Amyloid β peptide oligomers directly activate NMDA receptors

Amyloid beta (Aβ) oligomers accumulate in the brain tissue of Alzheimer disease patients and are related to disease pathogenesis. The precise mechanisms by which Aβ oligomers cause neurotoxicity remain unknown. We recently reported that Aβ oligomers cause intracellular Ca(2+) overload and neuronal death that can be prevented by NMDA receptor antagonists. This study investigated whether Aβ oligomers directly activated NMDA receptors (NMDARs) using NR1/NR2A and NR1/NR2B receptors that were heterologously expressed in Xenopus laevis oocytes. Indeed, Aβ oligomers induced inward non-desensitizing currents that were blocked in the presence of the NMDA receptor antagonists memantine, APV, and MK-801. Intriguingly, the amplitude of the responses to Aβ oligomers was greater for NR1/NR2A heteromers than for NR1/NR2B heteromers expressed in oocytes. Consistent with these findings, we observed that the increase in the cytosolic concentration of Ca(2+) induced by Aβ oligomers in cortical neurons is prevented by AP5, a broad spectrum NMDA receptor antagonist, but slightly attenuated by ifenprodil which blocks receptors with the NR2B subunit. Together, these results indicate that Aβ oligomers directly activate NMDA receptors, particularly those with the NR2A subunit, and further suggest that drugs that attenuate the activity of such receptors may prevent Aβ damage to neurons in Alzheimer?s disease.     

Triheteromeric NR1/NR2A/NR2B receptors constitute the major N-methyl-D-aspartate receptor population in adult hippocampal synapses

NMDA receptors (NMDARs), fundamental to learning and memory and implicated in certain neurological disorders, are heterotetrameric complexes composed of two NR1 and two NR2 subunits. The function of synaptic NMDARs in postnatal principal forebrain neurons is typically attributed to diheteromeric NR1/NR2A and NR1/NR2B receptors, despite compelling evidence for triheteromeric NR1/NR2A/NR2B receptors. In synapses, the properties of triheteromeric NMDARs could thus far not be distinguished from those of mixtures of diheteromeric NMDARs. To find a signature of NR1/NR2A/NR2B receptors, we have employed two gene-targeted mouse lines, expressing either NR1/NR2A or NR1/NR2B receptors without NR1/NR2A/NR2B receptors, and compared their synaptic properties with those of wild type. In acute hippocampal slices of mutants older than 4 weeks we found a distinct voltage dependence of NMDA R-mediated excitatory postsynaptic current (NMDA EPSC) decay time for the two diheteromeric NMDARs. In wild-type mice, NMDA EPSCs unveiled the NR1/NR2A characteristic for this voltage-dependent deactivation exclusively, indicating that the contribution of NR1/NR2B receptors to evoked NMDA EPSCs is negligible in adult CA3-to-CA1 synapses. The presence of NR1/NR2A/NR2B receptors was obvious from properties that could not be explained by a mixture of diheteromeric NR1/NR2A and NR1/NR2B receptors or by the presence of NR1/NR2A receptors alone. The decay time for NMDA EPSCs in wild type was slower than that for NR1/NR2A receptors, and the sensitivity of NMDA EPSCs to NR2B-directed NMDAR antagonists was 50%. Thus, NR2B is prominent in adult hippocampal synapses as an integral part of NR1/NR2A/NR2B receptors.     

Glutamate receptor properties of human mesencephalic neural progenitor cells: NMDA enhances dopaminergic neurogenesis in vitro

Human midbrain‐derived neural progenitor cells (NPCs) may serve as a continuous source of dopaminergic neurons for the development of novel regenerative therapies in Parkinson’s disease. However, the molecular and functional characteristics of glutamate receptors in human NPCs are largely unknown. Here, we show that differentiated human mesencepahlic NPCs display a distinct pattern of glutamate receptors. In whole‐cell patch‐clamp recordings, l ‐glutamate and NMDA elicited currents in 93% of NPCs after 3 weeks of differentiation in vitro. The concentration‐response plots of differentiated NPCs yielded an EC₅₀ of 2.2 μM for glutamate and an EC₅₀ of 36 μM for NMDA. Glutamate‐induced currents were markedly inhibited by memantine in contrast to 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) suggesting a higher density of functional NMDA than alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)/kainate receptors. NMDA‐evoked currents and calcium signals were blocked by the NR2B‐subunit specific antagonist ifenprodil indicating functional expression of NMDA receptors containing subunits NR1 and NR2B. In calcium imaging experiments, the blockade of voltage‐gated calcium channels by verapamil abolished AMPA‐induced calcium responses but only partially reduced NMDA‐evoked transients suggesting the expression of calcium‐impermeable, GluR2‐containing AMPA receptors. Quantitative real‐time PCR showed a predominant expression of subunits NR2A and NR2B (NMDA), GluR2 (AMPA), GluR7 (kainate), and mGluR3 (metabotropic glutamate receptor). Treatment of NPCs with 100 μM NMDA in vitro during proliferation (2 weeks) and differentiation (1 week) increased the amount of tyrosine hydroxylase‐immunopositive cells significantly, which was reversed by addition of memantine. These data suggest that NMDA receptors in differentiating human mesencephalic NPCs are important regulators of dopaminergic neurogenesis in vitro.     

Contribution of extrasynaptic N-methyl-d-aspartate and adenosine A1 receptors in the generation of dendritic glutamate-mediated plateau potentials

Thin basal dendrites can strongly influence neuronal output via generation of dendritic spikes. It was recently postulated that glial processes actively support dendritic spikes by either ceasing glutamate uptake or by actively releasing glutamate and adenosine triphosphate (ATP). We used calcium imaging to study the role of NR2C/D-containing N-methyl-d-aspartate (NMDA) receptors and adenosine A1 receptors in the generation of dendritic NMDA spikes and plateau potentials in basal dendrites of layer 5 pyramidal neurons in the mouse prefrontal cortex. We found that NR2C/D glutamate receptor subunits contribute to the amplitude of synaptically evoked NMDA spikes. Dendritic calcium signals associated with glutamate-evoked dendritic plateau potentials were significantly shortened upon application of the NR2C/D receptor antagonist PPDA, suggesting that NR2C/D receptors prolong the duration of calcium influx during dendritic spiking. In contrast to NR2C/D receptors, adenosine A1 receptors act to abbreviate dendritic and somatic signals via the activation of dendritic K⁺ current. This current is characterized as a slow-activating outward-rectifying voltage- and adenosine-gated current, insensitive to 4-aminopyridine but sensitive to TEA. Our data support the hypothesis that the release of glutamate and ATP from neurons or glia contribute to initiation, maintenance and termination of local dendritic glutamate-mediated regenerative potentials.     

Interleukin-2 inhibits NMDA receptor-mediated currents directly and may differentially affect subtypes

Using whole-cell patch-clamp recordings, this study investigated the effects of interleukin-2 (IL-2) on N-methyl-d-aspartate (NMDA) receptor-mediated currents (I(NMDA)) in rat cultured hippocampal neurons and human embryonic kidney (HEK) 293 cells expressing recombinant NMDA receptors. We found that IL-2 (0.01-1ng/ml) immediately and significantly decreased peak I(NMDA) in cultured neurons. Interestingly, the peak I(NMDA) induced in HEK 293 cells was also inhibited by IL-2. We also found that IL-2 differentially decreased the peak amplitudes of NR2A- and NR2B-containing NMDA receptor-mediated currents (I(NR2A) and I(NR2B)) by 54+/-5% and 30+/-4%, respectively. These results provide new evidence that IL-2 induces rapid inhibition of peak currents of NMDA receptor-mediated responses with possible NR1/NR2A and NR1/NR2B subtype-differentiation, and suggest that the inhibition is mediated by direct interaction between IL-2 and NMDA receptors.     

Characterization of Agonist-Induced Down-Regulation of NMDA Receptors in Cerebellar Granule Cell Cultures

Abstract: Exposure of cerebellar granule cells to NMDA in culture at 5 days in vitro, when cells are not yet vulnerable to NMDA, evoked a pronounced reduction in NMDA receptor activity, measured by NMDA-induced ⁴⁵Ca²⁺ influx, and counteracted the normal developmental increase in NMDA receptors. The effect was concentration and time dependent, the half-maximal effect being reached at about 45 µM and by 4–5 h. The decrease in NMDA receptor function was accompanied by a significant reduction in the protein level of the obligatory NMDA receptor subunit (NR) NR1. Both parameters remained at a low level as long as the agonist was present. However, receptor down-regulation was reversible, as receptor protein levels and NMDA responses were restored to control values upon NMDA removal, this process requiring protein synthesis. NMDA treatment also elicited a decrease in NR1, NR2A, and NR2B subunit messenger RNA (mRNA) levels. However, in comparison with NMDA receptor proteins, the decrease was faster, and NMDA receptor mRNA content recovered to control levels within 24 h in spite of the presence of NMDA. Concerning the mechanisms of agonist-induced regulation of NMDA receptor expression, it seems that protein kinase C-mediated protein phosphorylation is not involved, whereas inhibition of Ca²⁺/calmodulin-dependent kinase II/IV by KN-62 does depress NMDA receptor expression even in the absence of NMDA.     

Discovery of (R)-1-[2-hydroxy-3-(4-hydroxy-phenyl)-propyl]-4-(4-methyl-benzyl)-piperidin-4-ol: a novel NR1/2B subtype selective NMDA receptor antagonist

Starting from Ro-25-6981 as a lead compound, highly potent and selective NR1/2B subtype selective NMDA receptor antagonists, with low activity at alpha(1) adrenergic receptors were developed.     

NMDA Receptors on Non-Dopaminergic Neurons in the VTA Support Cocaine Sensitization

Background

The initiation of behavioral sensitization to cocaine and other psychomotor stimulants is thought to reflect N-methyl-D-aspartate receptor (NMDAR)-mediated synaptic plasticity in the mesolimbic dopamine (DA) circuitry. The importance of drug induced NMDAR mediated adaptations in ventral tegmental area (VTA) DA neurons, and its association with drug seeking behaviors, has recently been evaluated in Cre-loxp mice lacking functional NMDARs in DA neurons expressing Cre recombinase under the control of the endogenous dopamine transporter gene (NR1^DATCre mice).

Methodology and Principal Findings

Using an additional NR1^DATCre mouse transgenic model, we demonstrate that while the selective inactivation of NMDARs in DA neurons eliminates the induction of molecular changes leading to synaptic strengthening, behavioral measures such as cocaine induced locomotor sensitization and conditioned place preference remain intact in NR1^DATCre mice. Since VTA DA neurons projecting to the prefrontal cortex and amygdala express little or no detectable levels of the dopamine transporter, it has been speculated that NMDA receptors in DA neurons projecting to these brain areas may have been spared in NR1^DATCre mice. Here we demonstrate that the NMDA receptor gene is ablated in the majority of VTA DA neurons, including those exhibiting undetectable DAT expression levels in our NR1^DATCre transgenic model, and that application of an NMDAR antagonist within the VTA of NR1^DATCre animals still blocks sensitization to cocaine.

Conclusions/Significance

These results eliminate the possibility of NMDAR mediated neuroplasticity in the different DA neuronal subpopulations in our NR1^DATCre mouse model and therefore suggest that NMDARs on non-DA neurons within the VTA must play a major role in cocaine-related addictive behavior.     

The NMDA receptor NR2A subunit regulates proliferation of MKN45 human gastric cancer cells

The present study investigated proliferation of MKN28 and MKN45 human gastric cancer cells regulated by the N-methyl-d-aspartate (NMDA) receptor subunit. The NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (AP5) inhibited proliferation of MKN45 cells, but not MKN28 cells. Of the NMDA subunits such as NR1, NR2 (2A, 2B, 2C, and 2D), and NR3 (3A and 3B), all the NMDA subunit mRNAs except for the NR2B subunit mRNA were expressed in both MKN28 and MKN45 cells. MKN45 cells were characterized by higher expression of the NR2A subunit mRNA and lower expression of the NR1 subunit mRNA, but MKN28 otherwise by higher expression of the NR1 subunit mRNA and lower expression of the NR2A subunit mRNA. MKN45 cell proliferation was also inhibited by silencing the NR2A subunit-targeted gene. For MKN45 cells, AP5 or knocking-down the NR2A subunit increased the proportion of cells in the G₁ phase of cell cycling and decreased the proportion in the S/G₂ phase. The results of the present study, thus, suggest that blockage of NMDA receptors including the NR2A subunit suppresses MKN45 cell proliferation due to cell cycle arrest at the G₁ phase; in other words, the NR2A subunit promotes MKN45 cell proliferation by accelerating cell cycling.     

Increased expression of NR2A subunit does not alter NMDA-evoked responses in cultured fetal trisomy 16 mouse hippocampal neurons

The trisomy 16 (Ts16) mouse is an animal model for human trisomy 21 (Down's syndrome). The gene encoding the NR2A subunit of the NMDA receptor has been localized to mouse chromosome 16. In the present study, western blot analysis revealed a 2.5-fold increase of NR2A expression in cultured Ts16 embryonic hippocampal neurons. However, this increase did not affect the properties of NMDA-evoked currents in response to various modulators. The sensitivity of NMDA receptors to transient applications of NMDA, spermine, and Zn(2+) was investigated in murine Ts16 and control diploid cultured embryonic hippocampal neurons. Peak and steady-state currents evoked by NMDA were potentiated by spermine at concentrations < 1 mM, and inhibited by Zn(2+) in a dose-dependent and voltage-independent manner. No marked difference was observed between Ts16 and control diploid neurons for any of these modulators with regard to IC(50) and EC(50) values or voltage dependency. Additionally, inhibition by the NR2B selective inhibitor, ifenprodil, was similar. These results demonstrate that NMDA-evoked currents are not altered in cultured embryonic Ts16 neurons and suggest that Ts16 neurons contain similar functional properties of NMDA receptors as diploid control neurons despite an increased level of NR2A expression.     

Calcium influx through NMDA receptors, chronic receptor inhibition by ethanol and 2-amino-5-phosponopentanoic acid, and receptor protein expression

Chronic treatment of neurons with either ethanol or competitive and noncompetitive antagonists of NMDA receptors leads to enhanced expression of NMDA receptor density and function in neurons. The signal transduction pathways for such receptor up-regulation are not known. The focus of the present study was on the role of Ca2+ entry into neurons, either through receptor or voltage-gated channels, in the expression of the NMDA receptor subunit NR1 and the 71-kDa glutamate-binding protein (GBP) of a glutamate/NMDA receptor-like complex. Chronic inhibition of NMDA receptors in cortical neurons in primary cultures by either 100 mM ethanol or 100 microM 2-amino-5-phosphonopentanoic acid (2-AP5) increased the expression of NR1 and GBP. The effect of 2-AP5 on the expression of the two proteins was not additive with that of ethanol when neuronal cultures were treated with both agents at the same time. However, the effects of ethanol on NR1 and GBP expression were blocked by the simultaneous treatment with NMDA (50 microM). Activation or inhibition of other glutamate ionotropic receptors had no effect on the expression of NR1 and GBP. The inhibition of L- or N-type voltage-sensitive Ca2+ channels and voltage-gated Na+ channels also had little effect on the expression of either protein; neither did exposure of neurons to elevated extracellular Ca2+ concentrations (3 or 5 mM). On the other hand, treatment of neurons for 48 h with the intracellular Ca2+ chelator BAPTA-AM as well as partial chelation of extracellular Ca2+ with EGTA caused an up-regulation in NR1 and GBP expression. The enhanced expression of NR1 in neurons treated for 48 h with either ethanol or EGTA was correlated with increases in the activity of NMDA receptors demonstrated as a doubling of the NMDA-stimulated rise in intracellular free Ca2+ concentration. The effects of chronic administration of EGTA on both NR1 expression as well as NMDA receptor function were probably related to an acute inhibition by EGTA of NMDA-induced Ca2+ influx into neurons. It appears that the expression of both the NR1 subunit of NMDA receptors and the GBP of a receptor-like complex is regulated by intracellular Ca2+, especially that entering through NMDA receptor ion channels.     

AMPA receptor-dependent clustering of synaptic NMDA receptors is mediated by Stargazin and NR2A/B in spinal neurons and hippocampal interneurons

Under standard conditions, cultured ventral spinal neurons cluster AMPA- but not NMDA-type glutamate receptors at excitatory synapses on their dendritic shafts in spite of abundant expression of the ubiquitous NMDA receptor subunit NR1. We demonstrate here that the NMDA receptor subunits NR2A and NR2B are not routinely expressed in cultured spinal neurons and that transfection with NR2A or NR2B reconstitutes the synaptic targeting of NMDA receptors and confers on exogenous application of the immediate early gene product Narp the ability to cluster both AMPA and NMDA receptors. The use of dominant-negative mutants of GluR2 further showed that the synaptic targeting of NMDA receptors is dependent on the presence of synaptic AMPA receptors and that synaptic AMPA and NMDA receptors are linked by Stargazin and a MAGUK protein. This system of AMPA receptor-dependent synaptic NMDA receptor localization was preserved in hippocampal interneurons but reversed in hippocampal pyramidal neurons.     

An Endoplasmic Reticulum Retention Signal Located in the Extracellular Amino-terminal Domain of the NR2A Subunit of N-Methyl-d-aspartate Receptors

N-Methyl-d-aspartate (NMDA) receptors play critical roles in complex brain functions as well as pathogenesis of neurodegenerative diseases. There are many NMDA isoforms and subunit types that, together with subtype-specific assembly, give rise to significant functional heterogeneity of NMDA receptors. Conventional NMDA receptors are obligatory heterotetramers composed of two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits. When individually expressed in heterogeneous cells, most of the NR1 splice variants and the NR2 subunits remain in the endoplasmic reticulum (ER) and do not form homomeric channels. The mechanisms underlying NMDA receptor trafficking and functional expression remain uncertain. Using truncated and chimeric NMDA receptor subunits expressed in heterogeneous cells and hippocampal neurons, together with immunostaining, biochemical, and functional analyses, we found that the NR2A amino-terminal domain (ATD) contains an ER retention signal, which can be specifically masked by the NR1a ATD. Interestingly, no such signal was found in the ATD of the NR2B subunit. We further identified the A2 segment of the NR2A ATD to be the primary determinant of ER retention. These findings indicate that NR2A-containing NMDA receptors may undergo a different ER quality control process from NR2B-containing NMDA receptors.Ionotropic glutamate receptors (iGluRs)² mediate most of the excitatory neurotransmission in the central nervous system. They play key roles in complex brain functions as well as in the pathogenesis of neurodegenerative diseases. Based on pharmacological properties and sequence similarities, iGluRs can be grouped into three major subtypes: GluR1 to -4 subunits form α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, GluR5 to -7 and KA1 and -2 subunits make up kainate receptors, and NR1 together with NR2A to -D subunits comprise the NMDA receptors (1). All iGluR subunits share a unique membrane topology with a large extracellular NH₂-terminal domain, three transmembrane segments (TM1 (transmembrane domain 1), TM3, and TM4), a P-loop region, and a cytoplasmic COOH terminus (2, 3). Based on the sequence homology to bacterial periplasmic binding proteins, the NH₂-terminal domain of iGluRs can be divided into two domains in tandem: the amino-terminal domain (ATD), which includes the first 400 or so amino acids (4), and the following S1 domain preceding TM1, which forms the ligand-binding domain together with the extracellular loop between TM3 and TM4 (S2 domain) (5, 6).Among iGluRs, NMDA receptors are special in that conventional NMDA receptors are obligatory tetrameric membrane proteins composed of two glycine-binding NR1 and two glutamate-binding NR2 subunits. The NR1 subunit is essential for the formation of functional NMDA receptor channel, whereas the NR2 subunit modifies channel properties, such as current kinetics and channel conductance (1). The major NR1 splice variant and the NR2 subunits are retained in the ER when expressed alone in heterogeneous cells. Only when expressed together do they form functional receptors on the cell surface (7–9). In the last decade, enormous progress has been made in understanding the phenomenology and mechanisms of functional plasticity of NMDA receptors. However, much less is known about the mechanisms underlying the ER retention of NMDA receptor subunits. Previous studies focused on the COOH terminus have shown that the NR1a subunit contains an ER retention signal, RRR, in the C1 cassette, whereas a motif, HLFY, found in the NR2B subunit immediately following the TM4 (10) or, at least, the presence of any two amino acid residues after NR2 TM4 (11) is required for the export of NR1-NR2 complexes from the ER. Recently, novel ER retention signals were identified in the TM3 of both NR1 and NR2B subunits. In addition, TM3 of both NR1 and NR2B and TM4 of NR1 are necessary for masking ER retention signals found in TM3 (12).In the present study, we focused on the functional role of the ATD in the surface expression of NMDA receptors. Interestingly, we found an ER retention signal located in the ATD of the NR2A subunit but not in the corresponding domain of the NR2B. It is suggested that NR2A-containing NMDA receptors may undergo an ER quality control process different from that of NR2B-containing NMDA receptors.     

Fyn-mediated phosphorylation of NR2B Tyr-1336 controls calpain-mediated NR2B cleavage in neurons and heterologous systems

Cleavage of the intracellular carboxyl terminus of the N-methyl-d-aspartate (NMDA) receptor 2 subunit (NR2) by calpain regulates NMDA receptor function and localization. Here, we show that Fyn-mediated phosphorylation of NR2B controls calpain-mediated NR2B cleavage. In cultured neurons, calpain-mediated NR2B cleavage is significantly attenuated by blocking NR2B phosphorylation of Tyr-1336, but not Tyr-1472, via inhibition of Src family kinase activity or decreasing Fyn levels by small interfering RNA. In HEK cells, mutation of Tyr-1336 eliminates the potentiating effect of Fyn on calpain-mediated NR2B cleavage. The potentiation of NR2B cleavage by Fyn is limited to cell surface receptors and is associated with calpain translocation to plasma membranes during NMDA receptor activation. Finally, reducing full-length NR2B by calpain does not decrease extrasynaptic NMDA receptor function, and truncated NR1/2B receptors similar to those generated by calpain have electrophysiological properties matching those of wild-type receptors. Thus, the Fyn-controlled regulation of NMDA receptor cleavage by calpain may play critical roles in controlling NMDA receptor properties during synaptic plasticity and excitotoxicity.     

Ethanol sensitivity of NMDA receptors

NMDA receptors are ionotropic glutamate receptors assembled of subunits of the NR1 and of the NR2 family (NR2A–NR2D). The subunit diversity largely affects the pharmacological properties of NMDA receptors and, hence, gives rise to receptor heterogeneity. As an overall result of studies on recombinant and native NMDA receptors, ethanol inhibits the function of receptors containing the subunits NR2A and/or NR2B to a greater extent than those containing NR2C or NR2D. For example, in rat cultured mesencephalic neurons, NR2C expression was developmentally increased, whereas expression of NR2A and NR2B was decreased. These changes coincided with a developmental loss of sensitivity of NMDA responses to ethanol and ifenprodil, a non-competitive NMDA receptor antagonist that shows selectivity for NR2B-containing receptors. Also in rat locus coeruleus neurons, the low ethanol sensitivity of somatic NMDA receptors could be explained by a prominent expression of NR2C. The inhibitory site of action for ethanol on the NMDA receptor is not yet known. Patch–clamp studies suggest a target site exposed to or only accessible from the extracellular environment. Apparently, amino acid residue Phe⁶³⁹, located in the TM3 domain of NR1, plays a crucial role in the inhibition of NMDA receptor function by ethanol. Since this phenylalanine site is common to all NMDA and non-NMDA receptor (AMPA/kainate receptor) subunits, this observation is consistent with accumulating evidence for a similar ethanol sensitivity of a variety of NMDA and non-NMDA receptors, but it cannot explain the differences in ethanol sensitivity observed with different NR2 subunits.     

Extracellular mild acidosis decreases the Ca2+ permeability of the human NMDA receptors

NMDA receptors (NMDARs) are glutamate-gated ion channels involved in excitatory synaptic transmission and in others physiological processes such as synaptic plasticity and development. The overload of Ca²⁺ ions through NMDARs, caused by an excessive activation of receptors, leads to excitotoxic neuronal cell death. For this reason, the reduction of Ca²⁺ flux through NMDARs has been a central focus in finding therapeutic strategies to prevent neuronal cell damage.Extracellular H⁺ are allosteric modulators of NMDARs. Starting from previous studies showing that extracellular mild acidosis reduces NMDA-evoked whole cell currents, we analyzed the effects of this condition on the NMDARs Ca²⁺ permeability, measured as “fractional calcium current” (P_f, i.e. the percentage of the total current carried by Ca²⁺ ions), of human NMDARs NR1/NR2A and NR1/NR2B transiently transfected in HeLa cells. Extracellular mild acidosis significantly reduces P_f of both human NR1/NR2A and NR1/NR2B NMDARs, also decreasing single channel conductance in outside out patches for NR1/NR2A receptor. Reduction of Ca²⁺ flux through NMDARs was also confirmed in cortical neurons in culture. A comparative analysis of both NMDA evoked Ca²⁺ transients and whole cell currents showed that extracellular H⁺ differentially modulate the permeation of Na⁺ and Ca²⁺ through NMDARs.Our data highlight the synergy of two distinct neuroprotective mechanisms during acidosis: Ca²⁺ entry through NMDARs is lowered due to the modulation of both open probability and Ca²⁺ permeability. Furthermore, this study provides the proof of concept that it is possible to reduce Ca²⁺ overload in neurons modulating the NMDAR Ca²⁺ permeability.     

Expression of polyglutamine-expanded huntingtin induces tyrosine phosphorylation of N-methyl-D-aspartate receptors

In our previous studies, we found that expression of polyglutamine-expanded huntingtin in HN33 cells induced sensitization of N-methyl-D-aspartate (NMDA) receptors (Sun, Y., Savinainen, A., and Liu, Y. F. (2001) J. Biol. Chem. 276, 24713-24718). Following this study, we investigated whether tyrosine phosphorylation of NMDA receptors might contribute to the altered property of the receptors. Expression of polyglutamine-expanded huntingtin induced elevation of phosphorylated or activated Src and increased targeting of PSD-95 (post-synaptic density 95) and activated Src to cell surface membrane. Expression of the mutated huntingtin also induced tyrosine phosphorylation of NR2B (NMDA receptor 2B) subunits, and co-expression of PSD-95 enhanced the phosphorylation. Treatment of SU6656 (a specific Src inhibitor) or co-expression of a mutated NR2B subunit with mutations of all three major tyrosine phosphorylation sites significantly attenuated neuronal toxicity induced by the mutated huntingtin. Addition of AP-5 did not further inhibit the neuronal toxicity. Taken together, our studies show that polyglutamine-expanded huntingtin increases tyrosine phosphorylation of NMDA receptors via PSD-95 and Src, and increased tyrosine phosphorylation may contribute to the sensitization of the receptors mediated by polyglutamine-expanded huntingtin.     

Neuroprotective effects of vitexin by inhibition of NMDA receptors in primary cultures of mouse cerebral cortical neurons

The accumulation of glutamate can excessively activate the N-methyl-d-aspartate (NMDA) receptors and cause excitotoxicity. Vitexin (5, 7, 4-trihydroxyflavone-8-glucoside, Vit) is a c-glycosylated flavone which was found in the several herbs, exhibiting potent hypotensive, anti-inflammatory, and neuroprotective properties. However, little is known about the neuroprotective effects of Vit on glutamate-induced excitotoxicity. In present study, primary cultured cortical neurons were treated with NMDA to induce the excitotoxicity. Pretreatment with Vit significantly prevented NMDA-induced neuronal cell loss and reduced the number of apoptotic neurons. Vit significantly inhibited the neuronal apoptosis induced by NMDA exposure by regulating balance of Bcl-2 and Bax expression and the cleavages of poly (ADP-ribose) polymerase and pro-caspase 3. Furthermore, pretreatment of Vit reversed the up-regulation of NR2B-containing NMDA receptors and the intracellular Ca²⁺ overload induced by NMDA exposure. The neuroprotective effects of Vit are related to inhibiting the activities of NR2B-containing NMDA receptors and reducing the calcium influx in cultured cortical neurons.     

Antagonists of NMDA glutamate receptors selectively affect synaptic mechanisms of the nociceptive sensitization in the snail

The effects of N-methyl-D-aspartate (NMDA) glutamate receptor antagonists on the mechanisms of nociceptive sensitization were studied in LPl1 and RPl1 neurons of the semiintact preparation of a Helix lucorum snail. Application of sensitizing stimuli on the head part of the control preparation led to a depolarization of the membrane and increase in its excitability. A depression of responses of neurons evoked by tactile or chemical sensory stimulation during the short-term period and significant facilitation of responses during the long-term period of sensitization were observed. Sensitization performed under conditions of application of NMDA antagonists (AP5 or MK801) produced similar changes in membrane potential, membrane excitability, and neuronal responses evoked by tactile stimulation of the head or foot. However, the chemical stimulation of the head under these conditions evoked a significant depression of responses during the short- and long-term sensitization periods. The results suggest that the NMDA glutamate receptor antagonists selectively affect the plasticity induction mechanisms of the command neuron synaptic inputs, which mediate the chemical sensory stimulation from the snail's head.