Global cerebral ischemia and reperfusion alters NMDA receptor binding in canine brain

We employed a canine model to test whether binding to theN-methyl-d-aspartate (NMDA) class of glutamate receptor channels is altered by global cerebral ischemia and/or reperfusion. Ischemia was induced by 10-min cardiac arrest, followed by restoration of spontaneous circulation for periods of 0, 0.5, 2, 4, and 24 h. In vitro autoradiography was performed on frozen brain sections with three radioligands: [³H]glutamate (under conditions to label the NMDA site), [³H]glycine, and [³H]MK-801. Modest decreases in [³H]glutamate and [³H]MK-801 binding were seen in several regions of hippocampus, and parietal and temporal cortex at early times after reperfusion, with values returning toward control by 24 h. In the striatum, a different pattern was seen: [³H]glutamate and [³H]MK-801 binding increased 50–200% at 0.5–4 h after the start of reperfusion, returning toward control levels by 24 h. These increases correlate with findings of increased sensitivity to NMDA-stimulated release of dopamine from striatal tissue in the same model (Werling et al., 1993), and suggest that changes in tissue receptors may contribute to the selective vulnerability to ischemic damage during the first hours following reperfusion.     

Alterations of excitatory amino acid receptors in the brain of managese-treated mice

An excessive activation of excitatory amino acid (EAA) receptors has been associated with oxidative stress, which is considered the primary cause of manganese (Mn) poisoning neurotoxicity. Therefore, the EAA receptor distribution was analyzed by autoradiographic methods in several brain regions during Mn intoxication. We found that chronic treatment of mice with MnCl₂ during 8 wk significantly alters thel-[³H]glutamate (l-[³H]Glu) binding to total glutamate (Glu) receptors, as well as toN-methyl-d-aspartate (NMDA) and quisqualate (QA) receptor subtypes. A generalized decrease of 16–24% of thel-[³H]Glu binding to total Glu receptors was found in all cortex, hippocampus, basal ganglia (except globus pallidus), and cerebellum. Saturation studies showed a significant reduction of the maximal number of receptors (B _max) in Mn-treated mice, whereas the affinity (K _d) was not altered.l-[³H]Glu binding to NMDA sites was mainly decreased (10–21%) in a few cortical regions, basal ganglia (except globus pallidus), and hippocampus, whereas binding to QA receptor subtype was diminished (16–30%) in cortex, hippocampus, and cerebellum. The decrease of Glu receptor binding sites during Mn poisoning could reflect a receptor downregulation more than neuronal loss, since these reduction are moderate and diffuse. Thus, this down-regulation might mean a protection mechanism against an excitotoxic process associated with Mn toxicity.     

Differential responses of rat cerebral somatostatinergic and cholinergic cells to glutamate agonists

Reductions in cortical somatostatin (SRIH) and choline acetyltransferase (ChAT) are major biochemical deficits in Alzheimer disease (AD). SRIH and ChAT were measured in fetal rat cerebral neurons after exposure to the glutamate agonistsN-methyl-d-aspartate (NMDA), kainate (KA), and quisqualate (Q). NMDA (96 h incubation) stimulated SRIH release and content in a dose-dependent manner with aB _max of 10^?5 M and EC₅₀ of 2?3×10^?6 M. KA showed a small stimulation in SRIH levels at 10^?5 M, but produced marked inhibition at 10^?4 M. Q decreased both intracellular and secreted SRIH. KA (51–76% of basal) and Q (27–56% of basal) but not NMDA (91–114% of basal) also inhibited the incorporation of [³⁵S]methionine into proteins. In similar experiments 10^?4 M Q (23±9% of basal) and KA (20±3% of basal) but not NMDA (80±16% of basal) reduced ChAT levels in hypothalamic/septal cultures. These inhibitory actions on ChAT activity by KA and Q were reversed by γ-glutamyltaurine (GT) but not by 2-amino-5-phosphonopentanoic acid (AP5). Chronic NMDA exposure partially inhibited muscarinic acetylcholine receptor (mAChR) mediated inositol phospholipid (PI) turnover, whereas it was abolished after KA and Q pretreatment. These findings suggest that in cerebral cell cultures, NMDA has a stimulatory action on somatostatinergic neurons and non-NMDA receptor agonism could play an important role in EAA-mediated neural damage.     

The effect of glutamate receptor blockade on anoxic depolarization and cortical spreading depression.

We examined the effect of blockade of N-methyl-D-aspartate (NMDA) and non-NMDA subtype glutamate receptors on anoxic depolarization (AD) and cortical spreading depression (CSD). [K+]e and the direct current (DC) potential were measured with microelectrodes in the cerebral cortex of barbiturate-anesthetized rats. NMDA blockade was achieved by injection of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate [MK-801; 3 and 10 mg/kg] or amino-7-phosphonoheptanoate (APH; 4.5 and 10 mg/kg). Non-NMDA receptor blockade was achieved by injection of 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX; 10 and 20 mg/kg). MK-801 and APH blocked CSD, while NBQX did not. In control rats, the latency from circulatory arrest to AD was 2.1 +/- 0.1 min, while the amplitude of the DC shift was 21 +/- 1 mV, and [K+]e increased to 50 +/- 6 mM. All variables remained unchanged in animals treated with MK-801, APH, or NBQX. Finally, MK-801 (14 mg/kg) and NBQX (40 mg/kg) were given in combination to examine the effect of total glutamate receptor blockade on AD. This combination slightly accelerated the onset of AD, probably owing to circulatory failure. In conclusion, AD was unaffected by glutamate receptor blockade. In contrast, NMDA receptors play a crucial role for CSD.     

Polyamine effects uponN-methyl-D-aspartate receptor functioning: differential alteration by glutamate and glycine site antagonists

Polyamines such as spermidine potentiate activation of theN-methyl-D-aspartate (NMDA)-type excitatory amino acid receptor. The goal of the present study was to investigate interactions between the putative polyamine binding site and previously described sites for glutamate and glycine. Binding of the high-potency PCP receptor ligand [³H]MK-801 to well-washed rat brain membranes was used as an in vitro probe of NMDA receptor activation. Spermidine concentration-response studies were performed in the absence and presence of both glutamate and glycine, with and withoutD-(−)-2-amino-5-phosphonovaleric acid (D(−)AP-5) or 7-chlorokynurenic acid (7Cl-KYN). Incubation in the presence of spermidine alone induced a 20.4-fold increase in [³H]MK-801 binding with an EC₅₀ value of 13.3 μM. The mean concentration of spermidine which induced maximal stimulation of binding was 130 μM (n = 10,S.E.M.= 24.66,range= 25–250 μM). Glutamate (10 μM) decreased the EC₅₀ value for spermidine-induced stimulation of [³H]MK-801 binding to 3.4 μM. Glycine (10 μM) did not significantly alter either maximum spermidine-induced [³H]MK-801 binding or the EC₅₀ value for spermidine-induced stimulation of [³H]MK-801 binding. Incubation in the presence of the specific glutamate antagonistD(−)AP-5 attenuated [³H]MK-801 binding in a glutamate-reversible fashion. The competitive glycine antagonist 7Cl-KYN decreased maximum spermidine-induced [³H]MK-801 binding in a glycine-reversible fashion. In addition, 7Cl-KYN increased the EC₅₀ value for spermidine-induced stimulation of [³H]MK-801 binding whileD(−)AP-5 was without effect. These findings suggest that glutamate and glycine regulate the polyamine binding site differentially. PCP-like agents induce a psychotomimetic state closely resembling schizophrenia by inhibiting NMDA receptor-mediated neurotransmission. The ability of polyamines to modulate NMDA receptor functioning suggests a potential site for pharmacological intervention.     

Glucose deprivation potentiates toxicity of ouabain and glutamate in cortical neurons cultured for different time periods

The addition of glutamate (Glu) to culture medium for 24 hours induced the dose-dependent death of rat cortical neurons cultured for 9–10 days and did not affect neurons cultured for 4–5 days; this suggests that the later neurons are neurochemically immature. In mature cultures, glucose deprivation (GD) enhanced the toxic effect of low Glu concentrations by 15% and did not influence the toxicity of high concentrations of this neuromediator. In immature cultures, GD potentiated the Glu effect independent of the concentration of this neuromediator. Inhibition of Na⁺/K⁺ -ATPase induced the death of some of the neurons. In the presence of a normal level of glucose, ouabain decreased the viability of mature and immature neurons to 67 ± 4 % and 79 ± 5%, respectively, and its presence during GD diminished viability to 28 ± 4 % and 56 ± 3%, respectively. The toxicity of ouabain was substantially attenuated when ionotropic glutamate receptors were blocked by MK-801. GD alone caused no significant increase in the death of these cells, even after a 3-hour incubation. Thus, GD strongly increases the susceptibility of neurons to the toxicity mediated by the activation of the NMDA subtype of ionotropic Glu receptors, even in the case of neurochemically immature neurons.     

Neuroprotection against excitotoxicity by N-alkylglycines in rat hippocampal neurons

Excessive activation of glutamate receptors of the N-methyl-d-aspartate (NMDA) subtype is considered a relevant initial step underlying different neurodegenerative diseases. Recently, with the approval of memantine to treat Alzheimer dementia, NMDA receptors have regained clinical interest. Accordingly, the development and validation of NMDA receptor antagonists is being reconsidered. We recently identified a family of trialkylglycines that act as channel blockers of the NMDA receptor. Their neuroprotective activity against excitotoxic insults remains elusive. To address this issue, we first characterized the contribution of glutamate receptor sub-types to hippocampal death in culture as a function of days in culture in vitro (DIV). Whereas at 7 DIV neither NMDA nor glutamate produced a significant neuronal death, at 14 and 21 DIV, NMDA produced the death of 40% of the neurons exposed to this receptor agonist that was fully protected by MK-801. Similar results were obtained for l-glutamate at 14 DIV. In contrast, when neurons at 21 DIV were used, glutamate killed 51.1±4.9% of the neuronal population. This neuronal death was only partially prevented by MK-801, and fully abrogated by a combination of MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Glucose deprivation injured 37.1±9.2% of the neurons through a mechanism sensitive to MK-801. The family of recently identified N-alkylglycines tested protected neurons against NMDA and glucose-deprivation toxicity, but not against glutamate toxicity. Noteworthy, N-alkylglicines with a moderate protection against NMDA-induced toxicity strongly protected from β-amyloid toxicity. Collectively, these findings imply both NMDA and non-NMDA receptors in excitotoxicity of hippocampal neurons, and suggest that blockade of NMDA receptors alone may not suffice to efficiently abrogate neurodegeneration.     

MK-801 inhibition of nicotinic acetylcholine receptor channels.

MK-801 is a potent inhibitor of the NMDA subtype of glutamate receptors. Single-channel and macroscopic currents indicate that MK-801 also inhibits nicotinic acetylcholine receptors (nAChRs). MK-801 does not significantly increase desensitization of the nAChRs or compete for the ACh binding site. Although there is a slight inhibition of the closed nAChR, the main action of MK-801 is to enter and block the open channel. The voltage dependence for block is consistent with a single binding site within the channel that is 50% of the way through the membrane field. The IC50 for block is 3 microM at -70 mV for currents induced by 0.5 microM ACh. The data from both single-channel and macroscopic currents can be used to estimate a Kd (0) of 7 microM, which is about 40 times higher than the Kd (0) for MK-801 binding to the NMDA receptor. The relative potency of tricyclic compounds like MK-801 for various neurotransmitter systems points out that the pharmacologic action of these drugs could involve complicated interactions in vivo.     

Search for novel ligands selective at a polyamine recognition domain on theN-methyl-d-aspartate receptor complex using membrane binding techniques

Among over 60 polyamine derivatives tested, onlyN-(3-aminopropyl)octanediamine and bis-(3-aminopropyl)nonanediamine (TE393) markedly inhibited [³H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) binding at equilibrium in the presence of added spermidine (SPD) in “non-washed” rat brain synaptic membranes, without affecting that in the absence of added SPD. Although TE393 significantly potentiated [³H]MK-801 binding before equilibrium in the presence ofl-glutamic acid (Glu) alone or both Glu and glycine (Gly) added in “Triton-treated” membranes, the putative polyamine antagonists 1,10-decanediamine (DA10) and arcaine invariably inhibited binding irrespective of the addition of agonists. In the absence of added SPD, in addition, TE393 markedly enhanced abilities of both Glu and Gly to potentiate [³H]MK-801 binding before equilibrium. However, TE393 induced a rightward shift of the concentration-response curve of SPD for [³H]MK-801 binding before equilibrium. Moreover, TE393 was effective in potentiating binding of an antagonist but not an agonist radioligand to the NMDA domain and in inhibiting binding of an antagonist but not an agonist radioligand to the Gly domain. The potentiation of NMDA antagonist binding by TE393 occurred in a manner sensitive to prevention by arcaine but not by DA10. These results suggest that TE393 may be a novel ligand at the polyamine domain with an ability to interact with both the NMDA and Gly recognition domains in antagonist-preferring forms.     

[H]MK-801 binding sites in neonate rat brain

[³H]MK-801 binding sites are present in neonate rat brain as early as 3 days after birth. Immature hippocampus and cortex contain approximately one sixth the concentration of binding sites of the adult, while brainstem concentration is twice as high as that of adult. [³H]MK-801 binding is stimulated by glutamate and glycine and blocked by phencyclidine and Mg²⁺ both in 7-day-old neonate and adult, indicating that as early as 7 days postnatally, theN-methyl-d-asparatate-type glutamate receptor and MK-801 binding site are functionally coupled.     

MK-801 prevents hypobaric-ischemic neuronal degeneration in infant rat brain

Recent evidence implicates the endogenous excitatory amino acids, glutamate (Glu) and aspartate, in hypoxic/ischemic neuronal degeneration. In a preceding article (Ikonomidou et al., 1989) we described a new model for studying hypoxic/ischemic neuronal degeneration in the infant rat brain that entails unilateral common carotid artery ligation followed by exposure to a partial vacuum for 75 min. Promising features of this model include a low mortality rate and high incidence of acute brain damage disseminated over numerous brain regions. In addition, there is a striking similarity between the type of cytopathology characterizing this model of hypoxic/ischemic neuronal degeneration and that which has been described in infant animals treated with Glu. MK-801 is a powerful antagonist of the N-methyl-D-aspartate (NMDA) receptor ionophore complex (a subtype of Glu receptor). In the present study, after unilateral carotid artery ligation was performed on 10-d-old rat pups, they were treated either with MK-801 (1 mg/kg i.p.) or saline 15 min before exposure to the hypobaric condition. MK-801 exerted a strong neuroprotective effect without serious side effects; the majority of saline control animals sustained severe brain damage, whereas the majority of MK-801-treated pups had no brain damage. These and other recent findings suggest that the NMDA receptor may play an important role in hypoxic/ischemic neuronal degeneration in the immature brain and provide hope that NMDA antagonists such as MK-801 may be effective in preventing such degeneration.     

Ketamine and MK-801 prevent degeneration of thalamic neurons induced by focal cortical seizures

Ketamine and MK-801 act at phencyclidine receptors to block transmitter activity through the N-methyl-D-aspartate (NMDA) subtype of glutamate (GLU) receptor. These agents also block the potent excitotoxic actions of NMDA and are of interest for their potential ability to protect against neurodegenerative processes mediated by the excitotoxic action of endogenous Glu at NMDA receptors. Here we show that degeneration of thalamic neurons caused by persistent seizure activity in the corticothalamic tract (putative glutamergic transmitter pathway) is prevented by systemic administration of ketamine or MK-801, despite the failure of these agents to eliminate persistent electrographic seizure activity recorded from cortex and thalamus.     

Effects of MK-801 on glutamate-induced swelling of astrocytes in primary cell culture

The effects of glutamate and its agonists and antagonists on the swelling of primary astrocytes were studied. Glutamate (Glu), aspartate (Asp), homocysteate (HCA), and quisqualate (Quis) at 1 mM concentration caused a significant increase in astrocytic swelling as measured by the 3-0-methyl-[14C]-glucose, whereas kainate (KA), N-methyl-D-aspartate (NMDA), and receptor antagonists 2-amino-5-phosphonovaleric acid (APV), 2-amino-7-phosphonohepatanoic acid (APH), and kynurenic acid (Kynu) were not effective. This glial swelling was time-dependent since 1-hr or greater incubations with Glu or its agonists were needed to produce such an effect. Preincubation of glutamate or NMDA receptor anatogonists including Kynu, APH, and APV failed to ameliorate the Glu effects. However, MK-801, a noncompetitive NMDA antagonist, when added to the Glu-incubated astrocytes significantly reduced Glu-induced astrocytic swelling. MK-801 was also effective in reducing the astrocytic swelling induced by agonists including Asp, Quis, and HCA, suggesting that those agonists may share similar mechanisms of Glu in inducing astrocytic swelling. Since the cultured astrocytes lack the NMDA receptors, our data suggest that the observed beneficial effects of MK-801 on excitotoxin-induced swelling of astrocytes may be mediated by mechanisms other than NMDA receptors.     

Glutamate modulators as potential therapeutic drugs in schizophrenia and affective disorders

Severe psychiatric disorders such as schizophrenia are related to cognitive and negative symptoms, which often are resistant to current treatment approaches. The glutamatergic system has been implicated in the pathophysiology of schizophrenia and affective disorders. A key component is the dysfunction of the glutamatergic N-methyl-d-aspartate (NMDA) receptor. Substances regulating activation/inhibition of the NMDA receptor have been investigated in schizophrenia and major depression and are promising in therapeutic approaches of negative symptoms, cognition, and mood. In schizophrenia, add-on treatments with glycine, d-serine, d-alanine, d-cycloserine, d-amino acid oxidase inhibitors, glycine transporter-1 (GlyT-1) inhibitors (e.g., sarcosine, bitopertin) and agonists (e.g., LY2140023) or positive allosteric modulator (e.g., ADX71149) of group II metabotropic glutamate receptors (mGluRs) have been studied. In major depression, the NMDA receptor antagonists (e.g., ketamine, AZD6765), GluN2B subtype antagonists (e.g., traxoprodil, MK-0657), and partial agonists (e.g., d-cycloserine, GLYX-13) at the glycine site of the NMDA receptor have been proven to be effective in animal studies and first clinical trials. In addition, clinical studies of mGluR2/3 antagonist BCI-838 (a prodrug of BCI-632 (MGS0039)), mGluR2/3-negative allosteric modulators (NMAs) (e.g., RO499819, RO4432717), and mGluR5 NAMs (e.g., AZD2066, RO4917523) are in progress. Future investigations should include effects on brain structure and activation to elucidate neural mechanisms underlying efficacy of these drugs.     

Supporting evidence for negative modulation by protons of an ion channel associated with theN-methyl-D-aspartate receptor complex in rat brain using ligand binding techniques

The addition ofL-glutamic acid (Glu) alone, both Glu and glycine (Gly) or Glu/Gly/spermidine (SPD) was effective in potentiating[³H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) binding before equilibrium to an ion channel associated with theN-methyl-D-aspartate (NMDA) receptor complex in brain synaptic membranes extensively washed and treated with Triton X-100. The binding dependent on Glu almost linearly increased in proportion to decreasing proton concentrations at a pH range of 6.0 to 9.0 in external incubation medium, while a Gly-dependent portion of the binding increased with decreasing proton concentrations up to a pH of 7.5 with a plateau thereafter. In contrast, the SPD-dependent binding increased in proportion to decreasing proton concentrations up to a pH of 7.0 with a gradual decline thereafter. Similar profiles were also obtained with [³H]MK-801 binding at equilibrium, with an exception that significant binding of [³H]MK-801 was detected in the absence of any added agonists. The potency of SPD to potentiate [³H]MK-801 binding before equilibrium increased in proportion to decreasing proton concentrations, with those of both Glu and Gly being unchanged. In contrast, the ability of (+)MK-801 to displace [³H]MK-801 binding at equilibrium was not significantly affected by a decrement of external proton concentrations from pH 7.5 to pH 8.5 in the presence of Glu/Gly and Glu/Gly/SPD added. However, similar changes in external proton concentrations did not similarly affect binding of several radioligands for the NMDA and Gly domains on the receptor complex. Decreasing proton concentrations were effective in exponentially potentiating binding of [³H]SPD at a pH range of 6.0 to 9.0 without virtually altering [³H]D, L-α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid binding. In addition, [³H]kainic acid binding markedly decreased with decreasing proton concentrations only in the presence of Ca²⁺ ions. These results suggest that protons negatively modulate neuronal responses mediated by the NMDA receptor ionophore complex through interference with opening mechanisms of the channel domain without disturbing association processes of the endogenous agonists with the respective recognition domains in rat brain. Moreover, possible modulation by protons of responses mediated by the kainate receptor in the presence of Ca²⁺ ions at concentrations that occur in vivo is also suggested.     

Developmental maturation of the N-methyl- -aspartic acid receptor channel complex in postnatal rat brain

The N-methyl- -aspartate (NMDA) receptor plays an important role in developmental plasticity. Previous studies have reported differences between the NMDA receptor-channel complex in the rat pup brain and the adult brain. In the present study, modulation of the NMDA channel complex as a function of age was measured to determine when the temporal switching of the NMDA receptor from the immature form to the adult mature form takes place. [³H]MK-801 binding was measured in the rat forebrain from postnatal day 1 to day 21. Our data suggest the presence of two types of NMDA receptors — an immature type and a mature type. The immature NMDA receptor, seen during the early postnatal period (day 1–day 14) is highly sensitive to spermidine, -glutamate alone potentiates [³H]MK-801 binding, and glycine failed to potentiate an -glutamate-induced increase in [³H]MK-801 binding. During the late postnatal period (after day 14) spermidine alone did not increase [³H]MK-801 binding as potently as it did during the early postnatal period, high-affinity [³H]MK-801 binding was not seen in the presence of -glutamate alone, and -glutamate and glycine or -glutamate and spermidine or -glutamate, glycine and spermidine together, significantly increased [³H]MK-801 binding in a manner similar to that reported in the adult brain. Together, the pharmacology of the NMDA receptor during the early postnatal period differs from the adult-like receptor seen during the late postnatal period, and that in rats the apparent switching of the NMDA receptor from the immature type to the mature type takes place after the second postnatal week.     

Development of MK-801, kainate,AMPA, and muscimol binding sites and the effect of dark rearing in rat visual cortex

We used quantitative autoradiography to determine whether the development of glutamate receptors correlates with the plastic period for monocular deprivation in rat visual cortex. To study glutamate receptors, we incubated sections of rat visual cortex with tritiated (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10imine maleate (MK-801), tritiated kainate, and tritiated amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA). [³H]MK-801 is a noncompetitive ligand for the N-methyl-D-aspartate (NMDA) receptor. [³H]kainate and [³H]AMPA are competitive ligands for non-NMDA receptors. To compare glutamate binding sites with a nonglutamate binding site, we studied [³H]muscimol, which binds to γ-aminobutyric acid (GABA)_A receptors. [³H]MK-801 binding was maximal at postnatal day 26 (P26) and decreased in adulthood. [³H]AMPA binding was maximal at P18. [³H]kainate binding and [³H]muscimol binding were not age dependent. Dark rearing partially prevented the age-dependent decrease in [³H]MK-801 binding but had no effect on [³H]kainate or [³H]AMPA binding. Dark rearing decreased muscimol binding in adult animals. These results suggest that NMDA receptors, but not other glutamate receptors or GABA_A receptors, are likely to be critical for developmental plasticity in rat visual cortex. J. Comp. Neurol. 383:73–81, 1997. © 1997 Wiley-Liss, Inc.     

Prenatal ethanol exposure enhances glutamate release stimulated by quisqualate in rat cerebellar granule cell cultures

Effects of prenatal ethanol exposure on extracellular glutamate accumulation stimulated by glutamate receptor agonists were studied in rat cerebellar granule cell cultures. The prenatal exposure to ethanol was achieved via maternal consumption of a Sustacal^TM liquid diet containing either 5% ethanol or isocaloric sucrose (pair-fed) substituted for ethanol from gestation d 11 until the day of parturition. Neither the basal level of extracellular glutamate nor the increased accumulation of glutamate stimulated by KCl (40 mM) or by ionotropic glutamate receptor agonists,N-methyl-D-aspartate (NMDA) or kainate (KA) (100 μM each), in cells prepared from the ethanol-fed group was significantly different from that in cells prepared from the pair-fed group. Glutamate accumulation stimulated by quisqualate (QA, 100 μM) or bytrans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD, 250 μM) in the ethanol-fed group was higher than that in the pair-fed group by 116 and 36%, respectively. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 100 μM), an ionotropic QA receptor antagonist, the QA-induced accumulation of glutamate in the ethanol-fed group was still higher than that in the pair-fed group. In the presence of MK-801 (5 μM), an antagonist of the NMDA receptor, the enhanced accumulation of glutamate stimulated by either QA ort-ACPD was still observable in the ethanol-fed group as compared to the pair-fed group. Addition of (RS)-α-methyl-4-carboxyphenylglycine (MCPG, 500 μM), a selective antagonist of the metabotropic glutamate receptor, abolished the enhanced accumulation of glutamate stimulated by either QA ort-ACPD in the ethanol-fed group. Although immunoblotting of mGluR1 and mGluR2/3 did not show apparent differences between the pair-fed and the ethanol-fed groups, the overall results suggest that the effect of prenatal ethanol exposure was selectively through a pathway mediated by the metabotropic glutamate receptor.     

Laminar distribution of MK-801, kainate,AMPA, and muscimol binding sites in cat visual cortex: A developmental study

We used quantitative autoradiography to determine whether the development of glutamate receptors correlates with the sensitive period for monocular deprivation in the visual cortex. To study glutamate receptors, we incubated sections of cat visual cortex with tritiated (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10imine-maleate (MK-801), tritiated kainate, and tritiated amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA). [³H]MK-801 is a noncompetitive ligand for the N-methyl-D-aspartate (NMDA) receptor. [³H]kainate and [³H]AMPA are competitive ligands for non-NMDA receptors. We used [³H]muscimol, which binds to GABA_A receptors, so that we would have one control ligand that binds to a nonglutamate receptor. When all layers were combined, the results confirmed our previous studies with homogenate binding. [³H]MK-801 and [³H]kainate binding were significantly greater at 42 days than at earlier or later times. [³H]AMPA and [³H]muscimol binding did not show such a peak. This suggests that MK-801 and kainate binding sites are more likely to be involved in plasticity than are AMPA and muscimol binding sites. In layers 2/3, MK-801 had the greatest age-dependent changes; in layers 5 and 6, kainate binding changed most with age. This suggests that the mechanisms of plasticity may vary with cortical layer. © 1996 Wiley-Liss, Inc.