Neuroprotective effect of N-acetyl-aspartyl-glutamate in combination with mild hypothermia in the endothelin-1 rat model of focal cerebral ischaemia

Previously we showed that treatment with mild hypothermia (34 degrees C for 2 h) after a focal cerebral infarct was neuroprotective by reducing apoptosis in the penumbra (cortex), but not in the core (striatum) of the infarct. In this study we examined whether administration of N-acetyl-aspartyl-glutamate (NAAG) in combination with mild hypothermia could improve striatal neuroprotection in the endothelin-1 rat model. NAAG (10 mg/kg i.p.) was injected under normothermic (37 degrees C) or mild hypothermic conditions, either 40 min before or 20 min after the insult. NAAG reduced caspase 3 immunoreactivity in the striatum, irrespective of the time of administration and brain temperature. This neuroprotective effect could be explained, at least partially, by decreased nitric oxide synthase activity in the striatum and was blocked by the group II metabotropic glutamate receptor antagonist, LY341495. Hypothermia applied together with NAAG reduced both cortical and striatal caspase 3 immunoreactivity, as well as the overall ischaemic damage in these areas. However, no pronounced improvement was seen in total damaged brain volume. Extracellular glutamate levels did not correlate with the observed protection, whatever treatment protocol was applied. We conclude that treatment with NAAG causes the same degree of neuroprotection as treatment with hypothermia. Combination of the two treatments, although reducing apoptosis, does not considerably improve ischaemic damage.     

Modulation of Glutamate and Aspartate Release from Slices of Hippocampal Area CA1 by Inhibitors of Arachidonic Acid Metabolism

Abstract: Slices of hippocampal area CA1 were used to test inhibitors of arachidonic acid metabolism for their effects on glutamate/aspartate release from the CA3-derived Schaffer collateral, commissural, and ipsilateral associational terminals. Test compounds [3 µ M nordihydroguaiaretic acid (NDGA) and 1 µ M 3-[3-(4-chlorobenzyl)-3- tert -butylthio-5-isopropylindol-2-yl]-2,2-dimethyl-propanoic acid (MK-886)] that reduced the production and release of 5-lipoxygenase metabolites also selectively reduced the K⁺-evoked release of aspartate. In contrast, the cyclooxygenase inhibitor indomethacin (100 µ M ) selectively enhanced the release of glutamate. At a concentration (100 µ M ) that nonselectively depressed the release of arachidonic acid and its metabolites, NDGA markedly depressed the release of aspartate, glutamate, and GABA. An inhibitor of the 12-lipoxygenase and an inhibitor of nitric oxide synthase did not affect the K⁺-evoked release of any transmitter amino acid. These results suggest that a 5-lipoxygenase product selectively enhances aspartate release and a cyclooxygenase product selectively depresses glutamate release. They are also consistent with previous evidence that arachidonic acid and/or platelet-activating factor enhances the release and depresses the uptake of glutamate and aspartate. The K⁺-evoked release of excitatory amino acids is much more sensitive to modulation by lipid mediators than is GABA release. Activation of NMDA receptors may enhance the K⁺-evoked release of glutamate and aspartate from CA1 slices by stimulating the production and release of lipid modulators.     

Increased Nitric Oxide Synthase Activities and l-[3H]Arginine Uptake in Brain Following Portacaval Anastomosis

Abstract: Glutamatergic synaptic dysfunction has been proposed as a causal factor in portal-systemic encephalopathy. Increased in vitro and in vivo glutamate release and decreased glutamate binding to NMDA receptors were previously reported in the brains of portacaval-shunted rats. Such changes could lead to alterations in the second messenger systems coupled to glutamate receptors. As NMDA receptors have been shown to act via the nitric oxide/cyclic GMP second messenger system, we studied the activities of constitutive nitric oxide synthase (NOS), in the brains of rats following portacaval shunting. Results demonstrate that NOS activities are significantly increased in cerebellum (by 54%, p < 0.01), cerebral cortex (by 65%, p < 0.01), hippocampus (by 88%, p < 0.01), and striatum (by 64%, p < 0.01) of shunted rats compared with sham-operated controls. As l -arginine transport is a prerequisite for nitric oxide production, we also studied l -[³H]arginine transport into cerebellar and cerebral cortical synaptosomes prepared from the brains of portacaval-shunted and sham-operated rats. l -[³H]Arginine uptake was significantly increased (by ∼50%, p < 0.01) in both cerebellum and cortex. Increased NOS activities of neuronal and/or astrocytic origin and the resultant increased production of nitric oxide in brain could be the consequence of increased NMDA receptor activation following portacaval shunting. Furthermore, increased nitric oxide production could contribute to the increased cerebral blood flow consistently observed following portacaval shunting.     

Diethylmaleate decreased ascorbic acid release induced by cerebral ischemia in cerebral cortex of the anesthetized rat

The effect of diethylmaleate administration on ascorbic acid release following cerebral ischemia was investigated in anesthetized rat brain cortex. Cerebral ischemia, induced by ligating bilateral common carotid arteries and unilateral middle cerebral artery, significantly increased the extracellular ascorbic acid levels. Diethylmaleate (4 mmoles/kg, i.p.), which has been shown in earlier studies to decrease the ischemia-induced glutamate release, significantly reduced the ischemia-induced ascorbic acid release. The ischemia-induced ascorbic acid release was unaffected by perfusing NMDA receptor antagonist MK 801 (75 microM). Additionally, elevated extracellular glutamate levels, achieved by either externally applied glutamate solutions or by perfusing L-trans-pyrrolidine-2,4-dicarboxylate (PDC) (31.4 mM and 15.7 mM) to inhibit the glutamate uptake transporter, also significantly increased the extracellular ascorbic acid levels. These results suggested that ascorbic acid release in cerebral ischemia might be related to the elevated extracellular glutamate levels, which occurs following cerebral ischemia.     

Synergistic Effect of Selenium and Melatonin on Neuroprotection in Cerebral Ischemia in Rats

The synergistic scavenger effects of selenium and melatonin collectively we called Se-Mel was studied on the prevention of neuronal injury induced by ischemia/reperfusion. Male Wistar rats were treated with sodium selenite (0.1 mg/kg, i.p.) and melatonin (10 mg/kg, i.p.) 30 min before the middle carotid artery occlusion (MCAO) and immediately after MCAO to male Wistar rats and was continued for 3 days once daily at the interval of 24 h. Behavioral activity (spontaneous motor activity and motor deficit) was improved in Se-Mel-treated rats as compared to MCAO group rats. The level of glutathione and the activity of antioxidant enzymes was depleted significantly while the content of thiobarbituric acid reactive substances, protein carbonyl, and nitric oxide radical (NO^·) was increased significantly in MCAO group. Systemic administration of Se-Mel ameliorated oxidative stress and improves ischemia/reperfusion-induced focal cerebral ischemia. Se-Mel also inhibited inducible nitric oxide synthase expression in Se-Mel+MCAO group as compared to MCAO group rats. Thus, Se-Mel has shown an excellent neuroprotective effect against ischemia/reperfusion injury through an anti-ischemic pathway. In conclusion, we demonstrated that the pretreatment with Se-Mel at the onset of reperfusion, reduced post-ischemic damage, and improved neurological outcome following transient focal cerebral ischemia in male Wistar rat.     

Nitric Oxide Synthase Activity Endogenously Modulates NMDA Receptors

Abstract: We tested the possibility that endogenous nitric oxide synthase activity regulated NMDA receptors in primary cultured striatal neurons. We monitored NMDA-induced increase in intra-cellular Ca²⁺ levels with fura-2 ratio imaging, while nitric oxide synthase activity was either increased with l -arginihe (the natural substrate of nitric oxide synthase) or inhibited using nitro- l -arginine (a specific inhibitor of nitric oxide synthase). We found that the NMDA receptor effect was slowly but strongly diminished after an l -arginine (1 m M , 15 min) treatment ( l -arginine preincubation reduced the 100 μM NMDA-induced maximal effect by 30–50%). The l -arginine blockade of NMDA receptors was long-lasting but could be partially reversed by hemoglobin (100 μM , 10 min), which binds nitric oxide. This was not observed when the neurons were treated with l -arginine together with nitro- l -arginine. Our data strongly suggest that physiological nitric oxide synthase activity could regulate NMDA receptors.     

Green tea polyphenol (−)-epigallocatechin gallate reduces matrix metalloproteinase-9 activity following transient focal cerebral ischemia

Green tea polyphenol (?)-epigallocatechin gallate (EGCG) has been reported to reduce neuronal damage after cerebral ischemic insult. EGCG is known to reduce matrix metalloproteinase (MMP) activity. MMP can play an important role in the pathophysiology of neurological disorders including cerebral ischemia. The purpose of the current study was to investigate whether EGCG shows an inhibitory effect on MMP activity and neural tissue damage following transient focal cerebral ischemia. In the present study, C57BL/6 mice were subjected to 80 min of focal ischemia induced by middle cerebral artery occlusion (MCAO). Animals were killed 24 h after ischemia. EGCG (50 mg/kg) was administered intraperitoneally immediately after ischemia. Gelatin gel zymography showed an increase in the active form of MMP-9 after ischemia. EGCG reduced ischemia-induced up-regulation of the active form of MMP-9. In in situ zymography, EGCG reduced up-regulation of gelatinase activity induced by cerebral ischemia. Co-incubation with EGCG reduced gelatinase activity directly in postischemic brain section. In 2,3,5-triphenyltetrazolium chloride (TTC) assay, brain infarction was remarkable in the middle cerebral artery territory after focal cerebral ischemia. In EGCG-treated mice, infarct volume was significantly reduced compared with vehicle-treated mice. These results demonstrate that EGCG, a green tea polyphenol, may reduce up-regulation of MMP-9 activity and neuronal damage following transient focal cerebral ischemia. In addition to its antioxidant effect, MMP-9 inhibition might be a possible mechanism potentially involved in the neuroprotective effect of a green tea polyphenol, EGCG.     

Caveolin-1 regulates nitric oxide-mediated matrix metalloproteinases activity and blood-brain barrier permeability in focal cerebral ischemia and reperfusion injury

The roles of caveolin-1 (cav-1) in regulating blood-brain barrier (BBB) permeability are unclear yet. We previously reported that cav-1 was down-regulated and the production of nitric oxide (NO) induced the loss of cav-1 in focal cerebral ischemia and reperfusion injury. The present study aims to address whether the loss of cav-1 impacts on BBB permeability and matrix metalloproteinases (MMPs) activity during cerebral ischemia-reperfusion injury. We found that focal cerebral ischemia-reperfusion down-regulated the expression of cav-1 in isolated cortex microvessels, hippocampus, and cortex of ischemic brain. The down-regulation of cav-1 was correlated with the increased MMP-2 and -9 activities, decreased tight junction (TJ) protein zonula occludens (ZO)-1 expression and enhanced BBB permeability. Treatment of N(G) -nitro-L-arginine methyl ester [L-NAME, a non-selective nitric oxide synthase (NOS) inhibitor] reserved the expression of cav-1, inhibited MMPs activity, and reduced BBB permeability. To elucidate the roles of cav-1 in regulating MMPs and BBB permeability, we used two approaches including cav-1 knockdown in cultured brain microvascular endothelial cells (BMECs) in vitro and cav-1 knockout (KO) mice in vivo. Cav-1 knockdown remarkably increased MMPs activity in BMECs. Meanwhile, with focal cerebral ischemia-reperfusion, cav-1 deficiency mice displayed higher MMPs activities and BBB permeability than wild-type mice. Interestingly, the effects of L-NAME on MMPs activity and BBB permeability was partly reversed in cav-1 deficiency mice. These results, when taken together, suggest that cav-1 plays important roles in regulating MMPs activity and BBB permeability in focal cerebral ischemia and reperfusion injury. The effects of L-NAME on MMPs activity and BBB permeability are partly mediated by preservation of cav-1.     

Reduced neuronal nitric oxide synthase is involved in ischemia-induced hippocampal neurogenesis by up-regulating inducible nitric oxide synthase expression

Nitric oxide (NO), a free radical with signaling functions in the CNS, is implicated in some developmental processes, including neuronal survival, precursor proliferation, and differentiation. However, neuronal nitric oxide synthase (nNOS) -derived NO and inducible nitric oxide synthase (iNOS) -derived NO play opposite role in regulating neurogenesis in the dentate gyrus after cerebral ischemia. In this study, we show that focal cerebral ischemia reduced nNOS expression and enzymatic activity in the hippocampus. Ischemia-induced cell proliferation in the dentate gyrus was augmented in the null mutant mice lacking nNOS gene (nNOS−/−) and in the rats receiving 7-nitroindazole, a selective nNOS inhibitor, after stroke. Inhibition of nNOS ameliorated ischemic injury, up-regulated iNOS expression, and enzymatic activity in the ischemic hippocampus. Inhibition of nNOS increased and iNOS inhibitor decreased cAMP response element-binding protein phosphorylation in the ipsilateral hippocampus in the late stage of stroke. Moreover, the effects of 7-nitroindazole on neurogenesis after ischemia disappeared in the null mutant mice lacking iNOS gene (iNOS−/−). These results suggest that reduced nNOS is involved in ischemia-induced hippocampal neurogenesis by up-regulating iNOS expression and cAMP response element-binding protein phosphorylation.     

Effects of chronic L-NAME treatment on rat focal cerebral ischemia and cerebral vasoreactivity.

Nitric oxide has been shown to be involved in the regulation of cerebral blood flow and the consequences of cerebral ischemia. Short-term inhibition of its synthesis induces hypertension and increases the cortical infarct volume in focal ischemia. Our purpose was to investigate the influence of the long-term inhibition of nitric oxide synthase on infarct volume due to middle cerebral artery (MCA) occlusion and on the reactivity of cerebral arteries. Sprague Dawley rats were given N(omega)-nitro-L-arginine methyl ester (L-NAME) for 2 or 6 weeks and compared to untreated normotensive rats and untreated spontaneously hypertensive rats (SHRs). Brain nitric oxide synthase activity was measured by the 14C-L-arginine assay. Arterial blood pressure was measured in each group. Independently, the reactivity of MCA trees was studied in vitro by a perfusion technique. Cortical infarct volume was not significantly modified by either 2-week or 6-week L-NAME treatment, despite induced hypertension, whereas it was significantly higher in SHRs than in normotensive rats. The reactivity of the MCA tree was significantly affected by the treatment with a clearcut time-dependency. Compared to normotensive controls, contractility to noradrenaline and serotonin was reduced, more severely at 6 weeks, and while dilatation to acetylcholine and nitroprusside was moderately reduced at 6 weeks, dilatation to papaverine was then increased. A major difference of treated animals compared to SHRs was the decreased response to 5-hydroxytryptamine. We conclude that infarct expansion may be limited in treated animals by a progressive reduction in cerebral artery response to vasoconstrictory neurotransmitters, concomitant with augmented non-guanylate cyclase dilator responses (cf. papaverine) and some recovery of dilatation to acetylcholine.     

Neuroprotective effects of the mGlu5R antagonist MPEP towards quinolinic acid-induced striatal toxicity: involvement of pre- and post-synaptic mechanisms and lack of direct NMDA blocking activity

The aim of this work was to investigate the potential neuroprotective effects of the metabotropic glutamate receptor 5 (mGlu5R) antagonist 2-Methyl-6-(phenylethynyl)-pyridine (MPEP) towards quinolinic acid (QA)-induced striatal excitoxicity. Intrastriatal MPEP (5 nmol/0.5 micro L) significantly attenuated the body weight loss, the electroencephalographic alterations, the impairment in spatial memory and the striatal damage induced by bilateral striatal injection of QA (210 nmol/0.7 micro L). In a second set of experiments, we aimed to elucidate the mechanisms underlying the neuroprotective effects of MPEP. In microdialysis studies in naive rats MPEP (80-250 micro m through the dialysis probe) significantly reduced the increase in glutamate levels induced by 5 mm QA. In primary cultures of striatal neurons MPEP (50 micro m) reduced the toxicity induced by direct application of glutamate [measured as release of lactate dehydrogenase [LDH]). Finally, we found that 50 micro m MPEP was unable to directly block NMDA-induced effects (namely field potential reduction in corticostriatal slices, as well as LDH release and intracellular calcium increase in striatal neurons). We conclude that: (i) MPEP has neuroprotective effects towards QA-induced striatal excitotoxicity; (ii) both pre- and post-synaptic mechanisms are involved; (iii) the neuroprotective effects of MPEP do not appear to involve a direct blockade of NMDA receptors.     

Involvement of Nitric Oxide in Adenosine Release in the Developing and Adult Mouse Hippocampus

The novel type of neurotransmitter/neuromodulator nitric oxide (NO) is linked to activation of the N-methyl-D-aspartate (NMDA) class of glutamate receptors and has been shown to modify transmitter release in the brain. The inhibitory neuromodulator adenosine has been thought to act as an endogenous neuroprotectant against cerebral ischemia and neuronal damage. The effects of NO-generating compounds on the release of preloaded [3H]adenosine from hippocampal slices from developing (7-day-old) and adult (3-month-old) mice were investigated, using a superfusion system, under normal conditions and in vitro ischemia. The release of adenosine was markedly potentiated at both ages by the NO-producing compounds S-nitroso-N-acetylpenicillamine, sodium nitroprusside, and hydroxylamine. The evoked releases were reduced by the NO synthase inhibitors nitroarginine and 7-nitroindazole at both ages. They were also reduced by the inhibitor of soluble guanylyl cyclase 1H-(1,2,4-oxadiazolo(4,3a)quinoxalin-1-one (ODQ) in adults, indicating that the NO/cGMP pathway is involved in this release. Release of adenosine was also evoked when the cGMP levels were increased by superfusing slices with the phosphodiesterase inhibitor zaprinast. The markedly enhanced adenosine release under ischemic conditions was further potentiated by the ionotropic glutamate receptor agonists and NO-generating compounds, whereas zaprinast and ODQ had no effect, rendering unlikely the involvement of cGMP in the ischemic release. Moreover, NO was able to provoke substantial release of adenosine in the presence of NMDA under both normal and ischemic conditions, which could significantly add to the neuroprotective potential of this neuromodulator in both adult and developing hippocampus.     

Opioid Effects on 45Ca2+ Uptake and Glutamate Release in Rat Cerebral Cortex in Primary Culture

Abstract: Primary cultures of rat cortex, conveniently prepared from newborn animals, were used to study opioid effects on ⁴⁵Ca²⁺ uptake and glutamate release. ⁴⁵Ca²⁺ uptake, induced by treatment with glutamate or NMDA, was largely blocked by the NMDA antagonist MK-801. K⁺ depolarization-induced ⁴⁵Ca²⁺ uptake was also reduced by MK-801, indicating that the effect was mediated by glutamate release. Direct analysis verified that glutamate, and aspartate, were indeed released. Opioid peptides of the prodynorphin system were also released and these, or other peptides, were functionally active, because naloxone treatment increased glutamate release, as well as the ⁴⁵Ca²⁺ uptake induced by depolarization. Opioid agonists, selective for μ-, κ-, and δ-receptors, inhibited the ⁴⁵Ca²⁺ uptake induced by K⁺ depolarization. The combination of low concentrations of MK-801 and opioid agonists resulted in additive inhibition of K⁺- induced ⁴⁵Ca²⁺ uptake. The results indicate that this system may be useful as an in vitro CNS model for studying modulation by opioids of glutamate release and Ca²⁺ uptake under acute, and perhaps also chronic, opiate treatment.     

Serotonin 5-HT1A receptor activation prevents phosphorylation of NMDA receptor NR1 subunit in cerebral ischemia

The mechanisms involved in the neuroprotective effect of serotonin 5-HT1A receptor agonists on brain damage induced by ischemia remain to be fully elucidated. Given that serotonergic drugs may regulate N-methyl-D-aspartate (NMDA) receptor function, which is implicated in events leading to ischemia-induced neuronal cell death, this study sought to determine the effects of the selective 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), on the levels of NMDA receptor NR1 subunit in gerbil hippocampus after transient global cerebral ischemia. Pretreatment with 8-OH-DPAT (1 mg/kg) prevented the neuronal loss in CA1 subfield 72 h after ischemia. NMDA receptor NR1 levels in whole hippocampus were not affected 24 h after ischemia, but the levels of the subunit phosphorylated at the protein kinase A (PKA) site, pNR1(Ser897), were significantly increased, and this increase was prevented by the same 8-OH-DPAT dose, a probable consequence of the increased phosphatase 1 (PP1) enzyme activity found in ischemic gerbils pretreated with the 5-HT1A receptor agonist. The results suggest that NR1 subunit phosphorylation plays a role in the neuroprotective effect of 8-OH-DPAT on cell damage induced by global cerebral ischemia in the gerbil hippocampus and support the potential interest of 5-HT1A receptor activation in the search for neuroprotective strategies.     

Aminoguanidine Administration Ameliorates Hippocampal Damage After Middle Cerebral Artery Occlusion in Rat

The effects of a selective inducible nitric oxide synthase inhibitor aminoguanidine (AG) on neuronal cells survival in hippocampal CA1 region after middle cerebral artery occlusion (MCAO) were examined. Transient focal cerebral ischemia was induced in rats by 60 or 90 min of MCAO, followed by 7 days of reperfusion. AG treatment (150 mg/kg i.p.) significantly reduced total infarct volumes: by 70% after 90 min MCAO and by 95% after 60 min MCAO, compared with saline-treated ischemic group. The number of degenerating neurons in hippocampal CA1 region was also markedly lower in aminoguanidine-treated ischemic groups compared to ischemic groups without AG-treatment. The number of iNOS-positive cells significantly increased in the hippocampal CA1 region of ischemic animals, whereas it was reduced in AG-treated rats. Our findings demonstrate that aminoguanidine decreases ischemic brain damage and improves neurological recovery after transient focal ischemia induced by MCAO.     

Role of Nitric Oxide in Pathogenesis Underlying Ischemic Cerebral Damage

1. Based upon the intriguing report that nitric oxide synthase (NOS) inhibitor dose-dependently reverses N-methyl-D-aspartate (NMDA)-induced neurotoxicity observed in primary cortical cell cultures, many laboratories have investigated whether NOS inhibition is beneficial as a treatment for cerebral ischemia.2. Although the results are variable, it is likely thought that nitric oxide plays a key role in pathomechanism underlying ischemic brain damage.3. We review the experimental studies on effects of NOS inhibition on cerebral ischemia and measuring nitric oxide produced in the brain subjected to cerebral ischemia.4. Finally, the possibility of NOS inhibitors as a therapeutical tool is discussed.     

Tamoxifen inhibits nitrotyrosine formation after reversible middle cerebral artery occlusion in the rat

Tamoxifen (TAM), a widely used non-steroidal anti-estrogen, has recently been shown to be neuroprotective in a rat model of reversible middle cerebral artery occlusion (rMCAo). Tamoxifen has several potential mechanisms of action including inhibition of the release of excitatory amino acids (EAA) and nitric oxide synthase (NOS) activity. The question addressed in this study was whether TAM reduces ischemia-induced production of nitrotyrosine, considered as a footprint of the product of nitric oxide and superoxide, peroxynitrite. In rat brain, 2 h rMCAo produced a time-dependent increase in nitrotyrosine content in the cerebral cortex, as measured by Western blot analysis. Compared with vehicle, TAM significantly reduced nitrotyrosine levels in the ischemic cortex at 24 h. The neuronal (n)NOS inhibitor, 7-nitroindazole also tended to reduce nitrotyrosine, but this reduction was not statistically significant. Immunostaining for nitrotyrosine was seen in cortical neurons in the MCA territory and this immunostaining was reduced by TAM. In vitro, TAM and the calmodulin inhibitor trifluoperazine inhibited, with similar EC(50) values, the activity of recombinant nNOS as well as NOS activity in brain homogenates, measured by conversion of [(3)H]arginine to [(3)H]citrulline. There was marginal inhibition of recombinant inducible (i)NOS activity up to 100 microM TAM. These data suggest that TAM is an effective inhibitor of Ca(2+)/calmodulin-dependent NOS and the derived peroxynitrite production in transient focal cerebral ischemia and this may be one mechanism for its neuroprotective effect following rMCAo.     

Regulation of Kv4.2 channels by glutamate in cultured hippocampal neurons

Somatodendritic voltage-dependent K⁺ currents (Kv4.2) channels mediate transient A-type K⁺ currents and play critical roles in controlling neuronal excitability. Accumulating evidence has indicated that Kv4.2 channels are key regulatory components of the signaling pathways that lead to synaptic plasticity. In contrast to the extensive studies of glutamate-induced AMPA [(±) α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrate] receptors redistribution, less is known about the regulation of Kv4.2 by glutamate. In this study, we report that brief treatment with glutamate rapidly reduced total Kv4.2 levels in cultured hippocampal neurons. The glutamate effect was mimicked by NMDA, but not by AMPA. The effect of glutamate on Kv4.2 was dramatically attenuated by pre-treatment of NMDA receptors antagonist MK-801 [(5 S ,10 R )-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate] or removal of extracellular Ca²⁺. Immunocytochemical analysis showed a loss of Kv4.2 clusters on the neuronal soma and dendrites following glutamate treatment, which was also dependent on the activation of NMDA receptors and the influx of Ca²⁺. Furthermore, whole-cell patch-clamp recordings revealed that glutamate caused a hyperpolarized shift in the inactivation curve of A-type K⁺ currents, while the activation curve remained unchanged. These results demonstrate a glutamate-induced alteration of Kv4.2 channels in cultured hippocampal neurons, which might be involved in activity-dependent changes of neuronal excitability and synaptic plasticity.