D-aspartate (NMDA) receptors determine their importance in synaptic transmission, long-term synaptic plasticity, and developmental reorganization of synaptic circuits. To investigate the involvement of the C-terminal domain of the NR2B subunit in regulating channel properties and synaptic localization, we analyzed gene-targeted mice expressing C-terminally truncated NR2B subunits (NR2B(DeltaC/DeltaC) mice; Sprengel et al. [1998] Cell 92:279-89). Because homozygous NR2B(DeltaC/DeltaC) mice die perinatally, we studied embryonic neocortical neurons differentiating in culture. At early stages in vitro, neurons predominantly expressed NR1/NR2B receptors, as shown by the NR2B subunit-specific antagonist ifenprodil. At these nascent synapses, NMDA excitatory postsynaptic currents (EPSCs) in neurons from NR2B(DeltaC/DeltaC) mice showed a strong-amplitude reduction to 20% of control, but AMPA EPSCs were unaltered. Analysis of the MK-801 block of NMDA receptor-mediated whole-cell currents revealed a decreased peak open probability of NMDA receptor channels (to about 60%) in neurons from NR2B(DeltaC/DeltaC) mice, although their single channel conductance was unchanged. To study effects on synaptic targeting, we determined the fraction of synaptically localized NMDA receptors relative to the whole-cell NMDA receptor population. In neurons from NR2B(DeltaC/DeltaC) mice, the synaptic NMDA receptor fraction was drastically reduced, suggesting that the C-terminal domain of the NR2B subunit plays a major role in synaptic targeting of NMDA receptors at nascent synapses. With increasing time in culture, the reduction in NMDA EPSCs in neurons from NR2B(DeltaC/DeltaC) mice diminished. This is explained by the expression of additional NMDA receptor subtypes containing NR2A subunits at more mature synapses.