N-methyl-D-aspartate Receptor Subunits NR2A Research Articles

Triiodothyronine attenuates neurocognitive dysfunction induced by sevoflurane in the developing brain of neonatal rats

BackgroundWhilst concerns have been raised about the detrimental effects of general anaesthetics on the brain's development and function in the young, reports have indicated that thyroid hormones are able to promote neurogenesis in the developing brain. This present study aimed to investigate the effects of triiodothyronine (T3) on the neonatal rat brain, following sevoflurane exposure. MethodsPostnatal day 7 (P7) ratpups were treated with Triiodothyronine (T3) (1 µg/100 g body weight, i.p. injection, once/day for 3 days) after 2% sevoflurane exposure for 6 h. They were sacrificed at either P7 (immediately), P15 or P30 and their brains were harvested to assess cell death, proliferation in the hippocampus, N-methyl-D-aspartate (NMDA) receptor subunit A and B, and a post-synaptic protein (PSD-95 in the hippocampus,). Neuro-behavioral changes in other cohorts between P27 and P30 were evaluated with Morris water maze and open field tests. ResultsSevoflurane exposure caused cell death and suppressed the proliferation of astrocytes and neurons, as well as the dendritic growth of neurons in the hippocampus which were all reversed by the administration of T3. Moreover, cognitive function, including learning, memory, and adaptability to a new environment, were impaired by sevoflurane exposure, which was also negated by T3 treatment. Furthermore, sevoflurane decreased the expression of NMDA receptor subunits NR2A and NR2B, as well as PSD-95 in the hippocampus at P15 and those effects of sevoflurane were abolished by T3 administration. ConclusionsA potential therapeutic role of T3 in protecting general anesthetic induced neuronal injury in the developing brain is likely to occur through enhancing expression of PSD-95 and the NMDA NR2A and NR2B expression.

Neuroprotective effects of dexmedetomidine against isoflurane-induced neuronal injury via glutamate regulation in neonatal rats.

BackgroundConsiderable evidences support the finding that the anesthesia reagent isoflurane increases neuronal cell death in young rats. Recent studies have shown that dexmedetomidine can reduce isoflurane-induced neuronal injury, but the mechanism remains unclear. We investigated whether isoflurane cause neurotoxicity to the central nervous system by regulating the N-methyl-D-aspartate receptor (NMDAR) and excitatory amino acid transporter1 (EAAT1) in young rats. Furthermore, we examined if dexmedetomidine could decrease isoflurane-induced neurotoxicity.MethodsNeonatal rats (postnatal day 7, n=144) were randomly divided into four groups of 36 animals each: control (saline injection without isoflurane); isoflurane (2% for 4 h); isoflurane + single dose of dexmedetomidine (75 µg/kg, 20 min before the start of 2% isoflurane for 4 h); and isoflurane + dual doses of dexmedetomidine (25 µg/kg, 20 min before and 2 h after start of isoflurane at 2% for 4 h). Six neonates from each group were euthanatized at 2 h, 12 h, 24 h, 3 days, 7 days and 28 days post-anesthesia. Hippocampi were collected and processed for protein extraction. Expression levels of the NMDAR subunits NR2A and NR2B, EAAT1 and caspase-3 were measured by western blot analysis.ResultsProtein levels of NR2A, EAAT1 and caspase-3 were significantly increased in hippocampus of the isoflurane group from 2 h to 3 days, while NR2B levels were decreased. However, the -induced increase in NR2A, EAAT1 and caspase-3 and the decrease in NR2B in isoflurane-exposed rats were ameliorated in the rats treated with single or dual doses of dexmedetomidine. Isoflurane-induced neuronal damage in neonatal rats is due in part to the increase in NR2A and EAAT1 and the decrease in NR2B in the hippocampus.ConclusionDexmedetomidine protects the brain against the use of isoflurane through the regulation of NR2A, NR2B and EAAT1. However, using the same amount of dexmedetomidine, the trend of protection is basically the same.

Hippocampal Memory Recovery After Acute Stress: A Behavioral, Morphological and Molecular Study

Several studies have shown that a single exposure to stress may improve or impair learning and memory processes, depending on the timing in which the stress event occurs with relation to the acquisition phase. However, to date there is no information about the molecular changes that occur at the synapse during the stress-induced memory modification and after a recovery period. In particular, there are no studies that have evaluated—at the same time—the temporality of stress and stress recovery period in hippocampal short-term memory and the effects on dendritic spine morphology, along with variations in N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits. The aim of our study was to take a multidimensional approach to investigate concomitant behavioral, morphological and molecular changes induced by a single restraint stress exposure (2.5 h) and a recovery period of 6 and 24 h in rats. We found that acute stress elicited a reduced preference to explore an object placed in a novel position (a hippocampal-dependent task). These changes were accompanied by increased activity of LIM kinase I (LIMK; an actin-remodeling protein) and increased levels of NR2A subunits of NMDA receptors. After 6 h of recovery from stress, rats showed similar preference to explore an object placed in a novel or familiar position, but density of immature spines increased in secondary CA1 apical dendrites, along with a transient rise in GluA2 AMPA receptor subunits. After 24 h of recovery from stress, the animals showed a preference to explore an object placed in a novel position, which was accompanied by a normalization of NMDA and AMPA receptor subunits to control values. Our data suggest that acute stress produces reversible molecular and behavioral changes 24 h after stress, allowing a full reestablishment of hippocampal-related memory. Further studies need to be conducted to deepen our understanding of these changes and their reciprocal interactions.Adaptive stress responses are a promising avenue to develop interventions aiming at restoring hippocampal function impaired by repetitive stress exposure.

The effects of walnut supplementation on hippocampal NMDA receptor subunits NR2A and NR2B of rats

Objectives: Walnuts contain numerous selected dietary factors that have an impact on brain functions, especially learning and memory formation in the hippocampus. Hippocampal N-methyl d-aspartate receptors (NMDARs) are involved in the formation of cognitive functions. In this study, we aimed to investigate the molecular effects of walnut supplementation on the hippocampal expressions of NMDARs involved in cognitive functions and lipid peroxidation levels in rats.Methods: The male Sprague-Dawley rats (6 months old, n = 24) were fed with a walnut-supplemented diet (6% walnut diet, n = 12) and a control diet (rat food, n = 12) as ad libitum for 8 weeks. At the end of this period, NMDAR subunits NR2A and NR2B in the hippocampi were assayed by western blotting. Lipid peroxidation levels were measured using the thiobarbituric acid.Results: The expression of NR2A and NR2B was elevated in the walnut-supplemented rats compared with the control group (P < 0.05). In addition, the levels of lipid peroxidation in the walnut-supplemented group were significantly decreased compared with the control group.Discussion: We suggested that walnut supplementation may have protective effects against the decline of cognitive functions by regulating NMDAR and lipid peroxidation levels in the hippocampus. The study provides evidence that selected dietary factors (polyunsaturated fatty acids, melatonin, vitamin E, and flavonoids) within walnut may help to trigger hippocampal neuronal signal transduction for the formation of learning and memory.

Making of a Synapse: Recurrent Roles of Drebrin A at Excitatory Synapses Throughout Life.

Mature excitatory synapses are composed of more than 1500 proteins postsynaptically and hundreds more that operate presynaptically. Among them, drebrin is an F-actin-binding protein that increases noticeably during juvenile synaptogenesis. Electron microscopic analysis reveals that drebrin is highly enriched specifically on the postsynaptic side of excitatory synapses. Since dendritic spines are structures specialized for excitatory synaptic transmission, the function of drebrin was probed by analyzing the ultrastructural characteristics of dendritic spines of animals with genetic deletion of drebrin A (DAKO), the adult isoform of drebrin. Electron microscopic analyses revealed that these brains are surprisingly intact, in that axo-spinous synaptic junctions are well-formed and not significantly altered in number. This normal ultrastructure may be because drebrin E, the alternate embryonic isoform, compensates for the genetic deletion of drebrin A. However, DAKO results in the loss of homeostatic plasticity of N-methyl-D-aspartate receptors (NMDARs). The NMDAR activation-dependent trafficking of the NR2A subunit-containing NMDARs from dendritic shafts into spine head cytoplasm is greatly diminished within brains of DAKO. Conversely, within brains of wild-type rodents, spines respond to NMDAR blockade with influx of F-actin, drebrin A, and NR2A subunits of NMDARs. These observations indicate that drebrin A facilitates the trafficking of NMDAR cargos in an F-actin-dependent manner to mediate homeostatic plasticity. Analysis of the brains of transgenic mice used as models of Alzheimer's disease (AD) reveals that the loss of drebrin from dendritic spines predates the emergence of synaptic dysfunction and cognitive impairment, suggesting that this form of homeostatic plasticity contributes toward cognition. Two studies suggest that the nature of drebrin's interaction with NMDARs is dependent on the receptor's subunit composition. Drebrin A can be found co-clustering with NR2B-containing NMDARs at the plasma membrane, while NR2A-containing NMDARs co-traffic into the spine cytoplasm but do not co-cluster at the plasma membrane. Most recently, we encountered a physiological condition that supports this idea. When adolescent female rats are reared under a condition of restricted food access and ad libitum wheel access, they paradoxically become excessive runners, choosing to run, even during the limited hours of food availability. This behavioral pattern is termed activity-based anorexia (ABA) and has served as an animal model for anorexia nervosa. Those animals that exhibit the greatest ABA vulnerability, in that they lose the most amount of body weight and run with greatest exuberance to the point of risking their lives, exhibit the highest levels of NR2B-NMDARs and drebrin at the postsynaptic membrane of hippocampal pyramidal neurons. Those animals that exhibit the greatest resilience to ABA, in that they run minimally under such condition, thereby losing minimal amount of weight, exhibit the highest level of NR2A-NMDARs in the spine cytoplasm and lowest levels of drebrin at the postsynaptic membrane. This pattern suggests that drebrin has dual roles: retention of NR2A-NMDARs in the reserve pool and trafficking of NR2B-NMDARs to the postsynaptic membrane, ultimately contributing to an individual's reactivity to stress. Altogether, these observations indicate that drebrin is a protein that is important for synaptic plasticity and deserves the attention of neuroscientists studying the neurobiological basis of cognition and stress reactivity.

Neural Plasticity Associated with Hippocampal PKA-CREB and NMDA Signaling Is Involved in the Antidepressant Effect of Repeated Low Dose of Yueju Pill on Chronic Mouse Model of Learned Helplessness.

Yueju pill is a traditional Chinese medicine formulated to treat syndromes of mood disorders. Here, we investigated the therapeutic effect of repeated low dose of Yueju in the animal model mimicking clinical long-term depression condition and the role of neural plasticity associated with PKA- (protein kinase A-) CREB (cAMP response element binding protein) and NMDA (N-methyl-D-aspartate) signaling. We showed that a single low dose of Yueju demonstrated antidepressant effects in tests of tail suspension, forced swim, and novelty-suppressed feeding. A chronic learned helplessness (LH) protocol resulted in a long-term depressive-like condition. Repeated administration of Yueju following chronic LH remarkably alleviated all of depressive-like symptoms measured, whereas conventional antidepressant fluoxetine only showed a minor improvement. In the hippocampus, Yueju and fluoxetine both normalized brain-derived neurotrophic factor (BDNF) and PKA level. Only Yueju, not fluoxetine, rescued the deficits in CREB signaling. The chronic LH upregulated the expression of NMDA receptor subunits NR1, NR2A, and NR2B, which were all attenuated by Yueju. Furthermore, intracerebraventricular administration of NMDA blunted the antidepressant effect of Yueju. These findings supported the antidepressant efficacy of repeated routine low dose of Yueju in a long-term depression model and the critical role of CREB and NMDA signaling.

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

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

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

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

Instant and Lasting Down-Regulation of NR1 Expression in the Hippocampus is Associated Temporally with Antidepressant Activity After Acute Yueju.

Accumulating evidence indicated that N-methyl-D-aspartate (NMDA) receptors are involved in the pathophysiology of depression and implicated in therapeutic targets. NMDA antagonists, such as ketamine, displayed fast-onset and long-lasting antidepressant activity in preclinical and clinical studies. Previous studies showed that Yueju pill exerts antidepressant effects similar to ketamine. Here, we focused on investigating the association of acute and lasting antidepressant responses of Yueju with time course changes of NMDA receptor subunits NR1, NR2A, and NR2B expressions in the hippocampus, a key region regulating depression response. As a result, Yueju reduced immobility time in the forced swimming test from 30min to 5days post a single administration. Yueju acutely decreased NR1 and NR2B protein expression in the hippocampus, with NR2A expression unaltered. NR1 expression remained down-regulated 5days post Yueju administration, whereas NR2B returned to normal level in 24h. Yueju and ketamine similarly ameliorated the depression-like symptoms at least for 72h in learned helplessness test. They both reversed the up-regulated expression of NR1 in the learned helpless mice 1 or 3days post administration. Different from ketamine, the antidepressant effects of Yueju were not influenced by blockade of amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor. These findings served as preclinical evidence that Yueju may confer acute and long-lasting antidepressant effects by favorably modulating NMDA function in the hippocampus.

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

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

Upregulation of N-methyl-D-aspartate receptor subunits and c-Fos expressing genes in PC12D cells by nobiletin.

The N-methyl-D-aspartate (NMDA) receptor plays a key role in learning and memory. Our recent studies have shown that nobiletin from citrus peels activates the cAMP response element-binding protein (CREB) signaling pathway and ameliorates NMDA receptor antagonist-induced learning impairment by activating extracellular signal-regulated kinase. For the first time, we have shown that nobiletin significantly upregulated mRNA expression of the NMDA receptor subunits NR1, NR2A, and NR2B in PC12D cells. Furthermore, c-Fos mRNA expression also increased due to the action of nobiletin. Our results indicate that nobiletin modulates the expression of essential genes for learning and memory by activating the CREB signaling pathway, and suggest that this action mechanism of nobiletin plays a crucial role in improving NMDA receptor antagonist-induced learning impairment in model animals with dementia.

Early visual experience of background contrast affects the expression of NMDA-like glutamate receptors in the optic lobe of cuttlefish, Sepia pharaonis

Visual experience is essential for the normal development of the visual system in vertebrates. Previous studies have demonstrated that N-methyl-d-aspartate (NMDA) receptors play significant roles in the activity-dependent plasticity of the developing visual system. In cephalopods, numerous findings indicate that cuttlefish behaviors show experience-dependent plasticity and are subject to environmental influences during development. Substantial evidence also suggests that glutamatergic currents and NMDA-like receptor subunits NR2A/2B are present in the optic lobe of cuttlefish. In light of these earlier findings, the present study was aimed to examine if early visual experience of background contrast may exert its influence on behaviors by affecting NMDA receptor expression in the optic lobe of cuttlefish (Sepia pharaonis). The expression of NMDA receptors in the optic lobe of S. pharaonis was first confirmed by the 1-amino-4-guanidobutane (AGB) functional assay. Newly hatched animals were then separated into two cohorts, one reared in a uniform-gray background (low-contrast, or L group) and the other raised in a black/white checkerboard background (high-contrast, or H group). At Weeks 4, 8, and 12, the optic lobes of cuttlefish were subjected to immunohistochemical and Western blot analyses using an antibody against the NR2A subunit of NMDA receptor. Although immunoreactivity of NR2A in the optic lobes of L and H group cuttlefish appeared similar, relative expression of NR2A protein in two groups of cuttlefish optic lobes changed during development, being lower at Week 4 and higher at Weeks 8 and 12 in the H group when compared to those in the L group. This result indicates that the expression of NMDA-like receptors in the optic lobe is dependent on rearing background contrast during cuttlefish development. This finding thus provides a good correlate between the expression of NMDA-like receptors and the behavioral plasticity in developing cuttlefish.

Zinc reverses glycine-dependent inactivation of NMDARs in cultured rat hippocampal neurons

In the presence of glutamate and co-agonists, e.g., glycine, the N-methyl-D-aspartate receptor (NMDAR) plays an important role in physiological and pathophysiological brain processes. Previous studies indicate glycine could inhibit NMDAR responses induced by high concentration of NMDA in hippocampal neurons. The mechanism underlying this inhibitory impact, however, has been unclear. In this study, the whole-cell patch-clamp recording and Ca(2+) imaging with Fluo-3/AM under laser scanning confocal microscope were used to analyze the possible involvement of NMDAR subunits in this effect. We found that the peak current of NMDARs and Ca(2+) influx induced by high concentration of NMDA were reduced by treatment of glycine (0.03-10 μmol L(-1)) in a dose-dependent manner, and that the glycine-dependent inhibition of NMDAR responses, which were induced at 300 μmol L(-1) NMDA, was reversed by ZnCl(2) through the blocking of the NR2A subunit of NMDARs, but was less influenced by ifenprodil, a NR2B inhibitor. Our results suggest that the glycine-dependent inactivation of NMDARs is potentially modulated by the regulatory subunit NR2A.

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

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

Impairment of long-term depression in the anterior cingulate cortex of mice with bone cancer pain

The anterior cingulate cortex (ACC) has been shown to play an important role in pain-related perception and chronic pain. However, little is known about the molecular mechanisms involved. To address this issue, we analyzed excitatory synaptic transmission and long-term synaptic plasticity in layer II/III pyramidal neurons within the rostral ACC (rACC) from mice with bone cancer pain induced by intra-tibia implantation of osteolytic fibrosarcoma cells. Ex vivo whole-cell patch-clamp recordings from rACC neurons showed no significant alterations in presynaptic glutamate release probability and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated synaptic responses in mice with bone cancer pain. However, mechanical allodynia occurred in conjunction with decreased N-methyl-d-aspartate (NMDA)/AMPA ratio of synaptic currents elicited in bilateral rACC neurons. In addition, the induction of NMDA receptor-dependent long-term depression (LTD) at rACC synapses was impaired in rACC neurons of tumor-bearing mice. Western blot analysis revealed a significant decrease in the levels of NR1, NR2A, and NR2B subunits of NMDA receptors in the rACC under bone cancer pain condition. No significant changes in overall mRNA levels for any of the NMDA receptor subunits or calpain activity were observed in the rACC of tumor-bearing mice. These results indicate that tumor-induced injury or remodeling of primary afferent sensory nerve fibers that innervate the tumor-bearing bone may cause a persistent decrease in NMDA receptor expression in rACC neurons, resulting in a loss of LTD induction, thereby leading to long-term alterations of rACC activity and creating exaggerated pain behaviors.

Brain-Derived Antigens in Lymphoid Tissue of Patients with Acute Stroke

In experimental animals, the presence of brain-derived constituents in cervical lymph nodes has been associated with the activation of local lymphocytes poised to minimize the inflammatory response after acute brain injury. In this study, we assessed whether this immune crosstalk also existed in stroke patients. We studied the clinical course, neuroimaging, and immunoreactivity to neuronal derived Ags (microtubule-associated protein-2 and N-methyl d-aspartate receptor subunit NR-2A), and myelin-derived Ags (myelin basic protein and myelin oligodendrocyte glycoprotein) in palatine tonsils and cervical lymph nodes of 28 acute stroke patients and 17 individuals free of neurologic disease. Stroke patients showed greater immunoreactivity to all brain Ags assessed compared with controls, predominantly in T cell zones. Most brain immunoreactive cells were CD68(+) macrophages expressing MHC class II receptors. Increased reactivity to neuronal-derived Ags was correlated with smaller infarctions and better long-term outcome, whereas greater reactivity to myelin basic protein was correlated with stroke severity on admission, larger infarctions, and worse outcome at follow-up. Patients also had more CD69(+) T cells than controls, indicative of T cell activation. Overall, the study showed in patients with acute stroke the presence of myelin and neuronal Ags associated with lymph node macrophages located near activated T cells. Whether the outcome of acute stroke is influenced by Ag-specific activation of immune responses mediated by CD69 lymphocytes deserves further investigation.

Metabotropic Glutamate Receptor 5 Activation Enhances Tyrosine Phosphorylation of the &lt;i&gt;N&lt;/i&gt;-methyl-&lt;small&gt;D&lt;/small&gt;-aspartate (NMDA) Receptor and NMDA-Induced Cell Death in Hippocampal Cultured Neurons

The activation of group I metabotropic glutamate receptors (mGluRs), which are coupled with Gq-protein, initiates a variety physiological responses in different types of cells. While Gq-protein-coupled receptors can upregulate N-methyl-D-aspartate (NMDA) receptor function, group I mGluR-mediated regulations of NMDA receptor function are not fully understood. To determine biochemical roles of group I mGluRs in the regulation of the NMDA receptor, we have investigated changes in tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B induced by a selective mGluR5 agonist, (RS)-chloro-5-hydroxyphenylglycine (CHPG) in hippocampal neuronal cultures. Activation of mGluR5 by CHPG increased active-forms of Src. CHPG also enhanced tyrosine phosphorylation of NR2A and NR2B in hippocampal neuronal cultures. In addition, NMDA-induced cell death was enhanced by CHPG-induced mGluR5 stimulation at the concentration, which increased tyrosine phosphorylation of Src and NR2A/2B but did not induce cell death. This effect was inhibited by selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP). The results suggest that in hippocampal neurons, mGluR5 may regulate NMDA receptor activity, involving tyrosine phosphorylation of NR2A and NR2B and may be involved in NMDA receptor-mediated cell injury.

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

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

Chronic stress modulation of prefrontal cortical NMDA receptor expression disrupts limbic structure-prefrontal cortex interaction

Chronic stress causes various detrimental effects including cognitive and affective dysfunctions. Given the recent findings emphasizing the importance of information processing between the prefrontal cortex (PFC) and limbic structures on cognitive and affective functions, impairments of these functions caused by chronic stress may be associated with stress-induced adaptive and maladaptive responses in limbic structure-PFC interaction. In this study we have shown that chronic stress disrupts limbic structure-PFC interaction by modulating N-methyl-D-aspartate (NMDA) receptor expression in the PFC. We found that chronic stress decreased expression of NR1, NR2A and NR2B subunits of NMDA receptors in the PFC but not in the motor cortex. However, the reduction in NR2B subunits of NMDA receptors was larger in the dorsal part than the ventral part of PFC. In agreement with this observation, administration of the NMDA antagonist that was more selective for NMDA receptors containing NR2B subunits induced alterations of synchronous local field potentials between the PFC and limbic structures, synaptic plasticity induction in the limbic structure-PFC pathway, and spike firing of PFC neurons that were similar to those observed in the dorsal PFC of rats exposed to chronic stress. In contrast, administration of the NMDA antagonist that was not subunit-selective resulted in electrophysiological alterations resembling to those observed in the ventral PFC of rats exposed to chronic stress. These results suggest that chronic stress disrupts NMDA receptor-dependent limbic structure-PFC information processing.

Nanoparticle-rich diesel exhaust affects hippocampal-dependent spatial learning and NMDA receptor subunit expression in female mice

We investigated the effect of exposure to nanoparticle-rich diesel exhaust (NRDE) on hippocampal-dependent spatial learning and memory function-related gene expressions in female mice. Female BALB/c mice were exposed to clean air, middle-dose NRDE (M-NRDE), high-dose NRDE (H-NRDE) or filtered diesel exhaust (F-DE) for three months. A Morris water maze apparatus was used to examine spatial learning. The expression levels of the N-methyl-D-aspartate (NMDA) receptor subunit, proinflammatory cytokines and neurotrophin mRNAs in the hippocampus were then investigated using real-time RT-PCR. Mice exposed to H-NRDE required a longer time to reach the hidden platform and showed higher mRNA expression levels of the NMDA receptor subunit NR2A, the proinflammatory cytokine CCL3, and brain-derived neurotrophic factor (BDNF) in the hippocampus, compared with the findings in the control group. These results indicate that three months of exposure to NRDE affected spatial learning and memory function-related gene expressions in the female mouse hippocampus.