L-[3H]Glutamate binds to kainate-, NMDA- and AMPA-sensitive binding sites: an autoradiographic analysis

The anatomical distribution of L-[3H]glutamate binding sites was determined in the presence of various glutamate analogues using quantitative autoradiography. The binding of L-[3H]glutamate is accounted for by the presence of 3 distinct binding sites when measured in the absence of Ca2+, Cl- and Na+ ions. The anatomical distribution and pharmacological specificity of these binding sites correspond to that reported for the 3 excitatory amino acid binding sites selectively labelled by D-[3H]2-amino-5-phosphonopentanoate (D-[3H]AP5), [3H]kainate ([3H]KA) and [3H] alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA) which are thought to be selective ligands for the N-methyl-D-aspartate (NMDA), KA and quisqualate (QA) receptors, respectively.     

Quantitative autoradiography of binding sites for [H]AMPA, a structural analogue of glutamic acid

Binding sites for the potent glutamate agonist [³H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were localized in rat brain frozen sections by quantitative autoradiography. Highest levels of binding were seen in stratum radiatum and stratum oriens of the CA1 hippocampal subfield and in the dorsal subiculum. Substantially less but still high amounts of [³H]AMPA binding occurred in other hippocampal subfields and in rostral forebrain structures. The heterogeneous nature of [³H]AMPA binding is discussed in relation to [³H]glutamate binding visualized by similar methods. From these data it is suggested that [³H]AMPA may label a particular subclass of the glutamate receptor population which exhibits a high affinity for quisqualic acid.     

Binding sites for the glutamate-analogue [H]AMPA in cultured rat brainstem and spinal cord

By means of light microscopic autoradiography, binding sites for the glutamate-analogue [ [³H]AMPA [(RS)-α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid] were observed on the soma and processes of many brainstem and spinal neurons but not on glial cells. Unlabeled AMPA, glutamate or the glutamate antagonist glutamic acid diethyl ester at high concentrations inhibited or reduced binding of [³H]AMPA. Our data indicate that binding sites for [³H]AMPA exhibit characteristics expected for glutamate receptors.     

Distribution of [H]AMPA binding sites in rat brain as determined by quantitative autoradiography

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) is a potent neuroexcitatory compound which acts at the quisqualate class of excitatory amino acid receptors. In this study we describe the pharmacological characteristics and anatomical distribution of [³H]AMPA binding sites in rat brain using quantitative autoradiography. These binding sites exhibit the appropriate pharmacological characteristics and are found in high concentrations in the hippocampus, cerebral cortex (especially layers I–III), induseum griseum, and dorsal lateral septum. Intermediate concentrations are found in the corpus striatum and deeper layers of cerebral cortex. Lower concentrations are found in the diencephalon, midbrain and brainstem. These results demonstrate that [³H]AMPA binding sites are found throughout the CNS and suggest brain regions which may use quisqualate receptors as glutamate neurotransmitter receptors.     

Development of a micromethod to study the Na-independentl-[H]glutamic acid binding to rat striatal membranes. II. Effects of selective striatal lesions and deafferentations

An apparent single class of Na⁺-independentl-[³H]glutamate (l-[³H]Glu) binding sites was biochemically and pharmacologically identified on rat striatal tissue. The K_d value was 1.75 μM and the B_max3.89 nmol/g protein. In order to further elucidate the putative physiological role of these binding sites and to valid our binding assay, experiments were conducted to determine the anatomical location of thel-[³H]Glu binding sites in the striatum. Local injection of the neurotoxin, kainic acid into the striatum caused degeneration of target cells in the structure followed by an important decrease (-37%) in the number of these binding sites, with no significant change in the affinity constant. Lesions of the cortical frontal and parietal areas projecting to the striatum via putative glutamatergic fibers led, on the other hand, after 3 weeks to one month, to a significant increase (+23%) in the number ofl-[³H]Glu binding sites. The K_d value does not significantly change after decortication. Finally, suppression either of the nigrostriatal dopaminergic input or of the partially cholinergic thalamostriatal tract do not affect [³H]Glu binding to striatal tissue. These results suggest that about 40% of our [³H]Glu binding sites are located on striatal target cell sensitive to the neurotoxic effect of kainic acid. Therefore, they seem to be partly postsynaptic. The existence of a relation between these binding sites and the corticostriatal glutamatergic input was shown by the development of a supersensitivity response after suppression of this cortical input. These sites might therefore constitute one of the receptor subclass for Glu linked to the excitatory action of the corticostriatal afferent. Since degeneration of either the nigrostriatal dopaminergic or the thalamostriatal fibres did not affect the binding ofl-[³H]Glu characterized in our study, the binding sites will probably not occur at the presynaptic level on these nerve terminals in the striatum.     

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.     

l-aspartate binding sites in rat cerebellum: A comparison of the binding ofl-[H]aspartate andl-[H]glutamate to synaptic membranes

The binding ofl-[³H]aspartate to sonicated, extensively washed and preincubated cerebellar synaptic membranes was investigated. Binding was optimal under physiological conditions of pH and temperature, and attained equilibrium within 10 min. Binding was saturable, and Eadie-Hofstee analysis revealed interaction with a single population of binding sites (K_d = 874nM and B_max = 44pmol/mg protein), which displayed no cooperativity (Hill coefficient approx.= 1). Specific [³H]aspartate was readily and reversibly displaced by unlabelledl-aspartate (thed-isomer being less than half as active) with a half-life of dissociation of 32 sec. Quisqualate, 4-fluoroglutamate and 2-amino-4-phosphonobutyrate, which are good displacers of [³H]glutamate binding, were only weakly active against the aspartate system. The excitatory amino acid antagonists,dl-α-aminoadipate,dl-α-aminosuberate and HA-966 were effective displacers, but the proposed aspartate receptor-preferring agonist, N-methyl-d-aspartate was inactive. Kainic acid exhibited negligible affinity for the aspartate binding site, in common with that for glutamate.While freezing or cold storage of membranes resulted in diminished [³H]-aspartate binding, lyophilization was not only able to confer substantial stability, but induced a marked increase in affinity of the binding site.Differential effects of various cations on [³H]aspartate binding were observed — monovalent cations reduced, while divalent cations enhancedl-[³H]aspartate binding.     

Synchronized overproduction of AMPA,kainate, and NMDA glutamate receptors during human spinal cord development

Quantitative receptor autoradiography was used to map the distribution in the developing human spinal cord of the three types of ionotropic glutamate receptors. N-methyl-D-Aspartate (NMDA) receptors were labeled with [³H]glutamate, kainic acid (KA) receptors were labeled with [³H]KA, and α-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) receptors were labeled with [³H]AMPA. In the adult, labeling of all three receptor subtypes is largely restricted to the substantia gelatinosa (SG) in the dorsal horn, with very low level labeling elsewhere in the spinal gray matter. In marked distinction, in late fetal life, high level ligand binding is seen throughout the spinal gray matter. In early postnatal life, binding sites diminish in all regions, but least so in the SG, until the adult pattern emerges. Thus a coordinated transient high level of ionotropic glutamate receptor expression occurs within the developing spinal cord. Saturation analysis of ligand binding shows that the affinity of [³H]KA and [³H]AMPA binding is not developmentally regulated. In contrast, the affinity of [³H]glutamate binding to the NMDA receptor in the fetal ventral horn is three-fold greater than in the adult ventral horn. Thus, in addition to quantitative changes in glutamate receptor expression, qualitative changes occur in the expression of NMDA receptors during development. The distinct glutamate receptor phenotype of fetal and early postnatal spinal cord cells suggests that alterations in the excitable properties of these cells plays an important role in activity-dependent development and in susceptibility to excitotoxic injury. J. Comp. Neurol. 384:200-210, 1997. © 1997 Wiley-Liss, Inc.     

Non-NMDA glutamate receptor binding in canine brain after global cerebral ischemia and reperfusion

We employed a canine model to test the effects of global cerebral ischemia and reperfusion on binding to α-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA), kainate (KA), and metabotropic glutamate receptors. Ischemia was induced by 10 min of cardiac arrest, followed by restoration of spontaneous circulation for periods of 0, 0.5, 2, 4, and 24 h. Frozen sections were prepared from parietal and temporal cortex, hippocampus, and striatum, and in vitro autoradiography was performed with one of three radioligands: [³H]AMPA, [³H]KA, or [³H]glutamate (using conditions allowing specific labeling of the metabotropic binding site). In striatum, metabotropic binding was unchanged, whereas AMPA and KA binding decreased by 20–30% at 30 min postischemia, remaining depressed through 24h. In cortex, AMPA and metabotropic binding were decreased at several timepoints after ischemia and recirculation, particularly in parietal cortex, whereas KA binding was unaffected in this tissue. Binding to hippocampal regions was largely unchanged, except for a decrease in KA binding at 2 and 4 h postischemia. These findings contrast with results from parallel studies showing increased striatal binding to NMDA receptors following ischemia. Decreased binding to non-NMDA glutamate receptors in striatum and parietal cortex may serve to protect against damage mediated through these receptors.     

Evidence for cysteine sulfinate as a neurotransmitter

The Na⁺-independent binding ofl-[³H]cysteine sulfinate andl-[³H]cysteine sulfinate uptake were investigated in rat brain membranes and vesicles. Specific binding ofl-[³H]cysteine sulfinate was saturable and occurred by a single high affinity process with aK_b of 100nM± 9 and capacity (B_max) ± 0.22pmol/mg protein. Sodium ions were found to have a biphasic effect; low concentrations (in the range of 0.1–3 mM) induced a marked inhibition of the binding whereas higher concentrations (10–300 mM) resulted in a dose-dependent stimulation of binding. The inhibition potency, expressed as theK_i values of a wide range of compounds with known pharmacological activities was tested.l-Cysteine sulfinate was the most potent inhibitor being 3-fold more potent thanl-glutamate and 80 times more potent thanl-aspartate. The regional distribution of the binding ofl-[³H]cysteine sulfinate in the brain was found to be heterogeneous. These results provide the first evidence for an interaction of cysteine sulfinate with specific receptor sites on the synaptic membrane.The rate ofl-[³H]cysteine sulfinate uptake shows a biphasic dependence on the concentration ofl-cysteine sulfinate, corresponding to a high affinity (27.2 μM) and a low affinity (398 μM) transport system. The maximuml-[³H]cysteine sulfinate uptake is reached at 2 min. The reversibility of this transport was demonstrated. Thel-[³H]cysteine sulfinate uptake increases as a function of the sodium concentration. Chloride and potassium ions stimulate the uptake. The decrease or increase in the electrical membrane potential (†_Ψ) caused by replacing the chloride ions by the sulfate or sulfocyanate ions respectively leads to a decrease or increase in the rate of uptake. Increase in the extravesicular osmolarity leads to a decrease in the extent ofl-[³H]cysteine sulfinate accumulation. Amino acids with an acidic group in position ω were found to be potent inhibitors (the most potent beingl-aspartate). The length of the carbon chain also has a bearing on the inhibitory effect. The regional distribution ofl-[³H]cysteine sulfinate uptake in the brain was heterogeneous. These results demonstrate the existence of a high affinity system which may correspond to the transmitter inactivation.Binding and uptake sites are distinguishable as evidenced by the affinity constants, the ionic and pharmacological effects and the different regional distributions in the brain. Finally, these results give further evidence for a neurotransmitter role ofl-cysteine sulfinate.     

The quaking mouse: an epileptic mutant with alterations affecting the modulatory mechanisms of the NMDA receptor complex

The binding of [³H]glutamate and of [³H]1-(1-(2-thienyl)cyclohexyl)piperidine ([³H]TCP) has been examined in the genetically epileptic mutant mouse, quaking. The density of [³H]glutamate binding sites did not differ between the quaking mice and their controls of the same strain. In the absence of exogenous glutamate or glycine, the density of [³H]TCP binding sites was also similar in the two strains. In both the mutants and their controls, exogenously added glutamate, glycine and glutamate plus glycine dose-dependently increased the binding of [³H]TCP. In the 3 conditions, the modulation of [³H]TCP binding was significantly more efficient in the quaking mice than in the controls. Furthermore, in the presence of glutamate (10⁻⁵ M), the increase of the affinity of the ligand for the ion channel binding site was higher in the mutants than in the controls. These results suggest that the modulatory mechanisms of the

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receptor complex might be altered in these mutants. These alterations might be related to the previously observed anticonvulsant properties of NMDA receptor antagonists in the quaking mouse model of inherited epilepsy.     

Action of 3-((±)-2-car☐ypiperazin-4-yl)-propyl-1-phosphonic aci (CPP): a new and highly potent antagonist of N-methyl-d-aspartate receptors in the hippocampus

A new compound, 3-((±)-2-car☐ypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), has been evaluated as an excitatory amino acid receptor antagonist using electrophysiological assays and radioligand binding. In autoradiographic preparations, CPP reduces l-[³H]glutama binding in regions of the hippocampus rich in N-methyl-d-aspartate (NMDA) receptors, but not in regions richin kainate sites. In isolated membrane fraction preparations, CPP displaces l-[³H]glutamate binding to NMDA sites, but does not compete with the binding of selective kainate or quisqualate site ligands. CPP potently reduces depolarizations produced by application of NMDA but not depolarizations produced by quisqualate or kainate. Its order of potency against excitatory amino acid-induced responses in the hippocampus is NMDA > homocysteate > aspartate > glutamate > quisqualate. CPP has no efect on lateral perforant path responses or on inhibition of these responses by 2-amino-4-phosphonobutyrate. Finally, at doses that do not affect Schaffer collateral synpatic transmission, CPP reversibly blocks the induction of long-term potentiation of Schaffer synaptic responses. This new compounds is, therefore, a higly selective brain NMDA receptor blocker, and the most potent such by nearly an order of magnitude.     

Localization of [H]glutamate binding sites in rat adrenal medulla

A significant activity ofl-[³H]glutamic acid binding was detected in the membranous particulate preparations obtained from the rat adrenal glands. In vitro addition of sodium acetate (100 mM) resulted in a drastic enhancement of the binding to cerebral preparations, while inducing a significant inhibition of the adrenal binding. Quisqualic acid elicited a prominent suppression of the adrenal binding in a concentration-dependent manner to a slightly greater extent than the inhibition of cerebral binding. N-Methyl-d-aspartic acid exerted a significant stimulatory action on the adrenal binding without affecting the cerebral binding. The adrenal medullary part possessed more than 5-fold higher binding activity than that in cortical part. These results suggest the possible existence of [³H]glutamate binding sites in rat adrenal glands which are distinctly different from those in the central structures.     

A pharmacological characterization of the mGluR1α subtype of the metabotropic glutamate receptor expressed in a cloned baby hamster kidney cell line

The pharmacological specificity of the mGluR1α subtype of the metabotropic glutamate receptor (mGluR) was examined in a cloned baby hamster kidney cell line (BHK-ts13) measuring [³H]glutamate binding and inositol phosphate (PI) hydrolysis. PI-hydrolysis was maximally stimulated by quisqualate (1112±105%of basal), glutamate (1061±70%of basal), ibotenate (1097±115%of basal) andβ-N-methylamino-l-rmalanine (BMAA) (1010±104%of basal). In contrast, the maximal stimulation of PI-hydrolysis by(1S,3R)-1-amino-cyclopentane-1,3-dicar☐ylic acid (t-ACPD) was only673±78% of the basal level. The relative order of potency was quisqualate > glutamate > ibotenate > t-ACPD > BMAA. Agonist-stimulated PI-hydrolysis was attenuated (25±4%inhibition) byl-2-amino-3-phosphonopropionic acid and partially blocked (44±7%) by pertussis toxin treatment. Saturation binding studies with [³H]glutamate on membranes prepared from BHK-ts13 cells expressing the mGluR1α subtype showed that glutamate binds to a single affinity state of this receptor with a limited capacity (K_d = 296nM, B_max = 0.8pmol/mg protein). In competition experiments, [³H]glutamate was displaced by quisqualate, glutamate, ibotenate, t-ACPD and BMAA with a rank order of potency similar to that found for stimulation of PI-hydrolysis.     

Glycine-induced changes in synaptic efficacy in hippocampal slices involve changes in AMPA receptors

Brief applications of high glycine concentrations to hippocampal slices have been shown to produce long-lasting changes in synaptic efficacy. In the present study, we show that glycine application transiently and reversibly increases the amplitude and prolongs the duration of synaptic potentials mediated by (NMDA) receptors. The long-lasting changes in synaptic potentials mediated by AMPA receptors are correlated with changes in the binding of [³H]α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid ([³H]AMPA) to membranes prepared from glycine-treated slices. The changes in binding properties of AMPA receptors in adult slices are due to an increase in affinity of the agonist for the receptor. Furthermore, glycine-induced increases in [³H]AMPA binding and in synaptic potentials in adult hippocampal slices are markedly reduced in the presence of low extracellular calcium or of the phospholipase inhibitor bromophenacylbromide. Finally, glycine-induced potentiation of synaptic potentials is associated with an increased potency of the glutamate receptor antagonist, 6,7-dinitroquinoxaline (DNQX), to inhibit synaptic potentials. The results indicate that glycine-induced changes in synaptic efficacy are likely triggered by the activation of NMDA receptors and expressed by changes in the properties of AMPA receptors. As similar events underly long-term potentiation (LTP), this phenomenon might provide important clues to elucidate the molecular mechanisms involved in LTP maintenance.     

Development of a micromethod to study the Na-independentl-[H]glutamic acid binding to rat striatal membranes. I. Biochemical and pharmacological characterization

A micromethod was developed to measure the Na⁺-independentl-[³H]glutamic acid (Glu) binding to rat striatal membranes by using slightly purified membranes from very small tissue amounts, ranging from 0.2 to 0.5 mg wet tissue. The specific binding reached equilibrium in about 30 min incubation at 37 °C and was shown to be partly reversible. Scatchard's analysis of saturation data suggests the presence on striatal membranes of an apparent single homogeneous population of Na⁺-independent binding sites with K_d value 1.75 μM and B_max 3.89 nmol/g protein. Hill's plot of these data was linear, with slope not significantly different from unity, indicating the absence of cooperative interactions. Cl⁻ and Ca²⁺ ions were shown to severely influence thel-[³H]Glu binding to striatal tissue. Maximal activating effects were obtained in the presence of both ions, although Cl⁻ alone was shown to have a powerful stimulating action on the binding. Pharmacological studies suggested, however, the presence of at least two subpopulations of binding sites which bound quisqualic acid as well as ibotenic acid with differential affinities.l-aspartic acid andl-serine-O-sulfate were shown to be potent inhibitors of thel-[³H]Glu binding whendl-2-amino-4-phosphonobutyric acid (dl-APB) and glutamic acid diethylester (GDEE) competed with the binding but only at high concentrations. N-methyl-d-aspartic acid (NMDA),dl-2-amino-5-phosphonovaleric acid (dl-APV),d-α-aminoadipate (d-αAA) and kainic acid were shown not to significantly influence the binding of Glu to striatal membranes     

The expression and binding of kainate receptors is modified in different brain regions by glutamate neurotoxicity during postnatal rat development

Kainic acid receptor (KA-R) subunits are differentially expressed during brain development, and they modulate both neural growth and survival. High concentrations of glutamate in the brain can induce neuronal injury through these receptors, altering normal development. However, it is unclear whether KAR subunit expression itself is also modified by neonatal exposure to high glutamate. To analyze this, monosodium glutamate (4 mg/g of body weight) was subcutaneously administered on postnatal days 1, 3, 5 and 7, and the expression of GluR5, GluR6, KA1 and KA2, as well as [³H]-kainic acid (KA-R) binding, was evaluated on postnatal days 14, 21, 30 and 60 in different regions of rat brain. As a result, high levels of GluR5 expression associated with strong [³H]-kainic acid binding were observed on postnatal days 30 and 60 in the cerebral cortex of rats exposed to glutamate. Similarly, the changes induced by glutamate administration in the expression of the KA1 and KA2 subunits were paralleled by those of [³H]-kainic acid binding in the striatum at postnatal days 21 and 30. In contrast, while KAR subunits were over expressed in the hippocampus, no changes were observed in [³H]-kainic acid binding in adult rats that had been exposed to glutamate. Therefore, glutamate modifies both the expression of kainic acid receptor subunits and kainic acid binding in a determined spatial and temporal manner, which may be indicative of a regional susceptibility to glutamate neurotoxicity.     

l-aspartate binding sites in rat cerebellum: A comparison of the binding ofl-[3H]aspartate andl-[3H]glutamate to synaptic membranes

The binding ofl-[³H]aspartate to sonicated, extensively washed and preincubated cerebellar synaptic membranes was investigated. Binding was optimal under physiological conditions of pH and temperature, and attained equilibrium within 10 min. Binding was saturable, and Eadie-Hofstee analysis revealed interaction with a single population of binding sites (K_d = 874nM and B_max = 44pmol/mg protein), which displayed no cooperativity (Hill coefficient approx.= 1). Specific [³H]aspartate was readily and reversibly displaced by unlabelledl-aspartate (thed-isomer being less than half as active) with a half-life of dissociation of 32 sec. Quisqualate, 4-fluoroglutamate and 2-amino-4-phosphonobutyrate, which are good displacers of [³H]glutamate binding, were only weakly active against the aspartate system. The excitatory amino acid antagonists,dl-α-aminoadipate,dl-α-aminosuberate and HA-966 were effective displacers, but the proposed aspartate receptor-preferring agonist, N-methyl-d-aspartate was inactive. Kainic acid exhibited negligible affinity for the aspartate binding site, in common with that for glutamate.While freezing or cold storage of membranes resulted in diminished [³H]-aspartate binding, lyophilization was not only able to confer substantial stability, but induced a marked increase in affinity of the binding site.Differential effects of various cations on [³H]aspartate binding were observed — monovalent cations reduced, while divalent cations enhancedl-[³H]aspartate binding.     

Effect of aspartame on N-methyl-d-aspartate-sensitive l-[H]glutamate binding sites in rat brain synaptic membranes

Aspartame (l-aspartyl-l-phenylalanine methyl ester), an artificial low-calorie sweetener, was shown to dose-dependently inhibitl-[³H]glutamate binding to its N-methyl-d-aspartate-specific receptors.l-Aspartic acid, a major endogenous metabolite of aspartame, inhibited the binding more stronger than aspartame, while the other metabolites,l-phenylalanine and methanol, had no effect at the same concentration. Aspartame caused a significant change in the affinities ofl-[³H]glutamate binding without altering the V_max values of the binding, suggesting the inhibition is competitive. These in vitro findings suggested that aspartame may act directly on the N-methyl-d-aspartate-sensitive glutamate recognition sites in the brain synaptic membranes.