Effect of dopamine denervation and dopamine agonist administration on serine phosphorylation of striatal NMDA receptor subunits

Sensitization of striatal N-methyl-d-aspartate (NMDA) receptors has been implicated in the pathogenesis of the response alterations associated with dopaminomimetic treatment of parkinsonian animals and patients. To determine whether serine phosphorylation of NMDA receptor subunits by activation of Ca2+/calmodulin-dependent protein-kinase II (CaMKII) contributes to this process, we examined the effects of unilateral nigrostriatal ablation with 6-hydroxydopamine and subsequent treatment with levodopa, SKF 38393 (D1-preferring dopamine agonist), or quinpirole (D2-preferring agonist) on motor responses and phosphorylation states. Three weeks of twice-daily levodopa administration to rats shortened the duration of their rotational response to levodopa or SKF 38393 challenge, but prolonged the duration of quinpirole-induced rotation. At the same time, levodopa treatment elevated serine phosphorylation of striatal NR2A (p<0.02), but not that of NR2B subunits, without associated changes in subunit protein levels. Chronic treatment with SKF 38393 increased NR2A (p<0.0001) but decreased NR2B (p<0.004) serine phosphorylation. In contrast, chronic quinpirole treatment had no effect on NR2A but increased NR2B phosphorylation (p<0.0001). The acute intrastriatal injection of the CaMKII inhibitor KN93 (1.0 micrograms) not only normalized the levodopa-induced motor response alterations but also attenuated the D1 and D2 receptor-mediated serine phosphorylation of NR2A and NR2B subunits, respectively (p<0.02). These results suggest that a CaMKII-mediated rise in serine phosphorylation of NMDA receptor subunits induced by intermittent stimulation of D1 or D2 dopaminergic receptors contributes to the apparent enhancement in striatal NMDA receptor sensitivity and thus to the dopaminergic response plasticity in levodopa-treated parkinsonian rats.     

Acute ethanol affects phosphorylation state of the NMDA receptor complex: implication of tyrosine phosphatases and protein kinase A

Phosphorylation has been shown to regulate N-methyl-D-aspartic acid receptor (NMDAR) function. The inhibitory effect of ethanol on NMDAR function could be due, at least in part, to a change in NMDAR phosphorylation states. In order to investigate the effect of ethanol on phosphorylation of NR1 and NR2 subunits, NMDAR complexes were immunoprecipitated from cortical slices pre-exposed to ethanol. Acute ethanol, 100 and 200 mM, significantly decreased the tyrosine phosphorylation of NR2 subunits (Tyr-NR2). Treatment with a tyrosine phosphatase inhibitor reduced the inhibition of Tyr-NR2 phosphorylation caused by 100 mM ethanol. This suggests an involvement of tyrosine phosphatases in ethanol-induced inhibition of Tyr-NR2 phosphorylation. Slices pre-exposed to 100 and 200 mM ethanol exhibited a significant increase in the phosphorylation of NR1 by PKA at serine 897 (Ser897-NR1), which was blocked by a PKA inhibitor. Moreover, at 200 mM, ethanol produced a significant increase in PKA activity. Together, these results indicate that ethanol may increase Ser897-NR1 phosphorylation by activating PKA. However, ethanol did not affect phosphorylation of NR1 subunits by PKC at serine 896. We conclude that ethanol has the ability to modulate phosphorylation of both NR2 and NR1 subunits and these effects appear to implicate tyrosine phosphatases and PKA, respectively.     

Antiparkinsonian and antidyskinetic activity of drugs targeting central glutamatergic mechanisms

Motor dysfunction produced by the chronic non-physiological stimulation of dopaminergic receptors on striatal medium spiny neurons is associated with alterations in the sensitivity of glutamatergic receptors, including those of the N-methyl-D-aspartate (NMDA) subtype. Functional characteristics of these ionotropic receptors are regulated by their phosphorylation state. Lesioning the nigrostriatal dopamine system of rats induces parkinsonian signs and increases the phosphorylation of striatal NMDA receptor subunits on serine and tyrosine residues. The intrastriatal administration of certain inhibitors of the kinases capable of phosphorylating NMDA receptors produces a dopaminomimetic motor response in these animals. Treating parkinsonian rats twice daily with levodopa induces many of the characteristic features of the human motor complication syndrome and further increases the serine and tyrosine phosphorylation of specific NMDA receptor subunits. Again, the intrastriatal administration of selective inhibitors of certain serine and tyrosine kinases alleviates the motor complications. NMDA receptor antagonists, including some non-competitive channel blockers, act both palliatively and prophylactically in rodent and primate models to reverse these levodopa-induced response alterations. Similarly, in clinical studies dextrorphan, dextromethorphan, and amantadine have been found to be efficacious against motor complications. Recent observations in animal models further indicate that certain amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) antagonists alleviate, while others exacerbate, these complications. Thus, it appears that the denervation or intermittent stimulation of striatal dopaminergic receptors differentially activates signal transduction pathways in medium spiny neurons. These in turn modify the phosphorylation state of ionotropic glutamate receptors and consequently their sensitivity to cortical input. These striatal changes contribute to symptom production in Parkinson’s disease, and their prevention or reversal could prove useful in the treatment of this disorder.

     

Striatal neurons but not nigral dopaminergic neurons in neonatal primary cell culture express endogenous functional N-methyl-D-aspartate receptors

Developmental expression of N-methyl-D-aspartate (NMDA) receptor subunits were determined and compared in striatal and nigral neurons in neonatal primary cell cultures. In striatal neurons, NR1, NR2A and NR2B mRNAs and immunoreactivity, and NR2D mRNA were found and the maximal levels of NR1 mRNA and immunoreactivity expression were found at 6 day-in-vitro (DIV). NMDA receptors found at this stage in striatal neurons are likely to contain NR1 plus NR2A, NR2B and NR2D subunits. In nigral neurons, NR1 and NR2B mRNAs and immunoreactivity, and NR2D mRNA were found and the maximal level of NR1 immunoreactivity expression was found at 10 DIV. Unlike striatal neurons, NMDA receptors found in nigral neurons are likely to contain NR1 plus NR2B and NR2D subunits only. NMDA-induced toxicity assays showed that striatal neurons were most susceptible to cell death at around 10 DIV but nigral neurons were not susceptible to NMDA-induced cell death at all stages. In addition, patch clamp analysis revealed that functional NMDA receptors could only be found in striatal neurons but not in nigral dopaminergic neurons in vitro. The present results indicate that striatal and nigral neurons are programmed to express distinct NMDA receptor subunits during their endogenous development in cell cultures. Despite dopaminergic neurons in culture display NMDA receptor subunits, functional NMDA receptors are not assembled. The present findings have demonstrated that dopaminergic neurons in vitro may behave very differently to their counterparts in vivo in terms of NMDA receptor-mediated responses. Our results also have implications in transplantations using dopaminergic neurons in vitro in treatments of Parkinson's disease.     

Tyrosine phosphorylation of the N-methyl-D-aspartate receptor is enhanced in synaptic membrane fractions of the adult rat hippocampus

Hippocampal N-methyl-D-aspartate receptors (NMDARs) contribute to the expression of certain types of synaptic plasticity, such as long-term potentiation (LTP). It is well documented that tyrosine kinases increase NMDAR phosphorylation and potentiate NMDAR function. However, it is unclear how these phosphorylation changes result in enhanced NMDAR activity. We previously reported that NMDAR surface expression can be increased by LTP-inducing stimulation via tyrosine kinase-dependent mechanisms in the adult hippocampus [D.R. Grosshans, D.A. Clayton, S.J. Coultrap, M.D. Browning, Nat. Neurosci., 5 (2002) 27-33]. We therefore hypothesized that tyrosine phosphorylation of the NMDAR may enhance the trafficking of the receptor to the synaptic membrane. Here, we show that the stoichiometry of NR2A and NR2B tyrosine phosphorylation is significantly higher in synaptosomal membranes than intracellular microsomal/light membranes. Interestingly, NR2B tyrosine-1472, but not NR1 serine-896 or -897, phosphorylation is significantly higher in synaptosomal membranes than intracellular microsomal/light membranes. Furthermore, treatment of hippocampal slices with either a tyrosine phosphatase inhibitor or a tyrosine kinase inhibitor alters NMDAR tyrosine phosphorylation and produces a corresponding change in the concentration of NMDARs in the synaptosomal membrane fraction. Taken together, these data support the hypothesis that tyrosine phosphorylation may enhance NMDAR activity by increasing the number of NMDARs at the synaptic membrane.     

Effects of protein phosphatase inhibitors on the phosphorylation of spinal cord N-methyl-D-aspartate receptors following electroacupuncture stimulation in rats

The present study investigated the role of inhibitor of protein phosphatases 1 and 2A on the modulation of the phosphorylation of the spinal N-methyl-D-aspartate receptor (NMDAR) NR1 and NR2B subunits following electroacupuncture (EA) stimulation in rats. Bilateral 2Hz EA stimulations with 1.0 mA were delivered at those acupoints corresponding to Zusanli and Sanyinjiao to men via needles for 30 min. EA analgesia was slightly reduced by the intrathecal injection of calyculin A during EA stimulation. At 60 min after the termination of EA stimulation, the levels of c-fos, serine phosphorylation of NR1 and NR2B by Western analysis had increased in the L(4-5) segments of the spinal cord after EA treatment. These expressions were enhanced by the intrathecal injection of calyculin A and immunohistochemical analyses confirmed the significant increase of these proteins. As for the regional reaction of NMDAR subunits, a mean integrated optical density of phosphorylated NR1 and NR2B subunits was potentiated by calyculin A injections in the superficial laminae and neck region and superficial laminae and nucleus proprius, respectively. It can be concluded that protein phosphatase may play an important role in EA analgesia by modulating the phosphorylation state of spinal NMDAR subunits.     

Induced tolerance to glutamate neurotoxicity through down‐regulation of NR2 subunits of N‐methyl‐D‐aspartate receptors in cultured rat striatal neurons

We have previously shown differential vulnerabilities to glutamate (Glu) excitotoxicity mediated by the N‐methyl‐D‐aspartate (NMDA) receptor (NMDAR) between rat cortical and rat hippocampal neurons in culture. In this study, we evaluated the possible induced tolerance to NMDA neurotoxicity in cultured rat striatal neurons with prior sustained activation of NMDAR. Brief exposure to Glu or NMDA for 1 hr led to a significant decrease in cellular vitality determined 24 hr later in cultured rat striatal neurons, whereas no marked loss was seen in cellular survival after exposure to Glu or NMDA in striatal neurons previously cultured with Glu or NMDA. Sustained culture with Glu or NMDA invariably led to a significant decrease in protein levels of NR2, but not NR1, subunits without affecting their mRNA levels. Similar induced tolerance was seen to the excitotoxicity of NMDA in hippocampal neurons in a manner sensitive to an NMDAR antagonist. Prior culture with NMDA induced less effective alterations in both intracellular free Ca²⁺ levels and mitochondrial membrane potentials after the addition of NMDA in striatal neurons. However, calpain inhibitor‐I significantly prevented the decreased NR2B and NR2C protein levels in striatal neurons cultured with NMDA. These results suggest that prior tonic activation of NMDAR would induce tolerance to the excitotoxicity mediated by NMDAR through a mechanism related to calpain‐induced down‐regulation of particular NR2 subunits in rat striatal neurons. © 2010 Wiley‐Liss, Inc.     

Glycine site agonists of the N‐methyl‐d‐aspartate receptor and Parkinson's disease: A hypothesis

Limitations of current pharmacological approaches to Parkinson's disease (PD) highlight the need for the development of nondopaminergic therapeutic strategies. The potential role of glutamatergic neurotransmission modulators, including those active at the N‐methyl‐D‐aspartate receptor (NMDAR), is presently under investigation. Most literature proposes the use of NMDAR antagonists based on neurodegenerative theories of NMDAR function. Nevertheless, NMDAR antagonism has proven disappointing in clinical trials and may be associated with serious adverse events. More recent theories indicate that NMDAR target selectivity may be a cardinal prerequisite for efficacy, with present efforts being devoted primarily to development of NMDAR‐NR2B subunit antagonists. We propose a novel hypothesis according to which NMDAR stimulation, accomplished through allosteric modulation via the glycine modulatory site, may be beneficial in late‐phase PD. This hypothesis stems from: (1) meta‐analysis of randomized controlled trials performed in schizophrenia, indicating that glycine site agonists (eg, glycine, D‐serine) alleviate antipsychotic‐induced parkinsonian symptoms; (2) clinical observations indicating that NMDAR hypofunction is associated with motor disturbances; (3) results of a preliminary D‐serine trial in PD; (4) data indicating glycine efficacy in a rat tardive dyskinesia model; and (5) no evidence of excitotoxic damage following chronic high‐dose glycine nutritional supplementation. This hypothesis is discussed in the context of glycine site agonist effects on intrasynaptic NMDAR subunits and striatal synaptic plasticity. © 2013 Movement Disorder Society     

Astrocytic activation of A1 receptors regulates the surface expression of NMDA receptors through a Src kinase dependent pathway

Chemical transmitters released from astrocytes, termed gliotransmitters, modulate synaptic transmission and neuronal function. Using astrocyte-specific inducible transgenicmice (dnSNARE mice), we have demonstrated that inhibiting gliotransmission leads to reduced activation of adenosine A1 receptors (A1R) and impaired sleep homeostasis (Halassa et al. (2009) Neuron 61:213-219); Pascual et al. (2005) Science 310:113-116). Additionally, synaptic N-methyl-D-aspartate receptor (NMDAR) currents are reduced in these astrocyte-specific transgenic animals (Fellin et al. (2009) Proc Natl Acad Sci USA 106:15037-15042). Because of the importance of adenosine and NMDA receptors to sleep processes we asked whether there is a causal linkage between changes in A1R activation and synaptic NMDA receptors. We show that astrocytic dnSNARE expression leads to reduced tyrosine phosphorylation of Srckinase and NR2 subunits concomitant with the decreased surface expression of the NR2 subunits. To test the role of A1R signaling in mediating these actions, we show that incubation of wildtype (WT) slices with an A1R antagonist reduces tyrosine phosphorylation of Src kinase and NR2B, decreases the surface expression of the NR2B subunits and leads to smaller NMDA component of miniature EPSCs. In dnSNARE mice we could rescue WT phenotype by incubation in an A1R agonist:activation of A1 receptor led to increased tyrosine phosphorylation of Src kinase and NR2B subunits as well as increased the surface expression of the NR2B subunit and increased NMDA component of the synaptic mEPSC. These results provide the first demonstration that astrocytes can affect neuronal excitability on a long time scale by regulating the surface expression of NMDA receptors through the activation of specific intracellular signaling pathways.     

NMDA-complexes linked to spatial memory performance in the Barnes maze in CD1 mice

The N-methyl-d-aspartic acid receptor (NMDAR) is a well-documented key element in the formation of several memories including spatial, olfactory and contextual memory. Although receptor subunits have been linked to memory formation, data on the involvement of the NMDAR complexes is limited.In previous work CD1 mice were trained in the Barnes maze, a low-stress landmaze, and yoked controls were serving as controls. Hippocampal samples from this behavioural study were taken for comparing NMDAR complexes. Hippocampi were taken and stored until analysis at −80 °C.Membrane proteins were extracted from hippocampi using an ultracentrifugation step and applied on Blue Native gels that in turn were used for immunoblotting with antibodies against subunits NR1, NR2A and NR2B. The subunit content of the complexes was determined by denaturing two-dimensional gel electrophoresis and subsequent immunoblotting.An NMDAR complex with an apparent molecular weight between between146 and 242 kDa, probably representing an NR1 dimer was the only complex that was significantly different between trained and yoked animals. A series of NMDAR complexes containing modulatory subunits NR2A or NR2B or both were detected. All complexes contained the NR1subunit.The NR1 dimer complex level, increased in memory formation, may be directly or indirectly involved in the process of spatial memory formation in the CD1 mouse. The results are enabling and challenging further NMDAR studies, both, at the pharmacological and molecular level. Moreover, several NMDAR complexes in the CD1 mouse were shown to be mainly heteropolymers of subunits NR1, NR2A and NR2B, although other recently described subunits were not tested due to unavailability of specific antibodies.Determination of native receptor complexes rather than individual subunits is mandatory and provides the molecular basis for understanding mechanisms of spatial memory.     

Higher free D-aspartate and N-methyl-D-aspartate levels prevent striatal depotentiation and anticipate L-DOPA-induced dyskinesia

In Parkinson's disease (PD) progressive alteration of striatal N-methyl-d-aspartate receptors (NMDARs) signaling has emerged as a considerable factor for the onset of the adverse motor effects of long-term levodopa (l-DOPA) treatment. In this regard, the NMDAR channel blocker amantadine is so far the only drug available for clinical use that attenuates l-DOPA-induced dyskinesia (LID). In this study, we examined the influence of a basal corticostriatal hyper-glutamatergic transmission in the appearance of dyskinesia, using a genetic mouse model lacking d-Aspartate Oxidase (DDO) enzyme (Ddo^−/− mice). We found that, in Ddo^−/− mice, non-physiological, high levels of the endogenous free d-amino acids d-aspartate (d-Asp) and NMDA, known to stimulate NMDAR transmission, resulted in the loss of corticostriatal synaptic depotentiation and precocious expression of LID. Interestingly, the block of depotentiation precedes any change in dopaminergic transmission associated to 6-OHDA lesion and l-DOPA treatment. Indeed, lesioned mutant mice display physiological l-DOPA-dependent enhancement of striatal D1 receptor/PKA/protein phosphatase-1 and ERK signaling. Moreover, in line with synaptic rearrangements of NMDAR subunits occurring in dyskinetic animal models, a short l-DOPA treatment produces a dramatic and selective reduction of the NR2B subunit in the striatal post-synaptic fraction of Ddo^−/− lesioned mutants but not in controls. These data indicate that a preexisting hyper-glutamatergic tone at NMDARs in Ddo^−/− mice produce abnormal striatal synaptic changes that, in turn, facilitate the onset of LID.     

Hippocampal NMDA receptor subunits differentially regulate fear memory formation and neuronal signal propagation

Activation of NMDA receptors (NMDAR) in the hippocampus is essential for the formation of contextual and trace memory. However, the role of individual NMDAR subunits in the molecular mechanisms contributing to these memory processes is not known. Here we demonstrate, using intrahippocampal injection of subunit‐selective compounds, that the NR2A‐preferring antagonist impaired contextual and trace fear conditioning as well as learning‐induced increase of the nuclear protein c‐Fos. The NR2B‐specific antagonist, on the other hand, selectively blocked trace fear conditioning without affecting c‐Fos levels. Studies with cultured primary hippocampal neurons, further showed that synaptic and extrasynaptic NR2A and NR2B differentially regulate the extracellular signal‐regulated kinase 1 and 2/mitogen‐ and stress‐activated protein kinase 1 (ERK1/2/MSK1)/c‐Fos pathway. Activation of the synaptic population of NMDAR induced cytosolic, cytoskeletal, and perinuclear phosphorylation of ERK1/2 (pERK1/2). The nuclear propagation of pERK1/2 signals, revealed by upregulation of the downstream nuclear targets pMSK1 and c‐Fos, was blocked by a preferential NR2A but not by a specific NR2B antagonist. Conversely, activation of total (synaptic and extrasynaptic) NMDAR engaged receptors with NR2B subunits, and resulted in membrane retention of pERK1/2 without inducing pMSK1 and c‐Fos. Stimulation of extrasynaptic NMDAR alone was consistently ineffective at activating ERK signaling. The discrete contribution of synaptic and total NR2A‐ and NR2B‐containing NMDAR to nuclear transmission vs. membrane retention of ERK signaling may underlie their specific roles in the formation of contextual and trace fear memory. © 2009 Wiley‐Liss, Inc.     

The effect of transient global ischemia on the interaction of Src and Fyn with the N-methyl-D-aspartate receptor and postsynaptic densities: possible involvement of Src homology 2 domains.

Transient ischemia increases tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B in the rat hippocampus. The authors investigated the effects of this increase on the ability of the receptor subunits to bind to the Src homology 2 (SH2) domains of Src and Fyn expressed as glutathione-S-transferase-SH2 fusion proteins. The NR2A and NR2B bound to each of the SH2 domains and binding was increased approximately twofold after ischemia and reperfusion. Binding was prevented by prior incubation of hippocampal homogenates with a protein tyrosine phosphatase or by a competing peptide for the Src SH2 domain. Ischemia induced a marked increase in the tyrosine phosphorylation of several proteins in the postsynaptic density (PSD), including NR2A and NR2B, but had no effect on the amounts of individual NMDA receptor subunits in the PSD. The level of Src and Fyn in PSDs, but not in other subcellular fractions, was increased after ischemia. The ischemia-induced increase in the interaction of NR2A and NR2B with the SH2 domains of Src and Fyn suggests a possible mechanism for the recruitment of signaling proteins to the PSD and may contribute to altered signal transduction in the postischemic hippocampus.     

Wearing-off fluctuations in Parkinson's disease: Contribution of postsynaptic mechanisms

Wearing-off phenomenon that complicates levodopa therapy of Parkinson's disease has been attributed to a reduction in striatal dopamine storage due to the progressive degeneration of presynaptic dopaminergic terminals. To determine whether postsynaptic mechanisms also contribute to these response fluctuations, the duration of the antiparkinsonian response in parkinsonian patients grouped by disease severity was compared following discontinuation of a steady-state optimal-dose infusion of apomorphine. Although the plasma half-life of this dopamine receptor agonist remained constant, its mean efficacy half-time declined from 66 minutes in early, levodopa-naive patients to 33 minutes in advanced, complicated parkinsonians (p < 0.005). Since the motor effects of apomorphine do not depend on the presence of dopaminergic terminals, changes at the postsynaptic level undoubtedly contribute to the diminished response duration. The only slightly greater attenuation of levodopa's motor effects observed previously under similar conditions suggests these postjunctional alterations, possibly involving relatively plastic striatal dopaminoceptive systems, account for most of the shortening in the duration of levodopa action that underlie wearing-off fluctuations.     

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

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

Transient forebrain ischemia effects interaction of Src, FAK, and PYK2 with the NR2B subunit of N-methyl-D-aspartate receptor in gerbil hippocampus

Two different models of brain ischemia were used to examine the evoked changes in the tyrosine phosphorylation of NMDA receptor subunits 2A and 2B (NR2A and NR2B), as well as their interactions with non-receptor tyrosine kinases (NRTKs: FAK, PYK2 Src), and PSD-95 protein. Only short-term 5 min ischemia followed by 3 h reperfusion resulted in the elevated tyrosine phosphorylation of both investigated NMDA receptor subunits, but in contrast to previously published data, more pronounced in the case of NR2B. Concomitantly, an increased association of NR2B with FAK, PYK2, Src and PSD-95 has been observed. This sharp early reaction to brief ischemia was markedly attenuated during prolonged recovery (72 h) with almost complete return to control values. The initial recruitment of tyrosine kinases to NMDA receptor during the first 3 h of reperfusion is generally consistent with an active postischemic remodeling of PSD and may participate in the induction of the postischemic signal transduction pathway in gerbil hippocampus. In contrast, ischemia of longer duration (up to 30 min) caused an immediate decrease in the protein levels as well as tyrosine phosphorylation of both NR2A and NR2B subunits which was accompanied by the marked attenuation of the association with their investigated molecular partners--PSD-95 and NRTKs. This effect may be mimicked in vitro by Ca2+-dependent activation of endogenous calpains in purified PSD preparation suggesting irreversible deterioration of the synaptic signaling machinery during irreversible long-term ischemia.     

NR2B phosphorylation at tyrosine 1472 in spinal dorsal horn contributed to N-methyl-D-aspartate-induced pain hypersensitivity in mice

Calcium influx via N-methyl-D-aspartate (NMDA)-subtype glutamate receptors (NMDARs) regulates the intracellular trafficking of NMDARs, leading to long-lasting modification of NMDAR-mediated synaptic transmission that is involved in development, learning, and synaptic plasticity. The present study investigated the contribution of such NMDAR-dependent synaptic trafficking in spinal dorsal horn to the induction of pain hypersensitivity. Our data showed that direct activation of NMDARs by intrathecal NMDA application elicited pronounced mechanical allodynia in intact mice, which was concurrent with a specific increase in the abundance of NMDAR subunits NR1 and NR2B at the postsynaptic density (PSD)-enriched fraction. Selective inhibition of NR2B-containing NMDARs (NR2BR) by ifenprodil dose dependently attenuated the mechanical allodynia in NMDA-injected mice, suggesting the importance of NR2BR synaptic accumulation in NMDA-induced pain sensitization. The NR2BR redistribution at synapses after NMDA challenge was associated with a significant increase in NR2B phosphorylation at Tyr1472, a catalytic site by Src family protein tyrosine kinases (SFKs) that has been shown to prevent NR2B endocytosis. Intrathecal injection of a specific SFKs inhibitor, PP2, to block NR2B tyrosine phosphorylation eliminated NMDA-induced NR2BR synaptic expression and also attenuated the mechanical allodynia. These data suggested that activation of spinal NMDARs was able to accumulate NR2BR at synapses via SFK signaling, which might exaggerate NMDAR-dependent nociceptive transmission and contribute to NMDA-induced nociceptive behavioral hyperresponsiveness.     

Evaluation of behavioural effects of a selective NMDA NR1A/2B receptor antagonist in the unilateral 6-OHDA lesion rat model

The degeneration of the dopaminergic nigrostriatal pathway in Parkinson's disease (PD) is associated with altered transmission at striatal NMDA receptors containing NR2B subunits. We investigated a potential novel therapeutic compound, 4-trifluoromethoxy-N-(2-trifluoromethyl-benzyl)-benzamidine (BZAD-01), a selective NMDA NR1A/2B receptor antagonist for PD and compared it with levodopa, the standard treatment for PD. This study also evaluated whether combining levodopa and BZAD-01 gave better improvements of parkinsonian symptoms. Parkinsonism was induced by microinjection of the toxin, 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB) of 40 Sprague-Dawley rats. Parkinsonism and the efficacy of drugs were assessed using a battery of behavioural tests including balance beam, apomorphine-induced rotation, body axis bias or "curling", head position bias and disengage sensorimotor latency test. Immunohistochemistry was performed on post-mortem tissue to estimate the loss of dopaminergic neurons. The main effects were that BZAD-01 co-administration prevented chronic levodopa-induced potentiation of apomorphine rotation. However levodopa-treated rats were slower than either controls or BZAD-01-treated rats in the locomotor test. The improvement in the apomorphine rotation test suggests that BZAD-01 may be a useful adjunct to levodopa monotherapy.     

Altered expression and phosphorylation of N-methyl-D-aspartate receptors in piglet striatum after hypoxia-ischemia

The mechanisms for the profound degeneration of striatal neurons after hypoxia-ischemia in newborns are not understood. We hypothesized that this striatal neurodegeneration is related to N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity. Using a 1-week-old piglet model of hypoxia-ischemia, we evaluated whether the expression and phosphorylation of NMDA receptor subunits in striatum are modified with severity of evolving neuronal injury after hypoxia-ischemia. Protein levels of NR1, phosphorylated NR1 897serine, NR2A and NR2B in striatum were measured by immunoblotting after piglets underwent hypoxic-asphyxic cardiac arrest, cardiopulmonary resuscitation, and recovery for 3, 6, 12 or 24 h. In membrane fractions isolated from total striatum, mean NR1 and NR2A levels did not change significantly with time after hypoxia-ischemia compared to control; however, the levels of both NR1 and phosphorylated NR1 897serine correlated with neuronal injury in putamen, with higher levels associated with greater neuronal injury in individual animals. NR2B levels were increased at 24 h after hypoxia-ischemia. Astrocyte expression of NR2B was prominent after hypoxia-ischemia. We conclude that NMDA receptors are changed in striatum after neonatal hypoxia-ischemia and that abnormal NMDA receptor potentiation through increased NR1 phosphorylation may participate in the mechanisms of striatal neuron degeneration after hypoxia-ischemia.     

Estradiol potentiation of NR2B‐dependent EPSCs is not due to changes in NR2B protein expression or phosphorylation

The hormone, 17β‐estradiol (E2), influences the structure and function of synapses in the CA1 region of the hippocampus. E2 increases the density of dendritic spines and excitatory synapses on CA1 pyramidal cells, increases CA1 cells' sensitivity to excitatory synaptic input mediated by the NMDA receptor (NMDAR), enhances NMDAR‐dependent long‐term potentiation, and improves hippocampus‐dependent working memory. Smith and McMahon ( 2006 J Neurosci 26:8517–8522) reported that the larger NMDAR‐mediated excitatory postsynaptic currents (EPSCs) recorded after E2 treatment are due primarily to an increased contribution of NR2B‐containing NMDARs. We used a combination of electrophysiology, Western blot, and immunofluorescence to investigate two potential mechanisms by which E2 could enhance NR2B‐dependent EPSCs: An increase in NMDAR subunit protein levels and/or a change(s) in NR2B phosphorylation. Our studies confirmed the E2‐induced increase in NR2B‐dependent EPSC amplitude, but we found no evidence that E2 affects protein levels for the NR1, NR2A, or NR2B subunit of the NMDAR, nor that E2 affects phosphorylation of NR2B. Our findings suggest that the effects of E2 on NMDAR‐dependent synaptic physiology in the hippocampus likely result from recruitment of NR2B‐containing NMDARs to synapses rather than from increased expression of NMDARs or changes in their phosphorylation state. © 2010 Wiley‐Liss, Inc.