Glutamate Receptors In Brain Research Articles

Reevaluation of ACEA 1021 as an antagonist at the strychnine-insensitive glycine site of the N-methyl- d-aspartate receptor

Electrophysiological experiments were performed in vitro and in vivo to characterize the inhibitory effects of 6,7-dichloro-5-nitro-1,4-dihydro-2,3-quinoxalinedione (ACEA 1021; licostinel) on rat brain glutamate receptors. In vitro, ACEA 1021 was tested on N-methyl- d-aspartate (NMDA)-induced depolarizations in the neocortical slice preparation and on epileptiform activity in Mg 2+-free hippocampal slices, which is known to be NMDA receptor mediated. In both in vitro models, ACEA 1021 exhibited antagonistic effects on the NMDA receptor-mediated responses. Selectivity tests in the neocortical slice preparation, using NMDA, kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) showed that 10 μM ACEA 1021 reduced NMDA and kainate responses to 27.9 and 79.9% of the control value, respectively, whereas responses to AMPA were increased by 2.4% above the control value, thus showing that at this concentration ACEA 1021 acts preferentially at NMDA receptors. However, at 30 μM, all the NMDA-, AMPA- and kainate-induced responses were reduced. In vivo, ACEA 1021 was tested on NMDA-induced excitation in the CA1 region. After systemic administration of ACEA 1021, central effects were observed at 10 mg/kg i.v. in the CA1 region. These results indicate that ACEA 1021 is centrally active and inhibits NMDA receptor-mediated responses. Interestingly, selectivity tests in the CA1 region did not show clear differences in the action of ACEA 1021 on NMDA- and AMPA-induced excitations. Furthermore, ACh-induced excitations were also reduced. Thus, at low concentrations, ACEA 1021 seems to be a selective antagonist at the strychnine-insensitive glycine site of the NMDA receptor. However, at 30 μM in vitro and at 10 mg/kg in vivo, non-NMDA receptor-mediated actions of ACEA 1021 are observed. Our results suggest that these additional effects of ACEA 1021 may contribute to its anticonvulsive properties in mice as well as to its neuroprotective properties in animal models of cerebral ischemia.

Enhanced [3H]DOPA and [3H]dopamine turnover in striatum and frontal cortex in vivo linked to glutamate receptor antagonism.

We tested the hypothesis that blockade of NMDA glutamate receptors in brain enhances dopamine turnover. We blocked this class of glutamate receptors in the rat brain in vivo with dizocilpine (MK-801) and measured the accumulation of radiolabeled DOPA and its metabolites as functions of time after intravenous bolus injection. Using the time courses of the accumulated metabolites, we calculated the turnover constants of enzymes mediating dopamine synthesis and catabolism. Dizocilpine treatment for 8 days enhanced the rates of DOPA decarboxylation and dopamine oxidation (monoamine oxidation) 4- and 16-fold, respectively, in neostriatum and 10- and 3-fold, respectively, in frontal cortex. The findings are not inconsistent with the hypothesis that the psychotomimetic properties of dizocilpine may be the manifestation of denervation hypersensitivity linked to activation of key enzymes of dopamine turnover in striatum.

Expression of messenger RNAs encoding ionotropic glutamate receptors in rat brain: regulation by haloperidol

In situ hybridization was used to study the regional distribution of messenger RNAs encoding ionotropic glutamate receptor subtypes in the rat brain's dopaminergic cell body regions and their forebrain projection areas. Short oligonucleotide probes specific for the messenger RNAs encoding the flip or flop splice forms of the GluR1 and GluR2 AMPA ( α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) receptor subunits, or for the messenger RNAs encoding the N-methyl- d-aspartate R1 subunit, were used. Significant differences were seen in the relative messenger RNA levels, and the distribution of the flip and flop splice forms, of GluR1 and GluR2. In the dopaminergic cell groups of the substantia nigra pars compacta and the ventral tegmental area, the flip form of both GluR1 and GluR2 dominated over the flop form. Similarly, in the core division of the nucleus accumbens, GluR1 and GluR2 flip forms dominated over the flop forms. In contrast, in the accumbens shell, the GluR1 and GluR2 flop forms dominated over the flip forms. As a comparison to the AMPA receptor subunits, N-methyl- d-aspartate R1 messenger RNA was relatively evenly distributed in all the regions analysed. The results demonstrate a heterogeneous distribution of the flip and flop splice forms of GluR1 and GluR2 in the brain's dopaminergic pathways, which could contribute to physiological differences in regulation of the pathways by glutamatergic neurotransmission. We also studied regulation of glutamate receptor subunit expression in these regions by antipsychotic drugs, based on previous reports of altered levels of subunit immunoreactivity after drug treatment. Chronic administration of the typical antipsychotic drug, haloperidol, caused a small but significant induction of GluR2 flip messenger RNA in the dorsolateral caudate–putamen. This effect was not seen after chronic administration of the atypical antipsychotic drug, clozapine. Significant drug regulation of the other glutamate receptor subunits studied was not observed.

Identification of phosphatidic acid phosphatase type 2 (PAP-2) as an androgen-regulated gene in the human prostatic adenocarcinoma cell line LNCaP : W. Ulrix, J. Swinnen, W. Heyns, and G. Verhoeven. Laboratory for Experimental Medicine and Endocrinology, Faculty of Medicine, Onderwijs en Navorsing, Gasthuisberg, Catholic University of Leuven, Leuven, Belgium

Antibodies were made to synthetic peptides corresponding to residues 253–367, 757–771 and 877–889 of the published amino acid sequence of the rat brain glutamate receptor GluR1 subunit [Hollmann et al. (1989)Nature342, 643–648]. The peptides were synthesized both as multiple copies on a branching lysyl matrix (multiple antigenic peptides) and conventional linear peptides using solid-phase synthesis. Rabbits were immunized with these peptides either without conjugation (multiple antigenic peptides) or following coupling to ovalbumin with glutaraldehyde (monomeric peptides). The antibodies from immune sera were then purified by affinity chromatography using reactigel coupled monomeric peptides. All the rabbits produced good antipeptide responses, and were characterized by immunoprecipitation ofsolubilized α-amino-3-hydroxy-5-methylisoxazole-4-propionate and kainate binding activity and by their staining patterns on immunoblots.Antibody to peptide 253–267 specifically immunoprecipitated 12 ± 3, 50 ± 3 and 44 ± 4% of solubilized α-amino-3-hydroxy-5-methylisoxazole-4-propionate binding activity from cortex, hippocampus and cerebellum, respectively. Under identical conditions, antibody against the 877–889 peptide removed 23 ± 4, 9 ± 4 and 15 ± 9% of α-amino-3-hydroxy-5-methylisoxazole-4-propionate binding sites from these areas. On immunoblots of rat brain membrane samples separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, antibodies labelled a 105,000 mol. wt immunoreactive band. GluR1 was immunoaffinity-purified using subunit-specific antibodies against both N-terminal (253–267) and C-terminal (877–889) residues, covalently attached to protein A-agarose. Analysis of the purified product from each column showed a major immunoreactive band, recognized by both sera at 105,000 mol. wt and silver staining identified the same major protein. After exhaustive immunoprecipitation of solubilized membrane samples with antibody against the C-tenninal of the subunit, a subpopulation of GluR1 was labelled with antibodies specific for the N-terminal part of the receptor. These observations suggest that the GluR1 subunit consists of at least two isoforms possessing a common N-terminal region but a distinct C-terminus.Immunocytochemistry, using immunoperoxidase staining, was performed for the GluRl subunit in rat forebrain with antisera raised against the N-terminal (253–267) and the C-terminal parts (877–889) of the molecule. Both antisera gave a similar distribution of immunoreactivity at the light-microscopic level. Immunoreactivity for the GluR1 subunit was selectively distributed throughout the rat forebrain. The hippocampus, septum, amygdala and olfactory bulb exhibited the strongest immunoreactivity. Immunoreactivity was found in cell bodies and processes of neurons and also in tanicytes, a specialized glial cell type of the hypothalamus.The CA1 region of the hippocampus was particularly rich in GluR1 subunit. Electron-microscopic examination of the CA1 area revealed that immunoreactivity for the GluR1 subunit was present within the endoplasmic reticulum of neurons, at a majority of type I synapses established on dendritic spines, and also at extrasynaptic sites along the dendritic membrane of the pyramidal cells. Immunoreactivity obtained with the antiserum to the N-terminal part (253–267) detected epitopes at the extracellular face of plasma membrane, whereas the antiserum raised against the C-terminal part of the GluR1 subunit (877–889) recognized epitopes at the intracellular face of postsynaptic membranes.The results demonstrate that the antisera selectively react with GluR1 subunit(s) of the α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor. This subunit is distributed in an area and cell type specific manner in the forebrain and is present at synapses assumed to use excitatory amino acids as transmitters. Immunocytochemistry at the subcellular level suggests that the C-terminus of the subunit is located at the cytoplasmic face of the synaptic membrane contrary to the currently held model.

Stoichiometries of AMPA receptor subunit mRNAs in rat brain fall into discrete categories

In situ hybridization was used to estimate the relative concentrations of mRNAs encoding different subunits (GluR1-4) of alpha-amino 3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors in rat brain and to test the hypothesis that within-region expression profiles reflect a limited number of recurring patterns. Fractional subunit mRNA concentrations were calculated for 33 brain regions, and cluster analysis methods were applied to test for statistically meaningful groupings in the data. Four relatively homogeneous classes were identified and designated as AMPA receptor (AR) categories, numbered according to dominant subunit mRNAs. The AR-1 class (47% GluR1 mRNA) was expressed by structures near the mesodiencephalic border, including basal ganglia-related areas. The AR-2 class (57% GluR2 mRNA) was expressed in cortex and tectum. The AR-1,2 class (31% GluR1, 45% GluR2) was found in the largest number of regions, including such dissimilar cell fields as hippocampus and substantia nigra pars compacta. The AR-2,3 grouping (33% GluR2, 31% GluR3) was associated with the sensory relay and reticular thalamic nuclei. It is suggested that AR-1,2 and AR-2, the most closely related categories in clustering space, are largely telencephalic receptors with the former predominant in the subcortex and the latter in the cortex. The AR-2,3 class is associated with ascending sensory stations, whereas AR-1 appears to include several smaller categories expressed by specialized systems. If the balance of subunit mRNAs is reflected at the protein level, then the present data suggest that forebrain AMPA-type glutamate receptors can be classified into a limited number of recurring types.

Carbamoylation of Brain Glutamate Receptors by a Disulfiram Metabolite

S-Methyl-N,N-diethylthiolcarbamate sulfoxide (DETC-MeSO), a metabolite of the drug disulfiram, is a selective carbamoylating agent for sulfhydryl groups. Treatment of glutamate receptors isolated from mouse brain with DETC-MeSO blocks glutamate binding. In vivo, carbamoylated glutathione, administered directly to mice or formed by reaction of DETC-MeSO with glutathione in the blood, also blocks brain glutamate receptors. Carbamoyl groups appear to be delivered to brain glutamate receptors or to liver aldehyde dehydrogenase in vivo by a novel glutathione-mediated mechanism. Seizures caused by the glutamate analogs N-methyl-D-aspartate and methionine sulfoximine, or by hyperbaric oxygen, are prevented by DETC-MeSO, indicating that carbamoylation of glutamate receptors gives an antagonist effect. These observations offer an explanation for some of the previously reported neurological effects of disulfiram, such as its ability to prevent O2-induced seizures. Furthermore, some of the physiology of the disulfiram-ethanol reaction, that could not be accounted for based on the known inhibition of aldehyde dehydrogenase alone, may be explained by disulfiram's effect on glutamate receptors.

Expression and coupling to polyphosphoinositide hydrolysis of group I metabotropic glutamate receptors in early postnatal and adult rat brain.

We investigated the expression and coupling to the phospholipase C signal transduction pathway of metabotropic glutamate receptor (mGluR) subtypes by Western blot analysis and agonist-stimulated inositol monophosphate formation in several brain regions of postnatal day 9 (P9) and adult rats. In the cerebral cortex, hippocampus, corpus striatum, olfactory bulb, cerebellum and hypothalamus, the expression level of mGluR5 was greater at P9 than in adulthood. The mGluR5 signal was very low or absent in the adult cerebellum and hypothalamus. The expression of mGluR1a was slightly greater at P9 in the hypothalamus, hippocampus and olfactory bulb, whereas it substantially increased with age in the cerebellum, and did not change in the cerebral cortex and corpus striatum. mGluR1b and -1c were nearly undetectable by Western blot analysis. The expression level of mGluR5, but not that of mGluR1a, was significantly correlated with the extent of phosphoinositide hydrolysis stimulated by mGluR agonists in slices prepared from these brain regions. The mGluR antagonist cyclopropan[b]chromen-1a-carboxylic acid ethylester (CPCCOEt), potently antagonized responses mediated by mGluR1, but much less potently those mediated by mGluR5a in recombinant cells. CPCCOEt, at a concentration which efficiently blocks mGluR1 responses, did not substantially affect the polyphosphoinositide response in hippocampal or cerebellar slices from newborn animals, and antagonized only a minor component of the polyphosphoinositide response in adult hippocampal slices. CPCCOEt, however, prevented the small stimulation of polyphosphoinositide hydrolysis by mGluR agonists in adult cerebellar slices. We conclude that (i) the efficient mGluR-mediated polyphosphoinositide hydrolysis in 9-day-old rats is mediated by mGluR5; (ii) the increased expression of mGluR1 in the adult cerebellum does not substitute for the decline of mGluR5 expression in the ability to mediate polyphosphoinositide hydrolysis; and therefore (iii) mGluR1a might couple less efficiently than mGluR5 to polyphosphoinositide hydrolysis.

Purification of Human Double-stranded RNA-specific Editase 1 (hRED1) Involved in Editing of Brain Glutamate Receptor B Pre-mRNA

RNAs encoding subunits of glutamate-gated ion channel receptors are posttranscriptionally modified by RNA editing and alternative splicing. The change in amino acid sequence caused by RNA editing can affect both the kinetics and the permeability of the ion channel receptors to cations. Here, we report the purification of a 90-kDa double-stranded RNA-specific adenosine deaminase from HeLa cell nuclear extract that specifically edits the glutamine codon at position 586 in the pre-mRNA of the glutamate receptor B subunit. Site-specific deamination of an adenosine to an inosine converts the glutamine codon to that of arginine. Recently, a gene encoding a double-stranded-specific editase (RED1) was cloned from a rat brain cDNA library. Antibodies generated against the deaminase domain of its human homolog specifically recognized and inhibited the activity of the 90-kDa enzyme, indicating that we have purified hRED1 the human homolog of rat RED1. This enzyme is distinct from double-stranded RNA-specific adenosine deaminase which we and others have previously purified and cloned.

Purification and biochemical characterization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-sensitive L-glutamate receptors of pig brain.

Two preparations of glutamate receptors were purified from the synaptic junctions of pig brain by a combination of detergent solubilization, anion-exchange chromatography, wheat-germ agglutinin affinity chromatography and sedimentation through sucrose gradients. These preparations were enriched in specific L-[3H]glutamate binding activity (> 5000 pmol of glutamate binding sites/mg of protein), and the rank order of ligand affinity for binding to these preparations was: quisqualate > 6-cyano-7- nitroquinoxaline-2,3-dione > alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) > L-glutamate > kainate > > N-methyl-D-aspartate approximately L-2-amino-4-phosphonobutyrate. SDS/PAGE analysis revealed that more than 80% of the protein in either of these preparations appeared as a single protein band of 106 kDa. Two-dimensional gel electrophoresis further revealed that these 106 kDa proteins consisted of a series of acidic proteins which were recognized by antibodies against rat AMPA receptor subunits. These 106 kDa proteins were also recognized by wheatgerm agglutinin and concanavalin A; in addition, peptide N-glycosidase F treatment of these preparations decreased their size to 99 kDa. Our results suggest that the putative glutamate receptors isolated here are likely to belong to the AMPA subtype of glutamate receptors in pig brain. Using the purification procedure reported here, 5 micrograms of AMPA receptor proteins can be isolated from 250 g of pig brain tissue.

Secretion of Green Fluorescent Protein from Recombinant Baculovirus-Infected Insect Cells

Trichoplusia ni(High Five) andSpodoptera frugiperda(Sf21) cells were engineered for expression of epitope (Flag)-tagged signal peptide-green fluorescent protein (GFP) fusions to examine the suitability of GFP as a secretory marker. The recombinant baculovirus-infected cells became fluorescent, and the High Five cells but not Sf21 cells secreted GFP in the culture medium as detected by the presence in the culture supernatant of a Flag-immunoreactive 30-kDa species and the characteristic 510-nm GFP fluorescence peak. Signal peptides derived from ecdysteroid UDP-glucosyltransferase ofAutographa californicanuclear polyhedrosis virus and from rat brain glutamate receptor were both able to promote secretion of GFP. GFP may thus be used as a research tool in the study of the secretory process in insect cells both in cell biology and in biotechnological applications.

The enhancement and the inhibition of noradrenaune-induced cyclic amp accumulation in rat brain by stimulation of metabotropic glutamate receptors

1. 1. The actions of several metabotropic glutamate receptor agonists and antagonists on noradrenaline (NA)-stimulated [ 3H]-cyclic AMP accumulation were investigated in rat cerebral cortical slices. 2. 2. Quisqualate (QUIS), L-2-amino-3-phosphonopropionic acid (L-AP3) and glutamate (GLU) elicited concentration-dependent inhibition of (NA)-stimulated [ 3H]-cyclic AMP accumulation, with IC 50 values of 105 ± 29, 275 ± 36 and 944 ± 150 μM respectively. In contrast (Rs)-α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) (0.5 mM) and N-methyl-D-aspartic acid (NMDA) (0.5 mM) had no effect. 3. 3. (2S,3S,4S)-α-(Carboxycyclopropyl)glycine (L-CCGI), 1-Aminocyclo-pentane-lS,3R-dicarbo-xylate (1S,3R-ACPD), ibotenate (IBO) and (RS)-4-carboxy-3-hydroxy-phenylglycine (CHPG)elicited a concentration-dependent enhancement ofNA-stimulated [ 3H]-cyclic AMP accumulation, with EC 50 values of 2.5 ± 0.11, 42 ± 1.3, 97.8 ± 2.1 and 157 ± 13.4 μM, respectively. 4. 4. (S)-3-carboxy-4-hydroxyphenylglycine (3C4HPG) and (S)-4-carboxy-3-hydroxyphenyl-glycine (4C3HPG) produced a biphasic effect, at concentrations up to 100 and 500 μM, respectively, they significantly enhanced the action of NA (100 μM), at ImM concentration both compounds as well as α-methyl-4-carboxyphenylglycine (MCPG) produced a significant inhibition of NA-stimulated cyclic AMP accumulation. 5. 5. A putative mGluR antagonist — L-AP3, inhibited the 1S,3R-ACPD (100 μM) induced enhancement of the action of NA (100 μM) on [ 3H]-cyclic AMP accumulation in a biphasic manner with an IC 50 of 4.5 μM for the high affinity site, which represented 65% of the total and an IC 50 of 283 μM for the low affinity site. 6. 6. β-adrenoceptor antagonist propranolol inhibited the interaction between 1S,3R-ACPD (100 μM) and NA (100 μM) on [ 3H]-cyclic AMP accumulation by about 80%, with an IC 50 of 0.52 ± 0.011 μM, to the level observed after 1S,3R-ACPD alone. Prazosin, an α 1-adrenoceptor antagonist was more potent (IC 50 of 0.091 ± 0.012 μM) but less efficacious (60% inhibition) as an inhibitor of the interaction either between NA and 1S,3R-ACPD while yohimbine, na α 2-adrenoceptor antagonist (up to 1 μM) had no effect. 7. 7. Neither the protein kinase C inhibitor — staurosporine (10 μM) nor thapsigargin (1 μM), which depletes IP3 sensitive calcium stores, inhibited significantly the 1S,3R-ACPD (100 μM)-induced enhancement of the action of NA (100 μM) on [ 3H]-cyclic AMP accumulation. 8. 8. Adenosine deaminase (0.5 U/ml) abolished both the 1S,3R-ACPD (100 μM)-induced [ 3H]-cyclic AMP accumulation and the synergistic interaction of this compound with NA (100 μM). 9. 9. These results indicate the existence of different subtypes of metabotropic glutamate receptors in rat brain which either inhibit or enhance the NA-stimulated [ 3H]-cyclic AMP accumulation. The enhancement m cerebral cortical slices is mediated via receptors which are blocked with high affinity by L-AP3 and occurs via interactions with endogenous adenosine; the inhibition is mediated by receptors sensitive to quisqualate, L-AP3 and glutamate and may represent a predominant interaction between NA and excitatory amino acids (EAA), which in cerebral cortical slices is masked by excitatory effects.

RNA editing: how a message is changed

Considerable progress has been made in unraveling the mechanistic features of RNA editing processes in a number of genetic systems. Recent highlights include the identification of the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme as a zinc-dependent cytidine deaminase that binds to RNA, the demonstration that adenosines in brain glutamate receptor pre-mRNAs are converted into inosines and that double-stranded RNA A deamaminase (dsRAD), the candidate enzyme, is another zinc-dependent RNA nucleotide deaminase, and a mounting body of evidence for a cleavage-ligation mechanism for U insertion/deletion editing in kinetoplastid protozoa.

RNA Editing

RNA editing is a term describing a variety of novel mechanisms for the modification of nucleotide sequences of RNA transcripts in different organisms. These editing events include (a) the U-insertion and -deletion type of editing found in the mitochondrion of kinetoplastid protozoa, (b) the C-insertion editing found in the mitochondrion of Physarum, (c) the C-to-U substitution editing of the mammalian apoB mRNA, (d) a similar C-to-U substitution editing of mRNAs in higher plant mitochondria and chloroplasts and in tRNAs of marsupials and rats, (e) a diverse nucleotide substitution editing of tRNAs in Acanthomoeba mitochondria, and (f) the A-to-I type of editing found in the mammalian glutamate receptor subunits. These diverse phenomena involve several different enzymatic mechanisms. In several cases, duplex RNAs with internal or external guide sequences help determine the site specificity of editing. The A-to-I editing observed in RNAs encoding non-NMDA glutamate receptor subunits may be due to the actions of a double-stranded RNA-specific adenosine deaminase that is widespread in higher organisms. Although the function of many RNA editing events is unclear, the biological importance of RNA editing in other systems may prove as significant as the nucleotide modifications regulating the cation selectivity and electrophysiological profiles elaborated by non-NMDA glutamate receptors in the mammalian brain.

Inhibition of [3H]CGP 39653 binding to NMDA receptors by a P2 antagonist, suramin

Suramin, which has been used for more than 50 years as an anthelmintic in humans, has recently been shown to inhibit P2 purinoceptors in the CNS and to block glutamate-evoked excitatory potentials in hippocampus. We now report that suramin inhibits (IC50 ~ 100 µM) the binding of [3H]CGP 39653, a ligand specific for NMDA-type glutamate receptors in brain. Consequently, suramin at concentrations used sometimes in physiological experiments may act as both P2x and NMDA antagonist. Since both NMDA receptors and P2x-subtype of purinoceptors form Ca2+-selective channels, suramin may block Ca2+ entry into neurones depolarized by either ATP, l-glutamate or any other neurotransmitter or drug acting via P2x or NMDA receptors.

Inhibition of [3H]CGP 39653 binding to NMDA receptors by a P2 antagonist, suramin

Suramin, which has been used for more than 50 years as an anthelmintic in humans, has recently been shown to inhibit P2 purinoceptors in the CNS and to block glutamate-evoked excitatory potentials in hippocampus. We now report that suramin inhibits (IC50 approximately 100 microM) the binding of [3H]CGP 39653, a ligand specific for NMDA-type glutamate receptors in brain. Consequently, suramin at concentrations used sometimes in physiological experiments may act as both P2x and NMDA antagonist. Since both NMDA receptors and P2x-subtype of purinoceptors form Ca(2+)-selective channels, suramin may block Ca2+ entry into neurones depolarized by either ATP, L-glutamate or any other neurotransmitter or drug acting via P2x or NMDA receptors.

Minireview: Cell protective role of melatonin in the brain

In recent years an increasing amount of data has been published involving melatonin in the control of brain function. The pineal gland exerts a depressive influence on CNS excitability. This activity is linked to melatonin, since pharmacological doses of the hormone prevent seizures in several animal models. In addition, melatonin also has analgesic properties in these species. However, the sites and mechanism of melatonin action are not known. A role for the pineal gland and its hormone melatonin as a homeostatic system controlling brain excitability has been proposed, and GABA-containing neurons may be involved in some central action of melatonin. There is evidence supporting a role of melatonin in the regulation of the GABA-benzodiazepine receptor complex, and it appears that melatonin potentiates this inhibitory neurotransmitter system in brain. Melatonin does not bind to GABA or benzodiazepine binding sites themselves, because in vitro binding data showed that melatonin is a weak competitor of benzodiazepine binding in brain membranes at concentrations greater than 10(-5) M. The effect of melatonin on brain activity also involves the participation of corticotropic and opioid peptides, and the existence of an opioid-antiopioid homeostatic system is proposed, with the GABA-benzodiazepine receptor complex as an effector. Moreover, the interaction of melatonin with corticotropic peptides and mitochondrial benzodiazepine receptors may result in a participation of neurosteroids in the control of GABA activity and function. The most recently available data from biochemical and electrophysiological studies support the possibility that the anticonvulsant and depressive effects of melatonin on neuron activity may depend on its antioxidant and antiexcitotoxic roles, acting as a free radical scavenger and regulating brain glutamate receptors. The full characterization of the nuclear melatonin receptor explains the genomic effects of melatonin, opening a new perspective regarding actions and roles of melatonin as a cellular protector.

Inhibition of kainic acid binding to glutamate receptors by extracts of Gastrodia

S-(4-hydroxybenzyl)glutathione was isolated as the major principle responsible for the inhibition of the in vitro binding of kainic acid to brain glutamate receptors by water extracts of the plant Gastrodia elata. The affinity (IC 50 value) of the compound is slightly lower compared to glutamate and glutathione.

Possible dysfunction of ionotropic glutamate receptors in cerebellum of epileptic E1 mouse brain

Binding of [ 3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) to an ion channel domain on the N -methyl- d-aspartate (NMDA)-sensitive subclass of brain glutamate (Glu) receptors was highest in the hippocampus of the hereditary epileptogenic mutant E1 as well as its parent ddY strain mice, when determined before and at equilibrium in the presence of 3 different agonists at the respective domains on the NMDA receptor complex, including Glu, glycine (Gly) and spermidine (SPD). Cerebellar [ 3H]MK-801 binding before equilibrium was significantly lower in E1 mice than in ddY mice, while the binding was not significantly different from each other in other brain structures of both strains of mice. Kinetic analysis revealed that the association rate was significantly lower with [ 3H]MK-801 binding in the cerebellum of E1 mice than of ddY mice. In contrast to ddY mice, furthermore, Gly failed to potentiate cerebellar [ 3H]MK-801 binding before equilibrium in E1 mice, with SPD being active in significantly inhibiting the binding. However, saturation analysis revealed that the affinity and density of cerebellar [ 3H]MK-801 binding at equilibrium in E1 mice were not significantly different from those in ddY mice. In addition, epileptogenic E1 mice had significantly higher levels of [ 3H]SPD binding in all brain structures examined than ddY mice, whereas [ 3H] dl-α-amino-3-hydroxy-5-methylisoxazole-4-propionate binding was significantly lower in the cerebellum of E1 mice than of ddY mice. These results suggest that dysfunction of cerebellar Glu receptors may be at least in part responsible for a variety of abnormal symptoms observed in epileptic E1 mice.

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.

Immunohistochemical localization of metabotropic and ionotropic glutamate receptors in the mouse brain.

Immunohistochemical localization of metabotropic and ionotropic glutamate receptors in the mouse brain.