L-glutamate Binding Research Articles

Hydroxyindole- O-methyltransferase is another target for l-glutamate-evoked inhibition of melatonin synthesis in rat pinealocytes

Rat pinealocytes use l-glutamate as a modulator for melatonin synthesis. Upon binding of l-glutamate to the class II metabotropic glutamate receptor, norepinephrine (NE)-dependent formation of cAMP was inhibited, resulting in decreased serotonin- N-acetyltransferase (NAT) activity and melatonin output. Although l-glutamate at 1 mM caused 90% inhibition of melatonin synthesis, about 30% of the NAT activity remained, suggesting the presence of another target for l-glutamate. In this study, we found that l-glutamate also inhibits hydroxyindole- O-methyltransferase (HIOMT). The inhibition is reversible and dose-dependent: the maximal inhibition was obtained with more than 0.4 mM l-glutamate. Contrary to l-glutamate-evoked inhibition of NAT, agonists for class II metabotropic receptors such as (2 S,2′ R,3′ R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG IV) had no effect on HIOMT. Neither (2 S,3 S,4 S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG), an specific antagonist for class II mGluRs, nor dibutyryl cAMP restored the l-glutamate-evoked inhibition of HIOMT. Northern blot analyses revealed that l-glutamate significantly inhibits the expression of mRNA of NAT, but not that of HIOMT. These results indicated that HIOMT is an another target for l-glutamate due to its inhibition of melatonin synthesis, and the signaling pathway toward the inhibition is distinct from that of NAT.

3H- l-Glutamate Binding and 3H- d-Aspartate Release From Hippocampal Tissue During the Development of Pentylenetetrazole Kindling in Rats

SCHRÖEDER, H., A. BECKER, U. SCHRÖEDER AND V. HOELLT. 3H- l-Glutamate binding and K +-stimulated 3H- d-aspartate release from hippocampal tissue during the development of pentylenetetrazol-induced kindling in rats . PHARMACOL BIOCHEM BEHAV 62(2) 349–352, 1999.—Previous studies have proposed that there is an increase in the density of glutamate binding sites after pentylenetetrazol (PTZ) kindling, whereas the glutamate release is not altered. Little is known about the time course of these changes. Therefore, we studied 3H- l-glutamate binding to hippocampal membranes and K +-stimulated 3H- d-aspartate release from hippocampal slices of rats given PTZ 3, 7, and 13 times up to a fully kindling state. After three PTZ injections, amino acid release from hippocampal tissue slices was significantly enhanced in comparison to controls, whereas 3H- l-glutamate binding was not altered. After seven injections of PTZ, specific glutamate binding to hippocampal membranes tended to increase, and K +-stimulated 3H- d-aspartate release from rat hippocampal slices was normalized. The kindled state characterized by generalized clonic–tonic seizures was reached after 13 PTZ injections, and it was accompanied by an enhancement in the density of glutamate binding sites, whereas the chemically evoked amino acid release remained unchanged. It can be concluded that the amino acid release is increased in the early phase of PTZ kindling development, whereas after completion of kindling, the density of excitatory amino acid binding sites is enhanced.

Determination of membrane topology of glutamate receptors.

Glutamate receptors are large integral membrane proteins that belong to the class of ligand-gated ion channels. They constitute a family of receptors that are activated by binding of the neurotransmitter l-glutamate. Functional receptors are formed by assembly of several subunits around a central ion-conducting pore. Each subunit is thought to contain multiple membrane-spanning domains. A reliable membrane topology model, which defines the number of transmembrane segments and their orientation, is critical for understanding the relationship between the structure of these receptors and their function. This chapter describes some of the technical considerations of an approach that we and others have used to determine experimentally aspects of the membrane topology of ionotropic glutamate receptors (, , , , , , ). This strategy uses the fact that glycosylation of asparagine residues only occurs in extracellular domains, owing to the fact that the glycosyl transferases are localized to the lumen of the endoplasmatic reticulum. Demonstration of functional N-linked glycosylation therefore provides a definitive measure of extracellular localization. Alternative approaches that do not rely on alteration of N-glycosylation status have also provided information on membrane topology of glutamate receptors (, , ), but a detailed discussion of these methods is outside the scope of this chapter.

The effect of pentylenetetrazol kindling on synaptic mechanisms of interacting glutamatergic and opioid system in the hippocampus of rats

Endogenous opioids modulate processes of central excitability such as long-term potentiation and electrical kindling. Little is known about the neurochemical alterations in the interaction of the glutamatergic and opioid system in the development of pentylenetetrazol (PTZ) kindling in rats. Therefore, in the present study we investigated glutamate, DAMGO and naltrindole receptor binding, receptor protein expression by Western blot and ex vivo glutamate transmitter release in PTZ kindled rats. The specific 3 H -DAMGO and -naltrindole binding to hippocampal membranes displayed no significant changes in kindled rats compared to controls. In contrast, the 3 H - l-glutamate binding was significantly enhanced after completion of PTZ kindling. The expression of receptor protein for glutamate as well as the naloxone- and naltrindole-induced 3 H - d-aspartate release from hippocampal slices did not alter in any case as a consequence of PTZ kindling. The PTZ induced enhancement of the glutamate binding sites in the hippocampus was downregulated to control level by natrindole treatment of rats prior to each PTZ application. Furthermore, naltrindole pretreatment of rats significantly inhibited the development of seizure susceptibility. In contrast, naloxone was not able to alter the seizure activity induced by PTZ as well as the transmitter receptor binding. The results are discussed in the light of a modulating role of δ-opioid receptors in PTZ kindling.

Sensitivity and density of glutamate receptor subtypes in the hippocampal formation are altered in pentylenetetrazole - kindled rats

Kindling induced by 13 intraperitoneal injections of 40 mg/kg pentylenetetrazole (PTZ) over a period of 4 weeks resulted in a significant long-lasting increase in both the convulsive susceptibility of animals to the convulsant and the density of the specific [3H]-L-glutamate binding sites in the hippocampus. The quisqualate- and kainate-sensitive [3H]-L-glutamate binding sites were increased 24 h after the final PTZ injection, whereas the N-methyl-D-aspartate (NMDA)-sensitive sites had only a tendency to be enhanced. Furthermore, we investigated [3H]-L-glutamate binding on metabotropic receptors and found a significant increase in the hippocampus following PTZ kindling. In addition, in hippocampal tissue of kindled rats (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD)-stimulated inositol phosphate formation is increased. It can be concluded that the increase in metabotropic glutamate receptor (mGluR) density may be the expression of a specific enhancement in susceptibility of the glutamatergic systems to this excitatory amino acid developing in the course of PTZ-induced kindling.

Sequential ordered mechanism for the sodium-glutamate transport in intestinal brush border membrane vesicles

The glutamate/aspartate carrier localized in the brush-border membrane vesicles from enterocytes is known as a transport system catalyzing a sodium-substrate cotransport driven by the sodium gradient across the membrane. The kinetics of this transport system is studied by analogy with an enzymatic bi-substrate reaction. The results of this approach can be summarized as follows: (1) The dependence of the l-glutamate transport rate on the sodium concentration is sigmoidal, and the stoichiometry of the transport is 2 Na +/1 glutamate/1 carrier molecule. (2) The mechanism is sequential ordered, with l-glutamate binding after both the sodium cations. In addition, there is a very high degree of cooperativity between the two sodium binding sites.

Differential ontogenic pattern of metabotropic [3H]-l-glutamate receptors in normal and granule cell-deficient mouse cerebellum

[3H]-L-glutamate binding site distribution corresponding to metabotropic receptors was studied by autoradiography during normal and altered cerebellar ontogeny in mice treated on postnatal days (PND) 5 and 6 with the antimitotic methylazoxymethanol (MAM). Quisqualate (QA)-induced and (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine (L-CCG-I)-induced [3H]-L-glutamate binding inhibition allowed us to distinguish between group I and group II metabotropic receptor binding sites. In control cerebellar cortex, the QA-sensitive binding site density increases during development, while the L-CCG-I-sensitive binding site density decreases. In the deep cerebellar nuclei (DCN), both populations of binding sites decrease during ontogeny. The antimitotic treatment induces: (1) a slight but significant increase in the QA-sensitive binding sites in the DCN at PND 10 and in the cerebellar cortex beginning from PND 20; (2) a retarded decrease in the L-CCG-I-sensitive metabotropic receptor binding site density. These differences could be due to a retarded cell maturation and/or an over-expression of some postsynaptic receptors in the adult cerebellum in response to the afference deficiency.

Glycine stimulation of glutamate binding to chick retinal pigment epithelium.

The effect of glycine (Gly) and taurine (Tau) on the biochemical and pharmacological properties of [3H]L-glutamate ([3H] Glu) binding to membranes from primary cultures of chick retinal pigment epithelium (RPE), as well as from intact tissue during development was studied. Gly and Tau increase Bmax of [3H]Glu binding to a high affinity site (KB = 300 nM) in membranes from 16 days in vitro (immature) cultures; additionally, Gly discloses a low affinity Glu-binding site (KB = 970 nM) at this stage. In membranes from 25 days in vitro (mature) cultures, the high affinity site is no longer present and Tau has no effect on Glu-binding; Gly still stimulates binding to the low affinity site by four fold, with an EC50 = 200 microM. Pharmacological profile using specific excitatory amino acid (EAA) receptor agonists and antagonists suggests that at 16 days in vitro Glu binds preferentially to metabotropic Glu receptors (mGluRs), and at 25 days in vitro to ionotropic receptors different from neuronal ones. The stimulatory effect of Gly and Tau was also observed in intact RPE, and decreased with increasing embryonic age. Glu binding was also stimulated in membranes from chick retina, but not in those from rat brain. Results support the possibility of EAA participation in several aspects of RPE physiology, including phagocytosis and cell division.

Purification and characterization of a low molecular weight endogenous glutamate binding inhibitor (LGBI) in porcine brain

One of the endogenous substances which modulate glutamate receptor binding was isolated and highly purified from porcine brain. The purification involved extraction of brain tissue with doubled distilled water, followed by gel filtration, anion exchange, cation exchange, and several steps of C18 reverse-phase high performance liquid chromatography (HPLC). A low molecular weight glutamate binding inhibitor (LGBI) was purified to apparent homogeneity as judged from the elution profile of an HPLC column, in which a symmetrical peak was obtained when the eluate was monitored at 220 nm. The LGBI appears to be a small molecule (< 2 kD) that is heat- and acid/base-stable. The highly purified LGBI has no effect on GABAA and benzodiazepine receptor binding. The LGBI is not L-glutamate, L-aspartate or other negatively charged endogenous substances, since they are clearly separated from the LGBI in anion exchange chromatography. The inhibitory effect of the LGBI on [3H]L-glutamate binding is reversible, and it only changes the Bmax while the Kd remains the same. Since the membrane preparations used for [3H]L-glutamate binding assays for the detection of LGBI activity were enriched with quisqualate (QA)-sensitive subtypes, it was suggested that the LGBI could be a modulator of the QA receptor. Some amino acids which produce significant inhibition of glutamate binding activity were also compared with the LGBI, and they all showed no resemblance to the LGBI. The chemical structure of the LGBI remains to be determined.

Increases in NMDA receptor binding are specifically related to memory formation for a passive avoidance task in the chick: a quantitative autoradiographic study

One-day-old chicks ( Gallus domesticus) were trained on a one-trial passive avoidance task in which the aversive stimulus was an unpleasant tasting substance, methylanthranilate (MeA). Control birds were presented with a water (W) coated bead. Five minutes after training a group of MeA-trained chicks were given a brief sub-convulsive trans-cranial electric shock, which rendered half amnesic whilst the remainder were able to show recall for the aversive stimulus. Thirty minutes after training birds were killed and quantitative receptor autoradiography was used to determine NMDA sensitive [ 3H] l-glutamate binding in specific regions of the forebrain of: (i) MeA-trained chicks; (ii) water-control chicks; (iii) MeA-trained electroshocked chicks showing recall; and (iv) MeA-electroshocked chicks amnesic for the aversive stimulus. Increases (80% in the left lobus parolfactorius and 67% in the left intermediate medial hyperstriatum ventrale) in NMDA sensitive [ 3H] l-glutamate binding occurred in electro-shocked chicks which showed recall of the aversive experience but were absent in MeA-trained chicks rendered amnesic by electro-shock. The increased binding in electroshocked MeA-trained birds which showed recall was similar to that observed previously in MeA-trained birds (without electroshock), compared to water control birds, whereas binding levels in MeA-trained electroshocked amnesic birds were not different from those of water control birds. These data argue strongly that alterations in binding to glutamate receptor sub-types are specific to memory formation for the passive avoidance task.

Purification and pharmacological and immunochemical characterization of synaptic membrane proteins with ligand-binding properties of N-methyl-D-aspartate receptors.

A method was developed for the solubilization of approximately 50% of proteins in synaptic membranes that have ligand-binding characteristics of N-methyl-D-aspartate (NMDA) receptors. Affinity chromatographic separation of the solubilized proteins through L-glutamate-derivatized matrices and subsequent elution by NMDA-containing buffers led to the purification of four predominant proteins with estimated sizes of 67-70, 53-62, 41-43, and 28-36 kDa. The co-purification of NMDA-sensitive L-glutamate binding, dizocilpine-sensitive thienylcyclohexyl piperidine (TCP)-binding, and strychnine-insensitive glycine-binding proteins was achieved by this affinity chromatographic procedure. Glutamate, glycine, and the polyamine spermidine increased both the "on" rate and the equilibrium level of [3H]TCP binding to the isolated proteins. The group of proteins eluted by NMDA from the glutamate-derivatized matrices could be further purified through size exclusion chromatography of the NMDA-sensitive glutamate-binding from the dizocilpine-sensitive TCP-binding proteins. Polyclonal and monoclonal antibodies to the cloned NMDA receptor protein NMDAR1 did not react with any proteins in the solubilized membrane proteins or the purified fractions. However, immunoreaction of antibodies raised against a glutamate-binding protein and a phosphonoaminocarboxylic acid-binding protein indicated that these are two of the major proteins in the purified fractions. These studies indicate that these two proteins might be components of a complex that has some of the characteristics of NMDA receptors and that neuronal membranes may contain varieties of NMDA-like receptors composed of protein subunits that differ from the NMDAR1 and NMDAR2 receptor proteins.

Purification and Characterization of a High-Molecular-Weight Endogenous Glutamate-Binding Inhibitor in Porcine Brain.

A high-molecular-weight glutamate-binding inhibitor (HGBI) from porcine brain extract was purified to homogeneity. The results of this purification process show that glutamate receptor activity can be regulated by a high-molecular-weight protein, which inhibits [(3)H]L-glutamate binding to excitatory amino acid (EAA) receptors. The purified HGBI appears to be a protein with a molecular weight of approximately 70 kD. The purified HGBI is negatively charged, suggesting that it may contain acidic amino acids, and most likely, L-glutamate- and L-aspartate-enriched regions, responsible for its surface charge as well as for its binding to glutamate receptors. Inhibition of [(3)H]L-glutamate binding by the purified HGBI is reversible, and appears to change the binding kinetics. This endogenous ligand for glutamate receptors has unique characteristics separating it from all the other ligands found so far in the EAA receptor system. This HGBI represents a new class of modulator for the EAA receptor, thus further investigation of the function and structure of the HGBI should provide new understanding of the mechanisms of EAA-mediated neutrotransmission. Copyright 1994 S. Karger AG, Basel

Mutational analysis of the glycine-binding site of the NMDA receptor: Structural similarity with bacterial amino acid-binding proteins

Activation of the NMDA subtype of ionotropic glutamate receptors requires binding of both L-glutamate and the coagonist glycine. Site-directed mutagenesis of the NMDAR1 (NR1) subunit revealed that aromatic residues at positions 390, 392, and 466 are crucial determinants of glycine binding. Glutamate efficacy was little affected by mutations at these positions; however, inhibition of channel gating by the glycine antagonist 7-chlorokynurenic acid was drastically reduced. In addition, glutamine (Q387), valine (V666), and serine (S669) substitutions were found to reduce glycine efficacy. Since the mutated residues correspond to positions forming the binding site of homologous bacterial amino acid-binding proteins, a common amino acid-binding fold appears to be conserved from prokaryotic periplasmic proteins to glutamate receptors in the mammalian brain.

Specific [ 3H] l-glutamate binding and [ 3H] d-aspartate release in the hippocampus of rat after pentylenetetrazol kindling and long-term potentiation

In the present study, the time-course of changes in glutamate binding and aspartate release in the rat hippocampus following tetanization of perforant pathway and of pentylenetetrazol-induced kindling was investigated. The K + (48 mM)-stimulated [ 3H] d-aspartate release from hippocampal slices and the specific [ 3H] l-glutamate binding to crude synaptic membranes of the hippocampus were measured 1, 4 and 24 h after the last tetanization train or one, five and nine weeks after the last pentylenetetrazol injections, respectively. The tetanization of the fascia dentata was followed by a significant increase in K +-stimulated [ 3H] d-aspartate release from hippocampal slices only 1 h after the last tetanization train, but the specific [ 3 H] l-glutamate binding was enhanced 4 h afterwards. No further deviations from controls have been seen 24 h after tetanization. After pentylenetetrazol kindling, the K +-stimulated amino acid release from hippocampal slices was not changed. However, at one week as well as at five and nine weeks after the pentylenetetrazol kindling the specific [ 3H] l-glutamate binding in the hippocampus was significantly increased by about 50% compared to controls. From these findings it can be assumed that after long-term potentiation induction presynaptic transmitter release and post- (and/or pre-)synaptic glutamate binding are elevated with a different time course only in the early phase of potentiation. In contrast, kindling results in a long lasting increase in activity state of glutamatergic transmission. Therefore, it can be concluded that differences of the synaptic excitability following long-term potentiation and kindling are reflected by a different activation of glutamatergic system in the central nervous system.

Excitotoxins l-β-oxalyl-amino-alanine ( l-BOAA) and 3, 4, 6-trihydroxyphenylalanine (6-OH-DOPA) inhibit [ 3H]α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) binding in human hippocampus

Excitotoxins l-β-oxalyl-amino-alanine ( l-BOAA) and 3, 4, 6-trihydroxyphenylalanine (6-OH-DOPA) have been investigated with regard to their potency to inhibit [ 3H]α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) binding in human hippocampus in a quantitative autoradiographic study. With dissociation constants ( K D) of [ 3H]AMPA binding and inhibition concentrations (IC 50) of l-BOAA, 6-OH-DOPA and l-glutamate obtained from saturation and displacement experiments inhibition constants ( K i) for the inhibition of [ 3H]AMPA binding in individual hippocampal subregions could be calculated. They were between 5.2 ± 2.9 and 35.1 ± 39.9 μM for l-BOAA and 39.1 ± 26.8 and 59.4 ± 44.1 μM for 6-OH-DOPA. l-BOAA was equally potent as the endogenous agonist l-glutamate with K i's between 13.1 ± 3.9 and 21.4 ± 12.1 μM ( n = 4, mean±S.D.). Limbic system symptoms like cognitive deficits, mood disturbances and vivid dreams observed in patients with the motor neuron disease neurolathyrism may thus well be mediated by agonistic action of l-BOAA at AMPA glutamate receptors in hippocampus.

Regeneration of catalytic activity of glutamine synthetase mutants by chemical activation: Exploration of the role of arginines 339 and 359 in activity

In order to understand the nature of ATP and L-glutamate binding to glutamine synthetase, and the involvement of Arg 339 and Arg 359 in catalysis, these amino acids were changed to cysteine via site-directed mutagenesis. Individual mutations (Arg-->Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the Km values for the substrates ATP and glutamate were elevated substantially above the values for wild-type (WT) enzyme. Each cysteine was in turn chemically modified to an arginine "analog" to attempt to "rescue" catalytic activity by covalent modification; 2-chloroacetamidine (CA) (producing a thioether) and 2,2'-dithiobis (acetamidine)(DTBA) (producing a disulfide) were the reagents used to effect these chemical transformations. Upon reaction with CA, both R339C and R359C mutants showed a significant regain of catalytic activity (50% and 70% of WT, respectively) and a drop in Km value for ATP close to that for WT enzyme. With DTBA, chemically modified R339C had a greater kcat than WT glutamine synthetase, but chemically modified R359C only regained a small amount of activity. Modification with DTBA was quantitative for each mutant and each modified enzyme had similar Km values for both ATP and glutamate. The high catalytic activity of DTBA-modified R339C could be reversed to that of unmodified R339C by treatment with dithiothreitol, as expected for a modified enzyme containing a disulfide bond. Modification of each cysteine-containing mutant to a lysine "analog" was accomplished using 3-bromopropylamine (BPA).(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative analysis of the distributions of glutamatergic ligand binding sites in goldfish brain

Goldfish brain is a widely used model study or the study of the mechanism involved in neuronal regeneration and synaptic plasticity. Because of the proposed role of glutamate receptors in these processes we have investigated the anatomical localisations of [ 3H]AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionate), [ 3H]kainate, [ 3H]CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and [ 3H] l-glutamate binding sites in horizontal and sagittal sections. Binding sites for [ 3H] l-glutamate were the most widespread and both NMDA ( N-methyl- d-aspartate) and non-NMDA sensitive components were detected. The density of [ 3H]kainate binding was very high in the cerebellum compared to other regions and in comparison with the other radioligands used. Conversely, relatively low amounts of [ 3H]AMPA binding were present with the telencephalon being the most densely labelled structure. [ 3H]CNQX binding was most densely localised in the tectum with the cerebellum also possessing high binding. In addition, there was a small population of [ 3H]CNQX binding sites located in the telencephalon and lobus vagi that appeared insensitive to AMPA and kainate.

Actions of acromelic acid on nervous system L-glutamate receptors.

Acromelic acid, a naturally occurring kainoid, isolated from the mushroom Clitocybe acromelalga, is a weak displacer of [3H]L-glutamate binding to cockroach (Periplaneta americana) nerve cord membranes. Acromelic acid (1 mM) displaces approximately 60% of specifically bound [3H]L-glutamate. When applied by bath perfusion to the cell body membrane of the cockroach fast coxal depressor motor neurone, acromelic acid generated slow, prolonged, dose-dependent depolarizations at concentrations of 0.3 microM and above. Thus acromelic acid is among the most potent of the excitatory amino acids tested to date on insect neurones.

Distribution of seven major neurotransmitter receptors in the striate cortex of the new world monkey callithrix jacchus

The distribution of seven different binding sites for the transmitters l-glutamate ( l-glutamate binding sites and N-methyl- d-aspartate receptor), GABA (GABA A receptor), noradrenaline (α 1 receptor), acetylcholine (muscarinic M 1 and M 2 receptors) and serotonin (5-hydroxytryptamine 1 receptor) are analysed in the primary visual cortex (area 17) of the common marmoset, Callithrix jacchus, using quantitative autoradiography. All binding sites show a well-defined laminar pattern, which changes sharply at the cytoarchitectonic border to area 18. The quantitative data show that the distribution of different receptors is relatively invariant across the cortical layers. Almost all receptors show a maximum in supragranular layers, low densities in layers IVA/IVB and a second maximum in layer IVC. Statistical analysis of these similarities in laminar distribution patterns of different receptors (co-distribution) reveals, as in other brain regions and species, that l-glutamate binding sites are co-distributed with N-methyl- d-aspartate, GABA A, and muscarinic M 1 and M 2 receptors. This may reflect the structural basis of a possible interaction between these receptors and their respective transmitters on the level of single cortical layers. Further co-distributions are found between N-methyl- d-aspartate, GABA A and M 1, as well as between α 1 and M 1 and finally between M 1 and M 2 receptors. Since not all receptors are co-distributed, the similarities in laminar patterns reveal specific aspects of the neurochemical organization of the cortex when receptors of different transmitter systems are analysed in the same brain. A comparison with data from area 17 of human, rhesus and other monkeys reveals a very similar distribution pattern for most of the receptors investigated among the species. This means that not only the cytoarchitectonic structure of the striate cortex, but also the neurochemical organization of this area is highly conserved during primate evolution.

Autoradiographic localization of [ 3H]-l-glutamate binding sites in a model of cerebellar granule cell ectopia generated by methylazoxymethanol treatment

The distribution of [ 3H]glutamate binding sites was studied in a model of altered cerebellar development obtained by injecting methylazoxymethanol (MAM) in 5-day-old mice. In these mice, at the 25th postnatal day, cerebella were smaller than normal, stratification was normal except for the presence in some lobes of a thin ectopic granule cell layer in the middle of the molecular layer, the proportion of the distribution of [ 3H]glutamate binding sites between molecular and internal granule cell layers was maintained but site density of both quisqualate- and NMDA-sensitive types was increased in the two layers. In the molecular layer, this increase was uniform in spite of the presence of the ectopic cell layer. In the internal granular layer, the increase of quisqualate-sensitive and NMDA-sensitive [ 3H]glutamate binding sites is topographically segregated and the first corresponds to areas of lesser cellular density. These results show that MAM treatment induces persistent alterations of the cerebellar glutamatergic system, which consist of receptor over-expression, possibly due to deficit of innervation, reactive gliosis and immaturity of surviving granule cells.