Inhibition Of N-methylation Research Articles

Toluene exposure during brain growth spurt and adolescence produces differential effects on N-methyl- d-aspartate receptor-mediated currents in rat hippocampus

Toluene, an industrial organic solvent, is voluntarily inhaled as drug of abuse. Because inhibition of N-methyl- d-aspartate (NMDA) receptors is one of the possible mechanisms underlying developmental neurotoxicity of toluene, the purpose of the present study was to examine the effects of toluene exposure during two major neurodevelopmental stages, brain growth spurt and adolescence, on NMDA receptor-mediated current. Rats were administered with toluene (500 mg/kg, i.p.) or corn oil daily over postnatal days (PN) 4–9 (brain growth spurt) or PN 21–26 (early adolescence). Intracellular electrophysiological recordings employing in CA1 pyramidal neurons in the hippocampal slices were performed during PN 30–38. Toluene exposure during brain growth spurt enhanced NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) by electrical stimulation, but impaired the paired-pulse facilitation and NMDA response by exogenous application of NMDA. Toluene exposure during adolescence resulted in an increase in NMDA receptor-mediated EPSCs and a decrease in exogenous NMDA-induced currents, while lack of any effect on paired-pulse facilitation. These findings suggest that toluene exposure during brain growth spurt and adolescence might result in an increase in synaptic NMDA receptor responsiveness and a decrease in extrasynaptic NMDA receptor responsiveness, while only toluene exposure during brain growth spurt can produce presynaptic modulation in CA1 pyramidal neurons. The functional changes in NMDA receptor-mediated transmission underlying developmental toluene exposure may lead to the neurobehavioral disturbances.

Inhibition of N-methyl- d-aspartate receptor activity resulted in aberrant neuronal migration caused by delayed morphological development in the mouse neocortex

Embryonic and neonatal neocortical neurons already express functional N-methyl- d-aspartate (NMDA) receptors before they form synapses. To elucidate the role of NMDA receptors in neuronal migration in the developing neocortex, we visualized radially migrating neurons by transferring the enhanced green fluorescent protein (EGFP) gene into the ventricular zone (VZ) of the mouse neocortex using in utero electroporation at E15.5. Two days later, we prepared neocortical slices and examined the EGFP-positive cells using time-lapse imaging in the presence of the NMDA receptor antagonist Cerestat. The EGFP-positive cells generated in the VZ in the control slices exhibited a multipolar morphology, but within several hours they became bipolar (with a leading process and an axon-like process) and migrated toward the pial surface. By contrast, many of the multipolar cells in the Cerestat-treated slices failed to extend either process and become bipolar, and frequently changed direction, although they ultimately reached their destination even after Cerestat-treatment. To identify the molecules responding for mediating NMDA signaling during neuronal migration and the changes in morphology observed above, we here focused on Src family kinases (SFKs), which mediate a variety of neuronal functions including migration and neurite extension. We discovered that the activity of Src and Fyn was reduced by Cerestat. These findings suggest that NMDA receptors are involved in neuronal migration and morphological changes into a bipolar shape, and in the activation of Src and Fyn in the developing neocortex.

Dynamics of Learning in Cultured Neuronal Networks with Antagonists of Glutamate Receptors

Cognitive dysfunction may result from abnormality of ionotropic glutamate receptors. Although various forms of synaptic plasticity in learning that rely on altering of glutamate receptors have been considered, the evidence is insufficient from an informatics view. Dynamics could reflect neuroinformatics encoding, including temporal pattern encoding, spatial pattern encoding, and energy distribution. Discovering informatics encoding is fundamental and crucial to understanding the working principle of the neural system. In this article, we analyzed the dynamic characteristics of response activities during learning training in cultured hippocampal networks under normal and abnormal conditions of ionotropic glutamate receptors, respectively. The rate, which is one of the temporal configurations, was decreased markedly by inhibition of α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptors. Moreover, the energy distribution in different characteristic frequencies was changed markedly by inhibition of AMPA receptors. Spatial configurations, including regularization, correlation, and synchrony, were changed significantly by inhibition of N-methyl- d-aspartate receptors. These results suggest that temporal pattern encoding and energy distribution of response activities in cultured hippocampal neuronal networks during learning training are modulated by AMPA receptors, whereas spatial pattern encoding of response activities is modulated by N-methyl- d-aspartate receptors.

Cellular mechanisms underlying the effect of a single exposure to neonatal handling on neurotrophin-3 in the brain of 1-day-old rats

Neurotrophin-3 (NT-3) has an important role in brain development and is thus a good candidate molecule to be involved in the cellular mechanisms mediating the effects of early experiences on the brain. In the present work we employed the model of neonatal handling, which is known to affect the ability of the adult organism to respond to stressful stimuli, and determined its effects on NT-3 levels in the rat hippocampus and cortex 2, 4 and 8 h after handling on postnatal day 1. We also recorded maternal behavior during the 8 h following handling. At both the 4 and 8 h time-points there was an increase in NT-3 positive cells in field 1 of Ammon’s horn (CA1 area of the hippocampus) and parietal cortex of the handled animals. In the parietal cortex NT-3 levels increased with time following handling: at 8 h there were more NT-3 positive cells than at 4 h. During the 4 h following the end of handling, handled pups were subject to more maternal licking, indicating that the more intense maternal care could underlie the handling-induced increase in NT-3. In the hippocampus, the handling induced increase in NT-3 was cancelled by inhibition of N-methyl- d-aspartate (NMDA), AMPA/kainate, or GABA-A receptors, as well as L-type voltage-gated Ca 2+ channels. It thus appears that neonatal handling activates these neurotransmitter receptors and channels, leading to increased intracellular Ca 2+ and increased NT-3 expression. NT-3 can then activate downstream effectors and exert its morphogenetic actions and thus imprint the effects of handling on the brain.

Inhibition of N-methyl- N-nitrosourea-induced mammary carcinogenesis by molecular iodine (I 2) but not by iodide (I −) treatment : Evidence that I 2 prevents cancer promotion

We analyzed the effect of molecular iodine (I 2), potassium iodide (KI) and a subclinical concentration of thyroxine (T4) on the induction and promotion of mammary cancer induced by N-methyl- N-nitrosourea. Virgin Sprague-Dawley rats received short or continuous treatment. Continuous I 2 treated rats exhibited a strong and persistent reduction in mammary cancer incidence (30%) compared to controls (72.7%). Interruption of short or long term treatments resulted in a higher incidence in mammary cancer compared to the control groups. The protective effect of I 2 was correlated with the highest expression of the I −/Cl − transporter pendrin and with the lowest levels of lipoperoxidation expression in mammary glands. Triiodothyronine serum levels and Na +/I − symporter, lactoperoxidase, or p53 expression did not show any changes. In conclusion continuous I 2 treatment has a potent antineoplastic effect on the progression of mammary cancer and its effect may be related to a decrease in the oxidative cell environment.

Inhibition of N-methyl- d-aspartate receptors increases paraoxon-induced apoptosis in cultured neurons

Organophosphorus (OP) compounds, used as insecticides and chemical warfare agents, are potent neurotoxins. We examined the neurotoxic effect of paraoxon (O,O-diethyl O- p-nitrophenyl phosphate), an organophosphate compound, and the role of NMDA receptors as a mechanism of action in cultured cerebellar granule cells. Paraoxon is neurotoxic to cultured rat cerebellar granule cells in a time- and concentration-dependent manner. Cerebellar granule cells are less sensitive to the neurotoxic effects of paraoxon on day in vitro (DIV) 4 than neurons treated on DIV 8. Surprisingly, the N-methyl- d-aspartate (NMDA) receptor antagonist, MK-801, enhances paraoxon-mediated neurotoxicity suggesting that NMDA receptors may play a protective role. Pretreatment with a subtoxic concentration of N-methyl- d-aspartate (NMDA) [100 μM] protects about 40% of the vulnerable neurons that would otherwise die from paraoxon-induced neurotoxicity. Moreover, addition of a neuroprotective concentration of NMDA 3 h after treatment with paraoxon provides the same level of protection. Because paraoxon-mediated neuronal cell death is time-dependent, we hypothesized that apoptosis may be involved. Paraoxon increases apoptosis about 10-fold compared to basal levels. The broad-spectrum caspase inhibitor (Boc-D-FMK) and the caspase-9-specific inhibitor (Z-LEHD-FMK) protect against paraoxon-mediated apoptosis, paraoxon-stimulated caspase-3 activity and neuronal cell death. MK-801 increases, whereas NMDA blocks paraoxon-induced apoptosis and paraoxon-stimulated caspase-3 activity. These results suggest that activation of NMDA receptors protect neurons against paraoxon-induced neurotoxicity by blocking apoptosis initiated by paraoxon.

Screening of potential lactobacilli antigenotoxicity by microbial and mammalian cell-based tests

Antigenotoxicity is considered an important property for probiotic lactobacilli. The ability of non probiotic lactobacilli from dairy products and starters to inhibit two reference genotoxins: 4-nitroquinoline-1-oxide and N-methyl- N′-nitro- N-nitrosoguanidine was evaluated. The study was carried out using short-term assays with different targets, such as procaryotic cells (SOS-Chromotest for genotoxicity in Escherichia coli and Ames test for mutagenicity in Salmonella typhimurium) and eucaryotic cells (Comet assay for genotoxicity in Caco-2 enterocytes). A high proportion of strains inhibiting 4-nitroquinoline-1-oxide activity was found in Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus plantarum. Inhibition of N-methyl- N′-nitro- N-nitrosoguanidine activity occurred in only one L. acidophilus strain. All the strains with antigenotoxic properties also demonstrated antimutagenic activity and produced modifications in genotoxin spectroscopic profiles. Strain viability during and after genotoxin exposure was confirmed. Concordance of the results obtained with microbial and mammalian cell-based tests is underlined.

The effects of galanin on long-term synaptic plasticity in the CA1 area of rodent hippocampus

Learning and memory involve complex changes in neuronal excitability including long-lasting synaptic plasticity of glutamatergic synapses. The observation that the neuropeptide galanin affects performance in a number of behavioural models predicts that galanin should affect synaptic processes underlying learning. The present study in rat and mouse hippocampal slices now demonstrates that galanin inhibits long-term potentiation induced by both tetanic and theta-burst stimulation in both apical and basal dendrites of CA1 pyramidal neurones but does not affect long-term depression. This selective effect on long-term potentiation does not appear to be mediated through inhibition of N-methyl- D-aspartate or metabotropic glutamate receptor function, but likely resides downstream of receptor activation, possibly at the level of the kinase cascade that converts short-term into long-term potentiation. Our results indicate possible mechanisms by which the neuropeptide galanin may act at the molecular level to influence learning and memory in vertebrates.

Mechanisms for clearance of released N-acetylaspartylglutamate in crayfish nerve fibers: Implications for axon–glia signaling

Crayfish nerve fibers incubated with radiolabeled glutamate or glutamine accumulate these substrates and synthesize radioactive N-acetylaspartylglutamate (NAAG). Upon stimulation of the medial giant nerve fiber, NAAG is the primary radioactive metabolite released. Since NAAG activates a glial hyperpolarization comparable to that initiated by glutamate or axonal stimulation through the same receptor, we have proposed that it is the likely mediator of interactions between the medial giant axon and its periaxonal glia. This manuscript reports investigations of possible mechanisms for termination of NAAG-signaling activity. N-acetylaspartyl-[ 3H]glutamate was not accumulated from the bath saline by unstimulated crayfish giant axons or their associated glia during a 30-min incubation. Stimulation of the central nerve cord at 50 Hz during the last minute of the incubation dramatically increased the levels of radiolabeled glutamate, NAAG, and glutamine in the medial giant axon and its associated glia. These results indicate that stimulation-sensitive peptide hydrolysis and metabolic recycling of the radiolabeled glutamate occurred. There was a β-NAAG-, quisqualate- and 2-(phosphonomethyl)-pentanedioic acid-inhibitable glutamate carboxypeptidase II activity in the membrane fraction of central nerve fibers, but not in axonal or glial cytoplasmic fractions. Inactivation of this enzyme by 2-(phosphonomethyl)-pentanedioic acid or inhibition of N-methyl- D-aspartate (NMDA) receptors by MK801 reduced the glial hyperpolarization activated by high-frequency stimulation. These results indicate that axon-to-glia signaling is terminated by NAAG hydrolysis and that the glutamate formed contributes to the glial electrical response in part via activation of NMDA receptors. Both NAAG release and an increase in glutamate carboxypeptidase II activity appear to be induced by nerve stimulation.

Spreading depression induces expression of calcium-independent protein kinase C subspecies in ischaemia-sensitive cortical layers: regulation by N-methyl- d-aspartate receptors and glucocorticoids

Spreading depression is a wave of sustained depolarization challenging the energy metabolism of the cells without causing irreversible damage. In the ischaemic brain, sreading depression-like depolarization contributes to the evolution of ischaemia to infarction. The depolarization is propagated by activation of N-methyl- d-aspartate receptors, but changes in signal transduction downstream of the receptors are not known. Because protein phosphorylation is a general mechanism whereby most cellular processes are regulated, and inhibition of N-methyl- d-aspartate receptors or protein kinase C is neuroprotective, the expression of protein kinase C subspecies in spreading depression was examined. Cortical treatment with KCl induced an up-regulation of protein kinase Cδ and ζ messenger RNA at 4 and 8 h, whereas protein kinase Cα, β, γ and ϵ did not show significant changes. The gene induction was the strongest in layers 2 and 3, and was followed by an increased number of protein kinase Cδ-immunoreactive neurons. Protein kinase Cδ and ζ inductions were inhibited by pretreatment with an N-methyl- d-aspartate receptor antagonist, dizocilpine maleate, which also blocked spreading depression propagation, and with dexamethasone, which acted without blocking the propagation. Quinacrine, a phospholipase A 2 inhibitor, reduced only protein kinase Cζ induction. In addition, N G-nitro- l-arginine methyl ester, a nitric oxide synthase inhibitor, did not influence protein kinase Cδ or ζ induction, whereas 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione, an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor antagonist, and the cyclo-oxygenase inhibitors indomethacin and diclophenac tended to increase gene expression. The data show that cortical spreading depression induces Ca 2+-independent protein kinase C subspecies δ and ζ, but not Ca 2+-dependent subspecies, through activation of N-methyl- d-aspartate receptors and phospholipase A 2. Even though the signal pathway is similar to the induction described previously in ischaemia for genes implicated in delayed neuronal death, the gene inductions observed here are not necessarily pathogenetic, but may represent a general reaction to metabolic stress.

The p38 mitogen-activated protein kinase inhibitor SB203580 antagonizes the inhibitory effects of interleukin-1β on long-term potentiation in the rat dentate gyrus in vitro

Levels of the pro-inflammatory cytokine interleukin-1β are known to be elevated in patients with chronic disorders such as Alzheimer's disease. We have investigated the effects of interleukin-1β on long-term potentiation and N-methyl- d-aspartate receptor-mediated field potentials in the rat dentate gyrus in vitro utilizing field extracellular recordings obtained from the middle third of the molecular layer of the dentate gyrus. Presynaptic stimulation was applied to the commissural/association pathway at a frequency of 0.05 Hz and at a distance of 50 μm from the granule cell body layer. As previously reported, interleukin-1β (1 ng/ml) caused an inhibition of long-term potentiation (108±2% of baseline 1 h following application of tetanic stimulation compared with 145±5% in vehicle control slices). This action of interleukin-1β on long-term potentiation, as well as an inhibition of N-methyl- d-aspartate receptor-mediated field potentials, was attenuated by pre-treatment of slices with the p38 mitogen-associated protein kinase inhibitor SB203580 (1 μM). SB203580 alone had no significant affect on long-term potentiation, but did cause an increase in baseline synaptic transmission [107±2% of baseline, 1 h after SB203580 (1 μM) treatment]. The p42/44 mitogen-activated protein kinase cascade inhibitor PD98059 (50 μM) did not inhibit the interleukin-1β-induced inhibition of N-methyl- d-aspartate receptor-mediated field potentials. The cyclooxygenase inhibitor indomethacin (50 μM) was found to attenuate the interleukin-1β-induced effects on both long-term potentiation and N-methyl- d-aspartate receptor-mediated field potentials. The lipid second messenger analogue C2 ceramide (20 μM) was found to attenuate the expression of long-term potentiation (108±3% of baseline 1 h following tetanic stimulation), and this effect was not blocked by pre-treatment with SB203580. To investigate a possible role for interleukin-1β in the normal expression of long-term potentiation, the interleukin-1 receptor antagonist (25 ng/ml) was applied during the maintenance phase of long-term potentiation. This was found to depress the sustained expression of long-term potentiation (116±6% of baseline 1 h following tetanic stimulation). Our results indicate possible signalling mechanisms by which interleukin-1β at pathophysiological concentrations may serve to inhibit long-term potentiation, and also suggests a role for IL-1β in the physiological expression of synaptic plasticity in the rat dentate gyrus in vitro.

Fenamates protect neurons against ischemic and excitotoxic injury in chick embryo retina

Three fenamates (flufenamate, meclofenamate and mefenamate) were examined for their protective effect on neurons under ischemic (glucose/oxygen deprivation) or excitotoxic conditions, using the isolated retina of chick embryo as a model. Retinal damage was evaluated by histology and lactate dehydrogenase assay. Whole-cell recording was used to examine the direct effect of the fenamates on glutamate receptor-mediated currents. The fenamates protected the retina against the ischemic or excitotoxic insult. Part of the neuroprotection by the fenamates derived from inhibition of N-methyl- d-aspartate receptor-mediated currents. However, kainate receptor-mediated currents were not blocked by the fenamates, which nonetheless reduced kainate receptor-mediated retinal damage. Our results raise the possibility that fenamates may serve as lead structures in the development of novel therapeutic agents against brain ischemia.

PSA–NCAM Is Required for Activity-Induced Synaptic Plasticity

Hippocampal organotypic slice cultures maintained 10–20 days in vitro express a high level of the polysialylated embryonic form of neural cell adhesion molecule (NCAM) (PSA–NCAM). Treatment of the cultures with endoneuraminidase-N selectively removed polysialic acid (PSA) from NCAM and completely prevented induction of long-term potentiation (LTP) and long-term depression (LTD) without affecting cellular or synaptic parameters. Similarly, slices prepared from transgenic mice lacking the NCAM gene exhibited a decaying LTP. No inhibition of N-methyl- D-aspartic acid receptor–dependent synaptic responses was detected. Washout of the enzyme resulted in reexpression of PSA immunoreactivity which correlated with a complete recovery of LTP and LTD. This reexpression was blocked by TTX and low calcium and enhanced by bicuculline. Taken together, these results indicate that neuronal activity regulates the expression of PSA–NCAM at the synapse and that this expression is required for the induction of synaptic plasticity.

Stimulation and inhibition of N-methyl- D-aspartate receptors in rats: Developing a seizure model

The objective of this study was to develop an experimental rat hippocampal seizure model based on the stimulatory effects of N-methyl-D-aspartate and to determine the inhibitory effects of MK-801 on N-methyl-D-aspartate-induced seizures. Two separate experiments were performed. In the first experiment chemitrode-implanted rats were injected intracranially with increasing doses (5, 10, 20, and 30 micrograms) of N-methyl-D-aspartate into the hippocampus. Various electrophysiologic and behavioral parameters were examined to determine the dose required to reliably elicit hippocampal seizure activity without having toxic effects on the rats. In the second experiment rats were given an intraperitoneal injection of MK-801 (0.5 or 1 mg/kg), followed 20 minutes later by an intracranial injection of N-methyl-D-aspartate (20 or 30 micrograms). The ability of MK-801 to suppress N-methyl-D-aspartate-induced seizure activity was assessed in this experiment. Intrahippocampal injection of 20 micrograms of N-methyl-D-aspartate produced the shortest electrical seizure latency (193 +/- 72 seconds, p < 0.01). At this dose seizure was achieved in 80% (four of five of the animals, and the highest numbers of electrical seizures per animal were produced (2.2 +/- 0.8, p < 0.05). The group that received 30 micrograms of N-methyl-D-aspartate had a shorter latency, a longer duration of behavioral seizure and a higher number of behavioral seizures (p < 0.05). However, this group suffered a 60% (three of five) mortality rate. The addition of MK-801 significantly decreased the number of seizures per animal and the total seizure duration (p < 0.05). MK-801 also reduced the latency period. Intracranial injection of 20 micrograms of N-methyl-D-aspartate produced reliable hippocampal seizure activity without mortality. MK-801 at a dose of 1 mg/kg injected intraperitoneally had significant inhibitory effects on this seizure model.

Inhibition of N-methyl- l-aspartate activated ion current by desmethylimipramine

The tricyclic antidepressant desmethylimipramine (DMI) interacts with the NMDA receptor/ionophore complex; however, the site of the interaction has not been clearly established. Although evidence from receptor binding assays suggests that DMI interacts with the Zn 2+ binding site, other binding studies and electrophysiological studies suggest otherwise. Using the whole-cell patch clamp technique to record from cultured hippocampal neurons, we report that recovery of NMDA-activated current from block by DMI is time-dependent and this time-dependent component was not observed following preexporure of neurons to Zn 2+. These observations favor the hypothesis that DMI interacts at a binding site within the NMSA receptor/complex channel pore and not at the Zn 2+ binding site.