NR2B Subunit Research Articles

<p>Wnt3a Inhibitor Attenuates Remifentanil-Induced Hyperalgesia via Downregulating Spinal NMDA Receptor in Rats</p>

PurposeThe upregulation of spinal NMDA receptor is a crucial mechanism in remifentanil-induced hyperalgesia (RIH). Wnt3a/β-catenin pathway plays an important role in neuropathic pain. We hypothesized that wnt3a inhibitor (iwp-2) could downregulate the expression of NR2B subunit in NMDA receptor, in order to relieve RIH.Materials and MethodsThe study has 2 phases. The phase 1 study is designed by different doses of iwp-2 groups to create an appropriate iwp-2 dose used in RIH alleviation. The phase 2 study is designed to prove that the wnt3a inhibitor could downregulate the activation of the NR2B to inhibit RIH in rats. Thermal hyperalgesia (PWTL) and mechanical allodynia (PWMT) were evaluated after RIH. The area under the PWTL and PWMT curves (AUC) were calculated. The amount of activated NR2B subunit, c-fos, NF-κB, β-catenin, wnt3a and p-GSK-3β (Ser9) were detected in the lumbar spinal cord.ResultsRemifentanil infusion could induce overexpression of β-catenin and wnt3a in rats. Iwp-2 (60μM, 120μM, 180μM) could dose-dependently inhibit thermal hyperalgesia and mechanical allodynia in rats. In phase 2 study, both NR2B subunit antagonist Ro25-6981 and iwp-2 decreased the amount of activated NR2B, enhanced p-GSK-3β (Ser9), reduced β-catenin, c-fos and NF-κB in the lumbar spinal cord (p < 0.001). In comparison with the group iwp-2, the group of Ro25-6981 had more benefit in reversing hyperalgesia, including higher AUC value of PWTL (p = 0.022) and PWMT (p = 0.035).ConclusionRemifentanil exposure could induce overexpression of wnt3a and enhance the production of β-catenin in the spinal dorsal horn. Inhibition of wnt3a response was capable of attenuating RIH in alleviating hyperalgesia-related behavioral parameters, as well as reducing overexpression of c-fos, NF-κB, NR2B in spinal dorsal horn.

Intermittent Theta Burst Stimulation Improves the Spatial Cognitive Function of Rats with Chronic Hypertension-induced Cerebral Small Vessel Disease

We investigated whether intermittent theta burst stimulation (iTBS) can improve the spatial cognitive function of rats with hypertension-induced cerebral small vessel disease. To prove our hypothesis, stroke-prone renovascular hypertensive rats (RHRSPs) were treated with iTBS beginning at postoperative week 22. The Morris water maze was performed to assess spatial cognitive function. The expression of the N-methyl-d-aspartate receptor (NMDAR) subunits NR1, NR2A and NR2B, calcium/calmodulin-dependent protein kinase IIα (CaMKIIα), p-CaMKIIα and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit 1 (GluR1) in the hippocampus were evaluated by western blot analysis. The distribution of GluR1, glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule-1 (IBa-1) in the CA1 and CA3 regions and dentate gyrus (DG) of the hippocampus were evaluated by immunofluorescence analysis. Treatment with iTBS significantly improved the spatial cognitive function of RHRSPs, increased the expression of NR2B, p-CaMKIIα and GluR1 in the hippocampus, and decreased the proliferation of astrocytes and microglia. Our results showed that iTBS treatment had a beneficial effect on the cognitive impairments induced by cerebral small vessel disease, potentially through the activation of the NR2B-CaMKII pathway, an increase in GluR1 expression and the suppression of astrocyte and microglial activation.

Comparisons of Nociceptive Behavior and NMDA receptor‐Mediated Synaptic Transmission between Mice Lacking D‐Serine Production and Degradation Enzymes

D‐serine is the endogenous co‐agonist for the glycine‐binding site of the N‐methyl‐D‐aspartate (NMDA) glutamate receptor. D‐serine is produced by the enzyme serine racemase (SR) and is degraded by the enzyme D‐amino acid oxidase (DAO). Co‐activation of spinal NMDA receptors by D‐serine is known to play a significant role in the etiological mechanism of neuropathic and inflammatory pain. The present study aims to obtain further information on how D‐serine and its related enzymes (SR and DAO) are involved in the modulation of persistent pain.The experiments were performed on both SR knock‐out (SR‐KO) mice and the DAO‐mutant mice that lack the enzymatic activity of DAO. We have previously shown that SR‐KO mice have less than half of the D‐serine content in the spinal cord and that DAO‐mutant mice have a significantly elevated level of D‐serine.The DAO‐mutant and SR‐KO mice used in this study are kindly provided by R. Konno (International University of Health and Welfare, Tochigi, Japan).First, the nociceptive behavioral responses to formalin that is subcutaneously injected into a hind paw were analyzed. Results showed that the responses in the second phase, which are known be associated with NMDA receptor activation, were augmented both in SR‐KO mice and in DAO‐mutant mice.Second, tight‐seal whole‐cell recordings were made from neurons in the superficial dorsal horn (SDH) in the lumbar spinal slices that were prepared from SR‐KO mice or from DAO‐mutant mice. Non‐NMDA and NMDA receptor‐mediated excitatory postsynaptic currents (non‐NMDAEPSCs and NMDA‐EPSCs, respectively) were pharmacologically isolated. Results show that the ratio of the amplitudes of NMDA‐EPSCs to that of non‐NMDA‐EPSCs was significantly smaller in SR‐KO mice and larger in DAO‐mutant mice compared with each corresponding control group and that the time constant of decay time course of NMDA‐EPSCs was significantly longer in SR‐KO mice.Finally, quantitative real‐time RT‐PCR experiments were performed on RNA extracted from the SDH tissue. The results showed that the subunit composition of NMDA receptors altered in SR‐KO mice and DAO‐mutant mice. The most significant observation is that the expression level of the NR2B subunit of NMDA receptors increased in the SR‐KO mice.These observations might indicate that D‐serine and its related enzymes (SR and DAO) influence the activation level of NMDA receptors and their subunit composition and thus affect nociceptive synaptic transmission in the SDH of the spinal cord and influence nociceptive behavior.Support or Funding InformationThis work was supported by JSPS KAKENHI Grant Number JP18K16462 (EK).

Memantine treatment prevents okadaic acid induced neurotoxicity at the systemic and molecular levels

The present study was designed to investigate the effects of okadaic acid intracerebroventricular (ICV) injection on memory function and expression level of α7 subunit of nicotinic acetylcholine receptor (nAChR) and NR2B subunit of NMDA glutamate receptors in the hippocampus, as well as effect of the antidementic drug memantine on okadaic acid induced changes at systemic and molecular levels in rats. Okadaic acid was dissolved in artificial cerebrospinal fluid (aCSF) and injected ICV 200 ng/10 μl. Vehicle control received 10 μl of aCSF ICV bilaterally. Control and okadaic acid injected rats were divided into two subgroups: treated i.p. with saline or memantine (5 mg/kg daily for 13 days starting from the day of okadaic acid injection). Rats were trained in the dual-solution plus-maze task that can be solved by using place or response strategies. The Western immunoblotting was used to determine relative amount of hippocampal receptors protein levels. Obtained data revealed that okadaic acid ICV injected rats were severely impaired at acquiring the place version of the maze accompanied by significant decline in hippocampal α7 subunit of nACh receptors protein levels. Memantine treatment can prevent okadaic acid induced impairment of hippocampal-dependent spatial memory and accompanied by modulation of the expression level of α7 subunit of nACh and NR2B subunit of NMDA receptors in the hippocampus. Thus, our results support the presumption that α7 nACh receptors may play an important role in the cognitive enhancer effects of memantine and emphasize the role of cholinergic-glutamatergic interactions in memory.

Early Neurotoxic Effects of Inorganic Arsenic Modulate Cortical GSH Levels Associated With the Activation of the Nrf2 and NFκB Pathways, Expression of Amino Acid Transporters and NMDA Receptors and the Production of Hydrogen Sulfide.

Exposure to toxic metals and metalloids is an important cause of preventable diseases worldwide. Inorganic arsenic (iAs) affects several organs and tissues, causing neurobehavioral alterations in the central nervous system (CNS) that might lead to neurodegeneration. In this work, we wanted to explore the time- and dose-related changes on glutathione (GSH) levels in several regions of the CNS, such as the cortex, striatum, hippocampus, and cerebellum, to identify the initial cellular changes associated to GSH depletion due to iAs exposure. Mice received a single intraperitoneal injection containing 5 or 14 mg/kg sodium arsenite. Animals were killed at 2, 6, and 24 h. Significant depletion of GSH levels was observed in the cortex at 2 and 6 h, while on the striatum, hippocampus, or cerebellum regions, no significant changes were observed. GSH depletion in the cortex was associated with the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor kappa B (NFκB) pathways, which led to the upregulation of xCT, excitatory amino acid carrier 1 (EAAC1), glutamate/aspartate transporter (GLAST), and glial glutamate transporter 1 (GLT-1), and the activation of the transsulfuration pathways, which led to the overproduction of H2S in the cortex and increased levels of GSH in the cortex and cerebellum at 24 h. In the cortex, the N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B were also altered at 24 h. These early effects were not homogeneous among different brain regions and indicate early neurotoxic alterations in the cortex and cerebellum.

Proconvulsant effect of trans-cinnamaldehyde in pentylenetetrazole-induced kindling model of epilepsy: The role of TRPA1 channels

The transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily, is widely distributed in the central nervous system (CNS) and plays an important role in pain and inflammation. However, no data has been reported regarding the effects of TRPA1 on epileptic seizures. Thus, this study was designed to investigate the sub-chronic effect of trans-cinnamaldehyde (TCA), an agonist of TRPA1, in pentylenetetrazole (PTZ) induced kindling model via electrocorticography (ECoG). Furthermore, the expressions of cAMP response element binding protein (CREB), brain-derived neurotrophic factor (BDNF), and NMDA receptor subunit NR2B were measured using Western blotting. Rats were kindled by intraperitoneal (i.p.) PTZ (35 mg/kg) injections. After electrode implantation and healing period, 10 and 30 mg/kg TCA was given i.p. for 14 consecutive days. On the next day, ECoG recordings were obtained after the injection of PTZ (35 mg/kg, i.p.), and twenty-four hours later, rats were decapitated for molecular analyses. TCA, at a dose of 30 mg/kg, decreased the first myoclonic jerk latency and increased seizure duration and total spike activity. Additionally, both doses of TCA enhanced CREB, BDNF, and NR2B expressions, which were increased by the kindling. The evidence from this study suggests that long term activation of TRPA1 channels causes an exacerbated seizure activity. Moreover, PTZ-induced increases in CREB, BDNF, and NR2B levels were enhanced by the repeated administrations of TCA.

N-methyl-d-aspartate receptor subunit 2B on keratinocyte mediates peripheral and central sensitization in chronic post-ischemic pain in male rats

The spinal N-methyl-d-aspartate (NMDA) receptor, and particularly its NR2B subunit, plays a pivotal role in neuropathic pain. However, the role of peripheral NMDA receptor in neuropathic pain is less well understood. We first treated cultured human keratinocytes, HaCaT cells with NMDA or NR2B-specific antagonist, ifenprodil and evaluated the level of total and phosphorylated NR2B at 24 h using Western blot. Next, using the chronic post-ischemia pain (CPIP) model, we administered NMDA or ifenprodil subcutaneously into the hind paws of male rats. Nociceptive behaviors were assessed by measuring mechanical and thermal withdrawal thresholds. Expression and phosphorylation of NR2B on keratinocyte were analyzed at 6, 12, 18, and 24 h on day 1 (initiation of pain) as well as day 2, 6, 10 and 14 (development and maintenance of pain) after the ischemia. The level of peripheral sensitization-related proteins (nuclear factor-κB (NF-κB), extracellular regulated protein kinases (ERK), and interleukin-1β (IL-1β)) in epidermis and dorsal root ganglion (DRG) were evaluated by immunofluorescence and western blot. Central sensitization-related C-fos induction, as well as astrocytes and microglia activation in the spinal cord dorsal horn (SDH) were studied using immunofluorescence. Administration of NMDA upregulated NR2B phosphorylation on HaCaT cells. CPIP-induced mechanical allodynia and thermal hyperalgesia were intensified by NMDA and alleviated by ifenprodil. CPIP resulted in an early upregulation of NR2B (peaked at 24 h) and late phosphorylation of NR2B (peaked at 14d) in hindpaw keratinocytes. CPIP led to an upregulation and phosphorylation of NF-κB and ERK, as well as an increased IL-1β production in the ipsilateral skin and DRG. CPIP-associated c-fos induction in SDH persisted from acute to chronic stages after ischemia, while microglia and astrocyte activation were only observed in chronic phase. These CPIP-induced changes were also suppressed by ifenprodil administered subcutaneously in the hind paw. Our findings reveal a previously unrecognized role of keratinocyte NMDA receptor subunit 2B in peripheral and central nociceptive sensitization induced by CPIP.

Pioglitazone Attenuates Experimental Colitis-Associated Hyperalgesia through Improving the Intestinal Barrier Dysfunction

Impaired intestinal mucosal integrity during colitis involves the peroxisome proliferator-activated receptor-γ (PPARγ), an important anti-inflammatory factor in intestinal mucosa homoeostasis, which is a potential target in colitis. Recurrent chronic pain is a vital pathogenetic feature of colitis. Nevertheless, potential functions of PPARγ in the colitis-associated hyperalgesia remain unclear. This study aimed to investigate biological roles of pioglitazone in relieving colitis-associated pain hypersensitivity by a PPARγ tight junction protein-dependent mechanism during the course of dextran sodium sulfate (DSS)-induced intestinal inflammation. The DSS-induced colitis model was generated in C57BL/6 mice. Changes in colitis induced the injury of intestinal mucosal barrier and hyperalgesia after a 6-day treatment of pioglitazone (25 mg/kg, IP injection) were assessed through immunofluorescent, hematoxylin and eosin (H&E) staining, western blot analysis, and determination of paw withdrawal mechanical threshold. A significant reduction of paw withdrawal mechanical threshold occurred after DSS treatment. Follow-up data showed that systematic administration of PPARγ agonist pioglitazone ameliorated the DSS-induced colitis and the development of colitis-associated hyperalgesia by repairing the intestinal mucosal barrier. The tight junction proteins ZO-1 and Claudin-5 were upregulated by PPARγ signaling, which in turn promoted the improvement of intestinal barrier function. Moreover, pioglitazone inhibited phosphorylation of ERK and NF-κB in the colon and decreased the levels of inflammatory cytokines in both colon spine tissues. Furthermore, systemically pioglitazone treatment inhibited the activation of microglia and astrocytes, as well as DSS-induced phosphorylation of NR2B subunit in spinal cord, which was correspondingly consistent with the pain behavior. Pioglitazone ameliorates DSS-induced colitis and attenuates colitis-associated mechanical hyperalgesia, with improving integrity of the intestinal mucosal barrier by directly upregulating tight junction proteins. The PPARγ-tight junction protein signaling might be a potential therapeutic target for the treatment of colitis-associated chronic pain.

Evaluation of 11C-NR2B-SMe and Its Enantiomers as PET Radioligands for Imaging the NR2B Subunit Within the NMDA Receptor Complex in Rats.

[S-methyl-11C](±)-7-methoxy-3-(4-(4-(methylthio)phenyl)butyl)-2,3,4,5-tetrahydro-1H-benzo[d]azepin-1-ol (11C-NR2B-SMe) and its enantiomers were synthesized as candidates for imaging the NR2B subunit within the N-methyl-d-aspartate receptor with PET. Methods: Brains were scanned with PET for 90 min after intravenous injection of one of the candidate radioligands into rats. To detect any NR2B-specific binding of radioligand in brain, various preblocking or displacing agents were evaluated for their impact on the PET brain imaging data. Radiometabolites from brain and other tissues were measured ex vivo and in vitro. Results: Each radioligand gave high early whole-brain uptake of radioactivity, followed by a brief fast decline and then a slow final decline. 11C-(S)-NR2B-SMe was studied extensively. Ex vivo measurements showed that radioactivity in rat brain at 30 min after radioligand injection was virtually unchanged radioligand. Only less lipophilic radiometabolites appeared in plasma. High-affinity NR2B ligands, Ro-25-6981, ifenprodil, and CO101244, showed increasing preblocking of whole-brain radioactivity retention with increasing dose (0.01-3.00 mg/kg, intravenously). Five σ1 antagonists (FTC146, BD1407, F3, F4, and NE100) and 4 σ1 agonists ((+)-pentazocine, (±)-PPCC, PRE-084, and (+)-SKF10047) were ineffective preblocking agents, except FTC146 and F4 at a high dose. Two potent σ1 receptor agonists, TC1 and SA4503, showed dose-dependent preblocking effects in the presence or absence of pharmacologic σ1 receptor blockade with FTC146. Conclusion:11C-(S)-NR2B-SMe has adequate NR2B-specific PET signal in rat brain to warrant further evaluation in higher species. TC1 and SA4503 likely have off-target binding to NR2B in vivo.

4,5 caffeoylquinic acid and scutellarin, identified by integrated metabolomics and proteomics approach as the active ingredients of Dengzhan Shengmai, act against chronic cerebral hypoperfusion by regulating glutamatergic and GABAergic synapses

Dengzhan Shengmai (DZSM) is a proprietary Chinese medicine for remarkable curative effect as a treatment of cerebrovascular diseases, such as chronic cerebral hypoperfusion (CCH) and dementia based on evidence-based medicine, which have been widely used in the recovery period of ischemic cerebrovascular diseases. The purpose of this study was to investigate the active substances and mechanism of DZSM against CCH. Integrative metabolomic and proteomic studies were performed to investigate the neuroprotective effect of DZSM based on CCH model rats. The exposed components of DZSM in target brain tissue were analysed by a high-sensitivity HPLC-MS/MS method, and the exposed components were tested on a glutamate-induced neuronal excitatory damage cell model for the verification of active ingredients and mechanism of DZSM. Upon proteomic and metabolomic analysis, we observed a significant response in DZSM therapy from the interconnected neurotransmitter transport pathways including glutamatergic and GABAergic synapses. Additionally, DZSM had a significant regulatory effect on glutamate and GABA-related proteins including vGluT1 and vIAAT, suggested that DZSM could be involved in the vesicle transport of excitatory and inhibitory neurotransmitters in the pre-synaptic membrane. DZSM could also regulated the metabolism of arachidonic acid (AA), phospholipids, lysophospholipids and the expression of phospholipase A2 in post-synaptic membrane. The results of glutamate-induced neuronal excitatory injury cell model experiment for verification of active ingredients and mechanism of DZSM showed that there are five active ingredients, and among them, 4,5 caffeoylquinic acid (4,5-CQA) and scutellarin (SG) could simultaneously affect the GABAergic and glutamatergic synaptic metabolism as well as the related receptors, the NR2b subunit of NMDA and the α1 subunit of GABAA. The active ingredients of DZSM could regulate the over-expression of the NMDA receptor, enhance the expression of the GABAA receptor, resist glutamate-induced neuronal excitatory damage, and finally maintain the balance of excitatory and inhibitory synaptic metabolism dominated by glutamate and GABA. Furtherly, we compared the efficacy of DZSM, 4,5-CQA, SG and the synergistic effect of 4,5-CQA and SG, and the results showed that all the groups significantly improved cell viability compared with the model group (p < 0.001). The western blot results showed that DZSM, 4,5-CQA, SG and 4,5-CQA/SG co-administration groups could significantly regulate the expression of receptors (GABAA α1 and NR2b subunit of NMDA) and synaptic-related proteins, such as Sv2a, Syp, Slc17a7, bin1 and Prkca, respectively. These results proved DZSM and its active ingredients (4,5-CQA and SG) had the effect of regulating glutamatergic and GABAergic synapses. Finally, membrane potential FLIPR assay of 4,5-CQA and SG was used for GABRA1 activity test, and it was found that the two compounds could increase GABA-induced activation of GABRA1 receptor (GABA 10 μM) in a dose-dependent manner with EC50 value of 48.74 μM and 29.77 μM, respectively. Manual patch clamp method was used to record NMDA NR1/NR2B subtype currents, and scutellarin could cause around 10 % blockade at 10 μM (p＜0.05 compared with the control group). These studies provided definitive clues of the mechanism for the neuroprotective effect of DZSM for CCH treatment and the active compounds regulating glutamatergic and GABAergic synapses. Additionally, 4,5-CQA and SG might be potential drugs for the treatment of neurodegenerative disease related to CCH.

Dynamics of Spinal Inotropic Glutamate Receptors During the Development of Prolonged Postoperative Pain in the Rat.

Surgery can cause pain that lasts for weeks to months. Postoperative pain involves an early induction phase (hours to l days), and a longer maintenance phase. Experimental post-operative pain can be lowered by pre-operative spinal glutamate receptor antagonists as well as inhibitors of certain mitogen activated protein kinases (MAPKs). Here a model of postoperative pain in the rat, leading to 4 weeks of secondary mechanical allodynia-hyperalgesia, was used to examine changes in spinal inotropic glutamate receptors and MAPKs. Western blots show that GluR1subunits of AMPA receptors undergo rapid increases in phosphorylation at 4h post-op (2.3x naive), before tactile hyperalgesia is detectable, change to 1.6x naive at 2d post-op, when hyperalgesia is clearly present, and fall to sub-baseline levels (0.1x naive) at 14d, when hyperalgesia is maximum. The NR1 subunit of NMDA receptors are 1.7x, 1.8x, and 1.1x the level of naive at 4h, 2d and 14d, respectively, whilst the phosphorylated form occurs at 1.7x, 0.9x and 0.5x of naive at these times. NR2B subunit amounts follow a similar trend; 1.6x, 1.7x and 0.8x. Phosphorylation of the MAPK JNK was elevated to 1.45x, 1.35x and fell to 0.8x of naive at 4h, 2d and 14d postoperative, whilst pERK 1/2 phosphorylation remained unchanged. These results suggest that increases in total NMDA subunits and in phosphorylated NMDA and AMPA receptors, paralleled by elevated P~JNK, contribute to the induction phase of persistent post-operative pain, but are not involved in its maintenance.

Effects of glutamate-related drugs on anxiety and compulsive behavior in rats with obsessive-compulsive disorder

Objective: Altered glutamatergic neurotransmission has been implicated in the pathogenesis of obsessive-compulsive disorder (OCD). We examined the effects and potential mechanism of glutamate-related drugs on compulsive behavior in quinpirole (QNP)-sensitized rats, to deepen our understanding of the link between OCD and glutamate.Method: This study systematically compared the effects of the partial NMDA agonist D-Cycloserine and the NMDA antagonist NVP-AAMO77, Ro25-6981 on compulsive behavior using the elevated zero maze, open field, and marble burying tests in QNP-induced OCD model.Results: The competitive N-methyl-D-aspartate glutamate receptor (NMDAR) antagonists NVP-AAMO77 (5 mg/kg) and Ro25-6981 (5 mg/kg) significantly inhibited anxiety-like and compulsive behavior in rats. And D-Cycloserine at all doses showed significant suppression on anxiety-like and marble-burying behavior. Glutamic acid (Glu) levels, reflecting changes in the glutamatergic neurotransmission, were significantly decreased in rat hippocampus of the NVP-AAMO77 and D-Cycloserine-treated group compared to the saline-treated group. The levels of other amino acids were unaffected. Moreover, NVP-AAMO77 significantly decreased the expression of the subunit NR2A of the NMDAR, and Ro25-6981 suppressed the level of the subunit NR2B of the NMDAR, while D-Cycloserine decreased both the subunit NR2A and NR2B of the NMDAR.Conclusion: Collectively, these findings suggest a functional role of NMDARs in anxiety and compulsive behaviors, with NMDARs inhibition promoting anxiolytic-like and anti-compulsive responses. These findings suggest that D-cycloserine, NVP-AAMO77, and Ro25-6981 could be useful drugs for the treatment of OCD, which may be due to the suppression of NR2A- or NR2B- containing NMDAR.

Depletion of dietary phytoestrogens reduces hippocampal plasticity and contextual fear memory stability in adult male mouse

ABSTRACT Introduction: Phytoestrogens are non-steroidal estrogen analogues and are found primarily in soy products. They have received increasing attention as dietary supplements for estrogen deficiency and as modulators of endogenous estrogen functions, including cognition and emotion. In addition to modifying the levels of circulating sex hormones, phytoestrogens also exert direct effects on estrogen and androgen receptors in the brain and thus effectively modulate the neural circuit functions. Objective: The aim of this study was to investigate the long-term effects of low phytoestrogen intake (∼6 weeks) on the hippocampal plasticity and hippocampus-dependent memory formation in the adult C57BL/6 male mice. Methods and Results: In comparison to mice on a diet with normal phytoestrogen content, mice on low phytoestrogen diet showed a significant reduction in the phosphorylation of NR2B subunit, a molecular correlate of plasticity in the Schaffer collateral-CA1 synapse. We observed a profound decrease in long-term potentiation (LTP) in the ventral hippocampus, whereas no effect on plasticity was evident in its dorsal portion. Furthermore, we demonstrated that acute perfusion of slices with an estrogen analogue equol, an isoflovane metabolized from daidzein produced by the bacterial flora in the gut, was able to rescue the observed LTP deficit. Examining potential behavioral correlates of the plasticity attenuation, we found that mice on phytoestrogen-free diet display decreased contextual fear memory at remote but not at recent time points after training. Conclusions: Our data suggests that nutritional phytoestrogens have profound effects on the plasticity in the ventral hippocampus and ventral hippocampus-dependent memory.

Endothelin-1-Induced Ischemic Damage and Functional Impairment Is Mediated Primarily by NR2B-Containing NMDA Receptors.

Ischemic stroke accounts for 70-80% of stroke cases worldwide and survivors are frequently left with compromising sensorimotor deficits localized to one or more body regions. Most animal models of stroke involve transient or permanent occlusion of one or more major vessels such as the middle cerebral artery and are characterized by widespread damage to cortical and subcortical structures that result in deficits that can confound studies of neuroprotection and neurorehabilitation. Localized microinjections of the vasoconstricting peptide endothelin-1 (ET-1) into specific brain regions are becoming increasingly popular for such studies, but the pharmacology of endothelin-induced ischemic damage is poorly understood. To test the hypothesis that NMDA receptors, and particularly those containing the NR2B subunit, are involved in ET-1-mediated excitotoxicity and functional impairment, male CD1 rats (N = 32) were pre-treated with either the non-competitive NMDA antagonist MK-801 or the NR2B-selective antagonist Ro25-6981 (or vehicle) prior to unilateral microinjections of endothelin-1 into the somatosensory cortex and striatum. Rats were then tested using 4 established tests of sensory and/or motor function over 14days. Lesion volumes were quantified post-mortem using standard histology and image analysis. Results confirmed reproducible lesions and significant deficits in all tests in vehicle-treated rats that were significantly reduced in both drug groups but were not different between drugs, providing evidence that endothelin-induced ischemic damage is mediated almost exclusively by NR2B-containing NMDA receptors.

Nafamostat and sepimostat identified as novel neuroprotective agents via NR2B N-methyl-D-aspartate receptor antagonism using a rat retinal excitotoxicity model

In addition to its role in the treatment of pancreatitis, the serine protease inhibitor nafamostat exhibits a retinal protective effect. However, the exact mechanisms underlying this effect are unknown. In this study, the neuroprotective effects of nafamostat and its orally active derivative sepimostat against excitotoxicity were further characterised in vitro and in vivo. In primary rat cortical neurons, nafamostat completely suppressed N-methyl-D-aspartate (NMDA)-induced cell death. Intravitreal injection of nafamostat and sepimostat protected the rat retina against NMDA-induced degeneration, whereas the structurally related compounds, gabexate and camostat, did not. The neuroprotective effects of nafamostat and the NR2B antagonist ifenprodil were remarkably suppressed by spermidine, a naturally occurring polyamine that modulates the NR2B subunit. Both nafamostat and sepimostat inhibited [3H]ifenprodil binding to fractionated rat brain membranes. Thus, nafamostat and sepimostat may exert neuroprotective effects against excitotoxic retinal degeneration through NMDA receptor antagonism at the ifenprodil-binding site of the NR2B subunit.

NMDAR and JNK Activation in the Spinal Trigeminal Nucleus Caudalis Contributes to Masseter Hyperalgesia Induced by Stress.

It is commonly accepted that psychological stress is closely associated with the occurrence and development of chronic orofacial pain. However, the pathogenesis underlying this process has not been fully elucidated. In the present study, we explored the role of N-methyl-D-aspartate receptors (NMDARs) and Jun N-terminal kinase (JNK) mediated intercellular communication between neurons and astrocytes in the spinal trigeminal nucleus caudalis (Vc) in the induction of masseter hyperalgesia by psychological stress in rats. We found that subjecting rats to 14 days of restraint stress (8 h/d) caused a significant decrease in body weight gain, behavioral changes and marked masseter hyperalgesia in the rats. We also found that exposure to restraint stress for 14 days caused the expression of pJNK in astrocytes in the Vc to significantly increase, and intrathecally infusing a JNK inhibitor significantly prevented restraint stress-induced masseter hyperalgesia in the rats. In addition, after exposure to restraint stress for 14 days, the stressed group exhibited a noticeably increased expression level of pNR2B in neurons in the Vc. Then, we intrathecally injected MK-801 (an NMDAR inhibitor) and ifenprodil (a selective NR2B subunit antagonist) and observed that the two types of inhibitors not only alleviated masseter hyperalgesia but also significantly inhibited the phosphorylation of JNK in the Vc after restraint stress; this indicates that the effect of NMDAR antagonists may influence the activation of astrocytic JNK. Furthermore, inhibitors of neuronal nitric oxide synthase (nNOS) activation and guanylate cyclase (GC) inhibitor could not only inhibit the expression of pJNK in the Vc, but also effectively alleviate masseter hyperalgesia induced by restraint stress. Taken together, our results suggest that NMDAR activation could increase JNK phosphorylation in astrocytes after restraint stress, which may depend on the nNOS-GC pathway. The intercellular communication between neurons and astrocytes in the Vc may play a key role in the induction of masseter muscle hyperalgesia by psychological stress in rats.

MgO and ZnO nanoparticles anti-nociceptive effect modulated by glutamate level and NMDA receptor expression in the hippocampus of stressed and non-stressed rats

The anti-nociceptive mechanisms of MgO and ZnO nanoparticles have not been thoroughly investigated; in this study, we evaluated the effects of anti-nociceptive dose of MgO and ZnO NPs on glutamate level and NMDA receptor subunits expression (NR1, NR2 and NR2B) in the rat whole hippocampus with and without acute restraint stress. Adult rats were divided into control, MgO and ZnO NPs 5 mg/kg, the stress of 90 min alone and with MgO or ZnO NPs 5 mg/kg groups. All components injected intraperitoneally and the nociceptive response was measured with hot plate apparatus 90 min after injections or stress induction. Magnesium, zinc, glutamate levels and NMDA receptor subunits expression were measured in the animal hippocampus. MgO NPs, ZnO NPs and acute stress induced anti-nociceptive effect. MgO NPs observably decreased glutamate and increased magnesium levels and NR2B subunit expression. ZnO NPs decreased glutamate level. Stress elevated endogenous magnesium and zinc levels and also the NR2B expression, but did not change glutamate level. MgO and ZnO NPs in the presence of stress increased the glutamate level and ZnO NPs increased the zinc and the NR2A expression. Stress decreased endogenous magnesium in the hippocampus. MgO and ZnO NPs could affect pain perception by changing glutamate level in the whole hippocampus tissue, while ion level changes followed by injection could probably affect the gene expression in the presence and the absence of stress. It seems that stress indirectly could adverse nanoparticles effects on glutamate level and increase zinc ion releasing from ZnO NPs by activating the gene expression without affecting pain perception.

Development of Depotentiation in Adult-Born Dentate Granule Cells.

Activity-dependent synaptic plasticity, i.e., long-term potentiation (LTP), long-term depression (LTD) and LTP reversal, is generally thought to make up the cellular mechanism underlying learning and memory in the mature brain, in which N-methyl-D-aspartate subtype of glutamate (NMDA) receptors and neurogenesis play important roles. LTP reversal may be the mechanism of forgetting and may mediate many psychiatric disorders, such as schizophrenia, but the specific mechanisms underlying these disorders remain unclear. In addition, LTP reversal during the development of adult-born dentate granule cells (DGCs) remains unknown. We found that the expression of the NMDA receptor subunits NR2A and NR2B displayed dynamic changes during the development of postnatal individuals and the maturation of adult-born neurons and was coupled with the change in LTP reversal. The susceptibility of LTP reversal progressively increases with the rise in the expression of NR2A during the development of postnatal individual and adult-born neurons. In addition, NMDA receptor subunits NR2A, but not NR2B, mediated LTP reversal in the DGCs of the mouse hippocampus.

Memory Function, Neurological, and Immunological Biomarkers in Allergic Asthmatic Mice Intratracheally Exposed to Bisphenol A.

Bisphenol A (BPA) is a major constituent of plastic products, including epoxy resin containers, mobile phones, dental sealants, as well as electronic and medical equipment. BPA is recognized as an endocrine system-disrupting chemical which has toxic effects on the brain and reproductive system. However, little is known about the effects of co-exposure of BPA with allergens on the memory function and neurological as well as immunological biomarker levels. In this study, we examined the effects of intratracheal instillation of BPA on the memory function and neuroimmune biomarker levels using a mouse model of allergic asthma. Male C3H/HeJ Jcl mice were given three doses of BPA (0.0625 pmol, 1.25 pmol, and 25 pmol BPA/animal) intratracheally once a week, and ovalbumin (OVA) intratracheally every other week from 5 to 11 weeks old. At 11 weeks of age, a novel object recognition test was conducted after the final administration of OVA, and the hippocampi and hypothalami of the animals were collected after 24 h. The expression levels of the memory function-related genes N-methyl-D-aspartate (NMDA) receptor subunits, inflammatory cytokines, microglia markers, estrogen receptor-alpha, and oxytocin receptor were examined by real-time RT-PCR (real-time reverse transcription polymerase chain reaction) and immunohistochemical methods. Impairment of the novel object recognition ability was observed in the high-dose BPA-exposed mice with allergic asthma. In addition, the allergic asthmatic mice also showed downregulation of neurological biomarkers, such as NMDA receptor subunit NR2B in the hippocampus but no significant effect on immunological biomarkers in the hypothalamus. These findings suggest that exposure to high-dose BPA triggered impairment of memory function in the allergic asthmatic mice. This is the first study to show that, in the presence of allergens, exposure to high-dose BPA may affect memory by modulating the memory function-related genes in the hippocampus.

Tubulin-Dependent Transport of Connexin-36 Potentiates the Size and Strength of Electrical Synapses.

Connexin-36 (Cx36) electrical synapses strengthen transmission in a calcium/calmodulin (CaM)/calmodulin-dependent kinase II (CaMKII)-dependent manner similar to a mechanism whereby the N-methyl-D-aspartate (NMDA) receptor subunit NR2B facilitates chemical transmission. Since NR2B–microtubule interactions recruit receptors to the cell membrane during plasticity, we hypothesized an analogous modality for Cx36. We determined that Cx36 binding to tubulin at the carboxy-terminal domain was distinct from Cx43 and NR2B by binding a motif overlapping with the CaM and CaMKII binding motifs. Dual patch-clamp recordings demonstrated that pharmacological interference of the cytoskeleton and deleting the binding motif at the Cx36 carboxyl-terminal (CT) reversibly abolished Cx36 plasticity. Mechanistic details of trafficking to the gap-junction plaque (GJP) were probed pharmacologically and through mutational analysis, all of which affected GJP size and formation between cell pairs. Lys279, Ile280, and Lys281 positions were particularly critical. This study demonstrates that tubulin-dependent transport of Cx36 potentiates synaptic strength by delivering channels to GJPs, reinforcing the role of protein transport at chemical and electrical synapses to fine-tune communication between neurons.