Arlette Minet

Cloning of the rat brain cDNA encoding for the SLC-1 G protein-coupled receptor reveals the presence of an intron in the gene

In order to isolate new G protein-coupled receptors expressed in the cerebral cortex, a set of degenerate oligonucleotides corresponding to the third and seventh transmembrane segment were synthetized. Their use in PCR on rat brain cortex mRNA amplified several cDNA fragments. One of them, a 526 bp sequence, encoded for what was at that time an unknown G protein-coupled receptor. An oligonucleotide derived from the sequence was then used as a probe to isolate the receptor cDNA from a rat brain cDNA library. It encodes for a 353aa protein with seven transmembrane segments, three consensus N-glycosylation sites at the amino terminus and several potential phosphorylation sites in the intracellular loops. This protein shares 91% overall identity with a recently cloned human somatostatin-like receptor of 402aa named SLC-1. This suggests that we have cloned the rat orthologue of the human SLC-1. However, the extracellular N-terminus of the human receptor is 49 amino acids longer and shows 50% identity with the rat one. Because the human sequence was deduced from genomic DNA, we suspected the presence of an intron in the gene. This was confirmed by PCR using primers spanning the intron. On the basis of the sequence of a 128 kb fragment of chromosome 22 encompassing the SLC-1 gene, we were able to deduce a corrected amino acids sequence for the human receptor. So both rat and human SLC-1 receptors are 353aa long, with three consensus N-glycosylation sites. They share 96% identity at the amino acid level and are encoded by a gene containing one intron in the coding sequence.

Disrupting the melanin-concentrating hormone receptor 1 in mice leads to cognitive deficits and alterations of NMDA receptor function

In order to investigate the physiological properties of the melanin‐concentrating hormone (MCH) we have generated and used mice from which the MCH receptor 1 gene was deleted (MCHR1Neo/Neo mice). Complementary experimental approaches were used to investigate alterations in the learning and memory processes of our transgenic model. The ability of the knockout strain to carry out the inhibitory passive avoidance test was found to be considerably impaired although no significant differences were observed in anxiety levels. This impaired cognitive property prompted us to explore modifications in N‐methyl d‐aspartate (NMDA) responses in the hippocampus. Intracellular recordings of CA1 pyramidal neurons in hippocampal slices from the MCHR1Neo/Neo mice revealed significantly decreased NMDA responses. Finally, using in situ hybridization we found a 15% reduction in NMDAR1 subunit in the CA1 region. These results show for the first time a possible role for MCH in the control of the function of the NMDA receptor.

Immunocytochemical localization of ionotropic glutamate receptors subunits in the adult quail forebrain.

The excitatory amino acid glutamate is implicated in the central control of many neuroendocrine and behavioral processes. The ionotropic glutamate receptors are usually divided into the N‐methyl‐D‐aspartate (NMDA) and non‐NMDA (kainate and AMPA) subtypes. Subunits of these receptors have been cloned in a few mammalian species. Information available in birds is more limited. In quail, we recently demonstrated that glutamate agonists (kainate, AMPA, and NMDA) rapidly (within minutes) and reversibly decrease in vitro aromatase activity like several other manipulations affecting intracellular HCa2+ pools. Aromatase catalyzes the conversion of androgens into estrogens which is a limiting step in the control by testosterone of many behavioral and physiologic processes. Therefore, glutamate could control estrogen production in the brain, but the anatomic substrate supporting this effect is poorly understood. In quail, aromatase is mainly localized in the preoptic‐hypothalamic‐limbic system. We visualized here the distribution of the major ionotropic glutamate receptors in quail by immunocytochemical methods by using commercial primary antibodies raised against rat glutamate receptor 1 and receptors 2‐3 (GluR1, GluR2/3: AMPA subtype, Chemicon, CA), rat glutamate receptors 5‐7 (GluR5‐7: kainate subtype, Pharmingen, CA), and rat NMDA receptors (NMDAR1, Pharmingen, CA). Dense and specific signals were obtained with all antibodies. The four types of receptors are broadly distributed in the brain, and, in particular, immunoreactive cells are identified within the major aromatase cell groups located in the medial preoptic nucleus, ventromedial hypothalamus, nucleus striae terminalis, and nucleus taeniae. Dense specific populations of glutamate receptor‐immunoreactive cells are also present with a receptor subtype‐specific distribution in broad areas of the telencephalon. The distribution of glutamate receptors, therefore, is consistent with the idea that these receptors could be located at the surface of aromatase‐containing cells and mediate the rapid regulation of aromatase activity in a direct manner. J. Comp. Neurol. 428:577–608, 2000.

Astroglial Cells Express Large Amounts of GABAA Receptor Proteins in Mature Brain

Abstract: GABAA receptors were characterized in cellular fractions isolated from adult bovine brain. The fraction enriched in cortical astrocytes is very rich in high‐affinity binding sites for [3H]flunitrazepam and other “central‐type” benzodiazepine ligands. The amount of specific [3H]flunitrazepam binding was more than five times higher in the glial fraction than in synaptosomal and perikaryal fractions. [3H]Flunitrazepam was displaced by low concentrations of clonazepam and other specific ligands for central GABAA receptors. Specific binding sites for GABA, flunitrazepam, barbiturates, and picrotoxin‐like convulsants were characterized. Allosteric interactions between the different sites were typical of central‐type GABAA receptors. The presence of α‐subunit(s), as revealed by [3H]flunitrazepam photoaffinity labeling, was demonstrated in all brain fractions at molecular mass 51–53 kDa. Photoaffinity labeling was highest in the glial fraction. However, in primary cultured astrocytes from neonate rat cortex, no photoaffinity labeling was detected. Information obtained from astrocytes in culture should thus be taken with caution when extrapolated to differentiated astroglial cells. Our results actually show that, in mature brain, most of the fully pharmacologically active GABAA receptors are extrasynaptic and expressed in astroglia.

The Genetic Absence Epilepsy Rat from Strasbourg (GAERS), a Rat Model of Absence Epilepsy: Computer Modeling and Differential Gene Expression

Summary:  Purpose: We present results obtained by computer modeling of the thalamic network and differential gene expression analysis in a rat strain with absence epilepsy, the genetic absence epilepsy rat from Strasbourg (GAERS).

Expression of mRNA encoding alpha1E and alpha1G subunit in the brain of a rat model of absence epilepsy.

Low voltage-activated calcium channels are thought to play a key role in the generation of spike and waves discharges characteristic of absence epilepsy. Therefore, the expression level of mRNA encoding calcium channel alpha1E and alpha1G subunits was measured in the brain of genetic absence epilepsy rats from Strasbourg (GAERS). Using quantitative RT-PCR and in situ hybridization, no difference was found in alpha1G mRNA expression between GAERS and control animals, while a decreased expression of alpha1E was seen in the cerebellum and the brain stem of the GAERS. This phenomenon was not observed in young animals when the epileptic phenotype is not expressed.

Phenytoin dephosphorylates the alpha (−) catalytic subunit of (Na+, K+)-ATPase: A study in mouse, cat and human brain

Phenytoin, a potent antiepileptic drug, has been thought to stimulate Na+, K+ transport across cell membranes, but its influence on (Na+, K+)-ATPase activity remains highly controversial. We have investigated the effects of the drug on the phosphorylation level of (Na+, K+)-ATPase partially purified from mouse, cat and human brain. (Na+, K+)-ATPase catalytic subunits [alpha(+) and alpha(-)] were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis. Previous experiments had shown that phenytoin dephosphorylates the (Na+, K+)-ATPase catalytic subunit by +/- 50% in C57/BL mice. In the present study, we showed that phenytoin (10(-4) M) decreases the phosphorylation level of (Na+, K+)-ATPase catalytic subunit by the same value in cat and human cortex. Moreover, that effect is predominant on the alpha(-) subunit, thought to be the predominant enzymatic form in non-neuronal or glial cells. The results are thus favoring the hypothesis that phenytoin stimulates the brain (Na+, K+)-ATPase. They further suggest that phenytoin mainly activates the glial enzymatic form, providing central nervous system with an enhanced ability to regulate extracellular K+.

Effect of Milacemide on Audiogenic Seizures and Cortical (Na+, K+)-ATPase of DBA/2J Mice

SUMMARY: Milacemide (MLM, CP 1552 S, 2‐JV‐pentylaminoacetamide), a glycinamide derivative, is currently being evaluated clinically for antiepileptic activity. Anticonvulsant properties have been shown in various animal models, but the mechanism of action of MLM is unclear. We studied its activity in audiogenic seizures of DBA/2J mice. MLM was effective in inhibiting the convulsions induced by sound with a biphasic dose‐effect relation. The ED50 was 109 mg/kg orally against tonic extension. Higher doses were necessary to abolish clonic convulsion and running response. Because impaired cerebral (Na+, K+)‐ATPase activity is supposed to play a role in epileptogenesis, we tested MLM on in vitro cortical enzymatic activity of DBA/2J mice. Basal (Na+, K+)‐ATPase activity was unchanged by several concentrations of MLM in normal C57BL/6J and audiogenic DBA/ 2J mice. K+ activation (from 3 to 18 mM) of (Na+, K+)‐ATPase is abolished in DBA/2J mice as compared with C57BL/6J mice, suggesting impaired glial (Na+, K+)‐ATPase. In the presence of MLM (from 30 to 1000 mg/L), cortical (Na+, K+)‐ATPase of DBA/2J mice is activated by high concentrations of K+, as in C57BL/6J mice. Results suggest that the antiepileptic activity of MLM in audiogenic mice may be secondary to an activation of a deficient glial (Na+, K +)‐ATPase.

Unusual amino acids and monofluoroacetate from Dichapetalum michelsonii (Umutambasha), a toxic plant from Rwanda.

In the course of our investigations on Umutambasha in order to identify its convulsant principles, small quantities of monofluoroacetate were observed in stem bark, leaves, and fruits of this plant newly identified as Dichapetalum michelsonii Hauman. Conclusive evidence for a monofluoroacetate presence came from its isolation from the freeze-dried extract of stem bark. Three free unusual amino acids, named N-methyl-α-alanine, N-methyl-β-alanine, and 2,7-diaminooctan-1,8-dioic acid, described for the first time in a plant, and known trigonelline were also isolated from the stem bark of D. michelsonii. Structure elucidations were mainly achieved by spectroscopic methods (1H-NMR, 2D-NMR, MS) and by comparison with authentic references. These unusual amino acids were detected by a fast, reliable TLC analysis in all our batches of Umutambasha, suggesting that they could be used for identification purposes in case of human or livestock intoxications. Finally, EEG recordings and behavioural observations performed in mice suggested that the convulsive patterns produced by Umutambasha are the consequence of monofluoroacetate presence in D. michelsonii.