-Hydroxybutyric Acid and Diazepam Antagonize a Rapid Increase in GABAA Receptors 4 Subunit mRNA Abundance Induced by Ethanol Withdrawal in Cerebellar Granule Cells

Both benzodiazepines and -hydroxybutyric acid (GHB) are used to treat alcohol withdrawal syndrome. The molecular basis for this therapeutic efficacy was investigated with primary cultures of rat cerebellar granule cells. Long-term exposure of these cells to ethanol (100 mM, 5 days) reduced the abundance of mRNAs encoding the 2L and 2S subunits of the GABA type A receptor ( 32 and 23%, respectively) but failed to affect that of 1, 4, or 6 subunit mRNAs. Subsequent ethanol withdrawal resulted in decreases in the amounts of 1 ( 29%), 6 ( 27%), 2L ( 64%), and 2S ( 76%),subunit mRNAs that were maximal after 6 to 12 h. In contrast, 3 h after ethanol withdrawal, the abundance of the 4 subunit mRNA was increased by 46%. Ethanol withdrawal did not affect neuronal morphology but reduced cellular metabolic activity. The increase in 4 subunit was confirmed by functional studies showing a positive action of flumazenil in patch clamp recordings of GABA-stimulated currents after ethanol withdrawal. Diazepam (10 M) or GHB (100 mM) prevented the increase in the amount of the 4 subunit mRNA, the metabolic impairment, and the positive action of flumazenil induced by ethanol withdrawal but failed to restore the expression of the 1 and 2 subunits. The antagonism by GHB seems not to be mediated by a direct action at GABAAR because GHB failed to potentiate the effects of GABA or diazepam on Cl currents mediated by GABA type A receptor. Ethanol elicits its central effects through modulation of neurotransmission mediated by various receptors, especially that mediated by GABA type A receptors (GABAAR) (Crews et al., 1996; Mehta and Ticku, 1999a). GABAAR are heterogeneous in that they comprise various combinations of subunits (Barnard et al., 1998). The absence or presence of particular subunit isoforms in these receptors confers selectivity for certain drugs (Barnard et al., 1998). Different subunits also mediate distinct pharmacological actions of benzodiazepines, including sedative-hypnotic (Rudolph et al., 1999), anxiolytic, and myorelaxant (Low et al., 2000) effects. Long-term treatment either of rats or of cultured neurons with drugs that modulate GABAergic function, such as benzodiazepines (Holt et al., 1996; Follesa et al., 2001), barbiturates (Tyndale et al., 1997), and steroids (Yu et al., 1996; Concas et al., 1998; Follesa et al., 1998; Smith et al., 1998a,b; Follesa et al., 2000) affects the expression of the genes for various GABAAR subunits. Long-term ethanol administration also affects the subunit composition and, consequently the functional properties, of native GABAAR (Morrow et al., 1990; Mhatre et al., 1993; Devaud et al., 1997). The pharmacological profile of ethanol is highly similar to that of benzodiazepines. Long-term exposure to ethanol, like that to benzodiazepines, also results in the development of tolerance and dependence. The precise molecular mechanism by which prolonged ethanol consumption modifies GABAAR function, however, has remained unknown. Benzodiazepines are among the safest and most effective drugs used in the treatment of alcohol withdrawal syndrome. -Hydroxybutyric acid (GHB), a GABA metabolite naturally present in the brain that in pharmacological doses modulates a variety of neurotransmission systems (Gessa et al., 1968; Bernasconi et al., 1999), has also been proposed for use in the treatment of persons with this condition (Gallimberti et al., 1992; Addolorato et al., 1998). Although specific recognition sites for GHB have been identified in the brain (Benavides et al., 1982), it remains unclear whether all the effects of this compound are mediated by these sites. GHB reduces the self-administration of alcohol and suppresses alcohol withThis study was supported by Ministero dell’Istruzione dell’Universita e della Ricerca grant 2001055774. ABBREVIATIONS: GABAAR, GABA type A receptors; GHB, -hydroxybutyric acid; PCR, polymerase chain reaction; MBS, modified Barth’s solution; ANOVA, analysis of variance. 0026-895X/03/6304-896–907$7.00 MOLECULAR PHARMACOLOGY Vol. 63, No. 4 Copyright © 2003 The American Society for Pharmacology and Experimental Therapeutics 2071/1052893 Mol Pharmacol 63:896–907, 2003 Printed in U.S.A. 896 at A PE T Jornals on M ay 1, 2017 m oharm .aspeurnals.org D ow nladed from drawal signs in alcohol-preferring rats (Fadda et al., 1989). In humans, a single dose of GHB has been shown to suppress alcohol withdrawal symptoms for several hours, and repeated administration increases the number of days of abstinence from alcohol and reduces the number of drinks per day (Gallimberti et al., 1992). However, the molecular mechanism responsible for these effects is not known. We have recently shown that long-term exposure to and subsequent withdrawal of benzodiazepines, zaleplon, zolpidem, or neurosteroids result in selective changes in the expression of specific GABAAR mRNA and polypeptide subunits and in GABAAR function in cultured cerebellar granule cells (Follesa et al., 2000, 2001, 2002). In particular, discontinuation of long-term treatment of the cultured neurons with diazepam both resulted in a selective increase in the abundance of the 4 subunit mRNA and polypeptide and prolonged a decrease in the amounts of the 1 and 2 subunit mRNA and corresponding protein that was already apparent during long-term drug exposure (Follesa et al., 2001). These changes in mRNA and corresponding protein produced changes in receptor function (Follesa et al., 2001). Long-term diazepam administration produced a reduction in the efficacy of this drug in potentiating the GABA-evoked Cl currents (Follesa et al., 2000, 2001). In the same article, we demonstrated that withdrawal from diazepam or imidazenil was associated with both a reduced ability of diazepam to potentiate GABA action and the ability of flumazenil to potentiate GABA action. This effect of flumazenil in withdrawal cells resulted from the increase of the 4 subunit mRNA and corresponding protein (Follesa et al., 2000, 2001). Given that long-term ethanol administration and withdrawal elicit neurochemical and molecular effects similar to those induced by drugs able to activate GABAAR (Morrow et al., 1990; Mhatre et al., 1993; Devaud et al., 1997), we have now studied primary cerebellar granule cell cultures subjected to abrupt discontinuation of ethanol treatment and evaluated the effects of diazepam and GHB during ethanol withdrawal on the gene expression and function of the GABAAR. The use of this exemplified model system will provide new insights that might help to understand, the role played by single subunits of the GABAAR during withdrawal. Materials and Methods Cell Culture. Primary cultures of cerebellar neurons enriched in granule cells were prepared from cerebella of 8-day-old rats. After culture for 8 days, these cells contain the mRNAs for all 14 subunits of the GABAAR with an expression pattern similar to that apparent in the postnatal developing cerebellum but different from that observed in the adult rat cerebellum. Cells were plated (12.5 10 cells in 10 ml per dish) in 100-mm dishes that had been coated with poly(L-lysine) (10 g/ml; Sigma, St. Louis, MO). For the electrophysiological recording, cells were plated (3 10 cells in 1 ml) in multiwell plates containing, in each well, 12-mm round coverslips coated with poly(L-lysine). Cells from either type of plating were cultured in basal Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 2 mM glutamine, gentamicin (100 g/ml; Sigma), and 25 mM KCl. Such a high concentration of potassium was necessary to induce a persistent depolarization, which promotes the survival of granule cells. Cytosine arabinofuranoside (final concentration, 10 M; Sigma) was added to the cultures 18 to 24 h after plating to inhibit the proliferation of non-neuronal cells. After 3 days in culture, the cells were exposed for 5 days to ethanol at the indicated concentrations. In some ethanol-withdrawal experiments, the medium containing ethanol was then replaced with ethanol-free medium containing GHB (at the indicated concentrations) or diazepam (10 M). Ethanol was diluted in medium, GHB was dissolved in medium, and diazepam was dissolved in dimethyl sulfoxide and subsequently diluted in medium. Control cells were treated with the corresponding vehicle. The culture medium was replaced every day with fresh medium containing the indicated drug. Probe Preparation. The cDNA for each subunit of the GABAAR studied was prepared as described previously (Follesa et al., 1998) by reverse transcription and polymerase chain reaction (PCR). In brief, cDNA prepared from rat brain (1–10 ng) was amplified by PCR with TaqDNA polymerase (2.5 U; PerkinElmer, Boston, MA) in 100 l of standard buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2, and 0.01% gelatin) containing 1 M each of specific sense and antisense primers and 200 M of each deoxynucleoside triphosphate. The primer pairs for the various subunits of the GABAAR were designed to include cDNA sequences with the lowest degree of intersubunit homology (Follesa et al., 1998). The primers used to amplify the 6 subunit cDNA were 5 -GGGAAAAAGTCAATTGCTCAC-3 and 5 -CTCCTTATTAATCC-3 (upstream and downstream, respectively). The reaction was performed in a thermal cycler (Eppendorf) for 30 cycles of 94°C for 45 s, 60°C for 1 min, and 72°C for 1 min, with a final extension at 72°C for 15 min. The PCR products were separated by electrophoresis, visualized by staining with ethidium bromide, excised from the gel, purified, and cloned into the pAMP 1 cloning vector (Invitrogen). Escherichia coli DH5 was transformed with the resulting plasmids, which were subsequently purified from the bacteria and the cDNA inserts were sequenced with a Sequenase DNA sequencing kit (USB, Cleveland, OH). The determined nucleotide sequences were 100% identical to the respective previously published sequences. Plasmids containing the cDNA fragments corresponding to the various GABAAR subunits were linearized with restriction enzymes (Follesa et al., 1998) and then used as templates, together with the appropriate RNA polymerase (SP6 or T7) to generate [ -P]UTP-labeled cRNA probes for RNase protection assays. RNA Extraction and Measurement of GABAAR Subunit mRNAs. Total RNA was isolated from cultured cerebellar granule cells by the guanidine isothiocyanate method as described previously (Follesa et al., 1998) and quantified by measurement of absorbance at 260 nm. An RNase protection assay for the semiquantitative detection of the GABAAR 1, 4, 6, 2L, and 2S subunit mRNAs was performed as described previously (Follesa et al., 1998). In brief, 25 g of total RNA was dissolved in 20 l of hybridization solution containing 150,000 cpm of P-labeled cRNA probe for a specific GABAAR subunit mRNA (specific activity, 6 10 7 to 7 10 cpm/ g) and 15,000 cpm of P-labeled cyclophilin cRNA (1 10 cpm/ g). Given that cyclophilin is expressed widely among tissues, including the brain, and that its gene is most likely regulated in an “on or off” manner, we used cyclophilin mRNA as an internal standard for our measurements (Follesa et al., 1998). The hybridization reaction mixture was incubated overnight at 50°C and then subjected to digestion with RNase, after which RNA-RNA hybrids were detected by electrophoresis (on a sequencing gel containing 5% polyacrylamide and urea) and autoradiography. The amounts of GABAAR subunit mRNA and cyclophilin mRNA were determined by measuring the optical density of the corresponding bands on the autoradiogram with a densitometer (GS-700; Bio-Rad, Hercules, CA); this instrument is calibrated to detect saturated values, so that all our measurements were in the linear range. The data were normalized by dividing the optical density of the protected fragment for each receptor subunit mRNA by that of the respective protected fragment for cyclophilin mRNA. The amount of each receptor subunit mRNA was therefore expressed in arbitrary units. Metabolic Activity of Cerebellar Granule Cells. The metabolic activity of live cerebellar granule cells was measured with the resazurin (TOX-8) system (Magnani and Bettini, 2000). Resazurin (Sigma) is a dye that is blue in its oxidized form and red in -Hydroxybutyric Acid and Alcohol Withdrawal 897 at A PE T Jornals on M ay 1, 2017 m oharm .aspeurnals.org D ow nladed from its reduced form. Bioreduction of the dye by viable cells can thus be monitored spectrophotometrically and provides an indicator of cellular energy status. Cerebellar granule cells (7 10) were cultured in 24-well plates coated with poly(L-lysine) and containing 1 ml of minimum essential medium devoid of phenol red (Invitrogen) per well. They were treated for 5 days with ethanol (100 mM) and then subjected to ethanol withdrawal in the absence or presence of GHB or diazepam as described above. Four replica wells were used for each treatment. After ethanol withdrawal for 3 or 6 h, 100 l of TOX-8 stock solution was added to each well, and the plate was incubated for 2 h in the dark under standard conditions (37°C, humidified atmosphere containing 5% CO2). The absorbance of the dye was then measured at wavelengths of 600 and 690 nm. The absorbance of blank wells containing culture medium and the appropriate drug but lacking cells was also determined. The mean value of blank wells was subtracted from that of the experimental wells to yield net absorbance values. Metabolic activity was determined as the change in absorbance caused by resazurin reduction, and data are expressed as percentage change in metabolic activity relative to that of control cells (not treated with ethanol). To verify that changes in metabolic activity were not caused by cell death, we also counted with a hemocytometer the number of viable cells in each well after their removal with trypsin and staining with trypan blue. Whole-Cell Patch-Clamp Electrophysiological Recording. Immediately before recording, coverslips were transferred to a perfusion chamber (Warner Instruments, Hampden, CT), and cerebellar granule cells were visualized under a Nikon upright microscope equipped with Nomarski optics. Membrane potentials were clamped at 60 mV with a Axopatch 200-B amplifier (Axon Instruments, Union City, CA). The resting membrane potential for the recorded neurons was approximately 60 mV. Recording pipettes (borosilicate capillaries with filament, outer diameter 1.5 mm; Sutter Instruments, Novato, CA) were prepared with a two-step vertical puller (Sutter Instruments) and had resistances between 4 and 6 M . Pipette capacitance and series resistances were compensated, the latter at 60%. Currents through the patch-clamp amplifier were filtered (eight-pole bessel, 2 kHz) and digitized at 5.5 kHz using commercial software (pClamp 8.1; Axon Instruments). The external solution contained 130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.3, and 11 mM glucose (all chemicals from Sigma). The internal solution contained 140 mM CsCl, 2 mM MgCl2, 1 mM CaCl2, 10 mM EGTA, 10 mM HEPES, pH 7.3, and 2 mM 5 -ATP-Na2 (all chemicals from Fluka, Buchs, Switzerland). Drugs were applied with a fast-exchange flow-tube perfusion system driven by motor (Warner Instruments Co.). GABA was applied at a concentration of 1 to 3 M, which induced a current with an amplitude of 5 to 10% of the maximal response (EC5 10). Flumazenil (3 M) was applied at 30-s intervals. All experiments were performed at room temperature (23–25°C). Data were analyzed by pClampFit 8.01 (Axon Instruments, Union City, CA). Modulation of GABA-evoked Cl currents by flumazenil is presented as percentage change, [(I /I) 1] 100%, where I is the average of control responses obtained before application and after washout of drugs, and I is the average of agonist-induced response obtained from the same cell in the presence of drugs. Electrophysiological Recording Cloned GABAAR. The human 1, 2, and 2L subunit cDNAs were subcloned into the pCDM8 vector (Invitrogen) for nuclear injection. Oocytes were isolated from Xenopus laevis as described. A mixture of the three subunit cDNAs (0.5 ng each in a total volume of 30 nl) was injected into the animal pole of each oocyte (Colman, 1984). One to 4 days after injection, oocytes expressing recombinant 1 2 2L receptors were placed in a chamber (volume, 100 l) and perifused (2 ml/min) with modified Barth’s solution (MBS) comprising 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 10 mM HEPES-NaOH, pH 7.5, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, and 0.91 mM CaCl2. GABA was dissolved in MBS and applied for 30 s. Diazepam was dissolved in dimethyl sulfoxide and then diluted in MBS; the final concentration of solvent in MBS was 1% and did not affect GABA responses. GHB was dissolved in H2O and then diluted in MBS. Diazepam and GHB were each applied for 60 s before their coapplication for 30 s with a concentration of GABA that induced a current with an amplitude of 5 to 10% of the maximal response (EC5 10). Statistical Analysis. Data are presented as means S.E.M. and were subjected to analysis of variance (ANOVA) followed by Scheffé’s F test. A p value of 0.05 was considered statistically