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Groups of rats were maintained on a daily regimen of 22 h of water deprivation followed by a 2-h opportunity to take either water or a sweetened ethanol solution (ES). In one experiment, it was shown that previous morphine (M) dependence had no effect on initial daily intakes of fluids. After stable ES intakes were achieved, a variety of pharmacological manipulations were assessed for their effects on intake of the ES. Nalmefene, an opioid antagonist, dose-relatedly decreased intakes of ES, and was effective across days of injections. Fluoxetine (FX), a serotonergic reuptake inhibitor, also reduced ES intakes dose relatedly, and across days of injections, but the reduction was not as great as that seen with opioid antagonists. A small dose of M increased ES intakes when given in combination with an ineffective dose of FX, just as it does by itself. However, M had no effect on ES intakes in combination with an effective dose of FX. Pimozide (PIM), a dopaminergic antagonist, dose-relatedly decreased intakes of ES and water, and responding for positively reinforcing intracranial stimulation (ICS). When given in combination, M blunted PIM's reduction of ES intake, but had no effect on PIM's ability to decrease either intake of water or responding for ICS. Amphetamine did not reliably affect rats' intakes of ES across a range of doses. The data, in addition to previous work, lead to the idea that endogenous opioid systems are more salient, with respect to intake of alcoholic beverages, than the other tested neurotransmitter systems. Furthermore, the collective data suggest that a long-lasting opioid antagonist may be an effective pharmacological adjunct to other treatments for alcohol abuse and alcoholism.

The purpose of the current study was to ascertain whether ethyl nitrite could be detected in vitro from the reaction of ethanol with peroxynitrite, as well as after administration of ethanol to mice. Ethyl nitrite analyte was determined by using gas chromatography--mass spectrometry with headspace analysis with the use of a solid-phase microextraction device. Peroxynitrite was allowed to react with ethanol under a variety of conditions in vitro. Ethyl nitrite was generated when peroxynitrite was allowed to react with ethanol. Male, inbred short-sleep mice were injected intraperitoneally with either ethanol [5.2 g/kg; 15.0% (weight/volume) ethanol in saline] or a 50:50 mixture of deuterium-labeled ethanol (D5-ethanol) and ethanol. Blood samples, as well as whole brain and liver sections, were obtained from mice 30 min later for determination of ethanol, D5-ethanol, ethyl nitrite, and deuterium-labeled ethyl nitrite (D5-ethyl nitrite). Time courses for the appearance of ethyl nitrite in blood samples, as well as in whole brain and liver sections, obtained from mice were carried out. After ethanol administration, ethyl nitrite was detected and quantitated in mouse blood, brain, and liver. A small fraction of ethyl nitrite was present. When a 50:50 mixture of ethanol and D5-ethanol was given to animals, both ethyl nitrite and D5-ethyl nitrite were found in blood and brain in approximately the same ratio as that of ethanol and D5-ethanol. The level of D5-ethyl nitrite in liver was more than twice that of ethyl nitrite, indicating a possible isotope effect in the metabolism of ethyl nitrite. Ethyl nitrite is a new metabolite of ethanol in vivo. The mechanism of ethyl nitrite formation is most likely the reaction of ethanol with peroxynitrite generated in vivo from nitric oxide.

Generally, compounds discriminated by animals possess psychotropic effects in animals and humans. As with many other drugs of abuse, strength of the ethanol discriminative stimulus is dose related. The majority of studies show that doses close to 1.0 g/kg are close to the minimum at which the discrimination can be learned easily. Substitution studies suggest that anxiolytic, sedative, atactic, and myorelaxant effects of ethanol all play an important role in the formation of its intercoeptive stimulus. Low doses of ethanol produce more excitatory cues, similar to amphetamine-like subjective stimuli, whereas higher doses produce rather sedative/hypnotic stimuli similar to those elicited by barbiturates. Substitution studies have shown that the complete substitution for ethanol may be exerted by certain GABA-mimetic drugs acting through different sites within the GABA(A)-benzodiazepine receptor complex (e.g., diazepam, pentobarbital, certain neurosteroids), gamma-hydroxybutyrate, and antagonists of the glutamate NMDA receptor. Among the NMDA receptor antagonists both noncompetitive (e.g., dizocilpine) and competitive antagonists (e.g., CGP 40116) are capable of substituting for ethanol. Further, some antagonists of strychnine-insensitive glycine modulatory sites among the NMDA receptor complex (e.g., L-701,324) dose-dependently substitute for the ethanol discriminative stimulus. On the other hand, neither GABA-benzodiazepine antagonists nor NMDA receptor agonists produce contradictory effects (i.e., reduce the ethanol discriminative stimulus). There is influence of a particular training dose of ethanol on the substitution pattern of different compounds. For example, 5-HT(1B/2C) agonists substitute for intermediate (1.0 g/kg) but not higher (2.0 g/kg) ethanol training doses. Discrimination studies with ethanol and drugs acting on NMDA and GABA receptors consistently indicate asymmetrical generalization. For example, ethanol is able to generalize to barbiturates and benzodiazepines, but neither the benzodiazepine nor barbiturate response generalizes to ethanol. Only a few drugs are able to antagonize, at least to some extent, the discriminative stimulus of ethanol (e.g., partial inverse GABA-benzodiazepine receptor antagonist Ro 15-4513 and the opioid antagonist naloxone). The ethanol stimulus effect may be increased (i.e., stronger recognition) by N-cholinergic drugs (nicotine), dopaminergic drugs (apomorphine), and 5-HT3 receptor agonists (m-chlorophenylbiguanide). Thus, the ethanol stimulus is composed of the several components, with the NMDA receptor and GABA(A) receptor complex being of particular importance. This suggests that a drug mixture may be more capable of substituting for ethanol (or block its stimulus) than a single compound. The ability of drugs to substitute for the ethanol discriminative stimulus is frequently, although not preclusively, associated with the reduction of voluntary ethanol consumption. The examples of positive correlation are gamma-hydroxybutyrate, possibly memantine and certain serotonergic drugs such as fluoxetine. However, it remains uncertain to what extent the discriminative stimulus of ethanol can be seen as relevant in the understanding of the complex mechanisms of dependence.

Rats of two different ages (2 and 7 months) were treated with an ethanol-containing liquid diet for 24 days and change of the ceramide composition of gangliosides were studied in the brain synaptosomal, microsomal and myelin fractions. Greater differences were observed in the younger age, where ethanol treatment caused a significant increase of C20:1 LCB in GM1 ganglioside of synaptosomes and microsomes and in GD1a of myelin.

The drug discrimination paradigm was used to evaluate the behavioral differences in response to ethanol between three strains of rats, viz., Sprague-Dawley, N/Nih and Fawn-Hooded. This latter group is thought to have a genetically-transmitted diminished central serotonin function. Each group of rats was trained to discriminate between the stimulus properties of 600 mg/kg ethanol and its vehicle in a two-lever, food-motivated operant task. Results indicate that the Fawn-Hooded rats required a significantly longer time and a higher ethanol dose to reach criterion discrimination performance. Furthermore, the ED50 value of the Fawn-Hooded rats, once trained, was higher than the Sprague-Dawley or N/Nih rats. The possibility that a reciprocal relationship exists between lowered central serotonin concentrations and higher alcohol consumption is suggested and the hypothesis that the diminished ability to recognize the interoceptive stimuli produced by ethanol may result in larger amounts of ethanol being consumed is offered.

Microglia undergo maturation during the third trimester of human development (equivalent to the first 1-2 weeks of postnatal life in rodents), during which these cells may be particularly sensitive to insult. Alcohol exposure during this period can activate the neuroimmune system, an effect that may contribute to the pathophysiology of fetal alcohol spectrum disorders. Here, we investigated whether repeated alcohol exposure during the third trimester-equivalent in rats has a priming effect on the neuroimmune response to injection of bacterial lipopolysaccharide (LPS). Pups were exposed to alcohol in vapor chambers for 4 h daily from postnatal day (PD)2 to PD16 (peak blood alcohol concentrations ∼150 mg/dL). On PD17, rats were injected with either saline or LPS (50 μg/kg) and the frontal cortex, cerebellar vermis, and dentate gyrus were collected 2 h later. Messenger RNA (mRNA) levels for the pro-inflammatory agents interleukin 1β (IL-1β) and chemokine (C-C) motif ligand 2 (CCL2), as well as levels of the anti-inflammatory cytokine interleukin 10 (IL-10), were measured using reverse transcriptase polymerase chain reaction. LPS consistently increased IL-1β and CCL2 mRNA levels in the dentate gyrus, frontal cortex, and cerebellum of both male and female rats. Furthermore, the LPS-induced increase of IL-1β mRNA levels was significantly blunted in the frontal cortex of alcohol-exposed female rats. Conversely, LPS only minimally affected IL-10 mRNA expression and there were no significant differences between air- and alcohol-exposed rats. Taken together with the literature regarding the effect of third-trimester alcohol exposure on the neuroimmune system, our findings suggest that chronic exposure to lower levels is less disruptive to the neuroimmune system than binge-like exposure to high doses of alcohol.

Phospholipase C (PLC) activity was measured by the incorporation of [3H]-inositol into lipids and by the breakdown of [3H]-inositol-labelled phosphatidylinositols (PI) and polyphosphatidylinositols (PPI) to [3H]-inositol phosphates; phospholipase A2 (PLA2) activity by the breakdown of [3H]oleic acid-labelled phosphatidylcholine [( 3H]PC) to [3H]oleic acid and the enzymes of phospholipid base exchange (PLBE) by the incorporation of [14C]serine into membrane lipids. The activities of these enzymes in rat brain preparations were all increased by procedures which increase intracellular Ca2+, and were all inhibited to a varying extent by the presence of ethanol, 50 mM, in vitro. In contrast, the activities of PLA2 and PLBE enzymes were markedly increased in preparations from animals which had received ethanol chronically in vivo. Similarly, although the basal activity of PLC was only slightly increased in such preparations, depolarization induced the breakdown of a significantly greater fraction of radiolabelled PI than that which was obtained in control preparations. The results suggest compensatory alterations in the activity of Ca2+-activated enzymes of phospholipid metabolism in brain tissue during the continued presence of ethanol in vivo.

Chronic ethanol consumption in high doses is associated with constitutively elevated activity of the serum alcohol dehydrogenase I (ADH I) isoform, which demonstrates a high affinity not only for ethanol but also for a number of bioamine metabolites. Such excessive ADH activity is probably associated with disruptions in the metabolism of neurotransmitters (dopamine, serotonin, and norepinephrine) and subsequent long-term changes in the activity of their receptors. Ultimately, a stable depressive-like condition contributes to the development of patients' craving for ethanol intake, frequent disruptions during therapy, and low efficacy of treatment. We applied active immunization against ADH to investigate its efficacy in the reduction of excessive serum ADH activity and regulation of ethanol consumption by chronically ethanol-fed Wistar rats (15% ethanol, 4 months, free-choice method), and we analyzed its ability to influence the levels of bioamines in the brain. Immunization (2 injections, 2-week intervals) was performed using a combination of recombinant horse ADH isozyme as an antigen and 2% aluminum hydroxide-based adjuvant. The efficacy of immunization was demonstrated by the production of high titers of ADH-specific antibodies, which was consistent with the significantly reduced ADH activity in the serum of chronically ethanol-fed rats. On the 26th day after the first vaccine injection, we registered significantly lower levels of alcohol consumption compared to ethanol-fed control animals, and the difference reached 16% on the 49th day of the experiment. These observations were accompanied by data that showed reduced levels of ethanol preference in immunized rats. Chronic alcohol drinking led to a decrease in dopamine and DOPAL (a direct dopamine metabolite and a high-affinity ADH substrate) levels in the striatum,while immunization neutralized this effect. Additionally, we observed that inhibition of serum ADH activity caused a decrease in peak dopamine levels during acute alcohol intake in chronically ethanol-fed rats during ethanol withdrawal that was associated with reduced tyrosine hydroxylase activity in the striatum. The obtained data suggest a significant contribution of ADH to the changes in neurotransmitter systems during chronic alcohol consumption and make available new prospects for developing innovative strategies for treatment of excessive alcohol intake.