μM MK-801 Research Articles

Behavioral performance altering effects of MK-801 in zebrafish ( Danio rerio)

MK-801, a non-competitive NMDA-R antagonist, has been utilized in the analysis of mammalian learning and memory. The zebrafish is a novel vertebrate study species that has been proposed for the analysis of the mechanisms of learning and memory. Although learning paradigms have been developed for this species, psychopharmacological characterization of its behavioral responses is rudimentary. Before one attempts the analysis of the effects of MK-801 on learning and memory in zebrafish, one needs to know whether this drug affects motor function, perception and/or motivation, factors that may influence performance in learning tasks. Here we conduct dose response analyses investigating the effects of 0, 2, 20 and 100 μM MK-801 administered 24 h or 30 min before the behavioral test, or during the test. We analyze responses in the open tank to measure motor and posture patterns, in the light dark paradigm to evaluate visual perception, and in a group preference task to attempt to quantify motivation. Our results show a significant performance alteration only in the highest (100 μM) dose groups. These fish spent more time on the bottom of their tank, showed elevated Erratic movement, increased their clockwise and counterclockwise turning frequency, and reduced the time spent near a shoal stimulus, behavioral alterations that also depended upon the timing of drug administration. Thus, using the current delivery procedures and outbred zebrafish population, the highest dose that may not lead to significant performance deficits is 20 μM, a concentration we propose to use in a future learning study in zebrafish.

Direct and indirect control of orexin/hypocretin neurons by glycine receptors.

Hypothalamic hypocretin/orexin (hcrt/orx) neurons promote arousal and reward seeking, while reduction in their activity has been linked to narcolepsy, obesity and depression. However, the mechanisms influencing the activity of hcrt/orx networks in situ are not fully understood. Here we show that glycine, a neurotransmitter best known for its actions in the brainstem and spinal cord, elicits dose dependent postsynaptic Cl⁻ currents in hcrt/orx cells in acute mouse brain slices. The effect was blocked by the glycine receptor (GLyR) antagonist strychnine and mimicked by the GlyR agonist alanine. Postsynaptic GlyRs on hcrt/orx cells remained functional during both early postnatal and adult periods, and gramicidin-perforated patch-clamp recordings revealed that they progressively switch from excitatory to inhibitory during the first two postnatal weeks. The pharmacological profile of the glycine response suggested that developed hcrt/orx neurons contain α/β-heteromeric GlyRs that lack α2-subunits, whereas α2-subunits, whereas α2-subunits are present in early postnatal hcrt/orx neurons. All postsynaptic currents (PSCs) in developed hcrt/orx cells were blocked by inhibitors of GABA and glutamate receptors, with no evidence of GlyR-mediated PSCs. However, the frequency but not amplitude of miniature PSCs was reduced by strychnine and increased by glycine in ~50% of hcrt/orx neurons. Together, these results provide the first evidence for functional GlyRs in identified hcrt/orx circuits and suggest that the activity of developed hcrt/orx cells is regulated by two GlyR pools: inhibitory extrasynaptic GlyRs located on all hcrt/orx cells and excitatory GlyRs located on presynaptic terminals contacting some hcrt/orx cells.

Synergic induction of human periodontal ligament fibroblast cell death by nitric oxide and N-methyl-D-aspartic acid receptor antagonist

PurposeNitric oxide (NO) has been known as an important regulator of osteoblasts and periodontal ligament cell activity. This study was performed to investigate the relationship between NO-mediated cell death of human periodontal ligament fibroblasts (PDLFs) and N-methyl-D-aspartic acid (NMDA) receptor antagonist (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine hydrogen maleate (MK801).MethodsHuman PDLFs were treated with various concentrations (0 to 4 mM) of sodium nitroprusside (SNP) with or without 200 µM MK801 in culture media for 16 hours and the cell medium was then removed and replaced by fresh medium containing MTS reagent for cell proliferation assay. Western blot analysis was performed to investigate the effects of SNP on the expression of Bax, cytochrome c, and caspase-3 proteins. The differences for each value among the sample groups were compared using analysis of variance with 95% confidence intervals.ResultsIn the case of SNP treatment, as a NO donor, cell viability was significantly decreased in a concentration-dependent manner. In addition, a synergistic effect was shown when both SNP and NMDA receptor antagonist was added to the medium. SNP treated PDLFs exhibited a round shape in culture conditions and were dramatically reduced in cell number. SNP treatment also increased levels of apoptotic marker protein, such as Bax and cytochrome c, and reduced caspase-3 in PDLFs. Mitogen-activated protein kinase signaling was activated by treatment of SNP and NMDA receptor antagonist.ConclusionsThese results suggest that excessive production of NO may induce apoptosis and that NMDA receptor may modulate NO-induced apoptosis in PDLFs.

1-Methyl-1,2,3,4-tetrahydroisoquinoline and established uncompetitive NMDA receptor antagonists induce tolerance to excitotoxicity

The aim of this study was to establish the antagonistic effects of 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) on NMDA receptors and its neuroprotective abilities on primary cultures of rat cerebellar granule cells exposed for 30min to 250 or 100μM glutamate. Neuronal viability was tested after 24h with propidium iodide or calcein/ethidium homodimer-1 staining. The neuroprotective potential of 100, 250 or 500μM 1MeTIQ was compared with established uncompetitive NMDA receptor antagonists, 0.5 Μm MK-801, or 5μMmemantine. These substances were applied for 30min either together with glutamate, 24 or 48h before glutamate, or 0.5h, 1h and 3h after exposure to the excitotoxin. The results demonstrated that MK-801, memantine and 500μM 1MeTIQ induced an almost complete neuroprotection when co-applied with glutamate, but lower concentrations of 1MeTIQ were slightly less effective. Similar effects for 1MeTIQ and the established NMDA receptor antagonists were observed in the pretreatment experiments, even with a 48-h lag between the application of the tested substances and the excitotoxic challenge. In the post-treatment experiments, MK-801 and memantine and 500μM 1MeTIQ applied up to 3h after the exposure to glutamate significantly reduced the excitotoxic lesion, but 1MeTIQ in lower concentrations was ineffective. These results indicate that 1MeTIQ shares neuroprotective abilities with established uncompetitive NMDA receptor antagonists, which suggests that its inhibitory effect on NMDA receptors plays a key role in its anti-excitotoxic activity. Moreover, our data disclose a new mechanism of 1MeTIQ-evoked neuroprotection based on the induction of neuronal tolerance to excitotoxicity.

Neurotoxic mechanism of homocysteine in hippocampal neurons

Homocysteine (Hcy) is a neurotoxic amino acid that accumulates in several neurodegenerative disorders. We examined the consequences of treatment of cultured rat hippocampal neurons with Hcy and the effects of folate, S-adenosyl methionine (SAM) and MK-801 on Hcy-induced neuron apoptosis and the related molecular mechanisms were also observed. Primary hippocampal neurons were treated with 250 μM Hcy for 4 h resulted in apoptosis time-dependently. 100 μM S-adenosyl methionine (SAM) and 10 μM folate significantly repressed, although 1 μM MK-801, an NMDA receptor inhibitor did not, Hcy-induced neuron apoptosis. SAM and folate significantly repressed Hcy-induced neuron DNA damage. Hcy induced neuron calcium overload through activation of NMDA receptors. Hcy treatment also induced a significant increase in MDA level, but did not affect the neurons' total antioxidant capacity (T-AOC). Hcy (250 μM) significantly increased the expression of p53 and Bax while decreased the expression of Bcl-2. SAM and folate inhibited the changes of apoptosis-related protein expression induced by Hcy. These findings indicate that Hcy compromises neuronal homeostasis by not only DNA damage, but also neuron exitotoxicity, oxidative injury, apoptosis, and expression changes of apoptosis-related proteins.

Glutamate receptor-mediated taurine release from the hippocampus during oxidative stress

BackgroundHippocampal slices swell and release taurine during oxidative stress. The influence of cellular signalling pathways on this process is unclear. Glutamate signalling can facilitate volume regulation in other CNS preparations. Therefore, we hypothesize activation of taurine release by oxidative stress results from tissue swelling and is coupled to activation of glutamate receptors.MethodsRat hippocampi were incubated at room temperature for 2 hr in artificial cerebrospinal fluid (aCSF) equilibrated with 95% O2 plus 5% CO2. For some slices, 1 mM taurine was added to the aCSF to maintain normal tissue taurine content. Slices then were perfused with aCSF at 35° C and baseline data recorded before 2 mM H2O2 was added. For some studies, mannitol or inhibitors of glutamate receptors or the volume-regulated anion channel (VRAC) were added before and during H2O2 treatment. The intensity of light transmitted through the slice (the intrinsic optical signal, IOS) was determined at 1-min intervals. Samples of perfusate were collected at 2-min intervals and amino acid contents determined by HPLC. Data were analyzed by repeated measures ANOVA and post hoc Dunnett’s test with significance indicated for p<0.05.ResultsIOS of slices prepared without taurine treatment increased significantly by 3.3±1.3% (mean±SEM) during oxidative stress. Little taurine was detected in the perfusate of these slices and the rate of taurine efflux did not change during H2O2 exposure. The α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate antagonist, 25 µM CNQX, but not the N-methyl-D-aspartate (NMDA) receptor antagonist, 10 µM MK-801, inhibited the increase in IOS during H2O2 treatment. Taurine-treated slices exposed to H2O2 showed no change in IOS; however, taurine efflux increased by 335±178%. When these slices were perfused with hypertonic aCSF (350 mOsm) or exposed to the VRAC inhibitor, 20 µM DCPIB, no increase in the taurine efflux rate was observed during H2O2 exposure. Taurine-treated slices perfused with 10 µM MK-801 during H2O2 exposure showed a 4.6±1.9% increase in IOS but no increase in the taurine efflux rate.ConclusionsTaurine efflux via VRAC is critical for volume regulation of hippocampal slices exposed to oxidative stress. This increased taurine efflux does not result from direct activation of the taurine release pathway by H2O2. NMDA receptor activation plays an important role in taurine release during oxidative stress.

Nerve growth factor increases the sensitivity to zinc toxicity and induces cell cycle arrest in PC12 cells

Zinc is a basic trace element that plays important roles in brain and, consequently, its homeostasis needs to be critically controlled. High zinc concentrations in the interneuron synaptic space may induce neuronal death through mechanisms still partially solved. Undifferentiated pheochromocytoma (PC12) cells have been used to study zinc toxicity. As these cells can be differentiated into neuronal-like cells, the results obtained from differentiated cultures are more useful to understand zinc toxicity in neurons. In this paper, we show by flow cytometry that nerve growth factor (NGF) induces PC12 cells differentiation characterized by cell cycle arrest in the G1/G0 phase, similarly to that observed in serum-deprived cultures. Zinc induces cell death in NGF-differentiated PC12 cultures with an EC 50 value of 143 ± 14 μM, which reveals a higher sensitivity with respect to undifferentiated PC12 cultures (EC 50, 308 ± 32 μM) and a similar response to that obtained in hippocampal neurons (134 ± 12 μM). Thus, the differentiation process appeared responsible for such increase in sensitivity. To further support this tenet, when the NGF differentiation was impaired in presence of 10 μM MK-801, a selective blocker of the N-methyl- d-aspartate (NMDA) receptor that plays a role in the differentiation process, the higher sensitivity to zinc was reverted to an EC 50 value of 241 ± 26 μM. Flow cytometry experiments showed that NGF-differentiated PC12 cells in presence of zinc were positive for propidium iodide but not for annexin-V labeling. These results, together with data from fluorescent labeling of nuclear fragmentation, caspase-3 activation, and reactive oxygen species generation, support the view that zinc toxicity in NGF-differentiated PC12 cells takes place mainly through a necrotic process.

GMP prevents excitotoxicity mediated by NMDA receptor activation but not by reversal activity of glutamate transporters in rat hippocampal slices

Glutamate is the main excitatory neurotransmitter in the mammalian nervous system and is essential for its normal functions. However, overstimulation of glutamatergic system due to hyperactivation of NMDA receptors and/or impairment of glutamate reuptake system has been implicated in many acute and chronic neurological diseases. Regulation of extracellular glutamate concentrations relies on the function of glutamate transporters which can be reversed in situations related to excitotoxicity. Guanosine-5′-monophosphate (GMP), a guanine nucleotide which displays important extracellular roles, such as trophic effects to neurons and astrocytes, behaves as antagonist of glutamate receptors and is neuroprotective in hippocampal slices against excitotoxicity or ischemic conditions. Hippocampal slices exposed to 1 or 10 mM glutamate, or 100 μM NMDA with 10 μM glycine for 1 h and evaluated after 6 or 18 h, showed reduced cell viability and DNA fragmentation, respectively. Glutamate- or NMDA-induced cell death was prevented by 50 μM MK-801, but only NMDA-induced cell damage was prevented by GMP (1 mM). Glutamate-induced cell viability impairment and glutamate-induced l-[ 3H]glutamate release were both prevented by adding DL-TBOA (10 μM). Otherwise, NMDA-induced cell viability loss was not prevented by 10 μM of DL-TBOA and NMDA did not induce l-[ 3H]glutamate release. Our results demonstrate that GMP is neuroprotective when acting selectively at NMDA receptors. Glutamate-induced hippocampal slice damage and glutamate release were blocked by glutamate transporter inhibitor, indicating that glutamate-induced toxicity also involves the reversal of glutamate uptake, which cannot be prevented by GMP.

Arc/Arg3.1 Translation Is Controlled by Convergent N-Methyl-D-aspartate and Gs-coupled Receptor Signaling Pathways

Arc/Arg3.1 is an immediate early gene whose expression is necessary for the late-phase of long-term potentiation (LTP) and memory consolidation. Whereas pathways regulating Arc transcription have been extensively investigated, less is known about the role of post-transcriptional mechanisms in Arc expression. Fluorescence microscopy experiments in cultured hippocampal neurons revealed that Arc protein level was dramatically increased by activation of the cAMP-dependent protein kinase (PKA) pathway, which is implicated in long-term memory. A PKA-dependent increase in Arc protein level was observed after pharmacological or synaptic activation of N-methyl-D-aspartate (NMDA) receptors, which play a critical role in both LTP induction and learning. Arc protein was also up-regulated by activation of PKA through G(s)-coupled dopamine and beta-adrenergic receptors, which regulate the late-phase of LTP and memory. When agonists for the NMDA and G(s)-coupled receptors were co-applied, they had an additive effect on Arc protein expression. Interestingly, G(s)-coupled receptor stimulation was ineffective in the presence of an NMDA receptor antagonist, suggesting calcium influx through the NMDA receptor plays a gating role in this pathway. Stimulation of the cAMP/PKA pathway did not affect Arc mRNA level or protein stability, identifying translational efficacy as the main determinant of Arc protein expression level. It is concluded that efficient Arc translation requires NMDA receptor activity, whereas a further enhancement can be achieved with activation of G(s)-coupled receptors. These experiments have, therefore, revealed remarkable similarities in the signaling pathways that control Arc expression and those that regulate LTP, learning, and memory.

Interaction between Neuronal Depolarization and MK-801 in SH-SY5Y Cells and the Rat Cortex.

ObjectiveThe interaction between MK-801, a model of psychosis and KCl-induced depolarization or electroconvulsive shock (ECS), a therapeutic model of electroconvulsive therapy (ECT), was investigated in SH-SY5Y cells and the rat frontal cortex.MethodsSH-SY5Y cells were pretreated with 1 µM MK-801 for 15 min, followed by cotreatment with 100 mM KCl for 5 min. MK-801 was reintroduced after the KCl was washed out, and the samples were incubated before harvesting. For the experiments in rats, male Sprague-Dawley rats were treated with MK-801 followed by ECS. Immunoblot analyses of glycogen synthase kinase 3β (GSK3β) (Ser9), AKT (Ser473) and extracellular legulated kinase (ERK)1/2 in SH-SY5Y cells and the rat frontal cortex were performed.ResultsKCl-induced neuronal depolarization resulted in the transient dephosphorylation of AKT (Ser473) and GSK3β (Ser9), followed by increased phosphorylation of the enzymes in SH-SY5Y cells. Cotreatment with MK-801 and KCl inhibited the initial dephosphorylation of AKT and GSK3β produced by KCl-induced neuronal depolarization. Similarly, ECS resulted in the transient dephosphorylation of AKT (Ser473) and GSK3β (Ser9), whereas cotreatment with MK-801 inhibited the initial dephosphorylation of AKT (Ser473) and GSK3β (Ser9) produced by ECS in the rat frontal cortex. No significant interaction was observed between MK-801 and KCl in the dephosphorylation of ERK1/2.ConclusionThese results suggest that an antagonistic interplay between MK-801 and neuronal depolarization by KCl or ECS is involved the regulation of AKT (Ser473) and GSK3β (Ser9) phosphorylation.

Protection from inorganic mercury effects on the in vivo dopamine release by ionotropic glutamate receptor antagonists and nitric oxide synthase inhibitors

The possible role of ionotropics glutamate receptors on the HgCl 2-induced dopamine (DA) release from rat striatum was investigated by using in vivo brain microdialysis technique after administration of selective NMDA and AMPA/Kainate receptors antagonists dizocilpine (MK-801), D (−)-2-amino-5-phoshonopentanoic acid (AP5), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Moreover, we have also studied the effects of nitric oxide synthase (NOS) inhibitors l-nitro-arginine methyl ester ( l-NAME) and 7-nitro-indazol (7-NI) on HgCl 2-induced DA release. Intraestriatal infusion of 1 mM HgCl 2 increased striatal DA to 1717.2 ± 375.4% respect to basal levels. Infusion of 1 mM HgCl 2 in 400 μM MK-801 pre-treated animals produced an increase on striatal DA levels 61% smaller than that induced in non-pre-treated animals. In the case of AP5, this treatment reduced 92% the increase produced by HgCl 2 as compared to non-pre-treated rats. Nevertheless, the administration of CNQX did not produce any effect on HgCl 2-induced dopamine release. Intrastriatal infusion of 1 mM HgCl 2 in 100 μM l-NAME pre-treated animals produced an increase on extracellular DA levels 82% smaller than produced by HgCl 2 alone. In addition, the pre-treatment with 7-NI reduced 90% the increase produced by infusion of HgCl 2 alone in rats. Thus, HgCl 2-induced DA release could be produced at last in part, by overstimulation of NMDA receptors with NO production, since administration of NMDA receptor antagonists and NOS inhibitors protected against HgCl 2 effects on DA release.

Effect of NMDA receptor antagonist on proliferation of neurospheres from embryonic brain

The N-methyl-D-aspartate (NMDA) receptor, a subtype of ionotropic glutamate receptors, plays an important role in the regulation of neuronal development, learning and memory, and neurodegenerative diseases. NMDA receptor blockade enhances neurogenesis in the hippocampal dentate gyrus in vivo. The effect of NMDA receptor antagonist on proliferation of neural progenitor cells, however, remains to be determined. We investigated changes in the diameter and number of neurospheres derived from the embryonic rat brain after NMDA receptor blockade. Cortical progenitor cells were isolated from gestational day 18 fetal rats according to the Percoll density gradient method. Cultured spheres expressed neural progenitor markers, musashi-1 and nestin. Immunohistochemical analysis demonstrated that cells in Dulbecco's modified Eagle medium/F12 containing 1% fetal bovine serum on day 8 differentiated to MAP-2-positive neurons and GFAP-positive astrocytes. The expression of NR1 and NR2B subunits of the NMDA receptor in neurospheres was detected. Neither brief nor sustained exposure to NMDA altered the diameter and number of neurospheres. Brief exposure to 30 μM MK-801, an NMDA receptor antagonist, decreased the diameter of neurospheres. Sustained exposure to 30 μM MK-801 decreased the diameter and number of neurospheres. Our results provide evidence that MK-801 directly decreased proliferation of neural progenitor cells.

Simultaneous Monitoring of Ionophore- and Inhibitor-mediated Plasma and Mitochondrial Membrane Potential Changes in Cultured Neurons

Although natural and synthetic ionophores are widely exploited in cell studies, for example, to influence cytoplasmic free calcium concentrations and to depolarize in situ mitochondria, their inherent lack of membrane selectivity means that they affect the ion permeability of both plasma and mitochondrial membranes. A similar ambiguity affects the interpretation of signals from fluorescent membrane-permeant cations (usually termed "mitochondrial membrane potential indicators"), because the accumulation of these probes is influenced by both plasma and mitochondrial membrane potentials. To resolve some of these problems a technique is developed to allow simultaneous monitoring of plasma and mitochondrial membrane potentials at single-cell resolution using a cationic and anionic fluorescent probe. A computer program is described that transforms the fluorescence changes into dynamic estimates of changes in plasma and mitochondrial potentials. Exploiting this technique, primary cultures of rat cerebellar granule neurons display a concentration-dependent response to ionomycin: low concentrations mimic nigericin by hyperpolarizing the mitochondria while slowly depolarizing the plasma membrane and maintaining a stable elevated cytoplasmic calcium. Higher ionomycin concentrations induce a stochastic failure of calcium homeostasis that precedes both mitochondrial depolarization and an enhanced rate of plasma membrane depolarization. In addition, the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone only selectively depolarizes mitochondria at submicromolar concentrations. ATP synthase reversal following respiratory chain inhibition depolarizes the mitochondria by 26 mV.

Docosahexaenoic acid facilitates cell maturation and β-adrenergic transmission in astrocytes

The effects of docosahexaenoic acid (DHA; 22:6 n-3), a major omega-3 PUFA in the mammalian brain, on the structure and function of astrocytes were studied using primary cultures from rat cerebra. Gas-liquid chromatography of methyl esters of FAs isolated from cultures exposed to individual FAs, namely, stearic acid, linoleic acid, arachidonic acid, and DHA, showed alterations in the lipid profiles of the membranes, with a preferential incorporation of the FA to which the cells were exposed. Immunofluorescence studies demonstrated that unlike treatment with other FAs, after which the astrocytes remained as immature radial forms, DHA-treated astrocytes showed distinct differentiation, having morphology comparable to those grown in normal serum-containing medium. Receptor binding studies to determine the concentration of various neurotransmitter receptors showed that DHA selectively increased the number of beta-adrenergic receptors (beta-ARs) compared with FA-untreated controls, suggesting a greater role of DHA on beta-AR expression in membranes. This was also reflected by an increase in downstream events of the beta-AR pathways, such as the induction of protein kinase A and glycogen turnover by isoproterenol (ISP), a beta-AR agonist in DHA-treated cells. Moreover, ISP completely transformed DHA-treated cells into mature astrocytes bearing long processes, as in cells grown under normal conditions. Together, our observations suggest that DHA plays a unique role in facilitating some of the vital functions of astrocytes in the developing brain.

Inhibition of Metabotropic Glutamate Receptor Signaling by the Huntingtin-binding Protein Optineurin

Huntington disease is caused by a polyglutamine expansion in the huntingtin protein (Htt) and is associated with excitotoxic death of striatal neurons. Group I metabotropic glutamate receptors (mGluRs) that are coupled to inositol 1,4,5-triphosphate formation and the release of intracellular Ca(2+) stores play an important role in regulating neuronal function. We show here that mGluRs interact with the Htt-binding protein optineurin that is also linked to normal pressure open angled glaucoma and, when expressed in HEK 293 cells, optineurin functions to antagonize agonist-stimulated mGluR1a signaling. We find that Htt is co-precipitated with mGluR1a and that mutant Htt functions to facilitate optineurin-mediated attenuation of mGluR1a signaling. In striatal cell lines derived from Htt(Q111/Q111) mutant knock-in mice mGluR5-stimulated inositol phosphate formation is also severely impaired when compared with striatal cells derived from Htt(Q7/Q7) knock-in mice. In addition, we show that a missense single nucleotide polymorphism optineurin H486R variant previously identified to be associated with glaucoma is selectively impaired in mutant Htt binding. Although optineurin H486R retains the capacity to bind to mGluR1a, optineurin H486R-dependent attenuation of mGluR1a signaling is not enhanced by the expression of mutant Htt. Because G protein-coupled receptor kinase 2 (GRK2) protein expression is relatively low in striatal tissue, we propose that optineurin may substitute for GRK2 in the striatum to mediate mGluR desensitization. Taken together, these studies identify a novel mechanism for mGluR desensitization and an additional biochemical link between altered glutamate receptor signaling and Huntington disease.

Excitotoxic Injury to Mitochondria Isolated from Cultured Neurons

Neuronal death in response to excitotoxic levels of glutamate is dependent upon mitochondrial Ca2+ accumulation and is associated with a drop in ATP levels and a loss in ionic homeostasis. Yet the mapping of temporal events in mitochondria subsequent to Ca2+ sequestration is incomplete. By isolating mitochondria from primary cultures, we discovered that glutamate treatment of cortical neurons for 10 min caused 44% inhibition of ADP-stimulated respiration, whereas the maximal rate of electron transport (uncoupler-stimulated respiration) was inhibited by approximately 10%. The Ca2+ load in mitochondria from glutamate-treated neurons was estimated to be 167 +/- 19 nmol/mg protein. The glutamate-induced Ca2+ load was less than the maximal Ca2+ uptake capacity of the mitochondria determined in vitro (363 +/- 35 nmol/mg protein). Comparatively, mitochondria isolated from cerebellar granule cells demonstrated a higher Ca2+ uptake capacity (686 +/- 71 nmol/mg protein) than the cortical mitochondria, and the glutamate-induced load of Ca2+ was a smaller percentage of the maximal Ca2+ uptake capacity. Thus, this study indicated that Ca(2+)-induced impairment of mitochondrial ATP production is an early event in the excitotoxic cascade that may contribute to decreased cellular ATP and loss of ionic homeostasis that precede commitment to neuronal death.

N-Methyl-D-aspartate Receptor Subtype Mediated Bidirectional Control of p38 Mitogen-activated Protein Kinase

N-methyl-d-aspartate receptor (NMDAR) stimulation activates many downstream mechanisms involved in both cell survival and cell death. The manner in which the NMDAR regulates one of these pathways, the p38 mitogen-activated protein kinase (p38) pathway, is currently unknown. In the present study, we have defined a developmental-, concentration-, and time-dependent phosphorylation and subsequent dephosphorylation of p38. In cultured hippocampal neurons 7-8 days in vitro (DIV7-8), NMDAR stimulation leads to a concentration-dependent increase in p38 phosphorylation (phospho-p38). However, in more mature neurons (>DIV17) application of NMDA produces concentration-dependent effects, such that low concentrations result in sustained increases in phospho-p38 levels, and high concentrations dephosphorylate p38 within 5 min. Conantokin G, an antagonist of NR1/2A/2B and NR1/2B receptors, inhibits p38 phosphorylation, while NR1/2B-specific antagonists prevent the rapid dephosphorylation of p38 without affecting p38 activation. Furthermore, inhibition of calcineurin prevents the activation of p38, whereas inhibition of phosphoinositide 3-kinase (PI3K) prevents the rapid dephosphorylation of p38. Our results support the presence of subtype-dependent pathways regulating p38 activation and deactivation: one involves NR1/2A/2B receptors activating calcineurin and resulting in p38 phosphorylation, and the other utilizes NR1/2B receptors binding to and activating PI3K and leading to the dephosphorylation of p38 in a manner involving both NR1/2A/2B receptor activation and tyrosine phosphorylation of NR2B. The ability of NMDAR subtype-specific mechanisms to regulate p38 has implications for NMDAR-mediated synaptic plasticity, gene regulation, and excitotoxicity.

Seizures induced by microperfusion of glutamate and glycine in the hippocampus of rats pretreated with latrunculin A

Changes in the membrane distribution of N-methyl- d-aspartate (NMDA) glutamate receptors seem to produce dramatic modifications in neuronal excitability and other properties of the neuron. In order to determine in vivo if these effects are due to the binding of extracellular glutamate and glycine to NMDA extrasynaptic receptors, we perfused the hippocampus of freely moving rats with the actin depolymerizant agent latrunculin A (4 μM) through microdialysis probes. One month later, continuous microperfusion of glutamate (1 mM) or glycine (1 mM) was used to induce epileptic seizures in the animals pretreated with latrunculin A. Glutamate microperfusion induced seizures in 50% of the animals studied, and glycine induced seizures in 75% of the rats. However, no effect was observed on control rats, or on those animals previously treated with picrotoxin. Simultaneous microperfusion of 100 μM MK-801 significantly reduced the number and duration of seizures induced by both glutamate and glycine. This study demonstrates that the application of latrunculin A results in long-term changes in susceptibility to the epileptogenic action of glutamate and glycine.

Tissue plasminogen activator mediates amyloid-induced neurotoxicity via Erk1/2 activation

Tissue plasminogen activator (tPA) is the main activator of plasminogen into plasmin in the brain where it may have beneficial roles but also neurotoxic effects that could be plasmin dependent or not. Little is known about the substrates and pathways that mediate plasmin-independent tPA neurotoxicity. Here we show in primary hippocampal neurons that tPA promotes a catalytic-independent activation of the extracellular regulated kinase (Erk)1/2 signal transduction pathway through the N-methyl-D-aspartate receptor, G-proteins and protein kinase C. This results in GSK3 activation in a process that requires de novo synthesis of proteins, and leads to tau aberrant phosphorylation, microtubule destabilization and apoptosis. Similar effects are produced by amyloid aggregates in a tPA-dependent manner, as demonstrated by pharmacological treatments and in wt and tPA-/- mice neurons. Consistently, in Alzheimer's disease (AD) patients' brains, high levels of tPA colocalize with amyloid-rich areas, activated Erk1/2 and phosphorylated tau. This is the first demonstration of an intracellular pathway by which tPA triggers kinase activation, tau phosphorylation and neurotoxicity, suggesting a key role for this molecule in AD pathology.

Crush injury induces NMDA-receptor-dependent delayed nerve cell death in rat entorhinal–hippocampal slice cultures

The aim of the present study was to develop an in vitro model that produces an injury-induced delayed nerve cell death. We used entorhinal–hippocampal slices from 12-day-old rats, which were cultured for 14 days before experiments prior to a crush injury in the middle layers of the entorhinal cortex. Crush injury increased specific propidium iodide (PI) fluorescence at the site of primary injury at day 1. Specific PI fluorescence decreased over the following days, with a slower decrease taking place at days 4–7. At days 4–7, PI fluorescence increased in CA1 and CA3. Treatment with 30 μM MK-801 for 2 h at the time of crush injury prevented the increase in PI fluorescence at days 4–7 at the site of injury, in CA1, and in CA3. Crush injury induced TUNEL-positive cells at the site of injury at days 1 and 5. MK-801 markedly reduced the number of TUNEL-positive cells observed at day 5, but had no effect on the number of TUNEL-positive cells at day 1. The present data indicate that crush injury may induce two types of secondary cell death. The first, early, type of cell death is induced quickly and is maximal at day 1, after which the dead cells are gradually removed. The second, delayed, type of cell death appears at day 4. NMDA receptor antagonism prevents the induction of the delayed cell death but has no effect on the early cell death.