N-methyl-D-aspartate Receptor NR2A Research Articles

The Mammalian Circadian Clock Exhibits Chronic Ethanol Tolerance and Withdrawal-Induced Glutamate Hypersensitivity, Accompanied by Changes in Glutamate and TrkB Receptor Proteins.

Alcohol tolerance and withdrawal-induced effects are criteria for alcohol use disorders listed by the DSM-V. Although tolerance and withdrawal have been studied over many decades, there is still uncertainty regarding mechanistic distinctions that characterize these different forms of ethanol (EtOH)-induced plasticity. Previously, we demonstrated that the suprachiasmatic nucleus (SCN) circadian clock develops both acute and rapid tolerance to EtOH inhibition of glutamate-induced circadian phase shifts. Here, we demonstrate that chronic EtOH tolerance and withdrawal-induced glutamate hypersensitivity occur invitro and that rapid tolerance, chronic tolerance, and glutamate hypersensitivity have distinct cellular changes. We use single-unit extracellular electrophysiological recordings to determine whether chronic tolerance to EtOH inhibition of glutamatergic phase shifts and withdrawal-induced glutamate hypersensitivity develop in the SCN. We use Western blotting to compare phosphorylation state and total expression of N-methyl-D-aspartate (NMDA) receptor subunits and associated proteins in the SCN after mice were exposed to varying EtOH consumption paradigms. Chronic tolerance developed after a minimum of 8days of 4h/d EtOH access, as indicated by a decreased sensitivity to EtOH inhibition of glutamate-induced phase shifts. We also observed an increased sensitivity to glutamate-induced phase shifts in SCN tissue following withdrawal. We demonstrated an increase in the ratio of NR2B:NR2A NMDA receptor subunit expression after 21days, but not after 10days of EtOH drinking. This increase persisted during EtOH withdrawal, along with an increase in NR2B Y1472 phosphorylation, mature brain-derived neurotrophic factor, and phosphorylated TrkB. These results demonstrate that multiple tolerance forms and withdrawal-induced glutamate hypersensitivity occur in the SCN and that these different forms of EtOH-induced plasticity are accompanied by distinct changes in cellular physiology. Importantly, this study further demonstrates the power of using the SCN as a model system to investigate EtOH-induced plasticity.

Effect of dexmedetomidine on trafficking of N-methyl-D-aspartate receptors in spinal dorsal horn neurons during remifentanil-induced hyperalgesia in rats with incisional pain

Objective To investigate the effect of dexmedetomidine on trafficking of N-methyl-D-aspartate(NMDA)receptors in spinal dorsal horn neurons during remifentanil-induced hyperalgesia in the rats with incisional pain. Methods Twenty-four male Sprague-Dawley rats, aged 2-3 months, weighing 240-260 g, were randomly divided into 3 groups(n=8 each)using a random number table: control group(group C), remifentanil + incisional pain group(group R+ I), and dexmedetomidine + remifentanil + incisional pain group(group D+ R+ I). A 1 cm longitudinal incision was made in the plantar surface of the left hindpaw to establish the model of incisional pain in anesthetized rats.In group D+ R+ I, dexmedetomidine 50 μg/kg was injected subcutaneously at 30 min before establishment of the model.In R+ I and D+ R+ I groups, remifentanil was infused for 90 min at a rate of 1.2 μg·kg-1·min-1 at the same time as the model was established.In group C, normal saline was infused for 90 min at a rate of 0.12 ml·kg-1·min-1.The thermal paw withdrawal latency(TWL)and mechanical paw withdrawal threshold(MWT)were measured before administration of dexmedetomidine(T0), and at 2, 6, 24 and 48 h after stopping infusion of remifentanil(T1-4). The rats were sacrificed after the last behavioral testing, and the L4-6 segments of the spinal cord were removed for determination of the expression of NMDA receptor NR1, NR2A and NR2B subunits in membrane(m)and total(t)proteins in the spinal dorsal horn by Western blot analysis.The ratios of mNR1/tNR1, mNR2A/tNR2A and mNR2B/tNR2B were calculated. Results Compared with group C, the TWL was significantly shortened, the MWT was significantly decreased, the expression of mNR1, tNR1, mNR2B and tNR2B was significantly up-regulated, the ratios of mNR1/tNR1 and mNR2B/tNR2B were significantly increased(P 0.05). Compared with group R+ I, the TWL was significantly prolonged, the MWT was significantly increased, the expression of mNR1, tNR1, mNR2B and tNR2B was significantly down-regulated, the ratios of mNR1/tNR1 and mNR2B/tNR2B were significantly decreased(P 0.05). Conclusion The mechanism by which dexmedetomidine prevents remifentanil-induced hyperalgesia is related to reduction of trafficking of NR1 and NR2B subunits-containing NMDA receptors in spinal dorsal horn neurons of the rats with incisional pain. Key words: Dexmedetomidine; Piperidines; Receptors, N-methyl-D-aspartate; Pain; Postoperative complications; Hyperalgesia; Spinal cord

Effects of Neto1-siRNA on conditioned avoidance behavior and expression of NMDA receptor subunits in anterior cingulate cortex in rats with pain

Objective To evaluate the effects of Neto1-siRNA on pain-related negative affection and expression of Neto1 (Neuropilin and tolloid-like 1) and NMDA (N-methyl-D-aspartate) receptor NR2A and NR2B subunits in anterior cingulate cortex (ACC) in neuropathic pain (NP) model rats. Methods 40 healthy male Wistar rats were randomly divided into sham operated group (SO group, n=10) and chronic constriction injury of the sciatic nerve group(CCI group, n=10). Twenty-four days after injury LV-Neto1-RNAi (30160-1) was injected in ACC in rats of SO group and CCI group by stereotaxic apparatus to prepare siRNA SO group(n=10) and siRNA CCI group(n=10). The CCI model was established by the chronic constriction injury of the sciatic nerve. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of each groups were measured on 7th, 14th, 21th and 28th day after operation. The expression of Neto1, NR2A and NR2B subunit mRNA and protein in ACC by RT-PCR and Western Blotting respectively. Results Compared with SO group, TWL and MWT in CCI group were both decreased (P 0.05). In RT-PCR, expression of Neto1, NR2A and NR2B in CCI group was increased compared with SO group (P<0.05), and expression of Neto1 and NR2A was both decreased by Neto1-siRNA (P<0.05), while there was no difference of NR2B. In Western Blotting, expression of Neto1, NR2A and NR2B in CCI group was increased compared with SO group (P<0.05), and expression of Neto1 and NR2A was both decreased by Neto1-siRNA (P<0.05), while there was no difference of NR2B. Conclusion Decreased-expression of Neto1 in ACC may down-regulate NMDA receptor NR2A subunit in NP rats and does not affect pain-related negative affection. Key words: Pain; Anterior cingulate cortex; Conditioned avoidance behavior; Neuropilin and tolloid-like1 Protein; N-methyl-D-aspartate receptor

N-methyl-d-aspartate receptor NR2B subunit involved in depression-like behaviours in lithium chloride-pilocarpine chronic rat epilepsy model

Depression is a common comorbidity in patients with epilepsy with unclear mechanisms. This study is to explore the role of glutamate N-methyl-d-aspartate (NMDA) receptor NR1, NR2A and NR2B subunits in epilepsy-associated depression. Lithium chloride (Licl)-pilocarpine chronic rat epilepsy model was established and rats were divided into epilepsy with depression (EWD) and epilepsy without depression (EWND) subgroups based on forced swim test. Expression of NMDA receptor NR1, NR2A and NR2B subunits was measured by western blot and immunofluorescence methods. The immobility time (IMT) was significantly greater in Licl-pilocarpine model group than in Control group, which was also greater in EWD group than in EWND group. No differences of spontaneous recurrent seizure (SRS) counts over two weeks and latency were found between EWD and EWND groups. The number of NeuN positive cells was significantly less in Licl-pilocarpine model group than in Control group, but had no difference between EWD and EWND groups. The ratios of phosphorylated NR1 (p-NR1)/NR1 and p-NR2B/NR2B were significantly greater in the hippocampus in EWD group than in EWND group. Moreover, the expression of p-NR1 and p-NR2B in the CA1 subfield of hippocampus were both greater in Licl-pilocarpine model group than Control group. Selective blockage of NR2B subunit with ifenprodil could alleviate depression-like behaviours of Licl-pilocarpine rat epilepsy model. In conclusion, glutamate NMDA receptor NR2B subunit was involved in promoting depression-like behaviours in the Licl-pilocarpine chronic rat epilepsy model and might be a target for treating epilepsy-associated depression.

Possible Compensatory Mechanisms for Glutamatergic Disconnection Found in the Auditory Cortex in Schizophrenia

Possible Compensatory Mechanisms for Glutamatergic Disconnection Found in the Auditory Cortex in Schizophrenia

Enhancement of NMDA Receptor-Mediated Excitatory Postsynaptic Currents by gp120-Treated Macrophages: Implications for HIV-1-Associated Neuropathology

A plethora of prior studies has linked HIV-1-infected and immune activated brain mononuclear phagocytes (MP; blood borne macrophages and microglia) to neuronal dysfunction. These are modulated by N-methyl-D-aspartate receptor (NMDAR) antagonists and supporting their relevance for HIV-1-associated nervous system disease. The role of NMDAR subsets in HIV-1-induced neuronal injury, nonetheless, is poorly understood. To this end, we investigated conditioned media from HIV-1gp120-treated human monocyte-derived-macrophages (MDM) for its abilities to affect NMDAR-mediated excitatory postsynaptic currents (EPSC(NMDAR)) in rat hippocampal slices. Bath application of gp120-treated MDM-conditioned media (MCM) produced an increase of EPSC(NMDAR). In contrast, control (untreated) MCM had limited effects on EPSC(NMDAR). Testing NR2A NMDAR (NR2AR)-mediated EPSC (EPSC(NR2AR)) and NR2B NMDAR (NR2BR)-mediated EPSC (EPSC(NR2BR)) for MCM showed significant increased EPSC(NR2BR) when compared to EPSC(NR2AR) enhancement. When synaptic NR2AR-mediated EPSC was blocked by bath application of MK801 combined with low frequency stimulations, MCM retained its ability to enhance EPSC(NMDAR) evoked by stronger stimulations. This suggested that increase in EPSC(NMDAR) was mediated, in part, through extra-synaptic NR2BR. Further analyses revealed that the soluble factors with low (<3 kD) to medium (3-10 kD) molecular weight mediated the observed increases in EPSC(NMDAR). The link between activation of NR2BRs and HIV-1gp120 MCM for neuronal injury was demonstrated by NR2BR but not NR2AR blockers. Taken together, these results indicate that macrophage secretory products induce neuronal injury through extra-synaptic NR2BRs.

NADPH oxidase mediates the oxygen-glucose deprivation/reperfusion-induced increase in the tyrosine phosphorylation of the &lt;em&gt;N&lt;/em&gt;-methyl-D-aspartate receptor NR2A subunit in retinoic acid differentiated SH-SY5Y Cells

BackgroundEvidence exists that oxidative stress promotes the tyrosine phosphorylation of N-methyl-D-aspartate receptor (NMDAR) subunits during post-ischemic reperfusion of brain tissue. Increased tyrosine phosphorylation of NMDAR NR2A subunits has been reported to potentiate receptor function and exacerbate NMDAR-induced excitotoxicity. Though the effect of ischemia on tyrosine phosphorylation of NMDAR subunits has been well documented, the oxidative stress signaling cascades mediating the enhanced tyrosine phosphorylation of NR2A subunits remain unclear.ResultsWe report that the reactive oxygen species (ROS) generator NADPH oxidase mediates an oxidative stress-signaling cascade involved in the increased tyrosine phosphorylation of the NR2A subunit in post-ischemic differentiated SH-SY5Y neuroblastoma cells. Inhibition of NADPH oxidase attenuated the increased tyrosine phosphorylation of the NMDAR NR2A subunit, while inhibition of ROS production from mitochondrial or xanthine oxidase sources failed to dampen the post-ischemic increase in tyrosine phosphorylation of the NR2A subunit. Additionally, inhibition of NADPH oxidase blunted the interaction of activated Src Family Kinases (SFKs) with PSD-95 induced by ischemia/reperfusion. Lastly, inhibition of NADPH oxidase also markedly reduced cell death in post-ischemic SH-SY5Y cells stimulated by NMDA.ConclusionsThese data indicate that NADPH oxidase has a key role in facilitating NMDAR NR2A tyrosine phosphorylation via SFK activation during post-ischemic reperfusion.

Changes in total and surface N-methyl-D-aspartate receptor NR1, NR2A and NR2B subunits in spinal cord in a rat model of incisional pain-remifentanil-induced hyperalgesia

Objective To investigate the changes in total and surface N-methyl-D-aspartate receptor (NMDAR) NR1,NR2A and NR2B subunits in spinal cord in a rat model of incisional pain(IP)-remifentanil-induced hyperalgesia.Methods Thirty-two male SD rate in which caudal vein catheter were successfully placed were randomly divided into 4 groups ( n =8 each):control group(group C),remifentanil group(group R),IP group (group I) and IP + remifentanil group (group R + I).In groups R and C remifentanil 1.2 μg· kg-1 · min-1 or the same volume of normal saline was infused intravenously for60 main.In groups R + I and I,a 1 cm longitudinal incision was made in the plantar surface of left hindpaw and remifentanil 1.2 μg· kg-1 ·min-1 or the same volume of normal saline was infused intravenously for 60 min.Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured at 24 h before and 2,6,24,48 h after remifentanil or normal saline administration.The animals were sacrificed after last pain threshold measurement.The L4-6 segment of the spinal cord was isolated for determination of the expression of the total and surface NMDAR NR1,NR2A and NR2B subunits in spinal cord by Western blot.The ratio of surface NR2B/NR2A was calculated.Results Compared with group C,PWT was significantly decreased,PWL shortened,the expression of the total and surface NMDAR NR1,NR2A and NR2B subunits up-regulated and the ratio of surface NR2B/NR2A increased in groups 1,R and R + I( P ＜ 0.05).Compared with groups R and I,PWT was significantly decreased,PWL shortened,the expression of the total and surface NMDAR NR1,NR2A and NR2B subunits up-regulated and the ratio of surface NR2B/NR2A increased in group R + I( P ＜ 0.05).There was no significant difference in the total and surface NMDAR NR2A subunits among the four groups( P ＞ 0.05).Conclusion The up-regulation of total and surface NMDAR NR1 and NR2B subunits and the increased percentage of surface NR2B subunits in rats spinal cord may be involved in the development of incisional pain- remifentanil- induced hyperalgesia. Key words: Piperidines; Hyperalgesia; Receptors, N-methyl-D-aspartate; Spinal cord

Roles of the SH2 and SH3 domains in the regulation of neuronal Src kinase functions

Previous studies demonstrated that intra-domain interactions between Src family kinases (SFKs), stabilized by binding of the phosphorylated C-terminus to the SH2 domain and/or binding of the SH2 kinase linker to the SH3 domain, lock the molecules in a closed conformation, disrupt the kinase active site, and inactivate SFKs. Here we report that the up-regulation of N-methyl-D-aspartate receptors (NMDARs) induced by expression of constitutively active neuronal Src (n-Src), in which the C-terminus tyrosine is mutated to phenylalanine (n-Src/Y535F), is significantly reduced by dysfunctions of the SH2 and/or SH3 domains of the protein. Furthermore, we found that dysfunctions of SH2 and/or SH3 domains reduce auto-phosphorylation of the kinase activation loop, depress kinase activity, and decrease NMDAR phosphorylation. The SH2 domain plays a greater regulatory role than the SH3 domain. Our data also show that n-Src binds directly to the C-terminus of the NMDAR NR2A subunit in vitro, with a K(D) of 108.2 ± 13.3 nM. This binding is not Src kinase activity-dependent, and dysfunctions of the SH2 and/or SH3 domains do not significantly affect the binding. These data indicate that the SH2 and SH3 domains may function to promote the catalytic activity of active n-Src, which is important in the regulation of NMDAR functions.

Effects of ammonia and allopurinol on rat hippocampal NMDA receptors

Ammonia is considered to be the main agent responsible for hepatic encephalopathy which progressively leads to altered mental status. N-methyl-D-aspartate (NMDA) is an ionotropic glutamate receptor, which is involved in synaptogenesis, memory and neurotoxicity. The aim of this study was to investigate the effects of ammonia intoxication and allopurinol, a xanthine oxidase (XO) inhibitor, on NMDA receptor subunits, NR2A and NR2B, in the hippocampus of rats. Thirty-six male rats were divided into three groups (n = 12/group) as follows: (1)control group (phosphate buffered saline (PBS) solution); (2)ammonia group (ammonium acetate, 2.5 mmol/kg), (3)ammonia + allopurinol group (ammonium acetate, 2.5 mmol/kg, allopurinol, 50 mg/kg). Each rat received intraperitoneal injection for 28 days. Western Blotting technique was used for detecting NR2A and NR2B expressions. Both NR2A and NR2B subunit expressions decreased 27 and 11%, respectively, in ammonia group with respect to the control group. Ammonium acetate decreased significantly in NR2A subunit expressions in the hippocampus (p < 0.01). Administration of ammonia + allopurinol caused statistically significant increases in NR2A subunit expressions compared to the ammonia group (p < 0.001). The down-regulation of NMDA receptors caused by ammonium acetate suggest that these receptors may play role in the process of hepatic encephalopathy and using allopurinol may have some protective effects in ammonia toxicity.

Hippocampal N‐Methyl‐d‐Aspartate Receptor Subunit Expression Profiles in a Mouse Model of Prenatal Alcohol Exposure

Although several reports have been published showing prenatal ethanol exposure is associated with alterations in N-methyl-D-aspartate (NMDA) receptor subunit levels and, in a few cases, subcellular distribution, results of these studies are conflicting. We used semi-quantitative immunoblotting techniques to analyze NMDA receptor NR1, NR2A, and NR2B subunit levels in the adult mouse hippocampal formation isolated from offspring of dams who consumed moderate amounts of ethanol throughout pregnancy. We employed subcellular fractionation and immunoprecipitation techniques to isolate synaptosomal membrane- and postsynaptic density protein-95 (PSD-95)-associated pools of receptor subunits. We found that, compared to control animals, fetal alcohol-exposed (FAE) adult mice had: (i) increased synaptosomal membrane NR1 levels with no change in association of this subunit with PSD-95 and no difference in total NR1 expression in tissue homogenates; (ii) decreased NR2A subunit levels in hippocampal homogenates, but no alterations in synaptosomal membrane NR2A levels and no change in NR2A-PSD-95 association; and (iii) no change in tissue homogenate or synaptosomal membrane NR2B levels but a reduction in PSD-95-associated NR2B subunits. No alterations were found in mRNA levels of NMDA receptor subunits suggesting that prenatal alcohol-associated differences in subunit protein levels are the result of differences in post-transcriptional regulation of subunit localization. Our results demonstrate that prenatal alcohol exposure induces selective changes in NMDA receptor subunit levels in specific subcellular locations in the adult mouse hippocampal formation. Of particular interest is the finding of decreased PSD-95-associated NR2B levels, suggesting that synaptic NR2B-containing NMDA receptor concentrations are reduced in FAE animals. This result is consistent with various biochemical, physiological, and behavioral findings that have been linked with prenatal alcohol exposure.

Uncoupling the D1- N-Methyl-D-Aspartate (NMDA) Receptor Complex Promotes NMDA-Dependent Long-Term Potentiation and Working Memory

Although dopamine D1 receptors are involved in working memory, how D1 receptors contribute to this process remains unclear. Numerous studies have shown that D1 receptors have extensive functional interaction with N-methyl-D-aspartate (NMDA) receptor. Our group previously demonstrated that D1 receptors were able to regulate NMDA receptor functions through direct protein-protein interactions involving the carboxyl terminals of D1 receptors and NMDA receptor NR1a and NR2A subunits respectively. In this study, we explored the effects of the D1-NR1 interaction on NMDA receptor-dependent long-term potentiation (LTP) and working memory by using the TAT-conjugated interfering peptide (TAT-D1-t2). Miniature excitatory postsynaptic currents are recorded in rat hippocampal primary cultures. Coimmunoprecipitation and calcium/calmodulin-dependent protein kinase II (CaMKII) activity are measured in hippocampal slices and hippocampal neurons under the specified experimental conditions, respectively. Working memory was assessed using a delayed match-to-place protocol in the Morris Water Maze following administration of the TAT-D1-t2 peptide. Electrophysiology experiments showed that activation of D1 receptor upregulates NMDA receptor-mediated LTP in a CaMKII-dependent manner. Furthermore, D1 receptor agonist stimulation promotes the NR1-CaMKII coupling and enhances the CaMKII activity; and the D1 receptor-mediated effects can be blocked by the application of the TAT-D1-t2 peptide. Interestingly, animals injected with TAT-D1-t2 peptide exhibited significantly impaired working memory. Our study showed a critical role of NMDA-D1 direct protein-protein interaction in NMDA receptor-mediated LTP and working memory and implicated the involvement of CaMKII in this process.

Increased expression of the NR2A NMDA receptor subunit in the prefrontal cortex of rats reared in isolation

A hypofunction of the N-methyl-D-aspartate (NMDA) receptor has been implicated in the pathophysiology of schizophrenia. Compelling evidence of altered NMDA receptor subunit expression in the schizophrenic brain has not, however, so far emerged. Rats reared in isolation exhibit several characteristics, including disturbed sensory gating, which resemble those seen in schizophrenia. To explore the possibility that NMDA receptor dysfunction may contribute to the behavioral and neurochemical consequences of rearing rats in isolation, we compared NMDA receptor subunit expression in brains of rats which were housed in isolation and which displayed a deficit in prepulse inhibition of the acoustic startle response with that of socially housed controls. An initial microarray analysis revealed a 1.26-fold increase in NR2A transcript in the prefrontal cortex, but not in the nucleus accumbens, of rats reared in isolation compared with those housed socially. In contrast, NR1, NR2B, NR2C, NR2D, NR3A, and NR3B subunit expression was unchanged in either brain area. In a second cohort of animals, in situ hybridization revealed increased NR2A mRNA expression in the medial prefrontal cortex, an observation that was substantiated by increased [(3)H]CGP39653 binding suggesting that NR2A receptor subunit protein expression was also elevated in the medial prefrontal cortex of the same animals. No changes in expression of NR1 or NR2B subunits were observed at both mRNA and protein level. Altered NR2A subunit expression in the medial prefrontal cortex of rats reared in isolation suggests that NMDA receptor dysfunction may contribute to the underlying pathophysiology of this preclinical model of aspects of schizophrenia.

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.

Alterations in NMDA receptor subunit densities and ligand binding to glycine recognition sites are associated with chronic anxiety in Alzheimer's disease

Glutamatergic deficits are established neuropathological features of Alzheimer's disease (AD) and are known to correlate with cognitive impairments. In contrast, the role of glutamatergic alterations in behavioral and psychological symptoms of dementia (BPSD) is unclear. There is considerable preclinical evidence for the importance of glycine recognition sites (GlyRS) of N-methyl- d-aspartate (NMDA) receptors in the regulation of anxiety behaviors. This study aimed to correlate several glutamatergic measures with chronic anxiety in AD. Twenty-one AD patients assessed by the Neuropsychiatric Inventory (NPI) were divided into low anxiety (LA) and high anxiety (HA) subgroups. GlyRS and NMDA channel were measured by brain homogenate binding with [ 3H]MDL105,519 and [ 3H]MK-801, respectively. Densities of NMDA receptor NR2A, NR2B and alternate spliced NR1 subunits were quantified by immunoblotting. We found that the binding affinity to GlyRS was significantly higher in HA compared to LA, and this higher GlyRS affinity correlated with selective reduction of NR2A density as well as with elevated anxiety scores. Our observations suggest a novel mechanism whereby subunit specific changes in the NMDA receptor complex may be linked to chronic anxiety in AD via effects on GlyRS function. We propose that NR2A and GlyRS should be further assessed as novel targets of behavioral pharmacotherapy in AD.

Single‐channel analysis of a point mutation of a conserved serine residue in the S2 ligand‐binding domain of the NR2A NMDA receptor subunit

We have examined the function of a conserved serine residue (Ser670) in the S2 ligand-binding region of the NR2A N-methyl-d-aspartate (NMDA) receptor subunit, using recombinant NR1/NR2A receptors expressed in Xenopus laevis oocytes. Mutation of Ser670 to glycine (S670G) in NR2A reduced the potency of glutamate by 124-fold. Single-channel conductance and the duration of apparent open periods of NR2A(S670G) receptor mutants were, however, indistinguishable from wild-type NMDA receptors. NR1/NR2A(S670G) shut-time distributions were best described by a mixture of six exponential components, and the four shortest shut intervals of each distribution were considered to occur within a channel activation (burst). Bursts of single-channel openings were fitted with a mixture of four exponential components. The longest two components carried the majority of the charge transfer and had mean durations of 9.6 +/- 0.5 and 29.6 +/- 1.5 ms. The overall channel open probability during a burst was high (mean, 0.83 +/- 0.06). Consistent with a shortening of NMDA receptor-channel burst lengths was the observation of an increased deactivation rate of macroscopic currents evoked by brief applications of glutamate to outside-out membrane patches. Correlations between shut times and adjacent open times were observed in all data records. Noticeably, shorter than average openings tended to occur next to long closed periods, whereas longer than average openings tended to occur next to short closings. Our single-channel data, together with modelling using a kinetic scheme to describe channel activations, support our hypothesis that the S670G point mutation reduces the dwell time of glutamate in its binding site.

The role of NR2B containing NMDA receptor in place preference conditioned with morphine and natural reinforcers in rats

It has been reported that N-methyl-D-aspartate (NMDA) receptor is implicated in drug addiction and antagonists of the NMDA receptor complex can inhibit the development and expression of conditioned place preference (CPP) induced by several addictive drugs, implying that this class of compounds might be considered as candidate for the treatment of substance abuse. To explore this possibility, it is important to evaluate whether the inhibitory effect of NMDA receptor antagonists would be confined to behaviors produced by drugs of abuse only, but not by natural reinforcers. According to the quantitative changes of NMDA receptor subunits, including NR1, NR2A, and NR2B, induced by diverse types of reinforcers, we chose NR2B subunit as the target of research. Experimental results showed that (1) an augmented expression of NR2B subunit was revealed by Western blotting in the nucleus accumbens (NAc) and the hippocampus in rats with CPP induced by morphine, but not by natural rewards such as food, novel environment and social interaction. (2) Ifenprodil, an antagonist highly selective for NR2B subunit of the NMDA receptor, produced a dose-dependent reduction in CPP induced by morphine and novel environment, but not that by food consumption and social interaction. Taking together, these findings suggested that NR2B containing NMDA receptor may be more involved with morphine reward rather than natural rewards, and that antagonism of NR2B may have a potential for the treatment of morphine abuse.

Effect of δ-aminolevulinic acid treatment on N-methyl- d-aspartate receptor at different ages in the rat brain

We report here the effects of the chronic treatment with the oxidant agent δ-aminolevulinic acid (ALA) on the N-methyl- d-aspartate (NMDA) receptors in 4-, 12- and 24-month-old male Wistar rats. ALA was administered daily for 15 days (40 mg/kg i.p). The study was performed by membrane homogenate binding and autoradiography, using tritiated 5-methyl-10, 11-dihydro-5 H-dibenzo(a,d)cycloheptan-5,10-imine maleate ([ 3H]MK-801). [ 3H]MK-801 binding was significantly decreased in most areas studied (cortex and hippocampus) at all ages in treated rats with respect to their controls. Furthermore, Western blot assays were performed using antibodies against the NMDA receptor NR2A subunit, which is widely distributed in the brain, mainly in cortex and hippocampus. In cortex but not in hippocampus, the ALA treatment induced significant decreases in the amounts of NR2A subunit in 12- and 24-month-old animals. We conclude that chronic treatment with ALA is able to induce NMDA receptor decreases in an age-independent way and that NR2A subunit seems to be involved in these decreases in cerebral cortex, but not in the other structures studied.

Neuropsychiatric disturbances in SLE are associated with antibodies against NMDA receptors

To determine whether neuropsychiatric manifestations in patients with systemic lupus erythematosus (SLE) are influenced by antibodies against the human N-methyl-D-aspartate (NMDA) receptor types NR2a or NR2b. A decapeptide was synthesized containing a sequence motif present in the extracellular ligand-binding domain of NMDA receptors NR2a and NR2b, bound by the monoclonal murine anti-DNA antibody R4A. In an ELISA with the murine monoclonal R4v as positive control, plasma samples of 57 patients with SLE were examined for the anti-peptide (anti-NR2) antibody after the patients had been subjected to comprehensive psychological and cognitive testing. Poor performance on the Visual Paired Associates test (immediate), the Grooved Pegboard test, as well as high scores on the Beck Depression Inventory, and scales D-2 (depression), Pd-4 (psychopathic deviate), Sc-8 (schizophrenia), and Ma-9 (hypomania) of the MMPI-2 were significantly associated with elevated levels of anti-NR2 antibodies. The findings in several domains indicate an association between anti-NR2 antibodies and depressed mood in addition to decreased short-time memory and learning. Antibodies to NMDA receptors thus may represent one of several mechanisms for cerebral dysfunction in patients with SLE.

Long-term changes in glutamatergic synaptic transmission in phenylketonuria

The cellular mechanisms that underlie impaired brain function during phenylketonuria (PKU), the most common biochemical cause of mental retardation in humans, remain unclear. Acute application of L-Phe at concentrations observed in the PKU brain depresses glutamatergic synaptic transmission but does not affect GABA receptor activity in cultured neurons. If these depressant effects of L-Phe take place in the PKU brain, then chronic impairment of the glutamate system, which may contribute to impaired brain function, could be detected as changes in postsynaptic glutamate receptors. This hypothesis was tested by using a combination of liquid chromatography-mass spectrometry, patch-clamp, radioligand binding and western blot approaches in forebrain tissue from heterozygous and homozygous (PKU) Pah(enu2) mice. Brain concentrations of L-Phe were nearly six-fold greater in PKU mice (863.12 +/- 17.96 micromol/kg) than in their heterozygous counterparts (149.32 +/- 10.23 micromol/kg). This concentration is significantly higher than the K(B) of 573 microM for L-Phe to compete for N-methyl-D-aspartate (NMDA) receptors. Receptor binding experiments with [3H]MK-801 showed significant up-regulation of NMDA receptor density in PKU mice. Consistent with the depressant effects of L-Phe, expression of NMDA receptor NR2A and (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor Glu1 and Glu2/3 subunits was significantly increased, whereas expression of the NR2B subunit was decreased. There was no change in GABA alpha1 subunit expression. Given the role of the glutamatergic system in brain development and function, these changes may, at least in part, explain the brain disorders associated with PKU.