N-methyl-D-aspartate Receptor Subunit 2B Research Articles

Ethanol alters N-methyl-D-aspartate receptor regulation in the hippocampus of adolescent rats.

Our laboratory and others have shown that ethanol disrupts hippocampus-associated memory formation in adolescent rats, but the signaling pathway involved remains largely unknown. Phosphorylation of the N-methyl-D-aspartate receptor, particularly the N-methyl-D-aspartate receptor subunit 2B (NR2B), has been implicated in several ethanol effects and in memory formation. The present study investigated the effects of repeated ethanol exposure on the phosphorylation of the NR2B subunit and src protein in the hippocampus of adolescent rats. Rats were treated daily with ethanol or vehicle for 5 consecutive days and sacrificed at several time points after the last treatment. At 30 min postethanol treatment, levels of phosphorylated NR2B and src were significantly elevated; this was the same postethanol time point when adolescent rats showed impairments in their performance of memory tasks. Peak increases in both phosphorylated levels of NR2B and src were seen around 1 h post-treatment, after which levels of both phosphoproteins started to decline. Together, these data suggest that the time-dependent increase in hippocampal phosphorylated NR2B and src levels following repeated ethanol exposure may be responsible for hippocampus-associated memory impairments in adolescent rats.

The Src family kinase inhibitor dasatinib delays pain-related behaviour and conserves bone in a rat model of cancer-induced bone pain

Pain is a severe and debilitating complication of metastatic bone cancer. Current analgesics do not provide sufficient pain relief for all patients, creating a great need for new treatment options. The Src kinase, a non-receptor protein tyrosine kinase, is implicated in processes involved in cancer-induced bone pain, including cancer growth, osteoclastic bone degradation and nociceptive signalling. Here we investigate the role of dasatinib, an oral Src kinase family and Bcr-Abl tyrosine kinase inhibitor, in an animal model of cancer-induced bone pain. Daily administration of dasatinib (15 mg/kg, p.o.) from day 7 after inoculation of MRMT-1 mammary carcinoma cells significantly attenuated movement-evoked and non-evoked pain behaviour in cancer-bearing rats. Radiographic - and microcomputed tomographic analyses showed significantly higher relative bone density and considerably preserved bone micro-architecture in the dasatinib treated groups, suggesting a bone-preserving effect. This was supported by a significant reduction of serum TRACP 5b levels in cancer-bearing rats treated with 15 mg/kg dasatinib. Furthermore, immunoblotting of lumbar spinal segments showed an increased activation of Src but not the NMDA receptor subunit 2B. These findings support a role of dasatinib as a disease modifying drug in pain pathologies characterized by increased osteoclast activity, such as bone metastases.

Arginine vasopressin ameliorates spatial learning impairments in chronic cerebral hypoperfusion via V1a receptor and autophagy signaling partially

Chronic cerebral hypoperfusion (CCH) is a major factor contributing to neurological disorders and cognitive decline. Autophagy activation is believed to provide both beneficial and detrimental roles during hypoxic/ischemic cellular injury. Although arginine vasopressin (AVP) has been strongly involved in many behaviors, especially in learning and memory, the effects of AVP on CCH and their molecular mechanisms remain unclear. Here, to investigate whether there was neuroprotective effects of AVP on CCH through V1a receptor (an AVP receptor) signaling, permanent bilateral carotid arteries occlusion (two vessel occlusion, 2VO) was used to establish a rat model of CCH, and hypertonic saline (5.3%) was injected intraperitoneally to induce the secretion of AVP. Results showed that hypertonic saline effectively alleviated spatial learning and memory deficit, enhanced synaptic plasticity of CA3-CA1 hippocampal synapses, upregulated N-methyl-d-aspartate receptor subunit 2B (NR2B) and postsynaptic density protein 95 (PSD-95) surface expressions, reduced oxidative stress and increased Nissl bodies in 2VO model rats. These phenomena were significantly decreased by V1a receptor antagonist SR49059. Interestingly, hypertonic saline also upregulated autophagy in the hippocampus of 2VO rats partly through V1a receptor. These findings imply that AVP has a beneficial role for the treatment of cognitive impairments partly through V1a receptor signaling in CCH, which is possibly related to improving synaptic plasticity by promoting NR2B and PSD-95 externalization and by enhancing autophagy.

NMDA receptor 2B Subunit and BK Channel May Affect Mechanical Allodynia by Regulating the Activity of Enkephalinergic Neurons in the Spinal Dorsal Horn

Enkephalin (ENK)‐containing neurons in the spinal superficial dorsal horn (SDH) play an important role in regulating the transmission of nociceptive information. Using BAC transgenic mice, in which ENK‐containing neurons can be identified by green fluorescent protein expressed under the control of preproenkephalin gene promoter, we have observed that ENK‐containing neurons in the SDH express the NMDA receptor 2B (NR2B) subunit and large conductance calcium‐activated potassium (BK) channels. In experiments, we investigated the effects on nociceptive behavior of intrathecally administrating NR2B subunit antagonist and BK channel antagonist in peripheral nerve‐injured mice.The experiments were performed in 6‐ to 8‐week‐old male ICR mice. Partial sciatic nerve ligation was performed as described previously (PSL, Seltzer model). To assess the development of mechanical allodynia, we measured the frequency of withdrawal responses to 10 repetitive stimuli with von Frey filaments. Behavioral assessments were performed beginning 3 days prior to the ligation surgery. On post‐surgical day 7, effects on nociceptive behavior of the BK channel antagonist, charybdotoxin (CTX), and the NR2B antagonist, ifenprodil (IFN), were analyzed. Additionally, in order to clarify whether the actions of CTX and IFN are mediated by an alteration in the release of enkephalin, we investigated the effect of the selective δ‐opioid receptor antagonist naltrindole (NTI).CTX (1 pmol/10 μL per mouse), IFN (50 nmol/10 μL per mouse) and saline as a control (10 μL per mouse) was intrathecally injected according to the modified method originally described by Hylden. All behavioral experiments were performed in double‐blind fashion.PSL significantly increased the occurrence of withdrawal reflex to von Frey stimuli being indicative of the development of mechanical allodynia. Intrathecal CTX significantly reduced the occurrence of withdrawal reflex as compared to saline group. Additionally, intraperitoneal administration of NTI at the dose of 5 mg/kg body weight attenuated the effect on nociceptive behavior of CTX. Intrathecal IFN significantly reduced the occurrence of withdrawal reflex. This effect was also blocked by intraperitoneal NTI.These behavioral observations might suggest that NMDA receptors and BK channels inhibit the activity of enkephalin‐containing neurons in the spinal dorsal horn, which is speculated to be involved in peripheral nerve injury‐induced chronic pain state.Support or Funding InformationThis work was supported by Dokkyo Medical University Endowment Fund.

Inhibition of coactivator-associated arginine methyltransferase 1 modulates dendritic arborization and spine maturation of cultured hippocampal neurons

An improved understanding of the molecular mechanisms in synapse formation provides insight into both learning and memory and the etiology of neurodegenerative disorders. Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein methyltransferase that negatively regulates synaptic gene expression and inhibits neuronal differentiation. Despite its regulatory function in neurons, little is known about the CARM1 cellular location and its role in dendritic maturation and synapse formation. Here, we examined the effects of CARM1 inhibition on dendritic spine and synapse morphology in the rat hippocampus. CARM1 was localized in hippocampal post-synapses, with immunocytochemistry and electron microscopy revealing co-localization of CARM1 with post-synaptic density (PSD)-95 protein, a post-synaptic marker. Specific siRNA-mediated suppression of CARM1 expression resulted in precocious dendritic maturation, with increased spine width and density at sites along dendrites and induction of mushroom-type spines. These changes were accompanied by a striking increase in the cluster size and number of key synaptic proteins, including N-methyl-d-aspartate receptor subunit 2B (NR2B) and PSD-95. Similarly, pharmacological inhibition of CARM1 activity with the CARM1-specific inhibitor AMI-1 significantly increased spine width and mushroom-type spines and also increased the cluster size and number of NR2B and cluster size of PSD-95. These results suggest that CARM1 is a post-synaptic protein that plays roles in dendritic maturation and synaptic formation and that spatiotemporal regulation of CARM1 activity modulates neuronal connectivity and improves synaptic dysfunction.

Activation of N‐methyl‐D‐aspartate Receptor Inhibits Enkephalinergic Neurons in Mouse Spinal Superficial Dorsal Horn

Enkephalin (Enk)‐containing neurons are distributed at high concentration in the spinal cord superficial dorsal horn (SDH), where they play an important role in the transmission and modulation of nociceptive information. Recently, we performed physiological characterization of Enk‐containing neurons identified by green fluorescent protein fluorescence expressed under the control of the preproenkephalin gene promoter. We observed that Enk‐containing neurons in the SDH express the N‐methyl‐D‐aspartate receptor (NMDAR) 2B (NR2B) subunit and large conductance calcium activated potassium (BK) channels. In the present experiments, we investigated the involvement of NMDAR and BK channels in the regulation of the activity of Enk‐containing neurons in the SDH.The experiments were performed in 5‐ to 8‐week‐old male ICR mice. Under ketamine/xylazine anesthesia, the lumbar spinal cord was dissected, and transverse slices were prepared. Tight‐seal whole‐cell recordings were obtained from neurons in the SDH. L‐glutamate (Glu) was applied by pressure ejection through a glass pipette placed near the recorded neurons. After the electrophysiological experiments, the recorded neurons were sampled, and single cell real‐time RT‐PCR was performed to analyze the expression profile of genes of interest in single SDH neurons. In some experiments, the left sciatic nerve was partially ligated, and mechanical allodynia was assessed by von Frey test.Puff‐applied Glu evoked an inward current at a holding potential of −70 mV. Depolarizing the holding potential to 0 mV, outward current of long duration appeared subsequent to initial inward current. The NMDAR antagonist AP‐V abolished the outward current. The NR2B‐selective NMDAR antagonist ifenprodil reduced the outward current. The outward current was also abolished by the selective BK channels antagonist iberiotoxin. Single cell real‐time RT‐PCR analysis revealed that the neurons generating the outward current showed a significant tendency to express BK channels α‐subunit mRNA, which is consistent with the pharmacological results of a BK channels antagonist. Furthermore, single SDH neurons expressing the preproenkephalin mRNA also expressed the BK channel α‐subunit, NR1, and NR2B subunit mRNAs. The expression of NR2B increased significantly in sciatic nerve‐ligated mice.Our results suggest that Ca2+ influx through NMDAR may activate BK channels and thus inhibit Enk‐containing neurons in the SDH. NMDAR‐mediated inhibition of Enk‐containing neurons is speculated to be one of the possible cellular mechanisms of chronic pain induced by peripheral nerve injury.

Effects of Tumor Necrosis Factor Blocker on Salicylate-Induced Tinnitus in Mice.

Neuroinflammation is considered a novel mechanism for acute tinnitus. Here, we investigated the effects of a tumor necrosis factor (TNF) blocker on the gene expression of inflammatory-cytokine in the cochlea in a tinnitus animal model. Enbrel® (30 mg/kg, intraperitoneally (i.p.)) were administrated to the mice with the salicylate induced tinnitus for 3 days. Tinnitus score and mRNA expression levels of TNFR1, TNFR2, and N-methyl-d-aspartate receptor subunit 2B (NR2B) and its downstream regulatory element antagonist modulator (DREAM) in the cochlea of mice were measured and compared to the control. The tinnitus score significantly decreased in the Enbrel® treated group. The mRNA levels of both TNFR1 and TNFR2 were significantly lower in the treatment than in the control group. The mRNA levels of NR2B and DREAM followed a similar trend. we found that treatment with 30 mg/ kg Enbrel® decreased salicylate-induced behavior associated with tinnitus and reduced the mRNA expression levels of TNFR1/R2, NR2B, and DREAM in the cochlea of mice. These findings supported the hypothesis that neuroinflammation might be a novel mechanism for salicylate-induced tinnitus.

Low-anxiety rats are more sensitive to amphetamine in comparison to high-anxiety rats.

This study utilised the two injection protocol of sensitisation (TIPS) and the conditioned place preference test to validate and extend previous findings on the effects of amphetamine on positive reinforcement-related 50 kHz ultrasonic vocalisation (USV) in rats. We also examined changes in the expression of c-Fos and the NMDA receptor 2B (GluN2B) subunit, markers of neuronal activity and plasticity, in brain regions of rats in response to TIPS. We used low anxiety-responsive (LR) and high anxiety-responsive (HR) rats, which are known to exhibit different fear-conditioned response strengths, different susceptibilities to amphetamine in the TIPS procedure and different amphetamine-dependent 50 kHz USV responses. The LR rats, compared to the HR rats, not only vocalised much more intensely but also spent significantly more time in the amphetamine-paired compartment. After the second dose of amphetamine, the LR rats exhibited more c-Fos and GluN2B activation in layers II and III of the M1/M2 motor cortex area and prefrontal cortex (PRE, PRL, IL) and also presented with more GluN2B activation in the basal amygdala. These data reveal that HR and LR rats exhibit different levels of reactivity in the cortical-limbic pathway, which controls reward-related motivational processes. These findings contribute to the general hypothesis that heterogeneity in emotional processes is one of the causes of sensitisation to amphetamine and drug addiction.

Dexmedetomidine protects against learning and memory impairments caused by electroconvulsive shock in depressed rats: Involvement of the NMDA receptor subunit 2B (NR2B)-ERK signaling pathway

Cognitive impairment is a common adverse effect of electroconvulsive therapy (ECT) during treatment for severe depression. Dexmedetomidine (DEX), a sedative-anesthetic drug, is used to treat post-ECT agitation. However, it is not known if DEX can protect against ECT-induced cognitive impairments. To address this, we used chronic unpredictable mild stress (CUMS) to establish a model of depression for ECT treatment. Our Morris water maze and sucrose preference test results suggest that DEX alleviates ECT-induced learning and memory impairments without altering the antidepressant efficacy of ECT. To further investigate the underlying mechanisms of DEX, hippocampal expression of NR2B, p-ERK/ERK, p-CREB/CREB, and BDNF were quantified by western blotting. These results show that DEX suppresses over-activation of NR2B and enhances phosphorylation of ERK1/2 in the hippocampus of ECT-treated depressed rats. Furthermore, DEX had no significant effect on ECT-induced increases in p-CREB and BDNF. Overall, our findings suggest that DEX ameliorates ECT-induced learning and memory impairments in depressed rats via the NR2B-ERK signaling cascade. Moreover, CREB/BDNF seems not appear to participate in the cognitive protective mechanisms of DEX during ECT treatment.

Ginsenoside Rd Protects Against Cerebral Ischemia-Reperfusion Injury Via Decreasing the Expression of the NMDA Receptor 2B Subunit and its Phosphorylated Product.

Ginsenoside Rd (GSRd) is one of the active ingredients in ginseng. Recent studies have shown that GSRd can protect against cerebral ischemia through several pathways, one of which is mediated by the N-methyl-D-aspartate receptor (NMDAR). In this study, we aimed to investigate the effects of GSRd on the phosphorylation of the NMDAR 2B subunit (NR2B subunit) in cerebral ischemia. Ischemia-reperfusion injury (IRI) models induced by transient middle cerebral artery occlusion (MCAO) and oxygen glucose deprivation (OGD) were used to mimic in vivo or in vitro injury during cerebral ischemia. The models were pretreated or post-treated with GSRd after MCAO or OGD. As a vehicle control, 1,3-propanediol was used. The expression levels of the NR2B subunit and the phosphorylated NR2B subunit were determined using western blotting. GSRd significantly improved the behavior score, infarct volume, and viability of the cultured neurons after ischemia. GSRd inhibited the hyperphosphorylation of NR2B subunit and decreased the expression levels of NR2B subunit in cell membrane but did not change their levels in the total proteins after IRI. GSRd protected Sprague-Dawley rats and cultured neurons from IRI via inhibiting the hyperphosphorylation of NR2B subunit and decreasing its expression levels in cell membrane.

Chronic Administration of Benzo(a)pyrene Induces Memory Impairment and Anxiety-Like Behavior and Increases of NR2B DNA Methylation.

BackgroundRecently, an increasing number of human and animal studies have reported that exposure to benzo(a)pyrene (BaP) induces neurological abnormalities and is also associated with adverse effects, such as tumor formation, immunosuppression, teratogenicity, and hormonal disorders. However, the exact mechanisms underlying BaP-induced impairment of neurological function remain unclear. The aim of this study was to examine the regulating mechanisms underlying the impact of chronic BaP exposure on neurobehavioral performance.MethodsC57BL mice received either BaP in different doses (1.0, 2.5, 6.25 mg/kg) or olive oil twice a week for 90 days. Memory and emotional behaviors were evaluated using Y-maze and open-field tests, respectively. Furthermore, levels of mRNA expression were measured by using qPCR, and DNA methylation of NMDA receptor 2B subunit (NR2B) was examined using bisulfate pyrosequencing in the prefrontal cortex and hippocampus.ResultsCompared to controls, mice that received BaP (2.5, 6.25 mg/kg) showed deficits in short-term memory and an anxiety-like behavior. These behavioral alterations were associated with a down-regulation of the NR2B gene and a concomitant increase in the level of DNA methylation in the NR2B promoter in the two brain regions.ConclusionsChronic BaP exposure induces an increase in DNA methylation in the NR2B gene promoter and down-regulates NR2B expression, which may contribute to its neurotoxic effects on behavioral performance. The results suggest that NR2B vulnerability represents a target for environmental toxicants in the brain.

Septin 9 interacts with kinesin KIF17 and interferes with the mechanism of NMDA receptor cargo binding and transport.

Intracellular transport involves the regulation of microtubule motor interactions with cargo, but the underlying mechanisms are not well understood. Septins are membrane- and microtubule-binding proteins that assemble into filamentous, scaffold-like structures. Septins are implicated in microtubule-dependent transport, but their roles are unknown. Here we describe a novel interaction between KIF17, a kinesin 2 family motor, and septin 9 (SEPT9). We show that SEPT9 associates directly with the C-terminal tail of KIF17 and interacts preferentially with the extended cargo-binding conformation of KIF17. In developing rat hippocampal neurons, SEPT9 partially colocalizes and comigrates with KIF17. We show that SEPT9 interacts with the KIF17 tail domain that associates with mLin-10/Mint1, a cargo adaptor/scaffold protein, which underlies the mechanism of KIF17 binding to the NMDA receptor subunit 2B (NR2B). Significantly, SEPT9 interferes with binding of the PDZ1 domain of mLin-10/Mint1 to KIF17 and thereby down-regulates NR2B transport into the dendrites of hippocampal neurons. Measurements of KIF17 motility in live neurons show that SEPT9 does not affect the microtubule-dependent motility of KIF17. These results provide the first evidence of an interaction between septins and a nonmitotic kinesin and suggest that SEPT9 modulates the interactions of KIF17 with membrane cargo.

Melatonin pretreatment prevents isoflurane-induced cognitive dysfunction by modulating sleep–wake rhythm in mice

BackgroundSleep plays an important role in memory processing. However, its role in anesthesia-induced cognitive dysfunction was not revealed. Our study sought to investigate the connection between the cognition decline and sleep–wake rhythm disorders after long-term isoflurane anesthesia in mice. Also, we examined the effect of exogenous melatonin pretreatment on both cognitive function and circadian rhythm. Furthermore, we discussed whether NR2B (N-methyl–D-aspartate receptor 2B subunit)–CREB (cAMP-response element binding protein) signaling pathway was involved in this course. Methods2-month-old male C57/BL-6J mice were submitted to long-term anesthesia using 1% isoflurane from CT (Circadian Time) 14 to CT20. Melatonin pretreatment were conducted before anesthesia for 7 Days. Intellicage for mice and Mini-Mitter were applied to monitor spatial memory and gross motor activity which can reflect cognition and sleep–wake rhythm. Messenger RNA and protein expression of right hippocampus NR2B and CREB were examined by RT-PCR and Western blot. Results6h isoflurane anesthesia led to impaired spatial memory from Day 3 to Day 10 in mice accompanied by the disruption of sleep–wake rhythm. Meanwhile, the hippocampus CREB and NR2B expression declined in step. Melatonin pretreatment ameliorated disturbed sleep–wake cycle, improved isoflurane-induced cognitive dysfunction, and reversed the down-regulation of CREB and NR2B expression. ConclusionsOur data demonstrate that sleep–wake rhythm is involved in the isoflurane-induced cognition impairment and pretreatment of melatonin has a positive effect on circadian normalization and cognition reversal. Also, NR2B–CREB signaling pathway has a critical role in this process. This study provides us a new strategy for anesthesia-induced cognitive dysfunction therapy.

The role of GRIN2B in Tourette syndrome: Results from a transmission disequilibrium study

BackgroundPrevious studies have indicated that dopamine interacts with glutamatergic projection neurons and that N-methyl-d-aspartate (NMDA) receptors might be involved in the pathogenesis of Tourette syndrome (TS). In this study, we examined whether two functional polymorphisms (rs1805476 and rs1805502) in the 3′UTR of the NMDA receptor 2B subunit gene (GRIN2B) were associated with TS in Chinese Han trios. MethodsDNA samples collected from 261 TS nuclear families were genotyped by PCR and direct sequencing technology. Haplotype relative risk (HRR), transmission disequilibrium test (TDT) and Haplotype-based haplotype relative risk (HHRR) analyses were performed on the genotype data. ResultsWe found an over-transmission of the A allele in rs1805476 and the T allele in rs1805502 from parents to their affected children, using the HRR (rs1805476: HRR=0.696, χ2=4.161, P=0.041, 95% CI: 0.491–0.986; rs1805502: HRR=0.697, χ2=3.954, P=0.047, 95% CI: 0.488–0.995). There was also strong evidence for a linkage between polymorphisms and TS using the TDT (rs1805476: TDT=5.447, df=1, P=0.024; rs1805502: TDT=5.233, df=1, P=0.027). LimitationsThe sample is small and the current population is just limited to the Chinese Han population. ConclusionsThese data support the hypothesis that GRIN2B might play a major role in the pathogenesis of TS in Chinese Han trios. However, these results need to be replicated using larger datasets collected from different populations.

NR2B-dependent cyclophilin D translocation suppresses the recovery of synaptic transmission after oxygen–glucose deprivation

N-methyl d-aspartate receptor (NMDA) subunit 2B (NR2B)-containing NMDA receptors and mitochondrial protein cyclophilin D (CypD) are well characterized in mediating neuronal death after ischemia, respectively. However, whether and how NR2B and CypD work together in mediating synaptic injury after ischemia remains elusive. Using an ex vivo ischemia model of oxygen–glucose deprivation (OGD) in hippocampal slices, we identified a NR2B-dependent mechanism for CypD translocation onto the mitochondrial inner membrane. CypD depletion (CypD null mice) prevented OGD-induced impairment in synaptic transmission recovery. Overexpression of neuronal CypD mice (CypD+) exacerbated OGD-induced loss of synaptic transmission. Inhibition of CypD-dependent mitochondrial permeability transition pore (mPTP) opening by cyclosporine A (CSA) attenuated ischemia-induced synaptic perturbation in CypD+ and non-transgenic (non-Tg) mice. The treatment of antioxidant EUK134 to suppress mitochondrial oxidative stress rescued CypD-mediated synaptic dysfunction following OGD in CypD+ slices. Furthermore, OGD provoked the interaction of CypD with P53, which was enhanced in slices overexpressing CypD but was diminished in CypD-null slices. Inhibition of p53 using a specific inhibitor of p53 (pifithrin-μ) attenuated the CypD/p53 interaction following OGD, along with a restored synaptic transmission in both non-Tg and CypD+ hippocampal slices. Our results indicate that OGD-induced CypD translocation potentiates CypD/P53 interaction in a NR2B dependent manner, promoting oxidative stress and loss of synaptic transmission. We also evaluate a new ex vivo chronic OGD-induced ischemia model for studying the effect of oxidative stress on synaptic damage.

GluN2A/B ratio elevation induced by cortical spreading depression: electrophysiological and quantitative studies of the hippocampus.

Cortical spreading depression (CSD), an underlying mechanism of migraine aura, propagates to the hippocampus, and might explain hippocampusassociated symptoms during migraine attack. We hypothesised that this process is, some parts, mediated by NMDA receptors. By using a rat model, CSD was elicited by solid KCl for 45 minutes prior to electrophysiological and quantitative analyses. The result from electrophysiological study was the ratio of glutamate NMDA receptor 2A and 2B subunits (GluN2A/B). Total NMDA receptor response was isolated using an AMPA antagonist, prior to a GluN2B receptor antagonist. The GluN2A/B ratio was calculated by dividing the remaining NMDA-mediated field-excitatory synaptic potentials (fEPSP) with the subtracted difference of NMDAmediated fEPSP. Western blot analysis of the hippocampus was performed to confirm the quantitative change of GluN2A/B ratio. In hippocampal slice study (n = 12), the GluN2A/B ratio of hippocampal fEPSP was significantly increased in CSD group. Western blot analysis (n = 30) revealed an increase in GluN2A subunits and a decrease in GluN2B subunits in the hippocampus ipsilateral to the CSD induction. Our current study revealed that GluN2A/B ratio was shown to be elevated following CSD stimulation by increasing the total number of GluN2A while reducing the total number of GluN2B subunits. This ratio was demonstrated to be associated with synaptic plasticity of the hippocampus in numerous studies. In conclusion, we showed that CSD increased GluN2A/B ratio, in turn, would result in altered synaptic plasticity. Our findings provide a probable implication on the correlation of migraine aura and hippocampusassociated symptoms.

Association Study of N-Methyl-D-Aspartate Receptor Subunit 2B (GRIN2B) Polymorphisms and Schizophrenia Symptoms in the Han Chinese Population.

Schizophrenia (SZ) is a common and complex psychiatric disorder that has a significant genetic component. The glutamatergic system is the major excitatory neurotransmitter system in the central nervous system, and is mediated by N-methyl-D-aspartate (NMDA) receptors. Disturbances in this system have been hypothesized to play a major role in SZ pathogenesis. Several studies have revealed that the NMDA receptor subunit 2B (GRIN2B) potentially associates with SZ and its psychiatric symptoms. In this study, we performed a case–control study to identify polymorphisms of the GRIN2B gene that may confer susceptibility to SZ in the Han Chinese population. Thirty-four single nucleotide polymorphisms (SNPs) were genotyped in 528 paranoid SZ patients and 528 control subjects. A significant association was observed in allele and genotype between SZ and controls at rs2098469 (χ2 = 8.425 and 4.994; p = 0.025 and 0.014, respectively). Significant associations were found in the allele at rs12319804 (χ2 = 4.436; p = 0.035), as well as in the genotype at rs12820037 and rs7298664 between SZ and controls (χ2 = 11.162 and 38.204; p = 0.003 and 4.27×10-8, respectively). After applying the Bonferroni correction, rs7298664 still had significant genotype associations with SZ (p = 1.71×10-7). In addition, rs2098469 genotype and allele frequencies, and 12820037 allele frequencies were nominally associated with SZ. Three haplotypes, CGA (rs10845849—rs12319804—rs10845851), CC (rs12582848—rs7952915), and AAGAC (rs2041986—rs11055665—rs7314376—rs7297101—rs2098469), had significant differences between SZ and controls (χ2 = 4.324, 4.582, and 4.492; p = 0.037, 0.032, and 0.034, respectively). In addition, three SNPs, rs2098469, rs12820037, and rs7298664, were significantly associated with cognition factors PANSS subscores in SZ (F = 16.799, 7.112, and 13.357; p = 0.000, 0.017, and 0.000, respectively). In conclusion, our study provides novel evidence for an association between GRIN2B polymorphisms and SZ susceptibility and symptoms in the Han Chinese population.

Role of spinal MCP-1-ERK-KIF17/NR2B signaling pathway in maintenance of type 2 diabetic neuropathic pain in rats

Objective To explore the role of spinal monocyte chemoattractant protein-1 (MCP-1) -extracellular signal-regulated protein kinase (ERK) -kinesin superfamily motor protein 17 (KIF17) /N-methyl-D-aspartate receptor subunit 2B (NR2B) signaling pathway in the maintenance of type 2 diabetic neuropathic pain (DNP) in rats. Methods Type 2 diabetes mellitus was induced by a high-fat and high-sucrose diet and intraperitoneal streptozotocin (STZ) 35 mg/kg, and confirmed by fasting blood glucose level≥16.7 mmol/L 3 days later in male Sprague-Dawley rats aged 6 weeks.Type 2 DNP was confirmed when the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawl latency (TWL) measured on day 14 after STZ administration decreased to < 80% of the baseline value.The rats with type 2 DNP were randomly divided into 4 groups (n=36 each) using a random number table: type 2 DNP group (group DNP) , type 2 DNP + MCP-1 neutralizing antibody group (group DM) , type 2 DNP + ERK inhibitor group (group DE) and type 2 DNP + dimethyl sulfoxide group (group DD) . In DM, DE and DD groups, 0.1 ng/μl MCP-1 neutralizing antibody 10 μl, 0.5 μg/μl U0126 10 μl and 5 % dimethyl sulfoxide 10 μl were injected intrathecally, respectively, once a day for 14 consecutive days starting from 14 days after administration of STZ.Another 36 normal rats fed a common forage diet were adopted as control group (group C) . MWT and TWL were measured before STZ injection and at 1, 3, 7 and 14 days after STZ injection (T0-4) . Nine rats were sacrificed after measurement of pain thresholds at T1-4, and the lumbar segments (L4-6) of the spinal cord were removed for determination of the expression of phosphorylated ERK (p-ERK) , KIF17 and phosphorylated NR2B (p-NR2B) by Western blot. Results Compared with group C, the MWT was significantly decreased, the TWL was shortened, and the expression of p-ERK, KIF17 and p-NR2B was up-regulated at T1-4 in DNP, DM, DE and DD groups.Compared with group DNP, the MWT at T3-4 in group DM and at T2-4 in group DE was significantly increased, the TWL at T3-4 in group DM and at T2-4 in group DE was prolonged, and the expression of p-ERK, KIF17 and p-NR2B was down-regulated at T2-4 in DM and DE groups, and no significant changes were found in the parameters mentioned above in group DD. Conclusion Spinal MCP-1-ERK-KIF17/NR2B signaling pathway is involved in the maintenance of type 2 DNP in rats. Key words: Diabetes mellitus, type 2; Neuropathic pain; Chemokine CCL2; Extracellular Signal-Regulated MAP Kinases; Receptors, N-Methyl-D-Aspartate; Kinesin; Spinal cord

Salicylate-induced changes in immediate-early genes in the hippocampal CA1 area.

Studies have suggested that salicylate affects neuronal function via interactions with specific membrane channels/receptors. However, the effect of salicylate on activity and synaptic morphology of the hippocampal Cornu Ammonis (CA) 1 area remains to be elucidated. The activation of immediate-early genes (IEGs) was reported to correlate with neuronal activity, in particular activity-regulated cytoskeleton-associated protein and early growth response gene 1. The aim of the present study was to evaluate the expression of these IEGs, as well that of N-methyl D-aspartate (NMDA) receptor subunit 2B in rats following acute and chronic salicylate treatment. Protein and messenger RNA levels of all three genes were increased in rats following chronic administration of salicylate (300 mg/kg for 10 days), returning to baseline levels 14 days post-cessation of treatment. The transient upregulation of gene expression following treatment was accompanied by ultrastructural alterations in hippocampal CA1 area synapses. An increase in synaptic interface curvature was observed as well as an increased number of presynaptic vesicles; in addition, postsynaptic densities thickened and lengthened. In conclusion, the results of the present study indicated that chronic exposure to salicylate may lead to structural alteration of hippocampal CA1 neurons, and it was suggested that this process occurs through induced expression of IEGs via NMDA receptor activation.

Deletion of Adenosine A2A Receptors From Astrocytes Disrupts Glutamate Homeostasis Leading to Psychomotor and Cognitive Impairment: Relevance to Schizophrenia

BackgroundAdenosine A2A receptors (A2AR) modulate dopamine and glutamate signaling and thereby may influence some of the psychomotor and cognitive processes associated with schizophrenia. Because astroglial A2AR regulate the availability of glutamate, we hypothesized that they might play an unprecedented role in some of the processes leading to the development of schizophrenia, which we investigated using a mouse line with a selective deletion of A2AR in astrocytes (Gfa2-A2AR knockout [KO] mice]. MethodsWe examined Gfa2-A2AR KO mice for behaviors thought to recapitulate some features of schizophrenia, namely enhanced MK-801 psychomotor response (positive symptoms) and decreased working memory (cognitive symptoms). In addition, we probed for neurochemical alterations in the glutamatergic circuitry, evaluating glutamate uptake and release and the levels of key proteins defining glutamatergic signaling (glutamate transporter-I [GLT-I], N-methyl-D-aspartate receptors [NMDA-R] and α-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors [AMPA-R]) to provide a mechanistic understanding of the phenotype encountered. ResultsWe show that Gfa2-A2AR KO mice exhibited enhanced MK-801 psychomotor response and decreased working memory; this was accompanied by a disruption of glutamate homeostasis characterized by aberrant GLT-I activity, increased presynaptic glutamate release, NMDA-R 2B subunit upregulation, and increased internalization of AMPA-R. Accordingly, selective GLT-I inhibition or blockade of GluR1/2 endocytosis prevented the psychomotor and cognitive phenotypes in Gfa2-A2AR KO mice, namely in the nucleus accumbens. ConclusionsThese results show that the dysfunction of astrocytic A2AR, by controlling GLT-I activity, triggers an astrocyte-to-neuron wave of communication resulting in disrupted glutamate homeostasis, thought to underlie several endophenotypes relevant to schizophrenia.