Vol. 63, Issue 4, 896-907, April 2003

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

Paolo Follesa, Luisa Mancuso, Francesca Biggio, Maria Cristina Mostallino, Annalisa Manca, Maria Paola Mascia, Fabio Busonero, Giuseppe Talani, Enrico Sanna, and Giovanni Biggio

Department of Experimental Biology "Bernardo Loddo", University of Cagliari, Cagliari, Italy (P.F., L.M., F.B., A.M., F.B., G.T., E.S., G.B.); CNR Institute of Neuroscience, Section of Neuropsychopharmacology, Cagliari, Italy (M.C.M., M.P.M., G.B.)

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 µM) 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 GABA_AR because GHB failed to potentiate the effects of GABA or diazepam on Cl currents mediated by GABA type A receptor.