NR2B subunit blockade does not affect motor symptoms induced by 3-nitropropionic acid

Broad-spectrum N-methyl D-aspartate (NMDA) antagonists, although proposed in therapies for several pathologies including Huntington's disease (HD), can produce dramatic side-effects. Thus, the therapeutic potential of subunit selective NMDA receptor antagonists warrants investigation. Overactivation of NMDA receptors containing the NR2B subunit plays a pathogenic role in HD, suggesting a neuroprotective potential of selective NR2B blockade. In the present study, we investigated whether the selective NR2B receptor antagonist, R-(R*,S*)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidinepropanol, could also affect motor symptoms in mice intoxicated with 3-nitropropionic acid (3-NP), a phenotypic model of HD. NR2B subunit acute blockade had no effect on spontaneous activity, HD-like symptoms (clinical scale), and sensorimotor performances (beam task) in 3-NP intoxicated mice. These results suggest that selective NR2B antagonism has no acute symptomatic effect on motor and sensorimotor impairments due to 3-NP-induced striatal injury.     

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.     

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

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

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

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

Functional roles of NMDA receptor NR2A and NR2B subunits in the acute intoxicating effects of ethanol in mice

The present study examined the roles of NR2A and NR2B subunit-containing NMDA receptors in the mediation of the sedative/hypnotic effects of ethanol in mice. The ability of the competitive NMDA antagonist, CGP-37849 (0, 1, or 3 mg/kg), and the NR2B-selective antagonist, Ro 25-6981 (0, 3, or 10 mg/kg), to alter (3 g/kg) ethanol-induced sleep time was measured in C57BL/6J mice and NR2A knockout (KO) mice. The results show that pretreatment with either antagonist significantly potentiated the sedative/hypnotic effects of ethanol in C57BL/6J mice. These effects were not significantly altered in NR2A KO mice. Basal sleep time responses to ethanol were also normal in NR2A KO mice. These findings confirm a major role for NMDA receptors in the acute intoxicating actions of ethanol and provide tentative support for a prepotent role of the NR2B subunit in these effects.     

Discovery of 3-Substituted Aminocyclopentanes as Potent and Orally Bioavailable NR2B Subtype-Selective NMDA Antagonists

A series of 3-substituted aminocyclopentanes has been identified as highly potent and selective NR2B receptor antagonists. Incorporation of a 1,2,4-oxadiazole linker and substitution of the pendant phenyl ring led to the discovery of orally bioavailable analogues that showed efficient NR2B receptor occupancy in rats. Unlike nonselective NMDA antagonists, the NR2B-selective antagonist 22 showed no adverse affects on motor coordination in the rotarod assay at high dose. Compound 22 was efficacious following oral administration in a spinal nerve ligation model of neuropathic pain and in an acute model of Parkinson's disease in a dose dependent manner.     

Preferential inhibition by antidiarrheic 2‐methoxy‐4‐methylphenol of Ca2+ influx across acquired N‐methyl‐D‐aspartate receptor channels composed of NR1/NR2B subunit assembly

In our previous studies, particular phenolic ingredients, such as 2‐methoxy‐4‐methylphenol (2M4MP), of the antidiarrheic drug wood creosote significantly prevented cell death by both hydrogen peroxide and glutamate in cultured rat hippocampal neurons. In this study, we further evaluated the pharmacological properties of 2M4MP on Ca²⁺ influx across native and acquired N‐methyl‐D‐aspartate (NMDA) receptor (NMDAR) channels. The addition of 2M4MP significantly prevented the loss of cellular viability and the increase in intracellular free Ca²⁺ levels as determined by Fluo‐3 in cultured rat hippocampal neurons briefly exposed to NMDA. Brief exposure to NMDA also led to a marked increase in mitochondrial free Ca²⁺ levels determined by Rhod‐2, in addition to intracellular free Ca²⁺ levels, in HEK293 cells expressing either NR1/NR2A or NR1/NR2B subunit channels. The further addition of the general NMDAR channel blocker dizocilpine similarly inhibited the increase of intracellular Ca²⁺ levels by NMDA in both types of acquired NMDAR channels, whereas the NR2B subunit selective antagonist ifenprodil drastically inhibited the increase by NMDA in HEK293 cells expressing NR1/NR2B, but not NR1/NR2A, subunits. Similarly, 2M4MP significantly and selectively inhibited the NMDA‐induced influx of Ca²⁺ across acquired NR1/NR2B channels in a concentration‐dependent manner. Moreover, prior daily oral administration of 2M4MP significantly reduced the infarct volume in the ipsilateral cerebral hemisphere in rats with middle cerebral artery occlusion 1 day after reperfusion. These results suggest that 2M4MP may protect neurons from excitotoxicity through preferential inhibition of Ca²⁺ influx across NMDAR channels composed of NR1/NR2B subunits. © 2010 Wiley‐Liss, Inc.     

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

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

NR2B-containing NMDA receptors promote the neurotoxic effects of 3-nitropropionic acid but not of rotenone in the striatum

Decreased activity of mitochondrial complex I and II is implicated in the pathophysiology of progressive supranuclear palsy (PSP) and Huntington's disease (HD), respectively. Both disorders preferentially affect the nucleus striatum, a brain area particularly vulnerable to excitotoxic damage. To gain insights into the pathophysiology of neuronal degeneration during PSP and HD, here we studied the possible interplay between excitatory transmission and mitochondrial complex I and II inhibition in the development of striatal damage. By using in vitro neurophysiological recordings and cell swelling measures in corticostriatal slices, we found that stimulation of NMDA receptors significantly contributed to the neurotoxic effects of 3-nitropropionic acid (3-NP) but not of rotenone, selective inhibitors of mitochondrial complex II and I, respectively. We also found that blockade of a subset of NMDA receptors containing the NR2B subunit was sufficient to protect the striatum from the injurious effects of 3-NP, an effect unrelated to the prevention of membrane excitation by NMDA receptor stimulation. Pharmacological inhibition of dopamine receptors, conversely, failed to modulate both rotenone- and 3-NP-induced neuronal damage. Our results indicate that the cellular mechanisms leading to striatal neuronal death are different following inhibition of distinct mitochondrial complexes of the respiratory chain, implying that neuroprotective strategies in PSP and HD must significantly differ.     

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

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

A genetically modified mouse model probing the selective action of ifenprodil at the N-methyl-D-aspartate type 2B receptor

Selective antagonism of N-methyl-d-aspartate (NMDA) 2B subunit containing receptors has been suggested to have potential therapeutic application for multiple CNS disorders. The amino terminal NR2B residues 1 to 282 were found to be both necessary and sufficient for the binding and function of highly NR2B subunit specific antagonists like ifenprodil and CP-101,606. Using a genetic approach in mice, we successfully replaced the murine NR2B gene function by "knocking-in" (KI) a chimeric human NR2A/B cDNA containing the minimal domain abolishing ifenprodil binding into the endogenous NR2B locus. Patch-clamp recording from hippocampal cultures of the NR2B KI mice demonstrated that their NMDA receptors have reduced sensitivity to both ifenprodil and CP-101,606, as predicted, but also have a lower affinity for glycine. The NR2B KI mice exhibited normal locomotor activity making this ifenprodil-insensitive mouse model a valuable tool to test the specificity of NR2B selective antagonists in vivo.     

Lack of efficacy of NMDA receptor-NR2B selective antagonists in the R6/2 model of Huntington disease

N-methyl-d-aspartate receptor (NMDAR) mediated excitotoxicity is a probable proximate mechanism of neurodegeneration in Huntington disease (HD). Striatal neurons express the NR2B-NMDAR subunit at high levels, and this subunit is thought to be instrumental in causing excitotoxic striatal neuron injury. We evaluated the efficacy of 3 NR2B-selective antagonists in the R6/2 transgenic fragment model of HD. We evaluated ifenprodil (10 mg/kg; 100 mg/kg), RO25,6981 (10 mg/kg), and CP101,606 (30 mg/kg). Doses were chosen on the basis of pilot acute maximally tolerated dose studies. Mice were treated with subcutaneous injections twice daily. Outcomes included survival; motor performance declines assessed with the rotarod, balance beam task, and activity measurements; and post-mortem striatal volumes. No outcome measure demonstrated any benefit of treatments. Lack of efficacy of NR2B antagonists in the R6/2 model has several possible explanations including blockade of beneficial NMDAR mediated effects, inadequacy of the R6/2 model, and the existence of multiple proximate mechanisms of neurodegeneration in HD.     

Effects of a NR2B selective NMDA glutamate antagonist,CP‐101,606, on dyskinesia and parkinsonism

Glutamate antagonists decrease dyskinesia and augment the antiparkinsonian effects of levodopa in animal models of Parkinson's disease (PD). In a randomized, double‐blind, placebo‐controlled clinical trial, we investigated the acute effects of placebo and two doses of a NR2B subunit selective NMDA glutamate antagonist, CP‐101,606, on the response to 2‐hour levodopa infusions in 12 PD subjects with motor fluctuations and dyskinesia. Both doses of CP‐101,606 reduced the maximum severity of levodopa‐induced dyskinesia ～30% but neither dose improved Parkinsonism. CP‐101,606 was associated with a dose‐related dissociation and amnesia. These results support the hypothesis that glutamate antagonists may be useful antidyskinetic agents. However, future studies will have to determine if the benefits of dyskinesia suppression can be achieved without adverse cognitive effects. © 2008 Movement Disorder Society     

Alterations in regional brain metabolism in genetic and pharmacological models of reduced NMDA receptor function

A mouse line has been developed that expresses low levels of the NMDA R1 (NR1) subunit of the NMDA receptor [Cell 98 (1999) 427]. These NR1 hypomorphic mice represent an experimental model of reduced NMDA receptor function that may be relevant to the pathophysiology of schizophrenia. To further characterize the neurobiological phenotype resulting from developmental NMDA receptor hypofunction, regional brain metabolic activity was assessed by autoradiographic analysis of 14C-2-deoxyglucose (2-DG) uptake. In addition, ligand binding to NMDA, AMPA, and kainate receptors was measured by quantitative autoradiography. MK-801 binding to NMDA receptors was reduced markedly throughout the brain of the NR1 hypomorphic mice. However, no alteration in 3H-AMPA or 3H-kainate binding was apparent in any region examined. Neuroanatomically specific alterations in regional 2-DG uptake were observed in the NR1 hypomorphic animals. Reduced relative 2-DG uptake was observed in the medial prefrontal and anterior cingulate cortices. Altered patterns of 2-DG uptake were also found in neocortical regions, with selective reductions of uptake in layer 6 in frontal regions of somatosensory and motor cortices. These data indicate alterations in cortical circuitry in the NR1 hypomorphic animals and are consistent with functional imaging studies in chronic schizophrenia patients which typically show reduced frontal cortical metabolic activity. Reduced relative 2-DG uptake was also found in the caudate, accumbens, hippocampus, and select thalamic regions in the NR1-deficient mice. However, in many other brain regions no alteration in 2-DG uptake was observed. The alterations in 2-DG uptake in the NR1 hypomorphic mice were distinctly different compared to those observed after acute challenge with the selective NMDA antagonist MK-801 in wild-type mice. The altered patterns of brain 2-DG uptake in the NR1 hypomorphic mice found in the present work, together with the altered behavioral phenotypes previously described, suggest that the mice may provide a valuable model to study novel therapeutic strategies to counteract the neurobiological consequences of chronic developmental NMDA receptor hypofunction.     

Forebrain NR2B overexpression enhancing fear acquisition and long‐term potentiation in the lateral amygdala

N‐methyl‐d ‐aspartic acid (NMDA) receptor‐dependent long‐term potentiation (LTP) at the thalamus–lateral amygdala (T‐LA) synapses is the basis for acquisition of auditory fear memory. However, the role of the NMDA receptor NR2B subunit in synaptic plasticity at T‐LA synapses remains speculative. In the present study, using transgenic mice with forebrain‐specific overexpression of the NR2B subunit, we have observed that forebrain NR2B overexpression results in enhanced LTP but does not alter long‐term depression (LTD) at the T‐LA synapses in transgenic mice. To elucidate the cellular mechanisms underlying enhanced LTP at T‐LA synapses in these transgenic mice, AMPA and NMDA receptor‐mediated postsynaptic currents have been measured. The data show a marked increasing in the amplitude and decay time of NMDA receptor‐mediated currents in these transgenic mice. Consistent with enhanced LTP at T‐LA synapses, NR2B‐transgenic mice exhibit better performance in the acquisition of auditory fear memory than wild‐type littermates. Our results demonstrate that up‐regulation of NR2B expression facilitates acquisition of auditory cued fear memory and enhances LTP at T‐LA synapses.     

NR2 to NR3B subunit switchover of NMDA receptors in early postnatal motoneurons

The NR3B NMDA receptor subunit is selective to somatic motoneurons in the adult nervous system. Here we report its developmental expression in the mouse brain and spinal cord by in situ hybridization. NR3B mRNA was detected in few neural regions during embryonic and neonatal periods. It first appeared in motoneurons at postnatal day (P)10-P14, and attained the maximal level at P21 and adult stage. This developmental profile was reciprocal with that of NR2 subunits, of which NR2A mRNA was most predominant in embryonic and neonatal motoneurons and downregulated by P14. Interestingly, mRNA of the NR1 subunit, which is required for functional NMDA receptors, displayed a 'V'-shaped change, decreasing with the early postnatal decline of NR2 mRNAs and increasing with the subsequent appearance of NR3B mRNA. Therefore, the major regulatory subunit of NMDA receptors is likely to switch from NR2 to NR3B in somatic motoneurons during the early postnatal period.     

Role of neuronal NR2B subunit-containing NMDA receptor-mediated Ca2+ influx and astrocytic activation in cultured mouse cortical neurons and astrocytes

The excitatory neurotransmitter glutamate has been shown to mediate such bidirectional communication between neurons and astrocytes. In the present study, we determined the role of N-methyl-D-aspartate (NMDA) receptors on glutamate-evoked Ca(2+) influx into neurons and astrocytes. Either a nonselective NMDA receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) or selective NR2B subunit-containing NMDA receptor antagonists ifenprodil and (R,S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperid inepropanol (Ro25-6981) significantly inhibited the glutamate-evoked Ca(2+) influx into neurons, but not into astrocytes. Furthermore, we investigated whether NR2B subunit-containing NMDA receptor antagonists could suppress the astrocytic activation, as detected by glial fibrillary acidic protein (GFAP; as a specific marker of astrocyte)-like immunoreactivities in mouse cortical astrocytes. Here, we demonstrated that the increases in the level of GFAP-like immunoreactivities induced by glutamate were markedly suppressed by cotreatment with ifenprodil in cortical neuron/glia cocultures, but not in purified astrocytes. These results suggest that NR2B subunit-containing NMDA receptor plays a critical role in not only glutamate-evoked Ca(2+) influx into neurons, but also glutamate-induced astrocytic activation. Thus, glutamate-mediated pathway via NR2B subunit-containing NMDA receptor may, at least in part, contribute to neuron-to-astrocyte signaling.     

戊四氮点燃癫癎大鼠空间学习记忆改变及海马NR2B表达

目的观察戊四氮点燃癫癎大鼠空间学习记忆功能变化及海马NMDA2型受体(NR2)B亚单位(NR2B)表达,探讨二者的关系及PTZ致癎大鼠认知障碍发生的分子机制。方法采用戊四氮(PTZ)慢性癫癎(CE)模型,Y-迷宫对两组大鼠进行行为学检测,免疫组织化学方法观察两组大鼠海马CA3区NR2B表达的变化,反转录多聚酶链反应(RT-PCR)方法检测大鼠海马NR2B mRNA的表达。结果癫癎组大鼠空间学习记忆能力受损;其海马CA3区NR2B阳性细胞较对照组明显减少(P<0.01),同时伴有海马NR2B mRNA表达下降(P<0.01)。结论戊四氮点燃癫癎大鼠空间学习记忆受损可能与海马神经元NR2B的表达减少有关。     

Involvement of NMDA receptors in ryanodine receptor expression in dopaminergic neurons in the ventral tegmental area of mice with intermittent methamphetamine treatment

Excitatory synapses on dopaminergic neurons of the ventral tegmental area (VTA) represent an important role in psychostimulant-induced rewarding effect. This study investigated the regulation of ryanodine receptor (RyR) and N-methyl-D-aspartate (NMDA) receptor expression in mice under intermittent methamphetamine (METH) treatment using a place preference procedure. RyR-1 and -2 significantly increased in the VTA of mice with METH-induced place preference, whereas RyR-3 showed no changes. In addition, the levels of NR1, NR2A, and NR2B subunits were increased in the VTA. The METH-induced place preference was inhibited by intracerebroventricular pretreatment with MK-801, a noncompetitive NMDA receptor antagonist, and ifenprodil, a selective NR2B subunit-containing NMDA receptor antagonist, in a dose-dependent manner. Under these conditions, the increase of RyR-1 and -2 in the VTA was significantly blocked by ifenprodil. The immunohistochemical analysis revealed the colocalization of RyR-1 and -2 with NR2B subunits in dopaminergic neurons in the mouse VTA. These findings suggest that RyRs could be involved in the development of METH-induced place preference and that NR2B subunit-containing NMDA receptors in mice showing METH-induced place preference play an important role in expression of RyRs.     

Possible protection by notoginsenoside R1 against glutamate neurotoxicity mediated by N‐methyl‐D‐aspartate receptors composed of an NR1/NR2B subunit assembly

Notoginsenoside R1 (NTR1) is the main active ingredient in Panax notoginseng, a herbal medicine widely used in Asia for years. The purpose of this study was to investigate pharmacological properties of NTR1 on neurotoxicity of glutamate (Glu) in primary cultured mouse cortical neurons along with its possible mechanism of action. Wefound that NTR1 significantly protected neurons from the loss of cellular viability caused by brief exposure to 10 μM Glu for 1 hr in a dose‐dependent manner at concentrations from 0.1 to 10 μM, without affecting the viability alone. NTR1 significantly inhibited the increased number of cells positive to propidium iodide (PI) staining, increase of intracellular free Ca²⁺ ions, overproduction of intracellular reactive oxygen species, and depolarization of mitochondrial membrane potential in cultured neurons exposed to Glu, in addition to blocking decreased Bcl‐2 and increased Bax expression levels. We further evaluated the target site at which NTR1 protects neurons from Glu toxicity by using the acquired expression strategy of N‐methyl‐D ‐aspartate (NMDA) receptor subunits in human embryonic kidney 293 cells. We found that 10 μM NTR1 protected NR1/NR2B subunit expressing cells from cell death by 100 μM NMDA, but not cells expressing NR1/NR2A subunits, when determined by PI staining. These results suggest that NTR1 may preferentially protect neurons from Glu excitotoxicity mediated by NMDA receptor composed of an NR1/NR2B subunit assembly in the brain. © 2009 Wiley‐Liss, Inc.