Mobile NMDA receptors at hippocampal synapses

Glutamate receptors are concentrated in the postsynaptic complex of central synapses. This implies a highly organized and stable postsynaptic membrane with tightly anchored receptors. Recent reports of rapid AMPA receptor insertion and removal at synapses have challenged this view. We examined the stability of synaptic NMDA receptors on cultured hippocampal neurons using the open-channel blockers (+)-MK-801 and ketamine to tag synaptic NMDA receptors. NMDA receptor-mediated EPSCs showed an anomalous recovery following "irreversible" MK-801 block. The recovery could not be attributed to MK-801 unbinding or insertion of new receptors, suggesting that membrane receptors had moved laterally into the synapse. At least 65% of synaptic NMDA receptors were mobile. Our results indicate that NMDA receptors can move laterally between synaptic and extrasynaptic pools, providing evidence for a dynamic organization of synaptic NMDA receptors in the postsynaptic complex.     

Dopamine receptors regulate NMDA receptor surface expression in prefrontal cortex neurons

Interactions between dopamine (DA) and glutamate systems in the prefrontal cortex (PFC) are important in addiction and other psychiatric disorders. Here, we examined DA receptor regulation of NMDA receptor surface expression in postnatal rat PFC neuronal cultures. Immunocytochemical analysis demonstrated that surface expression (synaptic and non-synaptic) of NR1 and NR2B on PFC pyramidal neurons was increased by the D1 receptor agonist SKF 81297 (1 microM, 5 min). Activation of protein kinase A (PKA) did not alter NR1 distribution, indicating that PKA does not mediate the effect of D1 receptor stimulation. However, the tyrosine kinase inhibitor genistein (50 microM, 30 min) completely blocked the effect of SKF 81297 on NR1 and NR2B surface expression. Protein cross-linking studies confirmed that SKF 81297 (1 microM, 5 min) increased NR1 and NR2B surface expression, and further showed that NR2A surface expression was not affected. Genistein blocked the effect of SKF 81297 on NR1 and NR2B. Surface-expressed immunoreactivity detected with a phospho-specific antibody to tyrosine 1472 of NR2B also increased after D1 agonist treatment. Our results show that tyrosine phosphorylation plays an important role in the trafficking of NR2B-containing NMDA receptors in PFC neurons and the regulation of their trafficking by DA receptors.     

Effects of ammonia and allopurinol on rat hippocampal NMDA receptors

Ammonia is considered to be the main agent responsible for hepatic encephalopathy which progressively leads to altered mental status. N‐methyl‐D‐aspartate (NMDA) is an ionotropic glutamate receptor, which is involved in synaptogenesis, memory and neurotoxicity. The aim of this study was to investigate the effects of ammonia intoxication and allopurinol, a xanthine oxidase (XO) inhibitor, on NMDA receptor subunits, NR2A and NR2B, in the hippocampus of rats. Thirty‐six male rats were divided into three groups (n = 12/group) as follows: (1)control group (phosphate buffered saline (PBS) solution); (2)ammonia group (ammonium acetate, 2.5 mmol/kg), (3)ammonia + allopurinol group (ammonium acetate, 2.5 mmol/kg, allopurinol, 50 mg/kg). Each rat received intraperitoneal injection for 28 days. Western Blotting technique was used for detecting NR2A and NR2B expressions. Both NR2A and NR2B subunit expressions decreased 27 and 11%, respectively, in ammonia group with respect to the control group. Ammonium acetate decreased significantly in NR2A subunit expressions in the hippocampus (p < 0.01). Administration of ammonia + allopurinol caused statistically significant increases in NR2A subunit expressions compared to the ammonia group (p < 0.001). The down‐regulation of NMDA receptors caused by ammonium acetate suggest that these receptors may play role in the process of hepatic encephalopathy and using allopurinol may have some protective effects in ammonia toxicity. Copyright © 2010 John Wiley & Sons, Ltd.     

Calorie restriction modulates hippocampal NMDA receptors in diet-induced obese rats

Calorie restriction (CR) has attracted increased interest since CR enhances lifespan and alters age-related decline in hippocampal-dependent cognitive functions. Obesity is associated with poor neurocognitive outcome including impaired hippocampal synaptic plasticity and cognitive abilities such as learning and memory. N-Methyl-D-aspartate receptors (NMDARs) are linked to hippocampal-dependent learning and memory, which may be stabilized by CR. In the present study, we aimed to establish the effects of CR on NMDARs in CA1 region of hippocampus in obese and non-obese rats. In addition, malondialdehyde (MDA) levels were determined as a marker for lipid peroxidation (LPO) in hippocampus. Four groups were constituted as control group (C, n?=?9), obese group (OB, n?=?10), obese calorie-restricted group (OCR, n?=?9), and non-obese calorie-restricted group (NCR, n?=?10). OCR and NCR were fed with a 60% CR diet for 10 weeks. After 10 weeks of CR, the MDA levels significantly decreased in the calorie-restricted groups. Obesity caused significant decreases in NR2A and NR2B subunit expressions in the hippocampus. The hippocampal NR2A and NR2B levels significantly increased in the OCR group compared with the OB group (P?相似文献   

Calorie restriction modulates hippocampal NMDA receptors in diet-induced obese rats

Calorie restriction (CR) has attracted increased interest since CR enhances lifespan and alters age-related decline in hippocampal-dependent cognitive functions. Obesity is associated with poor neurocognitive outcome including impaired hippocampal synaptic plasticity and cognitive abilities such as learning and memory. N-Methyl-d-aspartate receptors (NMDARs) are linked to hippocampal-dependent learning and memory, which may be stabilized by CR. In the present study, we aimed to establish the effects of CR on NMDARs in CA1 region of hippocampus in obese and non-obese rats. In addition, malondialdehyde (MDA) levels were determined as a marker for lipid peroxidation (LPO) in hippocampus. Four groups were constituted as control group (C, n?=?9), obese group (OB, n?=?10), obese calorie-restricted group (OCR, n?=?9), and non-obese calorie-restricted group (NCR, n?=?10). OCR and NCR were fed with a 60% CR diet for 10 weeks. After 10 weeks of CR, the MDA levels significantly decreased in the calorie-restricted groups. Obesity caused significant decreases in NR2A and NR2B subunit expressions in the hippocampus. The hippocampal NR2A and NR2B levels significantly increased in the OCR group compared with the OB group (P?<?0.05). In contrast, the hippocampal NR2A and NR2B levels significantly decreased in the NCR group compared with the C group (P?<?0.05). Oxidative stress can be prevented by CR, and these data may provide a molecular and cellular mechanism by which CR may regulate NMDAR-mediated response against obesity-induced changes in the hippocampus.     

The effects of isoniazid on hippocampal NMDA receptors: Protective role of Erdosteine

Isoniazid (INH) has neurotoxic effects such as seizure, poor concentration, subtle reduction in memory, anxiety, depression and psychosis. INH-induced toxic effects are thought to be through increased oxidative stress, and these effects have been shown to be prevented by antioxidant therapies in various organs. Increased oxidative stress may be playing a role in these neurotoxic effects. N-methyl D-aspartat receptors (NMDA) are a member of the ionotropic group of glutamate receptors. These receptors are involved in a wide variety of processes in the central nervous system including synaptogenesis, synaptic plasticity, memory and learning. Erdosteine is a potent antioxidant and mucolytic agent. We aimed to investigate adverse effects of INH on rat hippocampal NMDAR receptors, and to elucidate whether erdosteine prevents possible adverse effects of INH. In the present study, compared to control group, NMDAR2A (NR2A) receptors were significantly decreased and malondialdehyde (MDA), end product of lipid peroxidation, production was significantly increased in INH-treated group. On the other hand, administration of erdosteine to INH-treated group significantly increased NR2A receptors and decreased MDA production. In conclusion, decreasing NR2A receptors in hippocampus and increasing lipid peroxidation correlates with the degree of oxidative effects of INH and erdosteine protects above effect of INH on NR2A receptors and membrane damage due to lipid peroxidation by its antioxidant properties.     

Long-term potentiation selectively expressed by NMDA receptors at hippocampal mossy fiber synapses

The mossy fiber to CA3 pyramidal cell synapse (mf-CA3) provides a major source of excitation to the hippocampus. Thus far, these glutamatergic synapses are well recognized for showing a presynaptic, NMDA receptor-independent form of LTP that is expressed as a long-lasting increase of transmitter release. Here, we show that in addition to this "classical" LTP, mf-CA3 synapses can undergo a form of LTP characterized by a selective enhancement of NMDA receptor-mediated transmission. This potentiation requires coactivation of NMDA and mGlu5 receptors and a postsynaptic calcium rise. Unlike classical LTP, expression of this mossy fiber LTP is due to a PKC-dependent recruitment of NMDA receptors specifically to the mf-CA3 synapse via a SNARE-dependent process. Having two mechanistically different forms of LTP may allow mf-CA3 synapses to respond with more flexibility to the changing demands of the hippocampal network.     

Activation of synaptic NMDA receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons

Long-term potentiation (LTP) of excitatory transmission in the hippocampus likely contributes to learning and memory. The mechanisms underlying LTP at these synapses are not well understood, although phosphorylation and redistribution of AMPA receptors may be responsible for this form of synaptic plasticity. We show here that miniature excitatory postsynaptic currents (mEPSCs) in cultured hippocampal neurons reliably demonstrate LTP when postsynaptic NMDA receptors are briefly stimulated with glycine. LTP of these synapses is accompanied by a rapid insertion of native AMPA receptors and by increased clustering of AMPA receptors at the surface of dendritic membranes. Both LTP and glycine-facilitated AMPA receptor insertion are blocked by intracellular tetanus toxin (TeTx), providing evidence that AMPA receptors are inserted into excitatory synapses via a SNARE-dependent exocytosis during LTP.     

Nicotine improves ethanol-induced memory impairment: The role of dorsal hippocampal NMDA receptors

AimsThe current study was undertaken to determine the role of dorsal hippocampal N-methyl-d-aspartate (NMDA) receptors in nicotine's effect on impairment of memory by ethanol.Main methodsAdult male mice were cannulated in the CA1 regions of dorsal hippocampi and trained on a passive avoidance learning task for memory assessment.Key findingsWe found that pre-training intraperitoneal (i.p.) administration of ethanol (0.5 and 1 g/kg) decreased memory retrieval when tested 24 h later. Pre-test administration of ethanol reversed the decrease in inhibitory avoidance response induced by pre-training ethanol. Similar to ethanol, pre-test administration of nicotine (0.125–0.75 mg/kg, s.c.) prevented impairment of memory by pre-training ethanol. In the animals that received ethanol (1 g/kg, i.p) before training and tested following intra-CA1 administration of different doses of NMDA (0.0005–0.005 µg/mouse), no significant change was observed in the retrieval latencies. Co-administration of the same doses of NMDA with an ineffective dose of nicotine (0.125 mg/kg, s.c.) significantly improved the memory retrieval and mimicked the effects of pre-test administration of a higher dose of nicotine. Pre-test intra-CA1 microinjection of MK-801 (0.25–1 µg/mouse), which had no effect alone, in combination with an effective dose of nicotine (0.75 mg/kg, s.c.) prevented the improving effect of nicotine on memory impaired by pre-training ethanol. Moreover, intra-CA1 microinjection of MK-801 reversed the NMDA-induced potentiation of the nicotine response.SignificanceThe results suggest the importance of NMDA glutamate system(s) in the CA1 regions of dorsal hippocampus for improving the effect of nicotine on the ethanol-induced amnesia.     

Activation of D1 dopamine receptors increases surface expression of AMPA receptors and facilitates their synaptic incorporation in cultured hippocampal neurons

Considerable evidence indicates that neuroadaptations leading to addiction involve the same cellular processes that enable learning and memory, such as long-term potentiation (LTP), and that psychostimulants influence LTP through dopamine (DA)-dependent mechanisms. In hippocampal CA1 pyramidal neurons, LTP involves insertion of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors into excitatory synapses. We used dissociated cultures to test the hypothesis that D1 family DA receptors influence synaptic plasticity in hippocampal neurons by modulating AMPA receptor trafficking. Brief exposure (5 min) to a D1 agonist increased surface expression of glutamate receptor (GluR)1-containing AMPA receptors by increasing their rate of externalization at extrasynaptic sites. This required the secretory pathway but not protein synthesis, and was mediated mainly by protein kinase A (PKA) with a smaller contribution from Ca2+-calmodulin-dependent protein kinase II (CaMKII). Prior D1 receptor stimulation facilitated synaptic insertion of GluR1 in response to subsequent stimulation of synaptic NMDA receptors with glycine. Our results support a model for synaptic GluR1 incorporation in which PKA is required for initial insertion into the extrasynaptic membrane whereas CaMKII mediates translocation into the synapse. By increasing the size of the extrasynaptic GluR1 pool, D1 receptors may promote LTP. Psychostimulants may usurp this mechanism, leading to inappropriate plasticity that contributes to addiction-related behaviors.     

Characterisation of the expression of NMDA receptors in human astrocytes

Astrocytes have long been perceived only as structural and supporting cells within the central nervous system (CNS). However, the discovery that these glial cells may potentially express receptors capable of responding to endogenous neurotransmitters has resulted in the need to reassess astrocytic physiology. The aim of the current study was to characterise the expression of NMDA receptors (NMDARs) in primary human astrocytes, and investigate their response to physiological and excitotoxic concentrations of the known endogenous NMDAR agonists, glutamate and quinolinic acid (QUIN). Primary cultures of human astrocytes were used to examine expression of these receptors at the mRNA level using RT-PCR and qPCR, and at the protein level using immunocytochemistry. The functionality role of the receptors was assessed using intracellular calcium influx experiments and measuring extracellular lactate dehydrogenase (LDH) activity in primary cultures of human astrocytes treated with glutamate and QUIN. We found that all seven currently known NMDAR subunits (NR1, NR2A, NR2B, NR2C, NR2D, NR3A and NR3B) are expressed in astrocytes, but at different levels. Calcium influx studies revealed that both glutamate and QUIN could activate astrocytic NMDARs, which stimulates Ca2+ influx into the cell and can result in dysfunction and death of astrocytes. Our data also show that the NMDAR ion channel blockers, MK801, and memantine can attenuate glutamate and QUIN mediated cell excitotoxicity. This suggests that the mechanism of glutamate and QUIN gliotoxicity is at least partially mediated by excessive stimulation of NMDARs. The present study is the first to provide definitive evidence for the existence of functional NMDAR expression in human primary astrocytes. This discovery has significant implications for redefining the cellular interaction between glia and neurons in both physiological processes and pathological conditions.     

6-Hydroxykynurenic acid and kynurenic acid differently antagonise AMPA and NMDA receptors in hippocampal neurones

6-Hydroxykynurenic acid (6-HKA), a derivative of kynurenic acid (KYNA) extracted from Ginkgo biloba leaves, was tested for its putative glutamate receptor (GluR) antagonism in comparison to the scaffold substance. The patch-clamp method together with fast-application techniques were used to estimate inhibition by 6-HKA and KYNA of agonist binding at NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (NMDARs and AMPARs) of CA1 pyramidal neurones. 6-Hydroxykynurenic acid proved to be a low-affinity antagonist. When comparing with KYNA, 6-HKA was less potent at NMDARs (IC(50) = 136 versus 59 microM), but showed a higher affinity to AMPARs (K(B) = 22 versus 172 microM). The replacement of 6-HKA and KYNA by glutamate was investigated on outside-out patches. Both antagonists competitively inhibited AMPAR responses and displayed fast unbinding kinetics, but the derivative was significantly slower displaced than KYNA (tau = 1.63 versus 1.22 ms). Our findings demonstrate that 6-hydroxylation considerably changes the pharmacological profile of KYNA. Among the 6-derivatives of KYNA, 6-HKA shows the highest affinity to AMPARS: Despite its relatively low lipophily, these properties might be of clinical relevance under conditions that compromise the integrity of the blood-brain barrier. Furthermore, 6-HKA should be a useful tool to analyse glutamate-mediated synaptic responses.     

Downregulation of hippocampal NMDA receptor expression by prenatal exposure to dioxin.

Gestational exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) has been implicated as causative to disparities between ethnic groups with respect to learning disabilities. Dioxin is an extremely toxic environmental pollutant that bioaccumulates in maternal adipose tissue, and is transferred to the developing organism during gestation and lactation. Long-term cognitive deficits have been reported following prenatal exposure to dioxin. N-methyl-D-aspartate (NMDA) receptors in the central nervous system (CNS) have been well known to play an important role in the activity-dependent synaptic plasticity underlying learning and memory and in CNS development including brain cell differentiation. Here, the effects of prenatal exposure to dioxin on the developmental expression profiles of rat hippocampal NMDA receptor subtype 1 mRNA and protein was examined. F-344 rats were exposed to 0 and 700 ng of dioxin/kg on gestational day 15. Real-time PCR and Western blot analysis clearly revealed that dioxin significantly downregulated NMDAR1 mRNA and protein expression during the first postnatal month. The study provides support to the hypothesis that NMDA receptors are important targets for dioxin-induced neurotoxicity in F1 preweaning pups. The results also support the concept that prenatal exposure to dioxin may contribute to the pathogenesis of diseases in the adult.     

Phencyclidine and some of its analogues have distinct effects on NMDA receptors of rat hippocampal neurons

Phencyclidine (PCP) is a dissociative anesthetic agent which blocks the excitatory effect of N-methyl-D-aspartate (NMDA) in the central nervous system. To investigate the role of the PCP reactive site in the control of NMDA activation of hippocampal pyramidal cells, we have examined the action of PCP and some of its analogues on the response properties of single NMDA receptors. Application of NMDA (5-15 microM) to outside-out patches of membrane elicited bursts of ion channel openings which were greatly reduced in frequency and duration in the presence of PCP (2.5-10 microM) or m-amino-PCP (2.5-10 microM), a behaviorally active derivative of PCP. These effects of PCP were reversed when the membrane potential was shifted from negative to positive values. Application of the behaviorally inactive agent 1-piperidino-cyclohexanecarbonitrile (greater than or equal to 220 microM) left NMDA-activated currents relatively unaltered. Treatment with another analogue, m-nitro-PCP (5-20 microM), resulted in an unexpected increase in frequency of openings. At a higher concentration (100-300 microM), however, m-nitro-PCP acted like PCP in reducing frequency of opening and channel life-time. Like PCP, these effects of m-nitro-PCP were reversed at positive potentials. Taken together, these results suggest that PCP and its derivatives block the open state of the NMDA channel. Moreover, the dual effect of m-nitro-PCP shows that excitability is not necessarily decreased by PCP analogues but may instead be enhanced depending on modifications of the PCP molecule.     

Proteolysis of filament proteins in glial and neuronal cells afterIn vivo stimulation of hippocampal NMDA receptors

An intrahippocampal injection of N-methyl-D-aspartate induced the appearance of degradation products of both the 68 kiloDalton neurofilament protein and the glial fibrillary acidic protein, as revealed by immunoblot techniques. The degradation of these two filament proteins was maximal at 10 days after the lession. The degradation patterns were similar to those induced with calpains or calcium in vitro. There were no degradation effects on the 200 kD neurofilament protein as tested with both mono- and polyclonal antibodies. Consequently, the neuronal degeneration after excessive activation of NMDA receptors appears to involve calcium activation of proteolytic enzymes. The effects on the glial proteins are probably secondary to neuronal damage but could be related to calcium dependent processes.     

In vivo neuroprotective role of NMDA receptors against kainate-induced excitotoxicity in murine hippocampal pyramidal neurons

Activation of NMDA receptors has been shown to induce either neuronal cell death or neuroprotection against excitotoxicity in cultured cerebellar granule neurons in vitro. We have investigated the effects of pretreatment with NMDA on kainate-induced neuronal cell death in mouse hippocampus in vivo. The systemic administration of kainate (30 mg/kg), but not NMDA (100 mg/kg), induced severe damage in pyramidal neurons of the hippocampal CA1 and CA3 subfields 3-7 days later, without affecting granule neurons in the dentate gyrus. An immunohistochemical study using an anti-single-stranded DNA antibody and TdT-mediated dUTP nick end labeling analysis both revealed that kainate, but not NMDA, induced DNA fragmentation in the CA1 and CA3 pyramidal neurons 1-3 days after administration. Kainate-induced neuronal loss was completely prevented by the systemic administration of NMDA (100 mg/kg) 1 h to 1 day previously. No pyramidal neuron was seen with fragmented DNA in the hippocampus of animals injected with kainate 1 day after NMDA treatment. The neuroprotection mediated by NMDA was prevented by the non-competitive NMDA receptor antagonist MK-801. Taken together these results indicate that in vivo activation of NMDA receptors is capable of protecting against kainate-induced neuronal damage through blockade of DNA fragmentation in murine hippocampus.