Transient Global Ischemia Alters NMDA Receptor Expression in Rat Hippocampus: Correlation with Decreased Immunoreactive Protein Levels of the NR2A/2B Subunits, and an Altered NMDA Receptor Functionality

Abstract: We investigated the gene expression levels, the immunoreactive protein prevalence, and the functional activity of N -methyl- d -aspartate (NMDA) receptor complexes at early times after severe global ischemia challenge in rats. The mRNA expression levels for the NR2A and NR2B subunits of NMDA receptors changed to different degrees within different subregions of the hippocampus after reperfusion with respect to sham-operated control. No significant change in expression was observed in the vulnerable CA1 subfield at or before 6 h after challenge for either receptor subunit, although changes in expression in other hippocampal subfields were observed. At 12 and 24 h after challenge, significant decreases in expression for both subunits were found in the vulnerable CA1 subfield, as well as in other hippocampal regions. At the protein level, a significant decrease in the amount of NR2A/NR2B immunoreactivity in the total hippocampus was observed at both 6 and 24 h after reperfusion compared with sham control. Electrophysiological assessment of single-channel NMDA receptor activity in the CA1 subfield indicates that the main conductance state of NMDA receptor channels is maintained 6 h after challenge, although by 18–24 h after challenge, this main conductance state is rarely observed. The NMDA receptor component of the excitatory postsynaptic field potential was found to be significantly diminished from sham control 24 h after challenge, such that only ∼10% of the sham response remained, but was not significantly altered from sham control at 6 h after challenge. These results indicate that decreases in the expression levels, the immunoreactive protein prevalence, and that alterations in the functionality of NMDA receptors occur in the hippocampus at early times after severe transient global ischemia.     

Key amino acid residues within the third membrane domains of NR1 and NR2 subunits contribute to the regulation of the surface delivery of N-methyl-D-aspartate receptors

N-methyl-d-aspartate (NMDA) receptors are glutamate ionotropic receptors that play critical roles in synaptic transmission, plasticity, and excitotoxicity. The functional NMDA receptors, heterotetramers composed mainly of two NR1 and two NR2 subunits, likely pass endoplasmic reticulum quality control before they are released from the endoplasmic reticulum and trafficked to the cell surface. However, the mechanism underlying this process is not clear. Using truncated and mutated NMDA receptor subunits expressed in heterologous cells, we found that the M3 domains of both NR1 and NR2 subunits contain key amino acid residues that contribute to the regulation of the number of surface functional NMDA receptors. These key residues are critical neither for the interaction between the NR1 and NR2 subunits nor for the formation of the functional receptors, but rather they regulate the early trafficking of the receptors. We also found that the identified key amino acid residues within both NR1 and NR2 M3 domains contribute to the regulation of the surface expression of unassembled NR1 and NR2 subunits. Thus, our data identify the unique role of the membrane domains in the regulation of the number of surface NMDA receptors.     

Synaptic activity-dependent developmental regulation of NMDA receptor subunit expression in cultured neocortical neurons

The biophysical properties of NMDA receptors are thought to be critical determinants involved in the regulation of long-term synaptic plasticity during neocortical development. NMDA receptor channel properties are strongly dependent on the subunit composition of heteromeric NMDA receptors. During neocortical development in vivo, the expression of the NMDA receptor 2A (NR2A) subunit is up-regulated at the mRNA and protein level correlating with changes in the kinetic and pharmacological properties of functional NMDA receptors. To investigate the developmental regulation of NMDA receptor subunit expression, we studied NR2 mRNA expression in cultured neocortical neurons. With increasing time in culture, they showed a similar up-regulation of NR2A mRNA expression as described in vivo. As demonstrated by chronic blockade of postsynaptic glutamate receptors in vitro, the regulation of NR2A mRNA was strongly dependent on synaptic activity. In contrast, NR2B mRNA expression was not influenced by activity blockade. Moreover, as shown pharmacologically, the regulation of NR2A mRNA expression was mediated by postsynaptic Ca(2+) influx through both NMDA receptors and L-type Ca(2+) channels. It is interesting that even relatively weak expression of NR2A mRNA was correlated with clearly reduced sensitivity of NMDA receptor-mediated whole-cell currents against the NR2B subunit-specific antagonist ifenprodil. Developmental changes in the expression of NR1 mRNA splice variants were also strongly dependent on synaptic activity and thus might, in addition to regulation of NR2 subunit expression, contribute to developmental changes in the properties of functional NMDA receptors. In summary, our results demonstrate that synaptic activity is a key factor in the regulation of NMDA receptor subunit expression during neocortical development.     

Glutamate Binding to the GluN2B Subunit Controls Surface Trafficking of N-Methyl-D-aspartate (NMDA) Receptors

Trafficking of NMDA receptors to the surface of neurons and to synapses is critical for proper brain function and activity-dependent plasticity. Recent evidence suggests that surface trafficking of other ionotropic glutamate receptors requires ligand binding for exit from the endoplasmic reticulum. Here, we show that glutamate binding to GluN2 is required for trafficking of NMDA receptors to the cell surface. We expressed a panel of GluN2B ligand binding mutants in heterologous cells with GluN1 or in rat cultured neurons and found that surface expression correlates with glutamate efficacy. Such a correlation was found even in the presence of dominant negative dynamin to inhibit endocytosis and surface expression correlated with Golgi localization, indicating differences in forward trafficking. Co-expression of wild type GluN2B did not enhance surface expression of the mutants, suggesting that glutamate must bind to both GluN2 subunits in a tetramer and that surface expression is limited by the least avid of the two glutamate binding sites. Surface trafficking of a constitutively closed cleft GluN2B was indistinguishable from that of wild type, suggesting that glutamate concentrations are typically not limiting for forward trafficking. YFP-GluN2B expressed in hippocampal neurons from GluN2B(-/-) mice rescued synaptic accumulation at similar levels to wild type. Under these conditions, surface synaptic accumulation of YFP-GluN2B mutants also correlated with apparent glutamate affinity. Altogether, these results indicate that glutamate controls forward trafficking of NMDA receptors to the cell surface and to synapses and raise the intriguing idea that NMDA receptors may be functional at intracellular sites.     

Growth Conditions Differentially Regulate the Expression of α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionate (AMPA) Receptor Subunits in Cultured Neurons

We have studied the expression of a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits in cultured cerebellar granule cells [7 days in vitro (DIV)] grown in medium containing different concentrations of K^± (10, 25, or 40 mM) with or without 100 μM N-methyl-D-aspartate (NMDA; added once after 2 DIV). All these conditions are known to influence maturation and survival of granule cells, as well as the functional expression of NMDA receptors during development in culture. The expression of both glutamate receptor (GluR) subunit 1 mRNA and receptor protein was low in cultures grown in 10 mM K^± (K10) and increased dramatically in cultures grown in 25 mM K^± (K25), with intermediate levels found in cultures grown in K10 and chronically exposed to NMDA (K10 ± NMDA). In cultures grown in 40 mM K^± (K40), the expression of GluR1 mRNA and receptor protein was lower than in K25 but still higher than in K10. GluR2 and -3 subunits were differently regulated by growth conditions, with their expression being higher in K10 and progressively reduced to the lowest levels in K40 (both mRNA and receptor proteins). GluR4 mRNA levels did not differ between K10 and K25, although they were reduced by chronic exposure to NMDA. To test how the differential expression of the various subunits affects the functional activity of AMPA receptors, we have measured AMPA-stimulated ^4SCa^2± influx and 40-[³H]phorbol 12, 13-dibutyrate binding in intact cells. Both functional parameters increased along with the K^± concentration and were maximal in K40, in coincidence with the lowest expression of the GluR2 subunits. These results indicate that functional diversity of AMPA receptors can be generated by the degree of chronic depolarization and/or exposure to NMDA in neurons developing in primary culture.     

Chronic ethanol exposure delays the 'developmental switch' of the NMDA receptor 2A and 2B subunits in cultured cerebellar granule neurons

Chronic ethanol treatment of cultured neurons from various brain areas has been found to increase NMDA receptor function and to alter the levels of some NMDA receptor subunit proteins. Because the cultured neurons are exposed to ethanol during a period when the NMDA receptor is undergoing developmental changes in subunit expression, we wished to determine whether ethanol treatment alters this developmental pattern. We found that 3 days of treatment of cerebellar granule neurons with ethanol, which was previously reported to increase NMDA receptor function, resulted in a delay in the 'developmental switch' of the NR2A and NR2B subunits, i.e. the developmental decrease in NR2B and increase in NR2A protein expression. As a result, the level of NR2B was higher, and that of NR2A was lower, in the ethanol-treated cells than in control cells. Cross-linking experiments showed that the changes in total receptor subunit proteins levels were reflected in cell-surface expressed proteins, indicating changes in the amount of functional receptors. These results were confirmed by a higher potency of glycine at the NMDA receptor in the ethanol-treated cells, as determined by NMDA/glycine-induced increases in intracellular Ca(2+). The results suggest that the mechanism by which ethanol alters NMDA receptor expression in cultured neurons, where receptors are undergoing development, differs from the mechanism of ethanol's effect on NMDA receptors in adult brain. Changes in the proportion of NR2A and NR2B subunits may contribute to effects of ethanol on neuronal development.     

Protein intake regulates the vasodilatory function of the kidney and NMDA receptor expression

Glycine infusion in normal rats causes an increase in renal plasma flow and glomerular filtration rate (GFR). Although the renal response to glycine infusion is well characterized, the mechanism initiating this vasodilation is unknown. We recently observed functionally active N-methyl-d-aspartate (NMDA) receptors in the kidney, located primarily in tubular structures. The mechanisms regulating activity of the NMDA receptor within the kidney are also unknown, as is its normal day-to-day functional role. Therefore, we hypothesize that dietary protein may impact the functional response to glycine infusion in both untreated rats and rats pretreated with angiotensin-converting enzyme (ACE) inhibitor and, furthermore, that renal NMDA receptors may be involved in the glycine response. Surprisingly, 2 wk of low-protein diet (8% protein vs. 21% protein in control diet) totally inhibited the glycine-induced vasodilation and GFR response. Associated with the absence of renal vasodilation, a significant reduction in proximal tubular reabsorption was observed during glycine infusion in low-protein-diet rats. In contrast to the disease models previously studied in our laboratory, administration of ACE inhibitors did not restore the glycine response in rats treated with low-protein diet. Western blots of normal- and low-protein-diet kidneys demonstrate that the newly described renal NMDA receptor is downregulated in rats fed a low-protein diet. Low-protein feeding results in loss of glycine-induced vasodilation and GFR responses associated with decreased renal NMDA receptor expression. Kidney NMDA receptor expression is conditioned by protein intake, and this receptor may play an important role in the kidney vasodilatory response to glycine infusion and protein feeding in rats.     

Significance of N-methyl-D-aspartate (NMDA) receptor-mediated signaling in human keratinocytes

Increasing data suggest that glutamate might act as a cell-signaling molecule in non-neuronal tissues such as the skin. Here we demonstrate the presence of functional N-methyl-D-aspartate (NMDA)-type glutamate receptors in human keratinocytes. NMDA receptor expression strongly reflects the degree of cell-to-cell contact. Wounding polarizes the expression of NMDA receptors in keratinocytes involved in re-epithelialization, and the process of re-epithelialization is inhibited by NMDA receptor activation. We also demonstrate that squamous cell carcinomas lack NMDA receptors. Our data suggest that Ca2+ entry through NMDA receptors influences the cycle of keratinocyte proliferation, differentiation, and migration during epithelialization. Moreover, NMDA receptor activation might play a role in contact-mediated inhibition of growth, a process that is absent during neoplastic pathology. This receptor may serve as a pharmacological target for modulating keratinocyte behavior and treating cutaneous disorders.     

Ionotropic glutamate receptors

In the brain, most fast excitatory synaptic transmission is mediated through L-glutamate acting on postsynaptic ionotropic glutamate receptors. These receptors are of two kinds—the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate (non-NMDA) and theN-methyl-D-aspartate (NMDA) receptors, which are thought to be colocalized onto the same postsynaptic elements. This excitatory transmission can be modulated both upward and downward, long-term potentiation (LTP) and long-term depression (LTD), respectively. Whether the expression of LTP/LTD is pre-or postsynaptically located (or both) remains an enigma. This article will focus on what postsynaptic modifications of the ionotropic glutamate receptors may possibly underly long-term potentiation/depression. It will discuss the character of LTP/LTD with respect to the temporal characteristics and to the type of changes that appears in the non-NMDA and NMDA receptor-mediated synaptic currents, and what constraints these findings put on the possible expression mechanism(s) for LTP/LTD. It will be submitted that if a modification of the glutamate receptors does underly LTP/LTD, an increase/decrease in the number of functional receptors is the most plausible alternative. This change in receptor number will have to include a coordinated change of both the non-NMDA and the NMDA receptors.     

Biochemical studies of the structure and function of theN-methyl-D-aspartate subtype of glutamate receptors

TheN-methyl-D-aspartate (NMDA) subtype of glutamate receptors plays a key role in synaptic transmission, synaptic plasticity, synaptogenesis, and excitotocity in the mammalian central nervous system. The NMDA receptor channel is formed from two gene products from two glutamate receptor subunit families, termed NR1 and NR2. Although the subunit composition of native NMDA receptors is incompletely understood, electrophysiological studies using recombinant receptors suggest that functional NMDA receptors consist of heteromers containing combinations of NR1, which is essential for channel activity, and NR2, which modulates the properties of the channels. The lack of agonists or antagonists selective for a given subunit of NMDA receptors has made it difficult to understand the subunit expression, subunit composition, and posttranslational modification mechanisms of native NMDA receptors. Therefore, most studies on NMDA receptors that examine regional expression and ontogeny have been focused at the level of the mRNAs encoding the different subunits using northern blotting, ribonuclease protection, andin situ hybridization techniques. However, the data from these studies do not provide clear information about the resultant subunit protein. To directly examine the protein product of the NMDA receptor subunit genes, the development of subunit-specific antibodies using peptides and fusion proteins has provided a good approach for localizing, quantifying, and characterizing the receptor subunits in tissues and transfected cell lines, and to study the subunit composition and the functional effects of posttranslational processing of the NMDA subunits, particularly the phosphorylation profiles of NMDA glutamate receptors.     

Rat lymphocytes express NMDA receptors that take part in regulation of cytokine production

Incubation of rat lymphocytes with homocysteine (HC) or homocysteic acid (HCA) was found to increase the stationary levels of free radicals in lymphocytes, the effect of both ligands being mediated by ionotropic receptors activated by N-methyl-D-aspactic acid (NMDA), the expression of which on rat lymphocyte membranes was earlier demonstrated. In agreement with these data, increase of free radicals in the lymphocyte cytoplasm is preceded by an increase in the intracellular calcium levels, activation of protein kinase C, nicotinamide adenine dinucleotide phosphate oxidase and/or nitric oxide synthase. Both HC and HCA increase the production of IFN-γ and TNF-α by lymphocytes and antagonist of NMDA receptors; MK-801 prevents this effect. The data presented show that rat lymphocyte membrane contains functionally active NMDA receptors, which regulate cytokine accumulation.     

Long-term potentiation as a mechanism of functional synapse induction in the developing hippocampus

During the first 2 days of postnatal development, CA1 hippocampal glutamatergic synaptic transmission is based almost exclusively on NMDA receptors and is non-functional at resting potential. Within the following days an increasing number of functionally mature synapses, containing both NMDA and AMPA receptors, were observed. We found that the maturation of the NMDA receptor-mediated synapses could be induced experimentally with a pairing protocol, a process termed functional synapse induction. Our data provide evidence that a LTP-like mechanism involved in the activity-dependent formation of functional glutamergic synapses in the developing hippocampus.     

Inducible expression and pharmacological characterization of recombinant rat NR1a/NR2A NMDA receptors

In this study, we have established a non-neuronal cell line stably and inducibly expressing recombinant NMDA receptors (NRs) composed of rat NR1a/NR2A subunits. EcR-293 cells were transfected with rat NR1a and NR2A cDNAs using the inducible mammalian expression vector pIND. Cell colonies resistant for the selecting agents were picked and tested for NR2A mRNA as well as protein expression using quantitative RT-PCR and flow cytometry based immunocytochemistry. Clonal cells expressing functional NMDA receptors were identified by measuring NMDA-evoked ion currents, and NMDA-induced increase in cytosolic free calcium concentration in whole-cell patch-clamp and fluorimetric calcium measurements, respectively. One clone named D5/H3, which exhibited the highest response to NMDA, was chosen to examine inducibility of the expression and for pharmacological profiling of recombinant NR1a/NR2A NMDA receptors. To check inducibility, NR2A subunit expression in D5/H3 cells treated with the inducing agent muristerone A (MuA) was compared with that in non-induced cells. Both NR2A mRNA and protein expression was several folds higher in cells treated with the inducing agent. As part of the pharmacological characterization, we examined the activation of the expressed NR1a/NR2A receptors as a function of increasing concentration of NMDA. NMDA-evoked concentration-dependent increases in cytosolic [Ca2+] with an EC50 value of 41 +/- 1 microM. In addition, whereas the NMDA response was concentration-dependently inhibited by the channel blocker MK-801 (IC50 = 58 +/- 6 nM), NR2B subunit selective NMDA receptor antagonists were ineffective. Thus, this cell line, which stably and inducibly expresses recombinant NR1a/NR2A NMDA receptors, can be a useful tool for testing NMDA receptor antagonists and studying their subunit selectivity.     

NMDA receptor expression and roles in human articular chondrocyte mechanotransduction

Mechanical forces influence articular cartilage structure by regulating chondrocyte activity. Mechanical stimulation results in activation of an alpha5beta1 integrin dependent intracellular signal cascade involving focal adhesion kinase and protein kinase C, triggering the release of interleukin-4 from the cell. In normal HAC the response to physiological mechanical stimulation is characterised by increased levels of aggrecan mRNA and a decrease in levels of mRNA for matrix metalloproteinase 3 (MMP-3), the net result of which would be to maintain and optimise cartilage structure and function. This protective/anabolic response is not seen when chondrocytes from osteoarthritic cartilage are subjected to an identical mechanical stimulation regime. Following the observation that the neurotransmitter substance P is involved in chondrocyte mechanotransduction the present study was undertaken to establish potential roles for glutamate receptors in the control of chondrocyte mechanical responses. Using immunohistochemistry and RTPCR normal and OA chondrocytes are shown to express NR1 and NR2a subunits of the NMDA receptor. Addition of NMDA receptor agonists to chondrocytes in primary culture resulted in changes in membrane potential consistent with expression of functional receptors. NMDA receptor antagonists inhibited the hyperpolarisation response of normal chondrocytes to mechanical stimulation but had no effect on the depolarisation response of osteoarthritic chondrocytes to mechanical stimulation. These studies indicate that at least one subset of the NMDA receptor family of molecules is expressed in cartilage and may have important modulatory effects on mechanotransduction and cellular responses following mechanical stimulation. Indeed the results suggest that there is an alteration of NMDA receptor signalling in OA chondrocytes, which may be critical in the abnormal response of OA chondrocytes to mechanical stimulation. Thus NMDA receptors appear to be involved in the regulation of human articular chondrocyte responses to mechanical stimulation, and in OA, mechanotransduction pathways may be modified as a result of altered activation and function of these receptors.     

Functional N-methyl-D-aspartate receptors in O-2A glial precursor cells: a critical role in regulating polysialic acid-neural cell adhesion molecule expression and cell migration

The capacity for long-distance migration of the oligodendrocyte precursor cell, oligodendrocyte-type 2 astrocyte (O-2A), is essential for myelin formation. To study the molecular mechanisms that control this process, we used an in vitro migration assay that uses neurohypophysial explants. We provide evidence that O-2A cells in these preparations express functional N-methyl-D-aspartate (NMDA) receptors, most likely as homomeric complexes of the NR1 subunit. We show that NMDA evokes an increase in cytosolic Ca2+ that can be blocked by the NMDA receptor antagonist AP-5 and by Mg2+. Blocking the activity of these receptors dramatically diminished O-2A cell migration from explants. We also show that NMDA receptor activity is necessary for the expression by O-2A cells of the highly sialylated polysialic acid- neural cell adhesion molecule (PSA-NCAM) that is required for their migration. Thus, glutamate or glutamate receptor ligands may regulate O- 2A cell migration by modulating expression of PSA-NCAM. These studies demonstrate how interactions between ionotropic receptors, intracellular signaling, and cell adhesion molecule expression influence cell surface properties, which in turn are critical determinants of cell migration.     

Expression of mRNAs Encoding Subunits of the NMDA Receptor in Developing Rat Brain

Abstract: Developmental changes in the levels of N -methyl- d -aspartate (NMDA) receptor subunit mRNAs were identified in rat brain using solution hybridization/RNase protection assays. Pronounced increases in the levels of mRNAs encoding NR1 and NR2A were seen in the cerebral cortex, hippocampus, and cerebellum between postnatal days 7 and 20. In cortex and hippocampus, the expression of NR2B mRNA was high in neonatal rats and remained relatively constant over time. In contrast, in cerebellum, the level of NR2B mRNA was highest at postnatal day 1 and declined to undetectable levels by postnatal day 28. NR2C mRNA was not detectable in cerebellum before postnatal day 11, after which it increased to reach adult levels by postnatal day 28. In cortex, the expression of NR2A and NR2B mRNAs corresponds to the previously described developmental profile of NMDA receptor subtypes having low and high affinities for ifenprodil, i.e., a delayed expression of NR2A correlating with the late expression of low-affinity ifenprodil sites. In cortex and hippocampus, the predominant splice variants of NR1 were those without the 5' insert and with or without both 3' inserts. In cerebellum, however, the major NR1 variants were those containing the 5' insert and lacking both 3' inserts. The results show that the expression of NR1 splice variants and NR2 subunits is differentially regulated in various brain regions during development. Changes in subunit expression are likely to underlie some of the changes in the functional and pharmacological properties of NMDA receptors that occur during development.     

Effects of Chronic Ethanol Treatment on the Expression of Calcium Transport Carriers and NMDA/Glutamate Receptor Proteins in Brain Synaptic Membranes

Abstract: Acute exposure to ethanol inhibits both the NMDA receptors and the Na/Ca-exchange carriers in neuronal membranes. This alters intraneuronal signaling pathways activated by Ca²⁺. Neurons exposed chronically to ethanol exhibit enhanced density and activity of NMDA receptors and increased maximal activity of the exchangers. In the present study, the expression of brain synaptic membrane proteins with ligand binding sites characteristic of NMDA receptors and of exchange carriers were determined after chronic ethanol administration (15 days) to rats. Such treatment caused an increase in the expression of the NMDAR1 receptor subunit, 15% above the levels in the pair-fed controls, as well as of three subunits of a complex that has properties characteristic of NMDA receptors, the glutamate, carboxypiperazinylphosphonate, and glycine binding proteins. Increases for the three binding proteins were 49, 50, and 62%, respectively. The expression of the 120-kDa exchanger proteins was increased by 14% and that of a 36-kDa exchanger-associated protein by 33%. Both the binding proteins and the exchangers returned to basal levels within 36–72 h after withdrawal from ethanol. No changes were detected in synaptic membrane Ca²⁺, Mg²⁺-ATPases. The enhanced expression of receptor and exchanger-associated proteins may explain the increases in the density and activity of NMDA receptors and exchange carriers after chronic ethanol treatment.     

Exposure to novel environment is characterized by an interaction of D1/NMDA receptors underlined by phosphorylation of the NMDA and AMPA receptor subunits and activation of ERK1/2 signaling, leading to epigenetic changes and gene expression in rat hippocampus

Interactions between dopamine and glutamate receptors are essential for prefrontal cortical (PFC) and hippocampal cognitive functions. The hippocampus has been identified as a detector of a novel stimulus, where an association between incoming information and stored memories takes place. Further to our previous results which showed a strong synergistic interaction of dopamine D1 and glutamate NMDA receptors, the present study is going to investigate the functional status of that interaction in rats, following their exposure to a novel environment. Our results showed that the “spatial” novelty induced in rat hippocampus and PFC (a) a significant increase in phosphorylation of NMDA and AMPA receptor subunits, as well as a robust phosphorylation/activation of ERK1/2 signaling, which are both dependent on the concomitant stimulation of D1/NMDA receptors and are both abolished by habituation procedure, (b) chromatin remodeling events (phosphorylation-acetylation of histone H3) and (c) an increase in the immediate early genes (IEGs) c-Fos and zif-268 expression in the CA1 region of hippocampus, which is dependent on the co-activation of D1/NMDA and acetylcholine muscarinic receptors. In conclusion, our results clearly show that a strong synergistic interaction of D1/NMDA receptor is required for the novelty-induced phosphorylation of NMDA and AMPA receptor subunits and for the robust activation of ERK1/2 signaling, leading to chromatin remodeling events and the expression of the IEGs c-Fos and zif-268, which are involved in the regulation of synaptic plasticity and memory consolidation.     

Subtype-specific enhancement of NMDA receptor currents by mutant huntingtin

Evidence suggests that NMDA receptor-mediated neurotoxicity plays a role in the selective neurodegeneration underlying Huntington's disease (HD). The gene mutation that causes HD encodes an expanded polyglutamine tract of >35 in huntingtin, a protein of unknown function. Both huntingtin and NMDA receptors interact with cytoskeletal proteins, and, for NMDA receptors, such interactions regulate surface expression and channel activity. To determine whether mutant huntingtin alters NMDA receptor expression or function, we coexpressed mutant or normal huntingtin, containing 138 or 15 glutamine repeats, respectively, with NMDA receptors in a cell line and then assessed receptor channel function by patch-clamp recording and surface expression by western blot analysis. It is interesting that receptors composed of NR1 and NR2B subunits exhibited significantly larger currents when coexpressed with mutant compared with normal huntingtin. Moreover, this effect was selective for NR1/NR2B, as NR1/NR2A showed similar currents when coexpressed with mutant versus normal huntingtin. However, ion channel properties and total surface expression of the NR1 subunit were unchanged in cells cotransfected with NR1/NR2B and mutant huntingtin. Our results suggest that mutant huntingtin may increase numbers of functional NR1/NR2B-type receptors at the cell surface. Because NR1/NR2B is the predominant NMDA receptor subtype expressed in medium spiny neostriatal neurons, our findings may help explain the selective vulnerability of these neurons in HD.     

The effects of NR2 subunit-dependent NMDA receptor kinetics on synaptic transmission and CaMKII activation

N-Methyl-D-aspartic acid (NMDA) receptors are widely expressed in the brain and are critical for many forms of synaptic plasticity. Subtypes of the NMDA receptor NR2 subunit are differentially expressed during development; in the forebrain, the NR2B receptor is dominant early in development, and later both NR2A and NR2B are expressed. In heterologous expression systems, NR2A-containing receptors open more reliably and show much faster opening and closing kinetics than do NR2B-containing receptors. However, conflicting data, showing similar open probabilities, exist for receptors expressed in neurons. Similarly, studies of synaptic plasticity have produced divergent results, with some showing that only NR2A-containing receptors can drive long-term potentiation and others showing that either subtype is capable of driving potentiation. In order to address these conflicting results as well as open questions about the number and location of functional receptors in the synapse, we constructed a Monte Carlo model of glutamate release, diffusion, and binding to NMDA receptors and of receptor opening and closing as well as a model of the activation of calcium-calmodulin kinase II, an enzyme critical for induction of synaptic plasticity, by NMDA receptor-mediated calcium influx. Our results suggest that the conflicting data concerning receptor open probabilities can be resolved, with NR2A- and NR2B-containing receptors having very different opening probabilities. They also support the conclusion that receptors containing either subtype can drive long-term potentiation. We also are able to estimate the number of functional receptors at a synapse from experimental data. Finally, in our models, the opening of NR2B-containing receptors is highly dependent on the location of the receptor relative to the site of glutamate release whereas the opening of NR2A-containing receptors is not. These results help to clarify the previous findings and suggest future experiments to address open questions concerning NMDA receptor function.