Pascale Montpied

Chronic ethanol and pentobarbital administration in the rat: Effects on GABAA receptor function and expression in brain

Chronic exposure of rats to ethanol significantly decrease GABAA receptor-mediated 36Cl- uptake in cerebral cortical synaptoneurosomes. Muscimol and pentobarbital stimulation as well as ethanol enhancement of muscimol-stimulated 36Cl- flux are significantly decreased following chronic ethanol inhalation. Repeated pentobarbital administration has a similar effect on muscimol and pentobarbital-stimulated 36Cl- uptake in cerebral cortical synaptoneurosomes. We have postulated that these adaptive response may be associated with an alteration of GABAA receptor gene expression. Chronic ethanol exposure resulted in a significant reduction in the levels of GABAA receptor alpha-subunit mRNAs. The most abundant mRNA species in the rat cerebral cortex were reduced 40-50% (4.4 Kb mRNA, 43%, 4.8 Kb mRNA, 47%). beta-Actin mRNA and poly(A)+ RNA levels were not significantly reduced following chronic ethanol exposure. Repeated pentobarbital administration had no effect on the level of the 4.4 and 4.8 Kb transcripts of alpha-subunit mRNAs in rat cerebral cortex. These data suggest that chronic ethanol exposure alters the level of mRNAs coding for the alpha-subunit of the GABAA receptor. This decrease may reflect an alteration of mRNA processing in the cell or an alteration in GABAA receptor gene expression.

Differential effects of chronic ethanol administration on GABAA receptor α1 and α6 subunit mRNA levels in rat cerebellum

Chronic ethanol exposure alters muscimol, pentobarbital, and benzodiazepine agonist and inverse agonist effects on the function of GABA(A) receptor-gated Cl(-) channels in the central nervous system (CNS). We have recently shown that prolonged ethanol inhalation reduces the expression of GABA(A) receptor alpha1 and alpha2 subunit mRNAs in the rat cerebral cortex, with no effect on the level of alpha3 subunit transcripts, glutamic acid decarboxylase mRNA levels, or poly(A)(+) RNA levels. In the present study, rats were administered alcohol by liquid diet for 2 weeks using a pair-fed design. GABA(A) receptor alpha subunit mRNA levels were quantified by Northern analysis using specific cRNA probes. GABA(A) receptor alpha1 subunit mRNA levels were reduced in the cerebral cortex to the same extent as previously reported following prolonged ethanol inhalation. In the cerebellum, chronic ethanol ingestion reduced the levels of GABA(A) receptor alpha1 subunit mRNAs (4.8 and 4.4 kb) by 20-30% and increased the levels of GABA(A) receptor a6 subunit mRNA (2.7 kb) by 45%. GABA(A) receptor alpha2 and alpha3 subunit mRNAs were not detected in the cerebellum. Glutamic acid decarboxylase mRNA levels as well as poly(A)(+) RNA levels were not significantly altered following chronic ethanol exposure by liquid diet. Acute ethanol administration had no effect on GABA(A) receptor a6 subunit mRNA levels. However, acute administration of both Ro15-4513 and its vehicle control altered GABA(A) receptor alpha6 subunit mRNA levels in the cerebellum. Since GABA(A) receptor alpha6 subunits contain recognition sites for Ro15-4513, an inverse agonist, and an ethanol antagonist, the elevation in the expression of these subunits following chronic ethanol ingestion may account for increased sensitivity to inverse agonists after chronic ethanol administration and possibly contribute to the withdrawal syndrome. These data also suggest that chronic ethanol exposure regulates GABA(A) receptor gene expression by differential effects on the synthesis of specific subunits of GABA(A) receptors in the CNS.

Expression of tyrosine hydroxylase in cerebellar Purkinje neurons of the mutant tottering and leaner mouse

In situ hybridization histochemistry, Northern blot analysis and immunohistochemistry were used to examine tyrosine hydroxylase (TH) mRNA concentrations and immunoreactivity in the locus coeruleus and cerebellum of the tottering (tg/tg), leaner (tgla/tgla), compound heterozygous (tg/tgla) and wild type control (+/+) mice, bred on a C57BL/6J background. Cerebellar Purkinje neurons, long considered to be GABAergic, showed high levels of TH mRNA in the caudal vermis and the lateral hemispheres of the cerebellum of tg/tg, tg/tgla, and tgla/tgla mice. Analysis of grain density over individual Purkinje cells showed significantly greater concentrations of TH mRNA in tg/tg, tg/tgla, and tgla/tgla mice as compared to +/+ wild type control mice. Comparison of adult (greater than or equal to 2 months) and young, pre-seizure (less than or equal to 3 weeks) mutant mice showed Purkinje cells densely labelled for TH mRNA at both ages, suggesting that TH gene expression in Purkinje cells is independent of the onset of seizures. Northern blot analysis confirmed the findings from the in situ hybridization studies, demonstrating a single band identical to TH mRNA. Immunohistochemistry confirmed the presence of TH protein in Purkinje cells of the caudal vermis and the lateral hemispheres of the cerebellum in both control and mutant mice. Quantitation of mRNA for TH and the coexisting neuropeptide, galanin, in the locus coeruleus detected no significant differences between adult tg/tg, tg/tgla and +/+ control mice. The present findings demonstrate that the classically GABAergic Purkinje cells in the cerebellum express low levels of TH, and that the mutant tottering and leaner strains of mice express extremely high levels of mRNA and protein for TH.

Regional Distribution of the GABAA/Benzodiazepine Receptor (α Subunit) mRNA in Rat Brain

Abstract: A human cDNA clone containing the 5’coding region of the GABAA/benzodiazepine receptor α subunit was used to quantify and visualize receptor mRNA in various regions of the rat brain. Using a [32P]CTP‐labelled antisense RNA probe (860 bases) prepared from the α subunit cDNA, multiple mRNA species were detected in Northern blots using total and poly A rat brain RNA. In all brain regions, mRNAs of 4.4 and 4.8 kb were observed, and an additional mRNA of 3.0 kb was detected in the cerebellum and hippocampus. The level of GABAA/benzodiazepine receptor mRNA was highest in the cerebellum followed by the thalamus = frontal cortex = hippocampus = parietal cortex = hypothalamus ≫ pons = striatum = medulla. In situ hybridization revealed high levels of α subunit mRNA in cerebellar gray matter, olfactory bulb, thalamus, hippocampus/dentate gyrus, and the arcuate nucleus of the hypothalamus. These data suggest the presence of multiple GABAA/benzodiazepine receptor α subunit mRNAs in rat brain and demonstrate the feasibility of studying the expression of genes encoding the GABAA/benzodiazepine receptor after pharmacological and/or environmental manipulation.

Multiple gabaa receptor α subunit mRNAs revealed by developmental and regional expression in rat, chicken and human brain

GABAA receptor α subunit transcripts were detected by Northern analysis of rat, chicken and human brain mRNA using a series of 32 P‐labelled antisense RNA probes derived from human α1 subunit cDNAs. These α subunit mRNAs differ in their distribution among various brain regions in the rat and at least one species is detected primarily in fetal brain. GABAA receptor αa subunit probes encoding the putative extracellular domain detect at least five α subunit transcripts in rat brain, whereas probes encoding the putative intracellular domain detect only two mRNAs. These data suggest the presence in brain of multiple GABAA receptor α subunits having homologous extracellular domains and whose expression is regionally and developmentally regulated. These α subunit transcripts may encode proteins that comprise GABAA isoreceptors differing in their pharmacological and physiological properties.

Repeated swim-stress reduces GABAA receptor α subunit mRNAs in the mouse hippocampus

The effects of brief repeated swim stress on the expression of GABAA receptor alpha 1 subunit mRNAs was investigated in the mouse. Adult male mice were exposed to repeated brief (10 min) swim-stress once daily for 7 or 14 days and the levels of GABAA receptor alpha subunit mRNAs were quantified in the hippocampus 24 h after the last session by Northern analysis. Repeated swim stress for 14 days resulted in a 47.3% +/- 6.5 and 39.8% +/- 7.6 decrease in the levels of the 4.8 kb and 4.4 kb GABAA receptor alpha 1 subunit mRNAs, respectively. While there was a trend toward a reduction in the level of GABAA receptor alpha 1 subunit mRNAs following 7 days of repeated swim stress, the latter did not reach statistical significance. In contrast, no significant alterations in the levels of glutamic acid decarboxylase or beta-actin mRNAs were observed at either time point. The reduction in GABAA receptor alpha 1 subunit mRNAs following repeated swim stress may underlie similar alteration(s) in hippocampal GABAA receptor density previously observed following repeated swim stress.

Failure of a protein synthesis inhibitor to modify glutamate receptor-mediated neurotoxicity in vivo

The delayed neuronal death (DND) resulting from brief forebrain ischemia has recently been reported to be markedly attenuated by parenteral administration of the reversible protein synthesis inhibitor, anisomycin. Previous work suggests that ischemia-induced DND is mediated by glutamate acting at one or more glutamate receptors, since glutamate receptor antagonists have been reported to reduce ischemia-induced DND. Consequently, we tested whether anisomycin could modify DND induced by direct intracerebral administration of the excitotoxins, N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxasole (AMPA) or kainic acid. Anisomycin, administered parenterally, in multiple doses did not alter DND induced by any of these excitotoxins, nor did combined parenteral and direct intracerebral injection of anisomycin protect against DND induced by AMPA. Thus, neurotoxicity induced by direct intracerebral administration of NMDA, AMPA or kainic acid does not appear to require de novo protein synthesis, and, therefore, is not likely to be mediated by the expression of a programmed cell death cascade.

Tyrosine Hydroxylase and Cholecystokinin mRNA Levels in the Substantia Nigra, Ventral Tegmental Area, and Locus Ceruleus Are Unaffected by Acute and Chronic Haloperidol Administration

Summary1.The studies described herein were designed to test the hypothesis that a neuroleptic, haloperidol, may alter the level of expression of the tyrosine hydroxylase and cholecystokinin genes in discrete brain regions.2.In situ hybridization was employed to quantitate changes in concentration of mRNA for tyrosine hydroxylase and cholecystokinin in the ventral tegmental area, substantia nigra, and locus ceruleus after acute or chronic treatment with haloperidol or vehicle.3.Haloperidol had no effect on the level of tyrosine hydroxylase or cholecystokinin mRNAs, in the ventral tegmentum, substantia nigra, or locus ceruleus, at either 3 or 19 days of drug administration.4.These data suggest that haloperidol administration does not alter the level of tyrosine hydroxylase or cholecystokinin mRNAs in midbrain dopamine neurons of the rat.

Glutamic acid decarboxylase mRNA in rat brain: regional distribution and effects of intrastriatal kainic acid ☆

Glutamic acid decarboxylase (GAD) mRNA was quantified in different regions of rat brain using an antisense RNA probe (ribo-probe) prepared from a cloned feline cDNA. In all brain regions studied a single band of GAD mRNA of approximately 3.7 kb was detected. The level of GAD mRNA was highest in the cerebellum, followed by the hypothalamus greater than thalamus greater than striatum greater than hippocampus greater than frontal cortex = parietal cortex greater than or equal to medulla = pons. Since GAD has been previously localized to intrinsic neurons of the striatum, we examined the effects of intrastriatal kainic acid administration on striatal GAD mRNA. The level of GAD mRNA in the kainic acid-lesioned striatum was reduced by 70-75% when compared to the contralateral (unlesioned) striatum. In contrast, the level of glutamine synthetase (an enzyme localized to glia) mRNA was increased approximately 290% in the kainic acid-lesioned striatum. There were no significant differences in GAD mRNA levels between the ipsilateral and contralateral cerebral cortices and hippocampi of rats injected with intrastriatal kainic acid.

NMDA receptor-mediated excitoprotection of cultured cerebellar granule neurons fails to alter glutamate-induced expression of c-fos and c-jun mRNAs.

Exposure of cultured cerebellar granule neurons to subtoxic concentrations of N-methyl-D-aspartate (NMDA) induces a state of excitoprotection when measured by subsequent exposure to toxic concentrations of glutamate. This NMDA-induced excitoprotective state is prevented by inhibitors of new RNA and protein synthesis. Since the neurotrophic and excitoprotective effects of NMDA in cerebellar granule neurons may involve changes in the expression of the immediate early genes c-fos and c-jun, we measured c-fos and c-jun mRNAs in cerebellar granule neurons after exposure to either toxic concentrations of glutamate or excitoprotective (subtoxic) concentrations of NMDA. Exposure of cerebellar granule neurons to toxic concentrations of glutamate induced a dramatic increase in c-fos and c-jun mRNAs which was not associated with a corresponding increase in c-fos and c-jun proteins as measured immunocytochemically. However, the increase in c-fos and c-jun mRNAs induced by toxic concentrations of glutamate was not altered by preexposing cerebellar granule neurons to NMDA, suggesting that increased expression of c-fos and c-jun mRNAs is not sufficient for glutamate toxicity of these neurons. Preexposure of cerebellar granule neurons to NMDA for 24 h, which induced a maximal excitoprotective state, resulted in a transient increase in c-fos, and to a lesser degree c-jun, mRNAs similar to that induced by toxic concentrations of glutamate. The induction of c-fos, but not that of c-jun, mRNA both by excitoprotective concentrations of NMDA and by neurotoxic concentrations of glutamate was blocked by the non-competitive NMDA receptor antagonist, MK-801.(ABSTRACT TRUNCATED AT 250 WORDS)