One of the pharmacological targets of ethanol is the GABA A receptor (GABAR), whose function and expression are altered after chronic administration of ethanol. The details of the changes differ between experimental models. In the chronic intermittent ethanol (CIE) model for alcohol dependence, rats are exposed to intermittent episodes of intoxicating ethanol and withdrawal, leading to a kindling-like state of behavioral excitability. This is accompanied by presumably causal changes in GABAR expression and physiology. The present study investigates further the effect of CIE on GABAR function and expression. CIE is validated as a model for human alcohol withdrawal syndrome (AWS) by demonstrating increased level of anxiety; diazepam improved performance in the test. In addition, CIE rats showed remarkably reduced hypnotic response to a benzodiazepine and a steroid anesthetic, reduced sensitivity to a barbiturate, but not propofol. Immunoblotting revealed decrease in ␣1 and ␦ expression and increase in ␥2 and ␣4 subunits in hippocampus of CIE rats, confirmed by an increase in diazepam-insensitive binding for ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo(1,5-␣)(1,4)benzodiazepine-3-carboxylate (Ro15-4513). Elevated mRNA levels were shown for the ␥2S and ␥1 subunits. Recordings in hippocampal slices from CIE rats revealed that the decay time of GABARmediated miniature inhibitory postsynaptic currents (mIPSCs) in CA1 pyramidal cells was decreased, and potentiation of mIPCSs by positive modulators of GABAR was also reduced compared with control rats. However, mIPSC potentiation by the ␣4-preferring benzodiazepine ligands bretazenil and Ro15-4513 was maintained, and increased, respectively. These data suggest that specific alterations in GABAR occur after CIE and may underlie the development of hyperexcitability and ethanol dependence.The molecular mechanisms involved in ethanol dependence and tolerance are poorly understood. Although a specific binding site has not been established for ethanol, several studies have shown that short-and long-term effects of ethanol involve an enhancement or a reduction of inhibitory synaptic transmission at the level of GABA A receptors (GABAR) function (Allan and

Alcohol withdrawal syndrome (AWS) symptoms include hyperexcitability, anxiety, and sleep disorders. Chronic intermittent ethanol (CIE) treatment of rats with subsequent withdrawal of ethanol (EtOH) reproduced AWS symptoms in behavioral assays, which included tolerance to the sleep-inducing effect of acute EtOH and its maintained anxiolytic effect. Electrophysiological assays demonstrated a CIE-induced long-term loss of extrasynaptic GABA A receptor (GABA A R) responsiveness and a gain of synaptic GABA A R responsiveness of CA1 pyramidal and dentate granule neurons to EtOH that we were able to relate to behavioral effects. After CIE treatment, the ␣4 subunit-preferring GABA A R ligands 4,5,6,7 tetrahydroisoxazolo[5,4-c]pyridin-3-ol, La 3ϩ , and Ro15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5␣][1,4]benzodiazepine-3-carboxylate) exerted decreased effects on extrasynaptic currents but had increased effects on synaptic currents. Electron microscopy revealed an increase in central synaptic localization of ␣4 but not ␦ subunits within GABAergic synapses on the dentate granule cells of CIE rats. Recordings in dentate granule cells from ␦ subunit-deficient mice revealed that this subunit is not required for synaptic GABA A R sensitivity to low [EtOH]. The profound alterations in EtOH sensitivity and ␣4 subunit localization at hippocampal GABA A Rs of CIE rats suggest that such changes in these and other relevant brain circuits may contribute to the development of tolerance to the sleep-inducing effects and long-term dependence on alcohol.

Previously, we reported (Cagetti, Liang, Spigelman, and Olsen, 2003) that chronic intermittent ethanol (CIE) treatment leads to signs of alcohol dependence, including anxiety and hyperactivity, accompanied by reduced synaptic ␥-aminobutyric acid (A) receptor (GABA A R) function and altered sensitivity to its allosteric modulators consistent with a measured switch in subunit composition. In this study, we separated the synaptic and extrasynaptic components of GABA A R activation in recordings from pyramidal CA1 cells of hippocampal slices and demonstrated marked differences in the responsiveness of synaptic and extrasynaptic GABA A Rs to agonists and allosteric modulators in control rats, and in the way they are altered following CIE treatment. Notably, tonic inhibition mediated by extrasynaptic GABA A Rs was differentially sensitive to the partial agonist gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol; THIP) and the allosteric modulator zolpidem, compared with the miniature inhibitory synaptic currents (mIPSCs) in the same cells from saline-treated rats. After CIE treatment, potentiation of tonic currents by diazepam and zolpidem was lost, whereas potentiation by the ␣4 subunit-preferring benzodiazepine Ro15-4513 (ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]-[1,4]benzodiazepine-3-carboxylate) and THIP was only partially reduced. Potentiation of synaptic GABA A R currents by zolpidem was eliminated after CIE, whereas THIP slightly inhibited mIPSCs from control rats and greatly enhanced them after CIE treatment. These results are consistent with ␣1 subunit decreases at synaptic and extrasynaptic GABA A Rs, whereas ␣4 subunits are increased at synaptic and decreased at extrasynaptic GABA A Rs. Behaviorally, THIP was active as a hypnotic and anxiolytic but not as an anticonvulsant against pentylenetetrazol seizures in control rats. Only slight tolerance was observed to the sleep time, but not to the anxiolytic, effect of THIP after CIE. Thus, differential alterations in synaptic and extrasynaptic GABA A Rs appear to play an important role in the brain plasticity of alcohol dependence, and withdrawal signs may be profitably treated with GABAergic drugs such as THIP, which does not show cross-tolerance with ethanol.

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. Reduced inhibition and sensitivity to neurosteroids in hippocampus of mice lacking the GABA A receptor ␦ subunit. J Neurophysiol 90: 903-910, 2003. First published April 17, 2003 10.1152/jn.01022.2002. The ␦ subunit of the ␥-aminobutyric acid (A) receptor (GABA A R) is expressed postnatally mostly in the cerebellum, thalamus, and dentate gyrus. Previous studies in mice with a targeted disruption of the ␦ subunit revealed a considerable attenuation of behavioral responses to neuroactive steroids but not to other neuromodulatory drugs. Here we show that ␦ subunit loss leads to a concomitant reduction in hippocampal ␣4 subunit levels. These changes were accompanied by faster decay of evoked inhibitory postsynaptic potentials (IPSPs) in dentate granule neurons of Ϫ/Ϫ mutants (decay ϭ 25 ms) compared with ϩ/ϩ controls ( ϭ 50 ms). Furthermore, the GABA A R-mediated miniature inhibitory postsynaptic currents (mIPSCs) also decayed faster in ␦-mutants ( ϭ 6.3 ms) than controls ( ϭ 7.2 ms) and had decreased frequency (controls, 10.5 Hz; mutants, 6.6 Hz). Prolongation of mIPSCs by the neuroactive steroid anesthetic, alphaxalone (1-10 M), was smaller in ␦-mutants (at 10 M, 65% increase) compared with ϩ/ϩ littermates (308% increase). In competition binding experiments, alphaxalone (0.03-1 M) modulation of [35 S]t-butylbicyclophosphorothionate binding was reduced in ␦-mutant brain homogenates, indicating that the decreased alphaxalone effects on mIPSCs were due to changes in the GABA A R protein. Faster decay of evoked IPSPs and mIPSCs in ␦-mutants suggests presence of the ␦ subunit at both synaptic and extrasynaptic GABA A Rs. Decreased synaptic and extrasynaptic inhibition likely contributes to the pro-epileptic phenotype of ␦-mutants. Reduced neurosteroid sensitivity might also contribute to seizure susceptibility. While the simplest explanation is that ␦ subunit-containing GABA A Rs represent the actual target of neurosteroids, it is possible that the behavioral and physiological sensitivity to neuroactive steroids is indirectly altered in the ␦ Ϫ/Ϫ mice.