S17ALCOHOL AND GABAA RECEPTORSS17.1EXTRASYNAPTIC GABAA-RS: PHARMACOLOGY AND REGULATION OF GENE EXPRESSIONS17.2ROLE OF DELTA SUBUNIT-CONTAINING GABAA-RS IN LOW DOSE ETHANOL ACTIONS ON THE BRAINS17.3THE ROLE OF GABAA-R SUBTYPES IN PUTATIVE ALCOHOL-ANTAGONISTIC EFFECTS OF RO15-4513S17.4PROTEIN KINASE C REGULATION OF ETHANOL ACTIONS AT GABAA-R

Drinking alcohol is associated with the disturbance of normal sleep rhythms, and insomnia is a major factor in alcoholic relapse. A number of studies have implicated GABAA receptors (GABAA-Rs) in the anxiolytic, amnestic, sedative and anesthetic effects of ethanol and other drugs including the benzodiazepines and the non-BZ hypnotics known as ‘Z-drugs’ such as Eszopiclone (Lunesta) and zolpidem (Ambien). The thalamus plays a pivotal role in sleep regulation and rhythmicity. Two distinct subtypes of synaptic GABAA receptors (GABAA-Rs) (alpha-1, beta-2 and gamma-2) and (alpha-3, beta-3 and gamma-2) are expressed in thalamocortical relay neurons and in interneurons of the RTN (reticular thalamic nucleus), respectively. In addition, an additional population of GABAA-Rs has recently been demonstrated to occur at extrasynaptic sites on thalamocortical relay neurons. These extrasynaptic GABAA-Rs (consisting mainly of alpha-4, beta-2 and gamma-2 subunits) are persistently activated by low concentrations of GABA and have distinct pharmacological properties that differentiate them from the synaptic GABAA-Rs in relay neurons. We compared the effects of ethanol and the Z-drugs on both synaptic and extrasynaptic GABAA-Rs of relay neurons in the thalamus. Neither of the two hypnotics had any effect on tonic inhibition in relay neurons. We found that ethanol (50 mM) elicits a sustained current in thalamocortical relay neurons from the mouse ventrobasal thalamus, and this current is associated with a decrease in neuronal excitability and firing rate in response to depolarization. The steady current induced by ethanol was totally abolished by gabazine and was absent in relay neurons from GABAA-R alpha-4 subunit knockout mice, indicating that the effect of ethanol is to enhance tonic GABA-mediated inhibition. Ethanol (50 mM) enhanced the amplitude of tonic inhibition by nearly 50%. Neither of the two Z-drug hypnotics affected tonic inhibition in relay neurons. The distinct patterns of subunit-selective activity on GABAA-Rs of thalamic neurons may contribute to some of the observed differences in the clinical effects of these three important hypnotic substances. # S17.2 ROLE OF DELTA SUBUNIT-CONTAINING GABAA-RS IN LOW DOSE ETHANOL ACTIONS ON THE BRAIN {#article-title-2} Our publications show that δ subunit-containing GABAR are sensitive to low-dose (5–30 mM) EtOH enhancement in vitro and in vivo , suggesting their relevance to EtOH action. This includes genetic evidnce that GABAR affect EtOH sensitivity in rats and the benziodiazepine aklcohol antagonist Ro15-4513 blocks EtOH action via δ-containing GABAR. δ-GABAR expression, which is notoriously difficult, is confirmed by high sensitivity to EtOH, and also muscimol and THIP, accounting for their high potency to block GABA radioligand binding and to enhance tonic inhibitory currents in neurons expressing the extrasynaptic δ-GABAR, and lost in δ subunit knockout (ko) mice. We reported acute and chronic EtOH-induced plastic changes in GABAR subunit levels and pharmacology in rats, which can account for behavioral changes corresponding to withdrawal and dependence. We demonstrated that the first EtOH-induced changes detectable are an endocytotic removal of neuronal cell surface GABAR α4 and δ subunits accompanying the loss of tonic inhibitory current and the loss of EtOH enhancement in hippocampal neurons, consistent with the δ-GABAR as first responders to EtOH. Similar changes in GABAR are found in cultured rat hippocampal neurons pre-exposed to EtOH. The δ and α4 ko mice have not shown dramatically reduced sensitivity to EtOH-induced behaviors. We showed that the α4 ko mouse shows reduced sensitivity to EtOH-modulated GABAR tonic inhibitory currents in hippocampal neurons, but increased sensitivity of synaptic GABAR, accompanied by elevated α2, α4 and γ2 subunits. α4 ko mice exhibited increased spatial contextual fear conditioning but no increased sensitivity to low-dose EtOH impairment of the hippocampal-specific function of immediate shock deficit contextual fear conditioning. CIE treatment of α4 ko mice showed a blunted withdrawal and tolerance to EtOH relative to wild-type consistent with a major role of the α4(δ) subunit in CIE-induced dependence mechanisms. # S17.3 THE ROLE OF GABAA-R SUBTYPES IN PUTATIVE ALCOHOL-ANTAGONISTIC EFFECTS OF RO15-4513 {#article-title-3} Ethanol produces its effects on the nervous system by acting on many molecular targets, the roles of which in behavior have remained poorly known. One of the most challenging targets has been the γ-aminobutyric acid (GABA) type A receptor, which is expressed as heterogeneous pentameric assembly of various subunits generated from almost 20 different genes. Importantly, the putative alcohol antagonist Ro 15-4513 is known to act on GABA-A receptors at γ2 subunit-dependent benzodiazepine sites and has been suggested to act also at a putative δ subunit-dependent Ro 15-4513/ethanol binding site. The data aiming at ascertaining the roles of these two types of GABA-A receptor for Ro 15-4513 actions using specific gene-modified mouse models (GABA-A receptor δ subunit knockout and transgenic GABAA receptor γ2 subunit F77I knock-in mouse lines) will be presented. The former mouse line has plenty of high-affinity [3H]Ro 15-4513 binding in widespread regions of the brain, while the latter mostly lacks the binding, especially in δ subunit-enriched regions of the cerebellum and thalamus. So far we have found that the antagonism of ethanol-induced sedation by Ro 15-4513 was unaffected in GABAA receptor δ subunit knockout mice, but blocked in the γ2F77I transgenic mice, suggesting that Ro 15-4513 action is mediated via γ2 subunit-dependent benzodiazepine sites. (This study was supported by the Finnish Foundation for Alcohol Studies.) # S17.4 PROTEIN KINASE C REGULATION OF ETHANOL ACTIONS AT GABAA-R {#article-title-4} There is controversy about the biological relevance of ethanol's actions at GABA-A receptors, since some labs have been unable to detect enhancement of receptor function using ethanol concentrations <50 mM (∼230 mg%). For years, some have postulated that post-translational modifications of receptors modulate their ethanol sensitivity. We have pursued this hypothesis by examining PKC-mediated phosphorylation of receptors and receptor-associated proteins. We determined that PKC epsilon decreases the function of synaptic GABA-A receptors through phosphorylation of at least two substrates: (i) GABA-A gamma2 subunits at Ser-327, which reduces the sensitivity of receptors to the positive allosteric effects of benzodiazepines and ethanol and (ii) the N -ethylmaleimide sensitive factor (NSF), at Ser-460 and Thr-461, which stimulates removal of GABA-A receptors from synapses. More recently, there has been interest in ethanol's actions at extrasynaptic GABA-A receptors since these may be particularly sensitive to ethanol concentrations <30 mM. We found that PKC delta phosphorylates GABA-A delta subunits and that this phosphorylation enhances the sensitivity of alpha4-delta-containing receptors to low concentrations of ethanol. Together our results indicate the importance of PKC-mediated phosphorylation in regulating the sensitivity of GABA-A receptors to ethanol. Dynamic changes in PKC phosphorylation that occur during intoxication may also contribute to acute functional tolerance to ethanol.