Analysis and degradation study of glyphosate and of aminomethylphosphonic acid in natural waters by means of polymeric and ion-exchange solid-phase extraction columns followed by ion chromatography-post-column derivatization with fluorescence detection

Excessive stimulation of the N-methyl-d-aspartate (NMDA)-type glutamate receptor has been implicated in the neuronal death resulting from focal hypoxia-ischemia. Certain neurosteroids, steroids synthesized de novo in the central nervous system (CNS), have been shown to modulate the action of neurotransmitters at their cellular receptors. Pregnenolone sulfate (PS) is an abundant neurosteroid that enhances the current evoked by NMDA. Using the Ca2+-sensitive fluorescent dye, Fluo-3, AM, and a trypan blue exclusion assay, we evaluated the ability of PS to modulate NMDA-induced changes in intracellular free calcium concentration ([Ca2+]i) and neuronal death in primary cultures of rat hippocampal neurons. The results demonstrate that PS potentiates NMDA-induced increases in [Ca2+]i by 150%. Further, PS exacerbates the MK-801-sensitive neuronal death produced by acute (PS EC50=37 microM) or chronic NMDA exposure, reducing the EC50 of NMDA from 13 to 4 microM under chronic exposure conditions, whereas pregnenolone is ineffective. Our results show that PS, or related sulfated neurosteroids, may play a role in the onset of excitotoxic neuronal death in vivo.     

Low-affinity kainate receptors and long-lasting depression of NMDA-receptor-mediated currents in rat superficial dorsal horn

In an in vitro spinal cord slice preparation whole cell electrophysiological recordings of rat superficial dorsal horn neurons responding differentially to glutamate (Glu) and N-methyl-D-aspartate (NMDA) were investigated systematically for the role of kainate (KA) receptors in modulating their activity. In these neurons, coapplication of Glu and NMDA, as well as application of Glu immediately before NMDA, induced long- and short-lasting depressions of NMDA-induced currents as well as depression of NMDA-receptor-mediated excitatory postsynaptic currents. KA applied before NMDA mimicked Glu-induced attenuating effects. Furthermore, the low-affinity KA receptor antagonist 5-nitro-6,7,8,9- tetrahydrobenzo[G]indole-2,3-dione-3-oxime potentiated Glu-induced NMDA-receptor-mediated currents in neurons responding differentially to Glu and NMDA. These results provide evidence for a novel mechanism, which may relate to classical long-term depression, involving low-affinity KA receptors in long-lasting modulation of NMDA-receptor-mediated currents. This implies a physiological role of KA receptors in long-term modulation of sensory transmission in the superficial dorsal horn of rat spinal cord.     

Antagonists for group I mGluRs attenuate excitotoxic neuronal death in cortical cultures

Activation of ion channel-linked glutamate receptors, especially N-methyl-D-aspartate (NMDA) receptors, mediates the excitotoxic effects of glutamate upon central neurons. We examined the hypothesis that activation of group I metabotropic glutamate receptors (mGluRs) would increase NMDA receptor-mediated cortical neuronal death. Addition of the selective group I mGluR agonists, dihydroxyphenylglycine (DHPG) or trans-azetidine-2,4-dicarboxylic acid (t-ADA) potentiated NMDA-induced neuronal death, and application of the group I mGluR-selective antagonist, aminoindan-1,5-dicarboxylic acid (AIDA), as well as the non-selective antagonists methyl-4-carboxyphenylglycine (MCPG) or 4-carboxyphenylglycine (4CPG) reduced NMDA- and kainate-induced neuronal death in murine cortical cultures. The pro-excitotoxic effect of group I mGluR activation may be mediated largely by enhancement of glutamate release, as DHPG potentiated high potassium-stimulated glutamate release, and the protective effects of both AIDA and MCPG were abolished when NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptors were blocked immediately after toxic NMDA receptor overstimulation. The present data support the possibility that antagonizing group I mGluRs may be a useful strategy for attenuating excitotoxic neuronal death in certain disease states.     

Glutamate receptor ion channel properties predict vulnerability to cytotoxicity in a transfected nonneuronal cell line

Excessive activation of glutamate receptors is thought to play a critical role in neuronal excitotoxicity. To compare the cytotoxic potential of different glutamate receptor subtypes and correlate receptor biophysical properties with cytotoxicity, we have expressed recombinant receptors in human embryonic kidney 293 (HEK-293) cells. Survival of transfected cells was analyzed under conditions of defined agonist concentration and exposure time. For HEK-293 cells transfected with N-methyl-D-aspartate (NMDA) receptors, the EC50 for NMDA-induced cytotoxicity was 300 microM. Experiments using ion substitution, or cells expressing mutant NMDA receptors with low calcium permeability, suggested that both calcium and sodium influx through NMDA receptors contributed to cytotoxicity. In contrast, cytotoxicity was not observed in cells transfected with calcium permeable alpha-amino 3-hydroxy-5-methyl-4-isoxazole propionate- or kainate-type glutamate receptors even at saturating agonist concentrations, unless inhibitors of agonist-dependent desensitization were included. These results directly demonstrate that calcium permeability and desensitization kinetics play important roles in determining the excitotoxic potential of different glutamate receptor subtypes.     

Dopaminergic modulation of NMDA-induced whole cell currents in neostriatal neurons in slices: contribution of calcium conductances

The present experiments were designed to examine dopamine (DA) modulation of whole cell currents mediated by activation of N-methyl-D-aspartate (NMDA) receptors in visualized neostriatal neurons in slices. First, we assessed the ability of DA, D1 and D2 receptor agonists to modulate membrane currents induced by activation of NMDA receptors. The results of these experiments demonstrated that DA potentiated NMDA-induced currents in medium-sized neostriatal neurons. Potentiation of NMDA currents occurred at three different holding potentials, although it was more pronounced at -30 mV. It was mediated by D1 receptors, because it was mimicked by D1 agonists and blocked by exposure to a D1 antagonist. Activation of D2 receptors produced inconsistent effects on NMDA-induced membrane currents. Either decreases, increases, or no effects on NMDA currents occurred. Second, we examined the contributions of intrinsic, voltage-dependent conductances to DA potentiation of NMDA currents. Blockade of K+ conductances did not prevent DA enhancement of NMDA currents. However, voltage-activated Ca2+ conductances provided a major contribution to DA modulation. The dihydropyridine L-type Ca2+ channel blockers, nifedipine, and methoxyverapamil (D-600), markedly reduced but did not totally eliminate the ability of DA to modulate NMDA currents. The D1 receptor agonist SKF 38393 also enhanced Ba2+ currents in neostriatal neurons. Together, these findings provide evidence for a complex interplay between DA, NMDA receptor activation and dihydropyridine-sensitive Ca2+ conductances in controlling responsiveness of neostriatal medium-sized neurons.     

The prevention and management of stroke

Ibogaine is an hallucinogenic indole alkaloid claimed to have anti-addictive properties. Although its mechanism of action is unknown, binding studies have indicated that the drug may interact with N-methyl-D-aspartate (NMDA) receptors. We further investigated the nature of the interaction between ibogaine and NMDA receptors in voltage clamp and binding studies, and sought to confirm that the drug has NMDA receptor blocking activity in vivo. In whole-cell recordings from cultured rat hippocampal neurons, ibogaine caused a slow, concentration-dependent block of NMDA-induced currents (IC50, 3.1 microM at -60 mV). In contrast, ibogaine failed to affect either kainate- or gamma-aminobutyric acid-evoked currents. The blockade of NMDA currents was use- and voltage-dependent, and the long lasting ibogaine block could be occluded by co-application of Mg2+. Ibogaine also inhibited equilibrium [3H]dizocilpine binding to NMDA receptors in rat forebrain membranes (IC50, 3.2 microM). We conclude that ibogaine is an open channel NMDA receptor antagonist. Administration of ibogaine to mice resulted in complete protection in the maximal electroshock test (ED50, 31 mg/kg, i.p.) and partial protection against NMDA-induced lethality, confirming that ibogaine can block NMDA receptors in vivo.     

Effect of nitrous oxide on excitatory and inhibitory synaptic transmission in hippocampal cultures

Nitrous oxide (N2O; laughing gas) has been a widely used anesthetic/analgesic since the 19th century, although its cellular mechanism of action is not understood. Here we characterize the effects of N2O on excitatory and inhibitory synaptic transmission in microcultures of rat hippocampal neurons, a preparation in which anesthetic effects on monosynaptic communication can be examined in a setting free of polysynaptic network variables. Eighty percent N2O occludes peak NMDA receptor-mediated (NMDAR) excitatory autaptic currents (EACs) with no effect on the NMDAR EAC decay time course. N2O also mildly depresses AMPA receptor-mediated (AMPAR) EACs. We find that N2O inhibits both NMDA and non-NMDA receptor-mediated responses to exogenous agonist. The postsynaptic blockade of NMDA receptors exhibits slight apparent voltage dependence, whereas the blockade of AMPA receptors is not voltage dependent. Although the degree of ketamine and Mg2+ blockade of NMDA-induced responses is dependent on permeant ion concentration, the degree of N2O blockade is not. We also observe a slight and variable prolongation of GABAA receptor-mediated (GABAR) postsynaptic currents likely caused by previously reported effects of N2O on GABAA receptors. Despite the effects of N2O on both NMDA and non-NMDA ionotropic receptors, glial glutamate transporter currents and metabotropic glutamate receptor-mediated synaptic depression are not affected. Paired-pulse depression, the frequency of spontaneous miniature excitatory synaptic currents, and high-voltage-activated calcium currents are not affected by N2O. Our results suggest that the effects of N2O on synaptic transmission are confined to postsynaptic targets.     

Pregnenolone sulfate modulates NMDA receptors, inducing and potentiating acute excitotoxicity in isolated retina

Pregnenolone sulfate (PS) acts as a positive allosteric modulator of N-methyl-D-aspartate (NMDA) receptor-mediated responses. In the retina, we previously observed that the synthesis of pregnenolone and PS increases after stimulation of NMDA receptors and blockade of the synthesis reduces retinal cell death. This study was carried out to explore in the isolated and intact retina the possible role of PS in NMDA-induced excitotoxicity. Lactate dehydrogenase (LDH) measurements and morphological analysis revealed that a 90-min exogenous application of PS at 0.1-500 microM concentrations potentiated NMDA-induced cell death and at 50-500 microM concentrations caused cytotoxicity. After 45 min, either NMDA or PS caused no significant LDH release; but their co-application resulted in a high degree of toxicity. In addition, we found that a mild NMDA insult developed into serious damage when even low PS concentrations (0.1-10 microM) were used. Toxicity-inducing and -potentiating effects were specific to PS modulatory action on NMDA receptors, in that they were blocked by 4-(3-phosphonopropyl)2-piperazinecarboxylic acid (CPP) and MK-801 but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and neither dehydroepiandrosterone sulfate nor pregnenolone caused LDH release. Prevention of degenerative signs was seen in retinae pretreated with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a Cl- channel blocker, thus indicating a Na+/Cl--dependent acute mode of excitotoxic cell death responsible for PS toxicity. The positive interaction between the neurosteroid and NMDA receptors was further proved by a PS dose-dependent increase in NMDA-induced stimulation of [3H] MK-801 binding to retinal membranes. The results suggest a crucial role of PS in retinal vulnerability and propose the toxicity-potentiating effects as an important key in linking NMDA-induced endogenous synthesis to acute excitotoxicity.     

Functional change of NMDA receptors related to enhancement of susceptibility to neurotoxicity in the developing pontine nucleus

The developing neurons have been reported to be extremely susceptible to toxicity of NMDA during a restricted developmental period. Pontosubicular neuronal necrosis is a typical type of perinatal human brain lesion and often coexists with other forms of cerebral hypoxic and ischemic injuries. To determine whether functional changes of NMDA receptors related to the susceptibility to NMDA toxicity are involved in developing neurons in the pontine nucleus, we have examined the lesion produced by in vivo direct injection of NMDA into the pontine nucleus of rats at postnatal days 1-30, recorded NMDA-induced whole-cell currents from neurons in the pontine nucleus in the developing rat brainstem slices, and performed in situ hybridization for NMDA receptor subunit mRNAs in the pontine nucleus. The susceptibility to NMDA neurotoxicity peaked near postnatal day 15, and the NMDA-induced currents showed prominent reduction of the voltage-dependent block by Mg2+ near postnatal day 15. The pontine nucleus near postnatal day 15 showed distinct expression of the NMDA receptor subunit NR2C mRNA. These results suggest that the susceptibility to NMDA neurotoxicity that is enhanced in the rat pontine nucleus near postnatal day 15 is mediated by the NMDA receptor channels that are relatively insensitive to Mg2+ and that the reduction in the sensitivity of NMDA receptors to Mg2+ correlates with the expression of the NR2C. We present the possibility that functional changes in the NMDA receptor channels play a crucial role in the occurrence of developmentally specific neuronal injury.     

Decahydroisoquinolines: novel competitive AMPA/kainate antagonists with neuroprotective effects in global cerebral ischaemia

In the present studies, we have evaluated the activity of a series of glutamate receptor antagonists from the decahydroisoquinoline group of compounds both in vitro and in vivo. Compound activity at alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate receptors was assessed using ligand binding to cloned iGluR2 and iGluR5 receptors and on responses evoked by AMPA and N-methyl-D-aspartate (NMDA) in the cortical wedge preparation. In vivo, compounds were examined for antagonist activity electrophysiologically in the rat spinal cord preparation and in the gerbil model of global cerebral ischaemia. Compounds tested were LY293558, which has been shown to protect in models of focal cerebral ischaemia, LY202157 (an NMDA antagonist), LY246492 (an NMDA and AMPA receptor antagonist), LY302679, LY292025, LY307190, LY280263, LY289178, LY289525, LY294486 (AMPA/kainate antagonists) and LY382884 (an iGluR5 selective antagonist). Results obtained support a role for AMPA receptors in cerebral ischemia. LY377770 (a mixed AMPA/iGluR5 antagonist and active isomer of LY294486) demonstrated good neuroprotection with a 2-h time window and may therefore be useful in the treatment of ischaemic conditions.     

Glutamatergic N2v cells are central pattern generator interneurons of the lymnaea feeding system: new model for rhythm generation

Electrophysiological and pharmacological methods were used to examine the role of glutamate in mediating the excitatory and inhibitory responses produced by the N2v rasp phase neurons on postsynaptic cells of the Lymnaea feeding network. The N2v --> B3 motor neuron excitatory synaptic response could be mimicked by focal or bath application of -glutamate at concentrations of >/=10(-3) M. Quisqualate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were potent agonists for the B3 excitatory glutamate receptor (10(-3) M), whereas kainate only produced very weak responses at the same concentration. This suggested that non-N-methyl--aspartate (NMDA), AMPA/quisqualate receptors were present on the B3 cell. The specific non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10(-5) M) blocked 85% of the excitatory effects on the B3 cell produced by focal application of glutamate (10(-3) M), confirming the presence of non-NMDA receptors. CNQX also blocked the major part of the excitatory postsynaptic potentials on the B3 cell produced by spontaneous or current-evoked bursts of spikes in the N2v cell. As with focal application of glutamate, a small delayed component remained that was CNQX insensitive. This provided direct evidence that glutamate acting via receptors of the non-NMDA, AMPA/quisqualate type were responsible for mediating the main N2v --> B3 cell excitatory response. NMDA at 10(-2) M also excited the B3 cell, but the effects were much more variable in size and absent in one-third of the 25 B3 cells tested. NMDA effects on B3 cells were not enhanced by bath application of glycine at 10(-4) M or reduction of Mg2+ concentration in the saline to zero, suggesting the absence of typical NMDA receptors. The variability of the B3 cell responses to NMDA suggested these receptors were unlikely to be the main receptor type involved with N2v --> B3 excitation. Quisqualate and AMPA at 10(-3) M also mimicked N2v inhibitory effects on the B7 and B8 feeding motor neurons and the modulatory slow oscillator (SO) interneuron, providing further evidence for the role of AMPA/quisqualate receptors. Similar effects were seen with glutamate at the same concentration. However, CNQX could not block either glutamate or N2v inhibitory postsynaptic responses on the B7, B8, or SO cells, suggesting a different glutamate receptor subtype for inhibitory responses compared with those responsible for N2v --> B3 excitation. We conclude that glutamate is a strong candidate transmitter for the N2v cells and that AMPA/quisquate receptors of different subtypes are likely to be responsible for the excitatory and inhibitory postsynaptic responses.     

Ca2+-mediated activation of c-Jun N-terminal kinase and nuclear factor kappa B by NMDA in cortical cell cultures

We examined the possibility that c-Jun N-terminal kinase (JNK) and nuclear factor kappaB (NF-kappaB) might be involved in intracellular signaling cascades that mediate NMDA-initiated neuronal events. Exposure of cortical neurons to 100 microM NMDA induced activation of JNK within 1 min. Activity of JNK was further increased over the next 5 min and then declined by 30 min. Similarly, ionomycin, a selective Ca2+ ionophore, induced activation of JNK. The NMDA-induced activation of JNK was abrogated in the absence of extracellular Ca2+, suggesting that Ca2+ entry is necessary and sufficient for the JNK activation. Immunohistochemistry with anti-NF-kappaB antibody demonstrated nuclear translocation of NF-kappaB within 5 min following NMDA treatment. NMDA treatment also enhanced the DNA binding activity of nuclear NF-kappaB in a Ca2+-dependent manner. Treatment with 3 mM aspirin blocked the NMDA-induced activation of JNK and NF-kappaB. Neuronal death following a brief exposure to 100 microM NMDA was Ca2+ dependent and attenuated by addition of aspirin or sodium salicylate. The present study suggests that Ca2+ influx is required for NMDA-induced activation of JNK and NF-kappaB as well as NMDA neurotoxicity. This study also implies that aspirin may exert its neuroprotective action against NMDA through blocking the NMDA-induced activation of NF-kappaB and JNK.     

Effects of the abused solvent toluene on recombinant N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors expressed in Xenopus oocytes

Previous studies have shown that toluene, which is commonly abused, depresses neuronal activity and causes behavioral effects in both animals and man similar to those observed for ethanol. In this study, the oocyte expression system was used to test the hypothesis that toluene, like ethanol, inhibits the function of ionotropic glutamate receptors. Oocytes were injected with mRNA for specific N-methyl-D-aspartate (NMDA) or non-NMDA subunits and currents were recorded using conventional two-electrode voltage clamp. To enhance the low water solubility of toluene, drug solutions were prepared by mixing toluene with alkamuls (ethoxylated castor oil) at a 1:1 ratio (v:v) and diluting this mixture to the appropriate concentration with barium-containing normal frog Ringer solution. Alkamuls, up to 0.1%, had no significant effects on membrane leak currents or on NMDA-induced currents. Toluene, up to approximately 9 mM, had only minor effects on membrane leak currents but dose-dependently inhibited NMDA-mediated currents in oocytes. The inhibition of NMDA receptor currents by toluene was rapid, reversible and the potency for toluene's effects was subunit dependent. The NR1/2B subunit combination was the most sensitive with an IC50 value for toluene-induced inhibition of 0.17 mM. The NR1/2A and NR1/2C receptors were 6- and 12-fold less sensitive with IC50 values of 1.4 and 2.1 mM, respectively. In contrast, toluene up to approximately 9 mM did not inhibit kainate-induced currents in oocytes expressing GluR1, GluR1(+)R2 or GluR6 subunits. These results suggest that some of the effects of toluene on neuronal activity and behavior may be mediated by inhibition of NMDA receptors.     

Dopaminergic modulation of early signs of excitotoxicity in visualized rat neostriatal neurons

Cell swelling induced by activation of excitatory amino acid receptors is presumably the first step in a toxic cascade that may ultimately lead to cell death. Previously we showed that bath application of N-methyl-D-aspartate (NMDA) or kainate (KA) produces swelling of neostriatal cells. The present experiments examined modulation of NMDA and KA-induced cell swelling by dopamine (DA) and its receptor agonists. Nomarski optics and infra-red videomicroscopy were utilized to visualize neostriatal medium-sized neurons in thick slices from rat pups (12-18 postnatal days). Increase in somatic cross-sectional area served as the indicator of swelling induced by bath application of glutamate receptor agonists. NMDA induced cell swelling in a dose-dependent manner. Activation of DA receptors in the absence of NMDA did not produce swelling. DA and the D1 receptor agonist SKF 38393, increased the magnitude of swelling produced by NMDA. This effect was reduced in the presence of the D1 receptor antagonist, SCH 23390. In contrast, activation of D2 receptors by quinpirole decreased the magnitude of NMDA-induced cell swelling. DA slightly attenuated cell swelling induced by activation of KA receptors. Quinpirole produced a significant concentration-dependent reduction in KA-induced swelling while SKF38393 increased KA-induced swelling, but only at a low concentration of KA. Together, these results provide additional support for the hypothesis that the direction of DA modulation depends on the glutamate receptor subtype, as well as the DA receptor subtype activated. One possible consequence of these observations is that endogenous DA may be an important contributing factor in the mechanisms of cell death in Huntington's disease.     

M100907, a selective 5-HT2A receptor antagonist and a potential antipsychotic drug, facilitates N-methyl-D-aspartate-receptor mediated neurotransmission in the rat medial prefrontal cortical neurons in vitro

The technique of intracellular recording was used to examine the effect of M100907 (formerly MDL 100907), a highly selective 5-HT2A receptor antagonist and a potential antipsychotic drug (APD), on N-methyl-D-aspartate (NMDA) and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated responses in pyramidal cells of the rat medial prefrontal cortex in in vitro brain slice preparations. Bath administration of M100907, but not its inactive stereoisomer M100009, produced a 350% to 550% increase of NMDA-induced responses in a concentration-dependent manner with an EC50 value of 14 nmol/L, reminiscent of the action of clozapine. M100907 did not alter AMPA responses. Moreover, M100907 significantly increased the amplitude and duration of excitatory postsynaptic potentials and currents evoked by electrical stimulation of the forceps minor. We have generated several lines of evidence indicating that M100907 enhances glutamate receptor-mediated neurotransmission in pyramidal cells of the medial prefrontal cortex by facilitating NMDA-induced release of excitatory amino acids. The robust potentiation of NMDA receptor-mediated neurotransmission may explain, at least partly, the potential antipsychotic action of this compound. Furthermore, if M100907 proves to be an effective APD and if our findings can be extended to other atypical APDs, which are known to possess a relatively high affinity to 5-HT2A receptors, they may account for the purported efficacy of atypical APDs in alleviating some negative symptoms such as cognitive and executive functions.     

NMDA-induced superoxide production and neurotoxicity in cultured rat hippocampal neurons: role of mitochondria

Excitotoxic mechanisms are believed to be involved in the death of neurons after trauma, epileptic seizures and cerebral ischaemia. We investigated the role of mitochondrial superoxide production in excitotoxic cell death of cultured rat hippocampal neurons. Brief exposure to the selective glutamate agonist N-methyl-D-aspartate (NMDA; 100-300 microM, 10 min) induced significant neuronal death, which was sensitive to cycloheximide (1 microM) and the caspase-1 inhibitor, acetyl-Tyr-Val-Ala-Asp-chloromethylketone (10 microM). Intracellular superoxide production was monitored semiquantitatively on sister cultures from the same platings using the oxidation-sensitive probe, hydroethidine. Brief exposures to toxic NMDA concentrations induced significant increases in superoxide production which correlated with the degree of neuronal injury. However, subtoxic NMDA exposures also produced moderate, yet statistically significant increases in superoxide production. Both NMDA-induced superoxide production and neurotoxicity were reduced by inhibition of mitochondrial electron transport using either sodium cyanide (1 mM), or a combination of rotenone (2 microM) and oligomycin (2 microM). The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP, 1 microM) mimicked the effect of NMDA on mitochondrial superoxide production. Both NMDA-induced superoxide production and neurotoxicity were potentiated by FCCP (1 microM). Exposure to FCCP alone (1-10 microM, 10 min), however, failed to produce any toxicity. Our data suggest that mitochondrial superoxide production per se is not sufficient to trigger the degeneration of cultured hippocampal neurons, but that manipulation of mitochondrial activity alters NMDA-induced superoxide production and neurotoxicity.     

Developmental change of the inhibition by lead of NMDA-activated currents in cultured hippocampal neurons

The inhibition of N-methyl-D-aspartate (NMDA)-activated current in cultured fetal rat hippocampal neurons by Pb2+ was investigated at various stages of cell development. Pb2+ selectively inhibited NMDA currents recorded from young cultured neurons. In the first week of culture, Pb2+ showed the most prominent inhibition, which was gradually attenuated in the following weeks. Pb2+'s action was selective for NMDA- as opposed to either kainate- or quisqualate-induced currents. The current-voltage relationship for NMDA-induced currents in the presence of Pb2+ revealed that the effect of this cation was voltage-independent, which suggested that the site of interaction of Pb2+ with the NMDA receptor/channel is located outside the membrane electric field. Single channel studies showed that Pb2+ reduced the frequency but not the lifetime of the NMDA-activated single channel currents. Further evaluation of the mechanism of action of Pb2+ on the NMDA receptor demonstrated that this cation is a noncompetitive antagonist of both NMDA and glycine. We have demonstrated that the NMDA-induced whole cell currents change along with cell development, and the effects of Pb2+ are also dependent upon age of culture. The NMDA-induced currents in cultured rat hippocampal neurons had two components, one that decayed rapidly and another that decayed slowly. The fast component was clearly observed at concentrations of glycine higher than 1 microM, whereas the slow component reached its maximum amplitude at the glycine concentration of 1 microM. Moreover, the rapidly decaying component of NMDA-evoked whole cell currents was predominant in young cultured neurons, and its contribution to the total current was reduced in old cultured neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ifenprodil prevents glutamate cytotoxicity via polyamine modulatory sites of N-methyl-D-aspartate receptors in cultured cortical neurons

Neuroprotective effects of ifenprodil, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, against glutamate cytotoxicity were examined in cultured rat cortical neurons. The viability of the cultures was markedly reduced by a 10-min exposure to glutamate followed by incubation with glutamate-free medium for 60 min. Ifenprodil and its derivative SL 82.0715 dose-dependently prevented cell death induced by glutamate. The NMDA antagonists MK-801 and 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid also prevented glutamate cytotoxicity with a potency similar to that of ifenprodil. Ifenprodil as well as MK-801 prevented NMDA-induced cytotoxicity, but did not affect kainate-induced cytotoxicity. Glutamate cytotoxicity was inhibited by removing extracellular Ca++ during and immediately after glutamate exposure. Ifenprodil and MK-801 reduced NMDA-induced Ca++ influx measured with rhod-2. Either spermidine, a polyamine modulatory site agonist, or glycine, a strychnine-insensitive glycine site agonist, potentiated NMDA- and glutamate-induced cytotoxicity. The protective effects of ifenprodil against NMDA- and glutamate-induced cytotoxicity were significantly reduced by spermidine, but not by glycine. These findings indicate that ifenprodil protects cortical neurons against glutamate cytotoxicity by selective antagonism of the polyamine modulatory site of the NMDA receptor complex.     

Differential assembly of coexpressed glutamate receptor subunits in neurons of rat cerebral cortex

In the rat, subunits of the glutamate receptor family fall into three pharmacologically distinct groups: alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid preferring receptors (Glu R1-4), kainate preferring receptors (Glu R5-7, KA 1, KA 2), and N-methyl-D-aspartate preferring receptors (NMDA R1, NMDA R2A-2D). In the present study, we demonstrate immunocytochemically that the majority of neurons in rat cerebral cortex coexpress members of all three groups of glutamate receptor subunits, Glu R2/3, Glu R5/6/7, and NMDA R1. Using immunoaffinity purified or immunoprecipitated alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, kainate and N-methyl-D-aspartate receptors, we show that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors containing Glu R1-4, kainate receptors containing Glu R6, Glu R7, and KA 2 and N-methyl-D-aspartate receptors containing NMDA R1 each form distinct protein complexes that do not share subunits. Our data indicate that a mechanism exists which allows for the specific assembly of selected glutamate receptor subunits into functionally and structurally distinct heteromeric receptors.     

Effects of amphetamine, butorphanol, and morphine pretreatment on the maintenance and reinstatement of cocaine-reinforced responding

1. Flupirtine (Katadolon) is a member of a class of triaminopyridines and is used as a nonopioid analgesic agent with muscle relaxant properties. 2. In situ experiments have revealed that flupirtine protects against ischemic-induced insults to the retina and brain. 3. Data derived from in vitro and in vivo studies suggest that flupirtine functions as a weak N-methyl-D-aspartate (NMDA) antagonist with little evidence that it acts on AMPA-kainate type glutamate receptors. 4. No evidence could be found from binding studies to suggest that flupirtine has an affinity for any of the characterized binding sites associated with the NMDA receptor. 5. Studies on cultured cortical neurons show that the NMDA-induced influx of 45Ca2+ is more readily decreased by flupirtine when a reducing agent (dithiothreitol) is present. However, when N'-ethylmaleimide, which is thought to alkylate the NMDA receptor redox site, is present, no obvious effect on the NMDA-induced influx of 45Ca2+ is produced by flupirtine. 6. Flupirtine is also known to counteract the production of reactive oxygen species caused by ascorbate/iron as well as to prevent apoptosis in cells lacking NMDA receptors induced by oxidative stress. 7. To explain all the experimental data, it is suggested that flupirtine affects the redox state/pH/electrons in the cell. The specific way by which flupirtine antagonizes the NMDA receptor might be by an action on the known redox site of the receptor.