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AbstractLarge conductance potassium (BK) channels are among the most sensitive molecular targets of ethanol and genetic variations in the channel-forming α subunit have been nominally associated with alcohol use disorders. However, whether the action of ethanol at BK α influences the motivation to drink alcohol remains to be determined. To address this question, we first tested the effect of systemically administered BK channel modulators on voluntary alcohol consumption in C57BL/6J males. Penitrem A (blocker) exerted dose-dependent effects on moderate alcohol intake, while paxilline (blocker) and BMS-204352 (opener) were ineffective. Because pharmacological manipulations are inherently limited by non-specific effects, we then sought to investigate the behavioral relevance of ethanol’s direct interaction with BK α by introducing in the mouse genome a point mutation known to render BK channels insensitive to ethanol while preserving their physiological function. The BK α K361N substitution prevented ethanol from reducing spike threshold in medial habenula neurons. However, it did not alter acute responses to ethanol in vivo, including ataxia, sedation, hypothermia, analgesia, and conditioned place preference. Furthermore, the mutation did not have reproducible effects on alcohol consumption in limited, continuous, or intermittent access home cage two-bottle choice paradigms conducted in both males and females. Notably, in contrast to previous observations made in mice missing BK channel auxiliary β subunits, the BK α K361N substitution had no significant impact on ethanol intake escalation induced by chronic intermittent alcohol vapor inhalation. It also did not affect the metabolic and locomotor consequences of chronic alcohol exposure. Altogether, these data suggest that the direct interaction of ethanol with BK α does not mediate the alcohol-related phenotypes examined here in mice.

The effect of low concentrations of ethanol on Na+,K(+)-ATPase activity, defined as ouabain-inhibitable 86Rb+ (K+) uptake, was investigated in a crude synaptosome preparation which was subject to minimal subcellular fractionation procedures. Moderate (20-30%) but potent (EC50 = 3.8 mM) stimulation of total ouabain (1 mM)-inhibitable K+ uptake by ethanol was observed following incubation periods of up to 20 min. The activity of the ethanol-induced component of K+ uptake was antagonized by nanomolar concentrations of ouabain. Thus, the moderate stimulation of total ouabain-inhibitable K+ uptake by ethanol was attributable to the activation of a component of K+ uptake which was very sensitive (VS; IC50 = 2.8 x 10(-10) M) to inhibition by ouabain. Slightly higher concentrations of ouabain (10(-9) - 10(-6.6) M) stimulated K+ uptake above control (no ethanol or ouabain) in both the absence and presence of ethanol. The selectivity of the VS-ethanol interaction was demonstrated by the lack of any ethanol effect on two other components of ouabain-inhibitable K+ uptake which accounted for inhibition of K+ uptake by concentrations of ouabain above 10(-6.6) M and were defined as sensitive (S; IC50 = 10(-6) M) and insensitive (I; IC50 = 10(-4) M) to ouabain. These results define the ethanol-inducible component of ouabain-inhibitable Na+,K(+)-ATPase activity and promote the view that changes in Na+,K(+)-ATPase-dependent ion translocation may contribute to ethanol intoxication in vivo.

The inhalation toxicity of an ethanol-gasoline mixture was investigated in rats. Groups of 15 male and 15 female rats were exposed by inhalation to 6130 ppm ethanol, 500 ppm gasoline or a mixture of 85% ethanol and 15% gasoline (by volume, 6130 ppm ethanol and 500 ppm gasoline), 6 h a day, 5 days per week for 4 weeks. Control rats of both genders received HEPA/charcoal-filtered room air. Ten males and ten females from each group were killed after 4 weeks of treatment and the remaining rats were exposed to filtered room air for an additional 4 weeks to determine the reversibility of toxic injuries. Female rats treated with the mixture showed growth suppression, which was reversed after 4 weeks of recovery. Increased kidney weight and elevated liver microsomal ethoxyresorufin-O-deethylase (EROD) activity, urinary ascorbic acid, hippuric acid and blood lymphocytes were observed and most of the effects were associated with gasoline exposure. Combined exposure to ethanol and gasoline appeared to exert an additive effect on growth suppression. Inflammation of the upper respiratory tract was observed only in the ethanol-gasoline mixture groups, and exposure to either ethanol and gasoline had no effect on the organ, suggesting that an irritating effect was produced when the two liquids were mixed. Morphology in the adrenal gland was characterized by vacuolation of the cortical area. Although histological changes were generally mild in male and female rats and were reversed after 4 weeks, the changes tended to be more severe in male rats. Brain biogenic amine levels were altered in ethanol- and gasoline-treated groups; their levels varied with respect to gender and brain region. Although no general interactions were observed in the brain neurotransmitters, gasoline appeared to suppress dopamine concentrations in the nucleus accumbens region co-exposed to ethanol. It was concluded that treatment with ethanol and gasoline, at the levels studied, produced mild, reversible biochemical hematological and histological effects, with some indications of interactions when they were co-administered.

Background: Dopamine concentrations in the nucleus accumbens fluctuate on phasic (subsecond) and tonic (over minutes) timescales in awake rats. Acute ethanol increases tonic concentrations of dopamine, but its effect on subsecond dopamine transients has not been fully explored.Methods: We measured tonic and phasic dopamine fluctuations in the nucleus accumbens of rats in response to ethanol (within‐subject cumulative dosing, 0.125 to 2 g/kg, i.v.).Results: Microdialysis samples yielded significant tonic increases in dopamine concentrations at 1 to 2 g/kg ethanol in each rat, while repeated saline infusions had no effect. When monitored with fast scan cyclic voltammetry, ethanol increased the frequency of dopamine transients in 6 of 16 recording sites, in contrast to the uniform effect of ethanol as measured with microdialysis. In the remaining 10 recording sites that were unresponsive to ethanol, dopamine transients either decreased in frequency or were unaffected by cumulative ethanol infusions, patterns also observed during repeated saline infusions. The responsiveness of particular recording sites to ethanol was not correlated with either core versus shell placement of the electrodes or the basal rate of dopamine transients. Importantly, the phasic response pattern to a single dose of ethanol at a particular site was qualitatively reproduced when a second dose of ethanol was administered, suggesting that the variable between‐site effects reflected specific pharmacology at that recording site.Conclusions: These data demonstrate that the relatively uniform dopamine concentrations obtained with microdialysis can mask a dramatic heterogeneity of phasic dopamine release within the accumbens.

Background:The ventral tegmental area (VTA) is involved in regulating ethanol drinking, and the posterior VTA seems to be a neuroanatomical substrate that mediates the reinforcing effects of ethanol in ethanol‐naïve Wistar and ethanol‐naïve alcohol‐preferring (P) rats. The objective of this study was to test the hypothesis that chronic ethanol drinking increases the sensitivity of the posterior VTA to the reinforcing effects of ethanol.Methods:Two groups of female P rats (one given water as its sole source of fluid and the other given 24‐hr free‐choice access to 15% ethanol and water for at least 8 weeks) were stereotaxically implanted with guide cannulae aimed at the posterior VTA. One week after surgery, rats were placed in standard two‐lever (active and inactive) operant chambers and connected to the microinfusion system. Depression of the active lever produced the infusion of 100 nl of artificial cerebrospinal fluid (CSF) or ethanol. The ethanol‐naïve and chronic ethanol‐drinking groups were assigned to subgroups to receive artificial CSF or 25, 50, 75, or 125 mg/dl of ethanol (n= 6–9/dose/group) to self‐infuse (FR1 schedule) during the 4‐hr sessions given every other day.Results:Compared with the infusions of artificial CSF, the control group reliably (p < 0.05) self‐infused 75 and 125 mg/dl of ethanol but not the lower concentrations. The ethanol‐drinking group had significantly (p < 0.05) higher self‐infusions of 50, 75, and 125 mg/dl of ethanol than artificial CSF during the four acquisition sessions; the number of infusions of all three doses was higher in the ethanol‐drinking group than in the ethanol‐naive group. Both groups decreased responding on the active lever when artificial CSF was substituted for ethanol, and both groups demonstrated robust reinstatement of responding on the active lever when ethanol was restored.Conclusions:Chronic ethanol drinking by P rats increased the sensitivity of the posterior VTA to the reinforcing effects of ethanol.

Background and Purpose— Ischemic stroke induces metabolic disarray. A central regulatory site, pyruvate dehydrogeanse complex (PDHC) sits at the cross-roads of 2 fundamental metabolic pathways: aerobic and anaerobic. In this study, we combined ethanol (EtOH) and normobaric oxygen (NBO) to develop a novel treatment to modulate PDHC and its regulatory proteins, namely pyruvate dehydrogenase phosphatase and pyruvate dehydrogenase kinase, leading to improved metabolism and reduced oxidative damage. Methods— Sprague–Dawley rats were subjected to transient (2, 3, or 4 hours) middle cerebral artery occlusion followed by 3- or 24-hour reperfusion, or permanent (28 hours) middle cerebral artery occlusion without reperfusion. At 2 hours after the onset of ischemia, rats received either an intraperitoneal injection of saline, 1 dose of EtOH (1.5 g/kg) for 2- and 3-hour middle cerebral artery occlusion, 2 doses of EtOH (1.5 g/kg followed by 1.0 g/kg in 2 hours) in 4 hours or permanent middle cerebral artery occlusion, and EtOH+95% NBO (at 2 hours after the onset of ischemia for 6 hours) in permanent stroke. Infarct volumes and neurological deficits were examined. Oxidative metabolism and stress were determined by measuring ADP/ATP ratio and reactive oxygen species levels. Protein levels of PDHC, pyruvate dehydrogenase kinase, and pyruvate dehydrogenase phosphatase were assessed. Results— EtOH induced dose-dependent neuroprotection in transient ischemia. Compared to EtOH or NBO alone, NBO+EtOH produced the best outcomes in permanent ischemia. These therapies improved brain oxidative metabolism by decreasing ADP/ATP ratios and reactive oxygen species levels, in association with significantly raised levels of PDHC and pyruvate dehydrogenase phosphatase, as well as decreased pyruvate dehydrogenase kinase. Conclusions— Both EtOH and EtOH+NBO treatments conferred neuroprotection in severe stroke by affecting brain metabolism. The treatment may modulate the damaging cascade of metabolic events by bringing the PDHC activity back to normal metabolic levels.

Impairment of motor coordination, or ataxia, is a prominent effect of alcohol ingestion in humans. To date, many models have been created to examine this phenomenon in animals. Evidence suggests that the tasks thought to measure this behavior in mice actually measure different components of this complex trait. We have characterized the parallel rod floor apparatus to quantify ethanol‐induced motor incoordination. Using genetically heterogeneous mice, we evaluated the influence of rod diameter and inter‐rod distance on dose‐related ethanol‐induced motor incoordination to select parameters that optimized testing procedures. We then used the DBA/2J and C57BL/6J inbred strains of mice to examine the effect of 2 g/kg of ethanol, by serially testing mice on two floor types, separated by 1 week. Finally, we tested eight inbred strains of mice on four floor types to examine patterns of strain sensitivity to 2 g/kg of intraperitoneal ethanol and determined the test parameters that maximized strain effect size. Motor incoordination varied depending on the floor type and strain. When data from strain 129S1/SvlmJ were removed from the analyses because of their extreme behavior, the greatest strain effect size was observed on one floor type during the first 10 min of testing after 2 g/kg of intraperitoneal ethanol. These findings suggest that the parallel rod floor apparatus provides a useful means for examining ethanol‐induced motor incoordination in mice but that specific testing procedures are important for optimizing detection of motor incoordination and genetic influences.