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Background Environmental lead ( Pb) exposure and alcohol abuse pose significant public health problems for our society. One of the proposed mechanisms of action of the developmental neurotoxicant Pb is related to its ability to affect antioxidant enzymes, including catalase ( CAT). Ethanol's ( Et OH) motivational effects are postulated to be mediated by the CAT-dependent acetaldehyde generated in the brain. The current study sought to investigate the role of this enzyme in the elevated EtOH intake previously reported in perinatally Pb-exposed rats. Methods Thirty-five-day-old male Wistar rats exposed to 220 ppm Pb during gestation and lactation were offered escalating Et OH solutions (2 to 10%) or water, 2 h/d for 28 days. Once baseline 10% Et OH intake was achieved, they were injected with (i) saline ( SAL), (ii) 3-amino 1,2,4 triazole (aminotriazole [AT], a CAT inhibitor, 250 mg/kg intraperitoneally [i.p.], 5 hours before the last 8 Et OH intake sessions), or (iii) 3-nitropropionic acid (3 NPA; a CAT activator, 20 mg/kg subcutaneously [s.c.], 45 minutes before the last 4 Et OH intake sessions). Rats were then sacrificed, blood collected, and brain regions harvested for CAT activity determination. Additional studies evaluated Et OH intake and CAT activity in response to 10 and 30 mg/kg 3 NPA. Both 3 NPA and AT were evaluated for striatal cytotoxicity. Results We observed that AT pretreatment blunted the increased Et OH intake, as well as the elevated CAT activity in blood, cerebellum, and hippocampus evidenced in the developmentally Pb-exposed rats that have consumed Et OH. Conversely, 20 mg/kg 3 NPA further increased voluntary Et OH intake in these animals as compared with controls, concomitantly with a slight elevation in CAT activity both in blood and in the striatum, associated with no changes in striatal cytotoxicity. Conclusions These results suggest a participation of CAT, and possibly acetaldehyde, in Pb-induced high Et OH intake, and open up new avenues to elucidate the mechanism that underlies the Pb and Et OH interaction. [ABSTRACT FROM AUTHOR]

Background : To clarify the role of brain acetaldehyde in the hypnotic effect of ethanol, we compared the ethanol-oxidizing capacity (rate of acetaldehyde accumulation) and catalase and aldehyde dehydrogenase activity in the brains of animals genetically selected for different sensitivities to the hypnotic effect of ethanol. Methods: We used high, low, or control alcohol-sensitive rats (HAS, LAS, and CAS) and short- and long-sleep mice (SS and LS), as well as SS × LS recombinant inbred mice with known strain differences in mean duration of ethanol-induced sleep. We studied the rate of accumulation of acetaldehyde from ethanol in brain homogenates of these animals and correlated those values with their hypnotic sensitivity to ethanol. Results: Acetaldehyde accumulation from ethanol was significantly higher in the brain homogenates from HAS rats and LS mice with high sensitivity to the hypnotic effect of ethanol in vivo, compared with LAS rats and SS mice with low sensitivity to ethanol. A correlation was found between the duration of ethanol-induced sleep and the in vitro rate of accumulation of ethanol-derived acetaldehyde in the brains of recombinant SS × LS mice strains. There was no correlation of sleep time with brain catalase levels. There were no line differences in brain catalase or aldehyde dehydrogenase or in alcohol or aldehyde dehydrogenase activity in livers of LAS, CAS, and HAS rats or in SS and LS mice. Conclusions: A correlation between the brain acetaldehyde accumulation, but not catalase levels, and the central effect of ethanol was demonstrated in animals genetically differing in initial sensitivity to the hypnotic effect of ethanol. [ABSTRACT FROM AUTHOR]

For many years, research from around the world has suggested that the neuroactive steroid (3α,5α)‐3‐hydroxypregnan‐20‐one (allopregnanolone or 3α,5α‐THP) may have therapeutic potential for treatment of various symptoms of alcohol use disorders (AUDs). In this critical review, we systematically address all the evidence that supports such a suggestion, delineate the etiologies of AUDs that are addressed by treatment with allopregnanolone or its precursor pregnenolone, and the rationale for treatment of various components of the disease based on basic science and clinical evidence. This review presents a theoretical framework for understanding how endogenous steroids that regulate the effects of stress, alcohol, and the innate immune system could play a key role in both the prevention and the treatment of AUDs. We further discuss cautions and limitations of allopregnanolone or pregnenolone therapy with suggestions regarding the management of risk and the potential for helping millions who suffer from AUDs. [ABSTRACT FROM AUTHOR]

Background: Binge alcohol drinking has been characterized as a key feature of alcoholism. The drinking‐in‐the‐dark (DID) preclinical model, a procedure that promotes high levels of ethanol (EtOH) intake in short periods of time, has been extensively used to investigate neuropharmacological and genetic determinants of binge‐like EtOH consumption. Using DID methodology, alcohol‐preferring strains of mice such as C57BL/6J (B6) mice consume enough EtOH to achieve blood concentrations (≥1.0 mg/ml) associated with behavioral intoxication (i.e., motor incoordination). DID procedures typically involve the use of socially isolated animals (single‐housed prior to and during the experiment). Previous research indicates that stress associated with social isolation can induce anxiety‐like behavior and promote increases in EtOH intake. The present study investigates the role of housing conditions in anxiety‐like behavior and binge‐like EtOH intake using a DID procedure. Methods: Male and female B6 mice were isolated or pair‐housed for a period of 6 weeks prior to evaluation of anxiety‐like (elevated plus maze, light and dark box, open field) and drinking (water, 10% sucrose, 10 to 30% EtOH) behavior. In order to measure intake, a variation of the standard DID procedure using a removable, transparent, and perforated plastic barrier strip (designed to temporarily divide the cage in 2) was introduced. This allowed for individual intake records (2‐hour test) of isolated and socially housed animals. Results: Increased anxiety‐like behavior and reduced sucrose consumption were found in isolated mice. The effects of housing conditions on EtOH intake were sex‐ and concentration‐dependent. In male mice, isolation increased 20 and 30% EtOH intake. In females, however, an increased intake of EtOH (30%) was found in socialized animals. No effects of housing or sex were found at EtOH 10%. Conclusions: Together with previous literature, the present study suggests that social isolation can promote anxiety‐associated behavior and produce sex‐dependent changes in binge‐like EtOH consumption. [ABSTRACT FROM AUTHOR]

Background: Adolescence is a critical period for neural development, and alcohol exposure during adolescence can lead to an elevated risk for health consequences as well as alcohol use disorders. Clinical and experimental data suggest that chronic alcohol exposure may produce immunomodulatory effects that can lead to the activation of pro‐inflammatory cytokine pathways as well as microglial markers. The present study evaluated, in brain and blood, the effects of adolescent alcohol exposure and withdrawal on microglia and on the most representative pro‐ and anti‐inflammatory cytokines and major chemokines that can contribute to the establishing of a neuroinflammatory environment. Methods: Wistar rats (males, n = 96) were exposed to ethanol (EtOH) vapors, or air control, for 5 weeks over adolescence (PD22‐PD58). Brains and blood samples were collected at 3 time points: (i) after 35 days of vapor/air exposure (PD58); (ii) after 1 day of withdrawal (PD59), and (iii) 28 days after withdrawal (PD86). The ionized calcium‐binding adapter molecule 1 (Iba‐1) was used to index microglial activation, and cytokine/chemokine responses were analyzed using magnetic bead panels. Results: After 35 days of adolescent vapor exposure, a significant increase in Iba‐1 immunoreactivity was seen in amygdala, frontal cortex, hippocampus, and substantia nigra. However, Iba‐1 density returned to control levels at both 1 day and 28 days of withdrawal except in the hippocampus where Iba‐1 density was significantly lower than controls. In serum, adolescent EtOH exposure induced a reduction in IL‐13 and an increase in fractalkine at day 35. After 1 day of withdrawal, IL‐18 was reduced, and IP‐10 was elevated, whereas both IP‐10 and IL‐10 were elevated at 28 days following withdrawal. In the frontal cortex, adolescent EtOH exposure induced an increase in IL‐1β at day 35, and 28 days of withdrawal, and IL‐10 was increased after 28 days of withdrawal. Conclusion: These data demonstrate that EtOH exposure during adolescence produces significant microglial activation; however, inflammatory markers seen in the blood appear to differ from those observed in the brain. [ABSTRACT FROM AUTHOR]

Background: Ethanol (EtOH), one of the most widely consumed substances of abuse, can induce brain damage and neurodegeneration. EtOH is centrally metabolized into acetaldehyde, which has been shown to be responsible for some of the neurophysiological and cellular effects of EtOH. Although some of the consequences of chronic EtOH administration on cell oxidative status have been described, the mechanisms by which acute EtOH administration affects the brain's cellular oxidative status and the role of acetaldehyde remain to be elucidated in detail. Methods: Swiss CD‐I mice were pretreated with the acetaldehyde‐sequestering agent d‐penicillamine (DP; 75 mg/kg, i.p.) or the antioxidant lipoic acid (LA; 50 mg/kg, i.p.) 30 minutes before EtOH (2.5 g/kg, i.p.) administration. Animals were sacrificed 30 minutes after EtOH injection. Glutathione peroxidase (GPx) mRNA levels; GPx and glutathione reductase (GR) enzymatic activities; reduced glutathione (GSH), glutathione disulfide (GSSG), glutamate, g‐L‐glutamyl‐L‐cysteine (Glut‐Cys), and malondialdehyde (MDA) concentrations; and protein carbonyl group (CG) content were determined in whole‐brain samples. Results: Acute EtOH administration enhanced GPx activity and the GSH/GSSG ratio, while it decreased GR activity and GSSG concentration. Pretreatment with DP or LA only prevented GPx activity changes induced by EtOH. Conclusions: Altogether, these results show the capacity of a single dose of EtOH to unbalance cellular oxidative homeostasis. [ABSTRACT FROM AUTHOR]

Background: Evidence supports a role for the circadian system in alcohol use disorders, but the impact of adolescent alcohol exposure on circadian timing later in life is unknown. Acute ethanol (EtOH) attenuates circadian photic phase‐resetting in adult, but not adolescent, rodents. However, nearly all studies have focused on males and it is unknown whether this adolescent‐typical insensitivity to EtOH persists into adulthood after adolescent drinking. Methods: Circadian activity was monitored in C57BL/6J mice receiving adolescent intermittent EtOH (AIE) exposure (15% EtOH and water every other day throughout adolescence) or water alone followed by 24 days wherein EtOH was not available (washout). Mice then received a challenge dose of EtOH (1.5 g/kg, intraperitoneal) or saline 15 minutes prior to a 30‐minute phase‐delaying light pulse and then were released into constant darkness (DD). To control for possible phase‐shifting by EtOH challenge alone, a separate group of mice underwent AIE exposure (or water‐only) and washout and then received an EtOH or saline injection, but did not receive a light pulse prior to DD. Results: Striking sex differences in nearly all measures of circadian photic entrainment were observed during adolescence but AIE effects were subtle and few. Only EtOH‐naïve adult male mice showed attenuated photic phase‐shifts with EtOH challenge, while all other groups showed normal phase‐resetting responses to light. AIE‐exposed females showed a persistent delay in activity offset. Conclusions: Adult male AIE‐exposed mice retained adolescent‐like insensitivity to EtOH‐induced suppression of photic phase‐resetting, suggesting AIE‐induced "lock‐in" of an adolescent behavioral phenotype. Adult AIE‐exposed females showed delayed initiation of the rest phase. Our results also indicate that intermittent EtOH drinking has subtle effects on circadian activity in mice during adolescence that differ from previously reported effects on adult males. The observed sex differences in circadian activity, EtOH consumption and preference, and responses to EtOH challenge merit future mechanistic study. Differential effects of adolescent drinking on circadian rhythms in adult mice. A) Adult female mice showed persistent delays in activity offset after exposure to adolescent intermittent ethanol (AIE), perhaps reflecting increased latency to sleep. B) Adult male mice with prior AIE exposure showed persistent insensitivity to acute ethanol‐induced attenuation of circadian phaseresetting to light. These data suggest that adolescent drinking affects circadian rhythms differently between the sexes, and may ultimately play a sex‐dependent role in motivation to drink in adulthood. [ABSTRACT FROM AUTHOR]

Background Ethanol (EtOH)-evoked oxidative stress, which contributes to myocardial dysfunction in proestrus rats, is mediated by increases in NADPH oxidase (Nox) activity, malondialdehyde ( MDA), and ERK1/2 phosphorylation. Whether these biochemical responses, which are triggered by alcohol-derived acetaldehyde in noncardiac tissues, occur in proestrus rats' hearts remains unknown. Therefore, we elucidated the roles of alcohol dehydrogenase ( ADH), cytochrome P4502E1 ( CYP2E1), and catalase, which catalyze alcohol oxidation to acetaldehyde, in these alcohol-evoked biochemical and hemodynamic responses in proestrus rats. Methods Conscious proestrus rats prepared for measurements of left ventricular ( LV) function and blood pressure ( BP) received EtOH (1.5 g/kg, intravenous [i.v.] infusion over 30 minutes) or saline 30 minutes after an ADH and CYP2E1 inhibitor, 4-methylpyrazole (4-MP) (82 mg/kg, intraperitoneal), a catalase inhibitor, 3- AT (0.5 g/kg, i.v.), their combination, or vehicle. LV function and BP were monitored for additional 60 minutes after EtOH or saline infusion before collecting the hearts for ex vivo measurements of LV reactive oxygen species ( ROS), Nox activity, MDA, and ERK1/2 phosphorylation. Results EtOH reduced LV function (d P/d tmax and LV developed pressure) and BP, and increased cardiac Nox activity, ROS and MDA levels, and ERK1/2 phosphorylation. Either inhibitor partially, and their combination significantly, attenuated these responses despite the substantially higher blood EtOH level, and the increased cardiac oxidative stress and reduced BP caused by 3- AT alone or with 4- MP. The inhibitors reduced cardiac MDA level and reversed EtOH effect on cardiac and plasma MDA. Conclusions EtOH oxidative metabolism plays a pivotal role in the EtOH-evoked LV oxidative stress and dysfunction in proestrus rats. Notably, catalase inhibition (3- AT) caused cardiac oxidative stress and hypotension. [ABSTRACT FROM AUTHOR]

Background In our companion article, we examined the role of MyD88-dependent signaling in ethanol (EtOH) consumption in mice lacking key components of this inflammatory pathway and observed differential effects on drinking. Here, we studied the role of these same signaling components in the acute sedative, intoxicating, and physiological effects of EtOH. Toll-like receptor 4 (TLR4) has been reported to strongly reduce the duration of EtOH-induced sedation, although most studies do not support its direct involvement in EtOH consumption. We examined TLR4 and other MyD88 pathway molecules to determine signaling specificity in acute EtOH-related behaviors. We also studied other GABAergic sedatives to gauge the EtOH specificity and potential role for GABA in EtOH's sedative and intoxicating effects in the mutant mice. Methods Loss of righting reflex ( LORR) and recovery from motor incoordination were studied following acute injection of EtOH or other sedative drugs in male and female control C57 BL/6J mice versus mice lacking CD14, TLR2, TLR4 (C57 BL/10ScN), or MyD88. We also examined EtOH-induced hypothermia and blood EtOH clearance in these mice. Results Male and female mice lacking TLR4 or MyD88 showed reduced duration of EtOH-induced LORR and faster recovery from EtOH-induced motor incoordination in the rotarod test. MyD88 knockout mice had slightly faster recovery from EtOH-induced hypothermia compared to control mice. None of the mutants differed from control mice in the rate of blood EtOH clearance. All of the mutants showed similar decreases in the duration of gaboxadol-induced LORR, but only mice lacking TLR4 were less sensitive to the sedative effects of pentobarbital. Faster recovery from diazepam-induced motor impairment was observed in CD14, TLR4, and MyD88 null mice of both sexes. Conclusions TLR4 and MyD88 were key mediators of the sedative and intoxicating effects of EtOH and GABAergic sedatives, indicating a strong influence of TLR4-MyD88 signaling on GABAergic function. Despite the involvement of TLR4 in EtOH's acute behaviors, it did not regulate EtOH consumption in any drinking model as shown in our companion article. Collectively, our studies demonstrate differential effects of TLR-MyD88 components in the acute versus chronic actions of EtOH. [ABSTRACT FROM AUTHOR]

Background Molecular and behavioral studies support a role for innate immune proinflammatory pathways in mediating the effects of alcohol. Increased levels of Toll-like receptors (TLRs) have been observed in animal models of alcohol consumption and in human alcoholics, and many of these TLRs signal via the MyD88-dependent pathway. We hypothesized that this pathway is involved in alcohol drinking and examined some of its key signaling components. Methods Different ethanol (EtOH)-drinking paradigms were studied in male and female control C57BL/6J mice versus mice lacking CD14, TLR2, TLR4 (C57BL/10ScN), or MyD88. We studied continuous and intermittent access 2-bottle choice (2BC) and 1-bottle and 2BC drinking-in-the-dark (DID) tests as well as preference for saccharin, quinine, and NaCl. Results In the 2BC continuous access test, EtOH intake decreased in male TLR2 knockout (KO) mice, and we previously reported reduced 2BC drinking in male and female CD14 KO mice. In the intermittent access 2BC test, EtOH intake decreased in CD14 KO male and female mice, whereas drinking increased in MyD88 KO male mice. In the 2BC-DID test, EtOH drinking decreased in male and female mice lacking TLR2, whereas drinking increased in MyD88 KO male mice. In the 1-bottle DID test, EtOH intake decreased in female TLR2 KO mice. TLR2 KO and CD14 KO mice did not differ in saccharin preference but showed reduced preference for NaCl. MyD88 KO mice showed a slight reduction in preference for saccharin. Conclusions Deletion of key components of the MyD88-dependent pathway produced differential effects on EtOH intake by decreasing (TLR2 KO and CD14 KO) or increasing (MyD88 KO) drinking, while deletion of TLR4 had no effect. Some of the drinking effects depended on the sex of the mice and/or the EtOH-drinking model. [ABSTRACT FROM AUTHOR]

Background Growing evidence supports a central role for the circadian system in alcohol use disorders, but few studies have examined this relationship during adolescence. In mammals, circadian rhythms are regulated by the suprachiasmatic nucleus, a biological clock whose timing is synchronized (reset) to the environment primarily by light (photic) input. Alcohol (ethanol [EtOH]) disrupts circadian timing in part by attenuating photic phase-resetting responses in adult rodents. However, circadian rhythms change throughout life and it is not yet known whether EtOH has similar effects on circadian regulation during adolescence. Methods General circadian locomotor activity was monitored in male C57BL6/J mice beginning in adolescence (P27) or adulthood (P61) in a 12-hour light, 12-hour dark photocycle for ~2 weeks to establish baseline circadian activity measures. On the day of the experiment, mice received an acute injection of EtOH (1.5 g/kg, i.p.) or equal volume saline 15 minutes prior to a 30-minute light pulse at Zeitgeber Time 14 (2 hours into the dark phase) and then were released into constant darkness (DD) for ~2 weeks to assess phase-resetting responses. Control mice of each age-group received injections but no light pulse prior to DD. Results While adults showed the expected decrease in photic phase-delays induced by acute EtOH, this effect was absent in adolescent mice. Adolescents also showed baseline differences in circadian rhythmicity compared to adults, including advanced photocycle entrainment, larger photic phase-delays, a shorter free-running (endogenous) circadian period, and greater circadian rhythm amplitude. Conclusions Collectively, our results indicate that adolescent mice are less sensitive to the effect of EtOH on circadian photic phase-resetting and that their daily activity rhythms are markedly different than those of adults. [ABSTRACT FROM AUTHOR]

The innate immune response in the central nervous system ( CNS) participates in both synaptic plasticity and neural damage. Emerging evidence from human and animal studies supports the role of the neuroimmune system response in many actions of ethanol (EtOH) on the CNS. Research studies have shown that alcohol stimulates brain immune cells, microglia, and astrocytes, by activating innate immune receptors Toll-like receptors (TLRs) and NOD-like receptors (inflammasome NLRs) triggering signaling pathways, which culminate in the production of pro-inflammatory cytokines and chemokines that lead to neuroinflammation. This review focuses on evidence that indicates the participation of TLRs and the inflammasome NLRs signaling response in many effects of EtOH on the CNS, such as neuroinflammation associated with brain damage, cognitive and behavioral dysfunction, and adolescent brain development alterations. It also reviews findings that indicate the role of TLR4-dependent signaling immune molecules in alcohol consumption, reward, and addiction. The research data suggest that overactivation of TLR4 or NLRs increases pro-inflammatory cytokines and mediators to cause neural damage in the cerebral cortex and hippocampus, while modest TLR4 activation, along with the generation of certain cytokines and chemokines in specific brain areas (e.g., amygdala, ventral tegmental area), modulate neurotransmission, alcohol drinking, and alcohol rewards. Elimination of TLR4 and NLRP3 abolishes many neuroimmune effects of EtOH. Despite much progress being made in this area, there are some research gaps and unanswered questions that this review discusses. Finally, potential therapies that target neuroimmune pathways to treat neuropathological and behavioral consequences of alcohol abuse are also evaluated. [ABSTRACT FROM AUTHOR]

Background Cyclic AMP-dependent protein kinase A ( PKA) signaling is a key target for the action of alcohol and may therefore play a role in the pathophysiology of alcohol withdrawal seizures ( AWSs). Here, we investigated the role of PKA activity with respect to increased seizure susceptibility in rats that were subjected to alcohol withdrawal. Methods Adult male Sprague Dawley rats received 3 daily doses of ethanol (EtOH) (or vehicle) for 4 consecutive days. Rats were then tested for susceptibility to acoustically evoked AWSs 3, 24, and 48 hours after the last alcohol dose. In separate experiments, the inferior colliculus ( IC) was collected at these same time points from rats subjected to alcohol withdrawal and control rats following alcohol withdrawal. PKA activity, catalytic C α ( PKAC α) protein, regulatory RII α ( PKARII α) protein, and RII β ( PKARII β) protein were measured in the IC. Lastly, in situ pharmacological studies were performed to evaluate whether inhibiting PKA activity in the IC suppressed AWSs. Results In the EtOH-treated group, AWSs were observed at the 24-hour time point, but not at the 3-hour or 48-hour time points. In the IC, PKA activity was significantly higher both 3 hours (i.e., before AWS susceptibility) and 24 hours after the last alcohol dose (when AWS susceptibility peaked) than in control rats. Consistent with these findings, protein levels of the PKAC α subunit were significantly increased in the IC both 3 and 24 hours after the last alcohol dose. Lastly, in situ inhibition of PKA activity within the IC suppressed AWSs. Conclusions The increase in PKA activity and PKAC α protein expression in the IC preceded the occurrence of AWSs, and inhibiting PKA activity within the IC suppressed acoustically evoked AWSs. Together, these findings suggest that altered PKA activity plays a key role in the pathogenesis of AWSs. [ABSTRACT FROM AUTHOR]

Background Novelty-seeking behavior is related to the reward system in the brain and can predict the potential for addiction. Alcohol use is prevalent in HIV-1-infected patients and adversely affects antiretroviral medication. The difference in vulnerability to alcohol addiction between HIV-1-infected and noninfected populations has not been fully investigated. This study was designed to determine whether HIV-1 proteins alter the effects of ethanol (EtOH) on novelty-seeking behavior using the HIV-1 transgenic ( HIV-1Tg) rat as the study model and to examine the molecular mechanisms responsible for this behavior. Methods Both HIV-1Tg and F344 control rats were tested for baseline novelty-seeking behavior, then received either EtOH (1 g/kg) at a concentration of 20% v/v or saline treatment for 13 days, and then were retested for novelty seeking. Quantitative real-time polymerase chain reaction was conducted to examine the differences in expression of 65 genes implicated in novelty seeking and alcohol addiction between strains and treatment groups. Results The HIV-1 proteins significantly enhanced baseline novelty-seeking behaviors in both the hole-board and open-field tests. Chronic EtOH treatment significantly increased baseline novelty-seeking behavior in both strains, but the effects of EtOH appeared to be more robust and prominent in HIV-1Tg rats. Strain-specific patterns of altered gene expression were observed for dopaminergic, cholinergic, and glutamatergic signaling in the nucleus accumbens, suggesting the effects of HIV-1 proteins on the brain's reward system. Chronic EtOH treatment was shown to greatly modulate the effects of HIV-1 proteins in these neurotransmitter systems. Conclusions Taken together, our findings indicate that HIV-1 proteins could modify novelty-seeking behavior at the gene expression level, and EtOH treatment may enhance this behavior in both strains but to a greater extent in HIV-1Tg rats. [ABSTRACT FROM AUTHOR]

Background There is controversy regarding the active agent responsible for alcohol addiction. The theory that ethanol (EtOH) itself was the agent in alcohol drinking behavior was widely accepted until acetaldehyde (AcH) was found in the brain. The importance of AcH formation in the brain is still subject to speculation due to the lack of a method to accurately assay the AcH levels directly. A highly sensitive gas chromatography mass spectrometry (GC-MS) method to reliably determine AcH concentration with certainty is needed to address whether neural AcH is indeed responsible for increased alcohol consumption. Methods A headspace gas chromatograph coupled to selected-ion monitoring MS was utilized to develop a quantitative assay for AcH and EtOH. Our GC- MS approach was carried out using a Bruker Scion 436- GC SQ MS. Results Our approach yields limits of detection of AcH in the nanomolar range and limits of quantification in the low micromolar range. Our linear calibration includes 5 concentrations with a least-square regression greater than 0.99 for both AcH and EtOH. Tissue analyses using this method revealed the capacity to quantify EtOH and AcH in blood, brain, and liver tissue from mice. Conclusions By allowing quantification of very low concentrations, this method may be used to examine the formation of EtOH metabolites, specifically AcH, in murine brain tissue in alcohol research. [ABSTRACT FROM AUTHOR]

Background A number of studies have shown that acetaldehyde synthesized in the brain is necessary to induce ethanol (EtOH) reinforcement in naïve animals ( acquisition phase). However, after chronic intake is achieved ( maintenance phase), EtOH intake becomes independent of acetaldehyde generation or its levels. Glutamate has been reported to be associated with the maintenance of chronic EtOH intake. The levels of brain extracellular glutamate are modulated by 2 glial processes: glutamate reabsorption via an Na+-glutamate transporter ( GLT1) and a cystine-glutamate exchanger. Chronic EtOH intake lowers GLT1 levels and increases extracellular glutamate. The administration of N-acetyl cysteine ( NAC), a precursor of cystine, has been shown to reduce the relapse of several drugs of abuse, while NAC has not been tested on chronic EtOH intake or on EtOH's influence on the motivation for another drug. These were investigated in the present study. Methods (i) Rats bred for their high EtOH intake were allowed access to 10% EtOH and water up to 87 days. NAC was administered (30 and 60 mg/kg daily, intraperitoneally) for 14 consecutive days, either during the acquisition phase or the maintenance phase of EtOH drinking. (ii) In additional experiments, rats were allowed EtOH (10%) and water access for 61 days, after which EtOH was replaced by saccharin (0.3%) to determine both if chronic EtOH consumption influences saccharin intake and whether NAC modifies the post chronic EtOH saccharin intake. Results NAC did not influence the acquisition ('first hit') of chronic EtOH intake, but greatly inhibited (60 to 70%; p < 0.0001) EtOH intake when NAC was administered to animals that were consuming EtOH chronically. NAC did not influence saccharin intake in naïve animals. In animals that had consumed EtOH chronically and were thereafter offered a saccharin solution (0.3%), saccharin intake increased over 100% versus that of EtOH-untreated animals, an effect that was fully suppressed by NAC. Conclusions N-acetyl cysteine, a drug approved for use in humans, markedly reduces chronic EtOH intake and abolishes the increased intake of saccharin stimulated by chronic EtOH drinking. [ABSTRACT FROM AUTHOR]

Background Ethanol is widely known for its depressant effects; however, the underlying neurobiological mechanisms are not clear. Calcium-activated anion channels ( CAACs) contribute to extracellular chloride levels and thus may be involved in regulating inhibitory mechanisms within the central nervous system. Therefore, we hypothesized that CAACs influence ethanol behavioral sensitivity by altering CAAC expression. Methods We assessed the role of CAACs in ethanol-induced loss of righting reflex ( LORR) and locomotor activity using intracerebroventricular infusions of several nonselective CAAC blockers. CAAC expression was determined after ethanol exposure. Results Ethanol-induced LORR (4.0 g/kg, intraperitoneally [i.p.]) was significantly attenuated by all 4 CAAC blockers. Blocking CAACs did not impact ethanol's low-dose (1.5 g/kg, i.p.) locomotor-impairing effects. Biochemical analysis of CAAC protein expression revealed that cortical Bestrophin1 (Best1) and Tweety1 levels were reduced as early as 30 minutes following a single ethanol injection (3.5 g/kg, intraperitoneally [i.p.]) and remained decreased 24 hours later in P2 fractions. Cortical Best1 levels were also reduced following 1.5 g/kg. However, CAAC expression was unaltered in the striatum following a single ethanol exposure. Ethanol did not affect Tweety2 levels in either brain region. Conclusions These results suggest that CAACs are a major target of ethanol in vivo , and the regulation of these channels contributes to select behavioral actions of ethanol. [ABSTRACT FROM AUTHOR]

Background The lung has a highly regulated system of innate immunity to protect itself from inhaled microbes and toxins. The first line of defense is mucociliary clearance, but if invaders overcome this, inflammatory pathways are activated. Toll-like receptors ( TLRs) are expressed on the airway epithelium. Their signaling initiates the inflammatory cascade and leads to production of inflammatory cytokines such as interleukin (IL)-6 and IL-8. We hypothesized that airway epithelial insults, including heavy alcohol intake or smoking, would alter the expression of TLRs on the airway epithelium. Methods Bronchoscopy with bronchoalveolar lavage and brushings of the airway epithelium was performed in otherwise healthy subjects who had normal chest radiographs and spirometry. A history of alcohol use disorders ( AUDs) was ascertained using the Alcohol Use Disorders Identification Test ( AUDIT), and a history of cigarette smoking was also obtained. Age, gender, and nutritional status in all groups were similar. We used real-time polymerase chain reaction (PCR) to quantitate TLR1 to 9 and enzyme-linked immune assay to measure tumor necrosis factor- α, IL-6, and IL-8. Results Airway brushings were obtained from 26 nonsmoking/non- AUD subjects, 28 smoking/non- AUD subjects, 36 smoking/ AUD subjects, and 17 nonsmoking/ AUD subjects. We found that TLR2 is up-regulated in AUD subjects, compared to nonsmoking/non- AUD subjects, and correlated with their AUDIT scores. We also measured a decrease in TLR4 expression in AUD subjects that correlated with AUDIT score. IL-6 and IL-8 were also increased in bronchial washings from AUD subjects. Conclusions We have previously demonstrated in normal human bronchial epithelial cells that in vitro alcohol exposure up-regulates TLR2 through a NO/ cGMP/ PKG-dependent pathway, resulting in up-regulation of inflammatory cytokine production after Gram-positive bacterial product stimulation. Our current translational study confirms that TLR2 is also up-regulated in humans with AUDs. [ABSTRACT FROM AUTHOR]

This review analyzes literature that describes the behavioral effects of 2 metabolites of ethanol (EtOH): acetaldehyde and salsolinol (a condensation product of acetaldehyde and dopamine) generated in the brain. These metabolites are self-administered into specific brain areas by animals, showing strong reinforcing effects. A wealth of evidence shows that EtOH, a drug consumed to attain millimolar concentrations, generates brain metabolites that are reinforcing at micromolar and nanomolar concentrations. Salsolinol administration leads to marked increases in voluntary EtOH intake, an effect inhibited by mu-opioid receptor blockers. In animals that have ingested EtOH chronically, the maintenance of alcohol intake is no longer influenced by EtOH metabolites, as intake is taken over by other brain systems. However, after EtOH withdrawal brain acetaldehyde has a major role in promoting binge-like drinking in the condition known as the 'alcohol deprivation effect'; a condition seen in animals that have ingested alcohol chronically, are deprived of EtOH for extended periods, and are allowed EtOH re-access. The review also analyzes the behavioral effects of acetate, a metabolite that enters the brain and is responsible for motor incoordination at low doses of EtOH. Also discussed are the paradoxical effects of systemic acetaldehyde. Overall, evidence strongly suggests that brain-generated EtOH metabolites play a major role in the early ('first-hit') development of alcohol reinforcement and in the generation of relapse-like drinking. [ABSTRACT FROM AUTHOR]

Background The molecular mechanisms of the acute hypotensive and indirectly assessed cardiac depressant effect of ethanol ( Et OH)-evoked myocardial depression and hypotension in female rats are not known. We tested the hypothesis that a time-dependent myocardial depression caused by Et OH is initiated by its direct and indirect (cardiac vagal dominance) effects and is exacerbated by gradual development of oxidative stress. Methods In conscious female rats, we directly measured left ventricular developed pressure ( LVDP), the maximal rise of ventricular pressure over time (d P/d tmax), blood pressure ( BP), heart rate ( HR), and sympathovagal activity following intragastric Et OH (1 g/kg) or water over 90 minutes. Catalytic activity of acetaldehyde ( ACA)-generating (alcohol dehydrogenase [ADH] and catalase) and eliminating aldehyde dehydrogenase [ALDH2] enzymes along with mediators of oxidative stress were measured in myocardial tissues collected at 30, 60, or 90 minutes after Et OH or water. Results Et OH reduced myocardial function ( LVDP and d P/d tmax) within 5 to 10 minutes before the steady fall in BP in conscious proestrus rats. Further, Et OH shifted the sympathovagal balance, analyzed by spectral analysis of high frequency and low frequency of interbeat intervals, toward vagal dominance. Prior vagal blockade (atropine) or antioxidant (tempol) treatment attenuated Et OH-evoked myocardial depression and hypotension. Ex vivo studies revealed time-dependent: (i) enhancement of ADH, but not ALDH2 activity (indicative of elevated ACA levels), (ii) increases in phosphorylated Akt and ERK1/2, NADPH-oxidase activity, reactive oxygen species, malondialdehyde, and 4-hydroxy-2-nonenal-modified proteins. These molecular responses along with reduced myocardial catalase activity were most evident at 90 minutes post- Et OH when the reductions in cardiac function and BP reached their nadir. Conclusions Vagal dominance and time-dependent myocardial oxidative stress along with the accumulation of cardiotoxic aldehydes mediate Et OH-evoked myocardial dysfunction and hypotension in conscious proestrus female rats. [ABSTRACT FROM AUTHOR]

Background Affiliative social interactions promote alcohol consumption, and alcohol also promotes affiliative behavior. Furthermore, for most species, moderate doses of ethanol ( Et OH) and social affiliation are each rewarding. However, animal studies of drug and Et OH reward typically test individuals in isolation. To address social dimensions of Et OH reward, this study tested Et OH-induced conditioned social preference in male C57 BL/6 mice with ( ORCHX+ T) and without ( ORCHX) testosterone. Methods ORCHX+T males received Et OH (0, 1, 2, or 3 g/kg) intraperitoneally and were paired 4× for 30 minutes each with 1 of 2 stimulus males: with the CS− stimulus male after saline injection and with the CS+ male following Et OH. After pairing, time spent with CS+ and CS− males was measured in a 10-minute test. Results ORCHX+T test males showed conditioned preference for the CS+ male in response to 3 g/kg Et OH (change in preference: +71.3 ± 30.0 s/10 min, p < 0.05), but not for 0, 1, or 2 g/kg. By contrast, ORCHX males did not demonstrate conditioned preference for 3 g/kg Et OH (+16.0 ± 24.3 s/10 min, p > 0.05). In separate groups of mice, stimulus males ( IS+) received Et OH during pairing to determine whether test mice prefer another intoxicated mouse. Both ORCHX+T and ORCHX test mice showed an increase in preference score for the IS+ mouse ( ORCHX+T: +68.1 ± 24.0 seconds; ORCHX: +58.9 ± 19.6 seconds, p < 0.05). Conclusions These data demonstrate that Et OH promotes social preference in male mice, as it does in females. Testosterone enhances this effect. [ABSTRACT FROM AUTHOR]

Background The cAMP-dependent protein kinase ( PKA) signaling transduction pathway has been shown to play an important role in the modulation of several ethanol (EtOH)-induced behavioral actions. In vivo, short-term exposure to EtOH up-regulates the cAMP-signaling cascade. Interestingly, different Ca2+-dependent cAMP- PKA cascade mediators play a critical role in the neurobehavioral response to EtOH, being of special relevance to the Ca2+-dependent adenylyl cyclases 1 and 8. We hypothesize an intracellular PKA activation elicited by EtOH administration, which may be regulated by a Ca2+-dependent mechanism as an early cellular response. Thus, the present work aims to explore the role of Ca2+ (internal and external) on the EtOH-activated PKA cascade. Methods Swiss male mice received an intraperitoneal injection of EtOH (0 or 4 g/kg), and brains were dissected following a temporal pattern (7, 15, 30, 45, 90, or 120 minutes). Either the enzymatic PKA activity or its fingerprint was analyzed on different brain areas (cortex, hypothalamus, hippocampus, and striatum). To explore the role of Ca2+ on the EtOH-activated PKA cascade, mice were pretreated with diltiazem (0 or 20 mg/kg), dantrolene (0 or 5 mg/kg), or 3,7- Dimethyl-1-(2-propynyl)xanthine (0 or 1 mg/kg) 30 minutes before EtOH (4 g/kg) administration. After 45 minutes of EtOH administration, brains were removed and dissected to measure the PKA activity or its fingerprint. Results Results from these experiments showed an EtOH-dependent activation of PKA in different brain areas. Manipulations involving a disruption of intracellular Ca2+ release from the endoplasmic reticulum resulted in a decreased EtOH-induced activation of PKA. On the contrary, extracellular-to-cytoplasm Ca2+ manipulations did not prevent the PKA activation by EtOH. Conclusions Altogether, these results show the critical role of stored Ca2+ as an intracellular mediator of different neurobiological actions of EtOH and provide further evidence of a possible new target for EtOH within the central nervous system. [ABSTRACT FROM AUTHOR]

Background Ethanol ( Et OH) abuse is a major health and economic concern, particularly for females who appear to be more sensitive to the rewarding effects of Et OH. This study compared sex differences to the rewarding and aversive effects of Et OH using place-conditioning procedures in rats. Methods Separate groups of adult (male, female, ovariectomized [ OVX] female) and adolescent (male and female) rats received Et OH (0, 0.5, 1.0, 2.0, or 2.5 g/kg, intraperitoneal) and were confined to their initially nonpreferred side of our conditioning apparatus for 30 minutes. On alternate days, they received saline and were confined to the other side. Following 5 drug pairings, the rats were retested for preference behavior. Separate cohorts of the same groups of rats were injected with a similar dose range of Et OH, and blood Et OH levels ( BELs) were compared 30 minutes later. Results Et OH produced rewarding or aversive effects in a dose-dependent manner. An intermediate dose of Et OH (1.0 g/kg) produced rewarding effects in adult female, but not in male or OVX female rats, suggesting that ovarian hormones facilitate the rewarding effects of Et OH. Similarly, this intermediate dose of Et OH produced rewarding effects in adolescent female, but not in male rats. The highest dose of Et OH (2.5 g/kg) produced aversive effects that were similar across all adult groups. However, the aversive effects of Et OH were lower in adolescents than adults, suggesting that adolescents are less sensitive to the aversive effects of Et OH. The aversive effects of Et OH did not vary across the estrous cycle in intact adult females. There were also no group differences in BELs, suggesting that our results are not related to Et OH metabolism. Conclusion Our results in rats suggest that human females may be more vulnerable to Et OH abuse due to enhanced rewarding effects of this drug that are mediated by the presence of ovarian hormones. [ABSTRACT FROM AUTHOR]

Background Hydrogen peroxide (H2O2) is the cosubstrate used by the enzyme catalase to form Compound I (the catalase-H2O2 system), which is the major pathway for the conversion of ethanol (EtOH) into acetaldehyde in the brain. This centrally formed acetaldehyde has been shown to be involved in some of the psychopharmacological effects induced by EtOH in rodents, including voluntary alcohol intake. It has been observed that different levels of this enzyme in the central nervous system (CNS) result in variations in the amount of EtOH consumed. This has been interpreted to mean that the brain catalase-H2O2 system, by determining EtOH metabolism, mediates alcohol self-administration. To date, however, the role of H2O2 in voluntary EtOH drinking has not been investigated. Methods In the present study, we explored the consequence of a reduction in cerebral H2O2 levels in volitional EtOH ingestion. With this end in mind, we injected mice of the C57BL/6J strain intraperitoneally with the H2O2 scavengers alpha-lipoic acid (LA; 0 to 50 mg/kg) or ebselen (Ebs; 0 to 25 mg/kg) 15 or 60 minutes, respectively, prior to offering them an EtOH (10%) solution following a drinking-in-the-dark procedure. The same procedure was followed to assess the selectivity of these compounds in altering EtOH intake by presenting mice with a (0.1%) solution of saccharin. In addition, we indirectly tested the ability of LA and Ebs to reduce brain H2O2 availability. Results The results showed that both LA and Ebs dose-dependently reduced voluntary EtOH intake, without altering saccharin consumption. Moreover, we demonstrated that these treatments decreased the central H2O2 levels available to catalase. Conclusions Therefore, we propose that the amount of H2O2 present in the CNS, by determining brain acetaldehyde formation by the catalase-H2O2 system, could be a factor that determines an animal's propensity to consume EtOH. [ABSTRACT FROM AUTHOR]

Background We tested the hypothesis that alterations of the phosphorylation/dephosphorylation profile of mitogen-activated protein kinases ( MAPKs) in the rostral ventrolateral medulla ( RVLM) underlie the pressor response elicited by ethanol ( Et OH) microinjection into the RVLM of spontaneously hypertensive rats ( SHRs). The studies were extended to determine whether acetaldehyde ( ACA), the primary oxidative product of Et OH, replicates the molecular effects of Et OH within the RVLM and the consequent pressor response. Methods Effects of Et OH or ACA on blood pressure ( BP) were evaluated in the absence or presence of selective JNK ( SP600125), ERK ( PD98059), p38 ( SB203580), or ser/thr phosphatases (okadaic acid [ OKA]) inhibitor. Results Intra- RVLM Et OH (10 μg/rat) or ACA (2 μg/rat) caused a similar ERK2-dependent pressor response because Et OH or ACA-evoked increases in BP and in RVLM p- ERK2 level were abolished after pharmacologic inhibition of ERK phosphorylation. SP600125 abrogated the pressor action of Et OH, but not ACA, thus implicating JNK in Et OH action on BP. Despite Et OH enhancement of p38 phosphorylation, pharmacological studies argued against a causal role for this kinase in Et OH-evoked pressor response. RVLM phosphatase catalytic activity was not influenced by Et OH or ACA. Interestingly, pharmacologic phosphatase inhibition ( OKA), which increased RVLM p- ERK2 and BP, abrogated the pressor effect of subsequently administered Et OH or ACA. Conclusions Enhancement of RVLM ERK2 phosphorylation constitutes a major molecular mechanism for the pressor response elicited by intra- RVLM Et OH or its metabolite, ACA, in conscious SHRs. Further, RVLM kinases dephosphorylation does not contribute to intra- RVLM Et OH- or ACA-evoked pressor response. [ABSTRACT FROM AUTHOR]

Background The main system of central ethanol (EtOH) oxidation is mediated by the enzyme catalase. By reacting with H2 O2, brain catalase forms compound I (the catalase- H2 O2 system), which is able to oxidize EtOH to acetaldehyde ( ACD) in the brain. We have previously shown that ACD regulates EtOH motivational properties and possesses reinforcing effects by itself. In this study, we investigate the effects of alpha-lipoic acid ( ALA), a scavenging agent for H2 O2, on oral EtOH self-administration. Methods To this end, we trained Wistar rats to orally self-administer EtOH (10%) by nose poking. The effect of intraperitoneal pretreatment with ALA was evaluated during (i) maintenance of EtOH self-administration, (ii) EtOH self-administration under a progressive ratio ( PR) schedule of reinforcement, and (iii) oral EtOH priming to induce reinstatement of EtOH seeking behavior. Moreover, we tested the effect of ALA on saccharin (0.05%) reinforcement, as assessed by oral self-administration. Results The results indicate that ALA dose-dependently reduced the maintenance, the break point of EtOH self-administration under a PR and the reinstatement of EtOH seeking behavior without suppressing saccharin self-administration. Conclusions These results support that ALA may have a potential use in alcoholism treatment. [ABSTRACT FROM AUTHOR]

Background Animals that have chronically consumed alcohol and are subsequently deprived of it markedly increase their intake above basal levels when access to alcohol is reinstated. Such an effect, termed the alcohol deprivation effect ( ADE), has been proposed to reflect (i) an obsessive-compulsive behavior, (ii) craving, or (iii) an increased reinforcing value of ethanol ( Et OH). It has been reported that acetaldehyde, a highly reinforcing metabolite of Et OH, is generated in the brain by the action of catalase. Recent studies show that the administration of an anticatalase (sh RNA)-encoding lentiviral vector into the brain ventral tegmental area ( VTA) of naïve rats virtually abolishes (85 to 95%) their Et OH intake. It is hypothesized that the antireinforcing effect of the anticatalase vector will also inhibit the ADE. Methods Two-month-old Wistar-derived UCh B alcohol drinker rats were offered free access to water and 10 and 20% Et OH for 67 days. Thereafter, the animals were deprived of Et OH for 15 days and were subsequently offered access to the Et OH solutions. At the start of the deprivation period, animals were microinjected a single dose of an anticatalase (or control) vector into the VTA. Et OH intake was measured on the first hour of Et OH re-exposure as well as on a 24-hour basis for 7 days. Results A marked ADE was observed when Et OH intake was measured on the first hour or 24 hours following Et OH re-exposure, compared to the corresponding controls. The administration of the anticatalase vector reduced ADE by 60 to 80% ( p < 0.001) on the first hour and by 63 to 80% ( p < 0.001) on the initial 24 hours of Et OH re-exposure (first and second ADE, respectively) without changing the total fluid intake, indicating a specific effect on Et OH drinking. Conclusions Ethanol intake associated with ADE-a binge-like drinking behavior-is markedly inhibited by the administration of an anticatalase vector into the VTA, which blocks the conversion of Et OH into acetaldehyde, strongly suggesting that the marked increased Et OH intake that follows an alcohol deprivation period is mediated by acetaldehyde and its reinforcing metabolite. [ABSTRACT FROM AUTHOR]

Background Published findings indicate that acetaldehyde ( ACD; the first metabolite of ethanol [ Et OH]) and salsolinol ( SAL; formed through the nonenzymatic condensation of ACD and dopamine [ DA]) can be formed following Et OH consumption. Both ACD and SAL exhibit reinforcing properties within the posterior ventral tegmental area (p VTA) and both exhibit an inverted ' U-shaped' dose-response curve. The current study was undertaken to examine the dose-response effects of microinjections of ACD or SAL into the p VTA on DA efflux in the nucleus accumbens shell ( Acb Sh). Methods For the first experiment, separate groups of male Wistar rats received pulse microinjections of artificial cerebrospinal fluid (a CSF) or 12-, 23-, or 90-μM ACD into the p VTA, while extracellular DA levels were concurrently measured in the Acb Sh. The second experiment was similarly conducted, except rats were given microinjections of a CSF or 0.03-, 0.3-, 1.0-, or 3.0-μ M SAL, while extracellular levels of DA were measured in the Acb Sh. Results Both ACD and SAL produced a dose-dependent inverted ' U-shaped' response on DA release in the Acb Sh, with 23-μ M ACD (200% baseline) and 0.3-μ M SAL (300% baseline) producing maximal peak responses with higher concentrations of ACD (90 μ M) and SAL (3.0 μ M) producing significantly lower DA efflux. Conclusions The findings from the current study indicate that local application of intermediate concentrations of ACD and SAL stimulated DA neurons in the p VTA, whereas higher concentrations may be having secondary effects within the p VTA that inhibit DA neuronal activity. The present results parallel the studies on the reinforcing effects of ACD and SAL in the p VTA and support the idea that the reinforcing effects of ACD and SAL within the p VTA are mediated by activating DA neurons. [ABSTRACT FROM AUTHOR]

Background: Striatopallidal medium spiny neurons have been viewed as a final common path for drug reward and the ventral pallidum as an essential convergent point for hedonic and motivational signaling in the brain. The medium spiny neurons are GABAergic, but they colocalize enkephalin. Purpose of this study was to investigate the role of the opioidergic mechanisms of the ventral pallidum in ethanol self-administration behavior. Methods: Effects of bilateral microinjections of μ-, δ-, and κ-opioid receptor agonists and antagonists into the ventral pallidum on voluntary ethanol consumption were monitored in alcohol-preferring Alko Alcohol (AA) rats using the 90-minute limited access paradigm. Results: Stimulation of μ-opioid receptors with DAMGO (0.01 to 0.1 μg) or morphine (1 to 10 μg) in the ventral pallidum decreased ethanol intake dose-dependently. Conversely, blocking μ-receptors with CTOP (0.3 to 3 μg) increased ethanol intake significantly. Unlike CTOP, DAMGO also increased locomotor activity. Consumption of ethanol was not modified significantly by a broad-spectrum opioid receptor antagonist naltrexone, by δ-opioid receptor agonist DPDPE or antagonist naltrindole, or by a κ-opioid receptor agonist U50,488H or antagonist nor-BNI. Conclusions: The study provides evidence for μ- but not δ- or κ-opioid receptors in the ventral pallidum playing a role in the regulation of voluntary ethanol consumption. Furthermore, present findings give support to earlier work, suggesting an essential role of pallidal opioidergic transmission in drug reward. [ABSTRACT FROM AUTHOR]

Acetaldehyde (ACD), the first metabolite of ethanol (EtOH) appears to be involved in many of the psychoactive effects of its parent compound, including EtOH-induced activation of the mesolimbic dopamine (DA) system, thereby suggesting that ACD may participate in EtOH motivational properties. -cysteine (Lcys), a thiol compound sequestering ACD, is able to prevent the behavioral effect of EtOH and ACD. Here we show that the stimulatory effect of both EtOH and ACD on the mesolimbic DA system is prevented by Lcys pretreatment. Male Wistar rats were implanted with a microdialysis probe in the nucleus accumbens shell (NAccs), and pretreated intraperitoneally with Lcys (30 mg/kg) before intragastric administration of EtOH (1 g/kg) or ACD (20 mg/kg) or before intraperitoneal administration of morphine (2.5 mg/kg). Pretreatment with Lcys prevented both EtOH and ACD-induced DA release in the NAccs without influencing morphine-induced DA release, suggesting that Lcys specifically affects EtOH-induced DA release possibly through ACD sequestering. Our results underscore the role of ACD on EtOH-induced stimulation of DA mesoaccumbens system and support the notion that thiol compounds such as Lcys, by modulating EtOH-derived ACD bioavailability, would blunt EtOH rewarding properties. [ABSTRACT FROM AUTHOR]

While the molecular entity responsible for the rewarding effects of virtually all drugs of abuse is known, that for ethanol remains uncertain. Some lines of evidence suggest that the rewarding effects of alcohol are mediated not by ethanol per se but by acetaldehyde generated by catalase in the brain. However, the lack of specific inhibitors of catalase has not allowed strong conclusions to be drawn about its role on the rewarding properties of ethanol. The present studies determined the effect on voluntary alcohol consumption of two gene vectors, one designed to inhibit catalase synthesis and one designed to synthesize alcohol dehydrogenase (ADH), to respectively inhibit or increase brain acetaldehyde synthesis. The lentiviral vectors, which incorporate the genes they carry into the cell genome, were (i) one encoding a shRNA anticatalase synthesis and (ii) one encoding alcohol dehydrogenase (rADH1). These were stereotaxically microinjected into the brain ventral tegmental area (VTA) of Wistar-derived rats bred for generations for their high alcohol preference (UChB), which were allowed access to an ethanol solution and water. Microinjection into the VTA of the lentiviral vector encoding the anticatalase shRNA virtually abolished (−94% p < 0.001) the voluntary consumption of alcohol by the rats. Conversely, injection into the VTA of the lentiviral vector coding for ADH greatly stimulated (2 to 3 fold p < 0.001) their voluntary ethanol consumption. The study strongly suggests that to generate reward and reinforcement, ethanol must be metabolized into acetaldehyde in the brain. Data suggest novel targets for interventions aimed at reducing chronic alcohol intake. [ABSTRACT FROM AUTHOR]

Background: The current therapies for alcohol abuse disorders are not effective in all patients, and continued development of pharmacotherapies is needed. One approach that has generated recent interest is the antagonism of ghrelin receptors. Ghrelin is a gut-derived peptide important in energy homeostasis and regulation of hunger. Recent studies have implicated ghrelin in alcoholism, showing altered plasma ghrelin levels in alcoholic patients as well as reduced intakes of alcohol in ghrelin receptor knockout mice and in mice treated with ghrelin receptor antagonists. The aim of this study was to determine the neuroanatomical locus/loci of the effect of ghrelin receptor antagonism on alcohol consumption using the ghrelin receptor antagonist, D-Lys3-GHRP-6. Methods: In Experiment 1 , male C57BL/6J mice were injected with saline 3 hours into the dark cycle and allowed access to 15% (v/v) ethanol or water for 2 hours in a 2-bottle choice experiment. On test day, the mice were injected with either saline or 400 nmol of the ghrelin receptor antagonist, D-Lys3-GHRP-6, and allowed to drink 15% ethanol or water for 4 hours. The preference for alcohol and alcohol intake were determined. In Experiment 2 , the same procedure was followed as in Experiment 1 but mice were only allowed access to a single bottle of 20% ethanol (v/v), and alcohol intake was determined. Blood ethanol levels were analyzed, and immunohistochemistry for c-Fos was carried out to investigate changes in neural activity. To further elucidate the mechanism by which D-Lys3-GHRP-6 affects alcohol intake, in Experiment 3 , the effect of D-Lys3-GHRP-6 on the neural activation induced by intraperitoneal ethanol was investigated. For the c-Fos studies, brain regions containing ghrelin receptors were analyzed, i.e. the perioculomotor urocortin population of neurons (pIIIu), the ventral tegmental area (VTA), and the arcuate nucleus (Arc). In Experiment 4 , to test if blood ethanol concentrations were affected by D-Lys3-GHRP-6, blood samples were taken at 2 time-points after D-Lys3-GHRP-6 pretreatment and systemic ethanol administration. Results: In Experiment 1 , D-Lys3-GHRP-6 reduced preference to alcohol and in a follow-up experiment ( Experiment 2 ) also dramatically reduced alcohol intake when compared to saline-treated mice. The resulting blood ethanol concentrations were lower in mice treated with the ghrelin receptor antagonist. Immunohistochemistry for c-Fos showed fewer immunopositive cells in the pIIIu of the antagonist-treated mice but no difference was seen in the VTA or Arc. In Experiment 3 , D-Lys3-GHRP-6 reduced the induction of c-Fos by intraperitoneal ethanol in the pIIIu but had no effect in the VTA. In the Arc, there was a significant increase in the number of c-Fos immunopositive cells after D-Lys3-GHRP-6 administration, but the antagonist had no effect on ethanol-induced expression of c-Fos. D-Lys3-GHRP-6-pretreatment also did not affect the blood ethanol concentrations observed after a systemic injection of ethanol when compared to saline-pretreated mice ( Experiment 4 ). Conclusions: These findings indicate that the action of ghrelin on the regulation of alcohol consumption may occur via the pIIIu. [ABSTRACT FROM AUTHOR]

Background: Ethanol exposure during early life has been shown to permanently alter the circadian expression of clock regulatory genes and the β-endorphin precursor proopiomelanocortin (POMC) gene in the hypothalamus. Ethanol also alters the stress- and immune-regulatory functions of β-endorphin neurons in laboratory rodents. Our aim was to determine whether the circadian clock regulatory Per2 gene modulates the action of ethanol on β-endorphin neurons in mice. Methods: Per2 mutant ( mPer2 Brdml) and wild type (C57BL/6J) mice were used to determine the effect of Per2 mutation on ethanol-regulated β-endorphin neuronal activity during neonatal period using an in vitro mediobasal hypothalamic (MBH) cell culture model and an in vivo milk formula feeding animal model. The β-endorphin neuronal activity following acute and chronic ethanol treatments was evaluated by measuring the peptide released from cultured cells or peptide levels in the MBH tissues, using enzyme-linked immunosorbent assay (ELISA). Results: Per2 mutant mice showed a higher basal level of β-endorphin release from cultured MBH cells and a moderate increase in the peptide content in the MBH in comparison with control mice. However, unlike wild type mice, Per2 mutant mice showed no stimulatory or inhibitory β-endorphin-secretory responses to acute and chronic ethanol challenges in vitro. Furthermore, Per2 mutant mice, but not wild type mice, failed to show the stimulatory and inhibitory responses of MBH β-endorphin levels to acute and chronic ethanol challenges in vivo. Conclusions: These results suggest for the first time that the Per2 gene may be critically involved in regulating β-endorphin neuronal function. Furthermore, the data revealed an involvement of the Per2 gene in regulating β-endorphin neuronal responses to ethanol. [ABSTRACT FROM AUTHOR]

Background and Methods: Ethanol (EtOH) activates hypothalamic–pituitary–adrenal (HPA) axis, resulting in adrenocorticotropin hormone, glucocorticoid release, and in modifications of the response of the axis to other stressors. The initial site of EtOH action within the HPA system seems to be the hypothalamus. Thus, to determine the mechanisms responsible for these effects, we investigated: (i) whether EtOH was able to release corticotrophic releasing hormone (CRH) from incubated hypothalamic explants; (ii) whether acetaldehyde (ACD), its first metabolite formed in the brain by catalase activity, might play a role in EtOH activity. To this aim, rat hypothalamic explants were incubated with: (i) medium containing EtOH at 32.6 × 103 μM; (ii) different concentration of ACD (1, 3, 10, and 30 μM); (iii) EtOH plus 3amino-1,2,4-triazole (3AT, 32 × 103 μM) an inhibitor of cerebral catalase; (iv) ACD plus D-penicillamine (DP, 50.3 × 103 μM) an ACD-trapping agent. CRH levels were evaluated by a radioimmunoassay. Results: Incubation with EtOH induced a 7-fold increase in CRH secretion, with respect to basal levels; ACD was able to stimulate CRH release in a dose-dependent manner; the inhibition of cerebral catalase by 3AT blocked EtOH-induced CRH outflow; the inactivation of ACD by DP reverted the ACD-stimulating effect on CRH secretion. Conclusions: These data show that both EtOH and acetaldehyde are able to increase hypothalamic CRH release from the rat hypothalamus and that acetaldehyde itself appears to be the mediator of EtOH activity. [ABSTRACT FROM AUTHOR]

Background: Epidemiological evidence of co-use of alcohol and areca nuts suggests a potential central interaction between arecoline, a major alkaloid of areca and a muscarinic receptor agonist, and ethanol. Moreover, the central cholinergic system plays an important role in the depressant action of ethanol and barbiturates. The purpose of this study was to investigate the effects of arecoline on pentobarbital- and ethanol-induced hypnosis in mice. Methods: Male ICR mice were tested for locomotor activity following acute systemic administration of ethanol alone, arecoline alone, or ethanol plus arecoline. For the loss of the righting reflex (LORR) induced by pentobarbital and ethanol, sleep latency and sleeping duration were evaluated in mice treated with arecoline alone or the combination of arecoline and scopolamine or methscopolamine. Results: Ethanol (1.0 to 3.0 g/kg, i.p.) reduced locomotor activity significantly and a declining trend was observed after treatment with arecoline (0.25 to 1.0 mg/kg, i.p.), but there were no synergistic effects of ethanol and arecoline on locomotor activity. The experiments on LORR demonstrated that arecoline (0.125 to 1.0 mg/kg, s.c.) shortened the duration of sleeping induced by ethanol (4.0 g/kg, i.p.), but not pentobarbital (45 mg/kg, i.p.). In addition, alterations of sleep latency were not obvious in both pentobarbital- and ethanol-induced LORR. Statistical analyses revealed that scopolamine (centrally acting), but not methscopolamine (peripherally acting), could antagonize the effect of arecoline on the duration of ethanol-induced LORR in mice. Conclusions: These results suggest that central muscarinic receptor is a pharmacological target for the action of arecoline to modulate ethanol-induced hypnosis. [ABSTRACT FROM AUTHOR]

Background: Acetaldehyde, the first metabolite of ethanol, has been suggested to be involved in many behavioral effects of ethanol. However, few studies have investigated the hypothermic effects of acetaldehyde or the contribution of acetaldehyde to ethanol-induced hypothermia. The aim of the present study is to better understand the hypothermic effects of acetaldehyde and the possible contribution of acetaldehyde in ethanol-induced hypothermia, especially under conditions leading to acetaldehyde accumulation. Methods: Female Swiss mice were injected intraperitoneally with ethanol and acetaldehyde and their rectal temperatures were measured with a digital thermometer at various time points after the injections. Experiment 1 compared the hypothermic effects of various acetaldehyde doses (0 to 300 mg/kg) with a reference dose of ethanol (3 g/kg). Experiment 2 tested the effects of a pretreatment with the aldehyde dehydrogenase (ALDH) inhibitor cyanamide (25 mg/kg) on ethanol- and acetaldehyde-induced hypothermia. In experiments 3 and 4, mice received a combined pretreatment with cyanamide and the alcohol dehydrogenase (ADH) inhibitor 4-Methylpyrazole (10 mg/kg) before the injection of ethanol or acetaldehyde. Results: Acetaldehyde at doses between 100 and 300 mg/kg induced significant hypothermic effects, but of shorter duration than ethanol-induced hypothermia. The inhibition of ALDH enzymes by cyanamide induced a strong potentiation of both ethanol- and acetaldehyde-induced hypothermia. The pretreatment with 4-MP prevented the potentiation of ethanol-induced hypothermia by cyanamide, but slightly increased the potentiation of acetaldehyde-induced hypothermia by cyanamide. Conclusions: The results of the present study clearly show that acetaldehyde has hypothermic properties in mice at least at relatively high concentrations. Furthermore, the accumulation of acetaldehyde following ALDH inhibition strongly enhanced the hypothermic effects of ethanol. These latter results confirm the hypothermic properties of acetaldehyde and show that acetate, the next step in ethanol metabolism, is not involved in these hypothermic effects. Finally, the experiment with 4-MP indicates that the potentiating effects of cyanamide are mediated by the peripheral accumulation of acetaldehyde, which then reaches the brain to induce a severe hypothermia. [ABSTRACT FROM AUTHOR]

Background: Behavioral sensitization is a result of neuroadaptation to repeated drug administration and is hypothesized to reflect an increased susceptibility to drug abuse. Proopiomelanocortin (POMC) derived peptides including β-endorphin and α-melanocyte stimulating hormone have been implicated in development of behavioral sensitization and the reinforcing effects of alcohol and other drugs of abuse. This study used a genetically engineered mouse strain that is deficient for neural POMC to directly determine if any POMC peptides are necessary for the development of ethanol-induced locomotor sensitization. Methods: Adult female mice deficient for POMC in neurons only ( Pomc −/− Tg/Tg, KO) and wildtype ( Pomc +/+ Tg/Tg, WT) littermates were injected once daily with either saline or ethanol (i.p.) for 12 to 13 days. On ethanol test day (day 13 or 14) all mice from both treatment groups received an i.p. injection of ethanol immediately before a 15-minute analysis of locomotor activity. Blood ethanol concentration (BEC) was measured on ethanol test day immediately following the test session. Baseline locomotor activity was measured for 15 minutes after a saline injection 2 days later in both groups. Results: There was no significant difference in BEC between genotypes (WT = 2.11 ± 0.06; KO = 2.03 ± 0.08 mg/ml). Both WT and nPOMC-deficient mice treated repeatedly with ethanol demonstrated a significant increase in locomotor activity on test day when compared to repeated saline-treated counterparts. In addition, mice of both genotypes in the repeated saline groups showed a significant locomotor stimulant response to acute ethanol injection. Conclusions: Central POMC peptides are not required for either the acute locomotor stimulatory effect of ethanol or the development of ethanol-induced locomotor sensitization. While these peptides may modulate other ethanol-associated behaviors, they are not essential for development of behavioral sensitization. [ABSTRACT FROM AUTHOR]

Background: Addictive drugs activate extracellular signal regulated kinase (ERK) in brain regions critically involved in their affective and motivational properties. The aim of this study was to demonstrate the ethanol-induced activation of ERK in the nucleus accumbens (Acb) and in the extended amygdala [bed nucleus of the stria terminalis lateralis (BSTL) and central nucleus of the amygdala (CeA)] and to highlight the role of dopamine (DA) D1 receptors in these effects. Methods: Ethanol (0.5, 1, and 2 g/kg) was administered by gavage and ERK phosphorylation was determined in the nucleus Acb (shell and core), BSTL, and CeA by immunohistochemistry. The DA D1 receptor antagonist, SCH 39166 (SCH) (50 μg/kg), was administered 10 minutes before ethanol (1 g/kg). Results: Quantitative microscopic examination showed that ethanol, dose-dependently increased phospho-ERK immunoreactivity (optical and neuronal densities) in the shell and core of nucleus Acb, BSTL, and CeA. Pretreatment with SCH fully prevented the increases elicited by ethanol (1 g/kg) in all brain regions studied. Conclusions: The results of this study indicate that ethanol, similar to other addictive drugs, activates ERK in nucleus Acb and extended amygdala via a DA D1 receptor-mediated mechanism. Overall, these results suggest that the D1 receptors/ERK pathway may play a critical role in the motivational properties of ethanol. [ABSTRACT FROM AUTHOR]

Background: Muscarinic acetylcholine receptors (mAChRs) are well positioned to mediate ethanol’s stimulant effects. To investigate this possibility, we examined the effects of scopolamine, a receptor subtype nonselective mAChR antagonist, on ethanol-induced stimulation in genotypes highly sensitive to this effect of ethanol. We also investigated whether the dopamine D1-like receptor antagonist, SCH-23390 or the dopamine D2-like receptor antagonist, haloperidol, could block the extreme stimulant response found following co-administration of scopolamine and ethanol. Methods: Scopolamine (0, 0.0625, 0.125, 0.25, or 0.5 mg/kg) was given 10 minutes prior to saline or ethanol (0.75 to 2 g/kg) to female FAST (Experiment I) or DBA/2J (Experiment II) mice that were then tested for locomotion for 30 minutes. In Experiments III and IV, respectively, SCH-23390 (0, 0.015, or 0.03 mg/kg) was given 10 minutes prior, and haloperidol (0, 0.08, or 0.16 mg/kg) was given 2 minutes prior, to scopolamine (0 or 0.5 mg/kg), followed 10 minutes later by saline or ethanol (1.5 g/kg) and female DBA/2J mice were tested for locomotion for 30 minutes. Results: FAST and DBA/2J mice displayed a robust enhancement of the locomotor effects of ethanol following pretreatment with scopolamine that was suggestive of synergism. SCH-23390 had no effect on the response to the scopolamine + ethanol drug combination, nor did it attenuate ethanol- or scopolamine-induced locomotor activity. Haloperidol, while attenuating the effects of ethanol, was not able to block the effects of scopolamine or the robust response to the scopolamine-ethanol drug combination. Conclusions: These results suggest that while muscarinic receptor antagonism robustly enhances acute locomotor stimulation to ethanol, dopamine receptors are not involved in the super-additive interaction of scopolamine and ethanol treatment. They also suggest that in addition to cautions regarding the use of alcohol when scopolamine is clinically prescribed due to enhanced sedative effects, enhanced stimulation may also be a concern. [ABSTRACT FROM AUTHOR]

Background: Experimental evidences suggest that acetaldehyde (ACD) contributes to the positive motivational properties of ethanol (EtOH) as assessed by the place conditioning paradigm; indeed, we found that by reducing ACD production and/or by using ACD-sequestrating agents, EtOH is deprived from its motivational properties. Thiol products, such as the amino acid cysteine, are known to be effective ACD-sequestering agents. Cysteine is able to covalently bind ACD thereby forming a stable, nontoxic 2-methyl-thiazolidine-4-carboxylic acid compound. Thus, we treated rats withl-cysteine before intragastric administration of EtOH or ACD. Methods: Male Wistar rats were pretreated intraperitoneally with saline orl-cysteine (10, 20, or 30 mg/kg), before intragastric administration of saline, EtOH (1 g/kg), or ACD (20 mg/kg). The specificity ofl-cysteine effect was addressed using morphine-induced conditioned place preference (cpp) (2.5 mg/kg, i.p.). Results: l-cysteine dose-dependently prevented both EtOH and ACD-induced cpp but did not interfere with morphine-induced cpp, suggesting thatl-cysteine specifically modulates the motivational properties of EtOH. Conclusion: The present results further underscore the role of EtOH-derived ACD in EtOH-induced motivational properties.l-cysteine, by binding EtOH-derived ACD, would deprive it of its rewarding properties and reduce its abuse liability. [ABSTRACT FROM AUTHOR]

Background: Systemic ethanol administration increases neuroactive steroid levels that increase ethanol sensitivity. Acetaldehyde is a biologically active compound that may contribute to behavioral and rewarding effects of ethanol. We investigated the role of acetaldehyde in ethanol-induced elevations of 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) levels in cerebral cortex. Methods: Male Sprague–Dawley rats were administered ethanol, and plasma acetaldehyde concentrations were measured by gas chromatography to determine relevant concentrations. Rats were then administered acetaldehyde directly, acetaldehyde plus cyanamide to block its degradation, or ethanol in the presence of inhibitors of ethanol metabolism, to determine effects on 3α,5α-THP levels in cerebral cortex. Results: Ethanol administration (2 g/kg) to rats results in a peak acetaldehyde concentration of 6-7 μM at 10 minutes that remains stable for the duration of the time points tested. Direct administration of acetaldehyde eliciting this plasma concentration does not increase cerebral cortical 3α,5α-THP levels, and inhibition of ethanol-metabolizing enzymes to modify acetaldehyde formation does not alter ethanol-induced 3α,5α-THP levels. However, higher doses of acetaldehyde (75 and 100 mg/kg), in the presence of cyanamide to prevent its metabolism, are capable of increasing cortical 3α,5α-THP levels. Conclusions: Physiological concentrations of acetaldehyde are not responsible for ethanol-induced increases in 3α,5α-THP, but a synergistic role for acetaldehyde with ethanol may contribute to increases in 3α,5α-THP levels and ethanol sensitivity. [ABSTRACT FROM AUTHOR]

Background: Disulfiram, an inhibitor of aldehyde dehydrogenase used in the treatment of alcoholism, is an effective medication when its intake is supervised by a third person. However, its therapeutic efficacy varies widely, in part due to the fact that disulfiram is a pro-drug that requires its transformation into an active form and because it shows a wide range of secondary effects which often prevent the use of doses that ensure full therapeutic effectiveness. In this preclinical study in rats we report the development of tolerance to disulfiram induced by the chronic ingestion of ethanol, an additional source of variation for the actions of disulfiram with possible therapeutic significance, We also addresses the likely mechanism of this effect. Methods: Wistar-derived rats bred for generations as high ethanol drinkers (UChB) were trained for either 3 days (Group A) or 30 days (Group B) to choose between ethanol (10% v/v) or water, which were freely available from 2 bottles on a 24-hour basis. Subsequently, animals in both groups were administered disulfiram or cyanamide (another inhibitor of aldehyde dehydrogenase) and ethanol intake in this free choice paradigm was determined. Animals were also administered a standard dose of 1 g ethanol/kg (i.p) and arterial blood acetaldehyde was measured. Results: Disulfiram (12.5 and 25 mg/kg) and cyanamide (10 mg/kg) markedly inhibited ethanol intake (up to 60 to 70%) in animals that had ethanol access for only 3 days (Group A). However both drugs were inactive in inhibiting ethanol intake in animals that had consumed ethanol for 30 days (Group B). Following the injection of 1 g ethanol/kg, arterial blood acetaldehyde levels reached levels of 150 and 300 μM for disulfiram and cyanamide respectively, values which were virtually identical regardless of the length of prior ethanol intake of the animals. Conclusions: Chronic ethanol intake in high-drinker rats leads to marked tolerance to the aversive effects of disulfiram and cyanamide on ethanol intake despite the presence of consistently high levels of blood acetaldehyde. These findings may have implications for the use of disulfiram for the treatment of alcoholism in humans. [ABSTRACT FROM AUTHOR]

Background: Acetaldehyde (ACD), the first metabolite of ethanol (EtOH), is produced peripherally by gastric and hepatic alcohol dehydrogenase (ADH) and centrally by brain catalase. In spite of the aversive properties classically ascribed to ACD, it has recently been suggested that ACD might mediate some of the motivational effects of EtOH. Accordingly, the relative role of ACD in the positive motivational properties of EtOH ingested is increasingly becoming the matter of debate. Thus, we studied the ability of intragastrically administered EtOH, ACD and EtOH-derived ACD to induce conditioned place preference (cpp) in rats. Methods: Wistar rats were pretreated intraperitoneally with saline, the peripheral competitive inhibitor of ADH, 4-methylpyrazole (4-MP, 22.5, 45 or 67.5 mg/kg) or with the selective ACD-sequestrating agent,d-penicillamine (DP, 25 or 50 mg/kg), before the intragastric administration of saline, EtOH (0.5, 1 or 2 g/kg) or ACD (10, 20, or 40 mg/kg). The specificity of 4-MP and DP effects was addressed using morphine-induced cpp (2.5 mg/kg). Results: Both, EtOH and ACD dose-dependently induced cpp; further, while EtOH-induced cpp was prevented by the administration of 4-MP and by DP, ACD-induced cpp was unaltered by 4-MP administration and prevented by DP. Both pretreatments did not interfere with morphine-induced cpp indicating that 4-MP and DP specifically modulate the motivational properties of EtOH and ACD. Conclusion: The ability of 4-MP and DP to decrease EtOH-induced cpp suggests that a reduction of ACD levels is crucial in depriving EtOH from its motivational properties as indexed by the cpp procedure. In addition, this conclusion is supported by the inefficacy of 4-MP in preventing ACD-induced cpp, and by its blockade observed after administration of the selective ACD sequestrating agent DP. The present results underscore the role of EtOH-derived ACD in EtOH-induced motivational properties as well as its abuse liability. [ABSTRACT FROM AUTHOR]

Background: Energy drinks (ED) are a widely used group of beverages known for their stimulant effects on central nervous system (CNS). The main components of ED are caffeine, taurine, carbohydrates, glucuronolactone, inositol, niacin, pantenol, and β-complex vitamins. The studies evaluating the effects of ED describe improvements in attention and/or reaction times and indices of alertness. It has been also shown that combination of caffeine and glucose, fundamental constituents of ED, can ameliorate deficits in cognitive performance and subjective fatigue during extended periods of cognitive demand. Moreover, the associated ingestion of alcohol and ED has recently been observed to be becoming more and more widespread. Methods: With the aim to know the habits and uses of students, we administered a questionnaire containing questions regarding ED drinking alone or in association with alcoholic beverages. Five hundred students of the School of Medicine of the University of Messina were interviewed, and 450 filled the questionnaire. Results: A total of 56.9% of students declared using ED. A great part of users (48.4%) associate frequently ED and alcohol. In particular, 35.8% of ED + alcohol users have used ED + alcohol more than 3 times in the last month. Distinguishing the users into 2 groups (users of ED + alcohol and users of both ED and ED + alcohol), we observed in the second group a major use of cocktail containing a mix of ED and alcoholic beverages. This difference between the 2 groups is less represented about the ingestion of ED + alcohol in the night. Conclusions: Our data indicate that association of ED + alcohol is very popular among students. This behavior can be dangerous. In fact, the combination of ED + alcoholic drinks can reduce adversive symptoms of alcohol intoxication including the depressant effects. As consequence, users of ED + alcoholic beverages might not feel the signs of alcohol intoxication, thus increasing the probability of accidents and/or favoring the possibility of development of alcohol dependence. [ABSTRACT FROM AUTHOR]

Background: It is widely accepted that, in addition to removing acetaldehyde produced during the metabolism of ethanol, mitochondrial aldehyde dehydrogenase (ALDH2) functions in the pathway by which aldehyde metabolites of the monoamines dopamine (DA) and serotonin (5-HT) are converted to their acidic metabolites. Moreover, studies of ALDH2 inhibitors used for treating alcoholism suggest that their antidipsotropic effects may be related to inhibition of monoamine metabolism. Therefore, we examined the hypothesis that altered brain monoamine metabolism is related to the influence of ALDH2 on behavioral responses to ethanol. Methods: Mice were generated with a gene-trap mutation of the ALDH2 gene. ALDH2 mRNA was absent in ALDH2−/− mice. Western blot analysis of liver mitochondria confirmed the absence of ALDH2 protein in the ALDH2−/− mice. Wild-type and ALDH2-deficient mice were tested for the effects of different doses of ethanol on locomotor activity, ataxia, and a 2-bottle ethanol–water preference test. Results: Wild-type and ALDH2+/− mice preferred ethanol to water. However, ALDH2−/− mice drank significantly less ethanol than wild-type or ALDH2+/− mice. Locomotor activity and ataxia were significantly more affected by ethanol in ALDH2−/− mice than in wild-type or ALDH2+/− mice. There was no effect of genotype on levels of 5-HT, DA, or their precursors or metabolites in several brain regions, as measured by HPLCec. Conclusions: The results indicate that: (1) the effect of the mutant genotype on behavioral responses to ethanol is unrelated to altered brain monoamine metabolism and (2) ALDH2 is not required for the metabolism of brain monoamines in vivo. [ABSTRACT FROM AUTHOR]

Background: Differences in ethanol metabolizing enzymes expressed in brain have been suggested to contribute to the significant differences in ethanol (alcohol) preference between inbred C57BL/6 and DBA/2 mouse strains. Methods: We have utilized 2 different platforms of oligonucleotide microarray technology (CodeLink UniSet I BioArray from G.E. Healthcare and MG U74A v2.0 from Affymetrix) to simultaneously assess expression of alcohol and acetaldehyde metabolizing enzymes in the whole brain of naïve (no exposure to alcohol) C57BL/6 and DBA/2 mice. Results: There were no significant differences between the 2 strains of mice in gene expression intensity for alcohol dehydrogenases (ADH), catalase, and a number of the cytochrome P450 family of genes, which can be involved in ethanol catabolism. However, significantly higher expression of mRNA for aldehyde dehydrogenase 2 (ALDH2), an isoform mainly responsible for the catabolism of acetaldehyde, was observed in whole brains of DBA/2 mice with both platforms. Aldehyde dehydrogenase 2 protein was also higher in DBA/2 brain. Expression of aldehyde dehydrogenase 1A1 (ALDH1A1) mRNA was found to be higher in brains of DBA/2 mice, when measured with the CodeLink platform, but not when measured with Affymetrix arrays or quantitative reverse transcriptase–real-time polymerase chain reaction (qRT-PCR). The ALDH1A1 protein, however, reflected the results obtained with the CodeLink arrays and was higher in DBA/2 brain, compared with brains of C57BL/6 mice. In contrast, the expression intensity for the aldehyde dehydrogenase 7A1 (ALDH7A1) mRNA and protein was significantly higher in C57BL/6 mice than DBA/2 mice. These expression differences are consistent with more rapid metabolism of acetaldehyde in brains of DBA/2 mice. Conclusions: The use of 2 different microarray platforms provides important cross-validation of many results, and some discrepancies can be resolved with qRT-PCR and immunoblotting. The expression differences that were validated may affect alcohol/aldehyde metabolism in brain and/or alcohol preference in the 2 strains of mice. [ABSTRACT FROM AUTHOR]

Background: The exact enzymatic mechanisms of ethanol oxidation in the brain are still unclear. The catalase-mediated oxidation of ethanol was demonstrated in rat brain using incubation of brain homogenates with catalase inhibitors. The role of the alcohol dehydrogenase (ADH) or cytochrome P450–dependent system in this process is possible, but has not been confirmed. The objective of the study was to determine the contribution of the different enzymatic pathways to ethanol oxidation in brain homogenates from mice and rats. Methods: Three approaches were used to investigate the enzymatic mechanisms of ethanol oxidation in the brain of rats and mice: (1) preincubation of brain homogenates with inhibitors of the ethanol-metabolizing enzymes (catalase, CYP2E1, ADH, and ALDH); (2) utilization of mice with genetic deficiency in ethanol-metabolizing enzymes (catalase, CYP2E1, or both enzymes); and (3) determination of ethanol oxidation in brain subcellular fractions known to have differential activity of ethanol-metabolizing enzymes. The ethanol-derived acetaldehyde (AC) and acetate were determined in brain samples by gas chromatography. Results: The catalase inhibitors sodium azide (5 mM) and aminotriazole (5 mM) as well as CYP2E1 inhibitors diallyl sulfide (2 mM) and β-phenethyl isothiocyanate (0.1 mM) lowered significantly the accumulation of the ethanol-derived AC and acetate in brain homogenates. The ADH inhibitor 4-methyl pyrazole (5 mM) significantly decreased the acetate but not the AC accumulation. Ethanol-derived AC accumulation in brain homogenates of acatalasemic mice was 47% of the control value, 91% in CYP2E1-null mice, and 24% in double mutants (with deficiency of both catalase and CYP2E1). The highest levels of ethanol oxidation were found in microsomal and peroxisomal subcellular brain fractions, where CYP2E1 and catalase are located, respectively. Conclusions: Catalase is the key enzyme of ethanol oxidation in the brain of rodents: it may be responsible for about 60% of the process. CYP2E1 plays an important role in ethanol oxidation in the rodent brains. Alcohol dehydrogenase plays a minor role, if any, in this process. Aldehyde dehydrogenase plays the crucial role in the further oxidation of ethanol-derived AC in the brain homogenates. [ABSTRACT FROM AUTHOR]

Background: Pharmacological studies have implicated the endogenous opioid system in mediating alcohol intake. Other evidence has shown that alcohol administration can influence endorphinergic and enkephalinergic activity, while very few studies have examined its effect on dynorphinergic systems. The aim of the present study was to investigate the effect of alcohol administration or a mechanical stressor on extracellular levels of dynorphin A1–8 in the rat nucleus accumbens—a brain region that plays a significant role in the processes underlying reinforcement and stress. Methods: Male Sprague–Dawley rats were implanted with a microdialysis probe aimed at the shell region of the nucleus accumbens. Artificial cerebrospinal fluid was pumped at a rate of 1.5 μL/min in awake and freely moving animals and the dialysate was collected at 30-minute intervals. In one experiment, following a baseline period, rats were injected intraperitoneally with either physiological saline or 1 of 3 doses of alcohol, 0.8, 1.6, or 3.2 g ethanol/kg body weight. In a second experiment, following a baseline period, rats were applied a clothespin to the base of their tail for 20 minutes. The levels of dynorphin A1–8 in the dialysate were analyzed with solid-phase radioimmunoassay. Results: Relative to saline-treated controls, an alcohol dose of 1.6 and 3.2 g/kg caused a transient increase in the extracellular levels of dynorphin A1–8 in the first 30 minutes of alcohol administration. However, the effect resulting from the high 3.2 g/kg dose was far more pronounced and more significant than with the moderate dose. There was no effect of tail pinch on dynorphin A1–8 levels in the nucleus accumbens. Conclusions: In this experiment, a very high dose of alcohol was especially capable of stimulating dynorphin A1–8 release in the nucleus accumbens. Dynorphin release in the accumbens has been previously associated with aversive stimuli and may thus reflect a system underlying the aversive properties of high-dose alcohol administration. However, the lack of effect of tail-pinch stress in the present study suggests that dynorphin A1–8 is not released in response to all forms of stressful/aversive stimuli. [ABSTRACT FROM AUTHOR]

Background: Alcohol-preferring alko alcohol (AA) rats are more susceptible to morphine-induced behavioral and neurochemical sensitization than alcohol nonpreferring alko nonalcohol (ANA) rats. Alko alcohol rats sensitized to morphine, however, do not show enhanced acquisition of ethanol drinking. The purpose of the present study was to clarify further interactions between morphine-induced behavioral sensitization and voluntary ethanol drinking in the AA rats. Methods: Alko alcohol rats drinking ethanol in a limited 6-hour access paradigm were sensitized to morphine with repeated injections of morphine (5–15 mg/kg). Injection days alternated with days of ethanol access. Controls had access only to water and/or were given injections of saline. After a 5-day washout period from ethanol and morphine, the rats were challenged with morphine or saline and subsequent ethanol drinking or locomotor activity was recorded. Results: Ethanol intake was suppressed during the repeated treatment with morphine, and the morphine-treated rats did not differ in ethanol intake from the controls when given access to ethanol after the washout. Intake of ethanol was, however, increased when the rats were challenged with morphine [1 or 10 mg/kg, subcutaneously (s.c.)], while in the controls an increase in ethanol intake was seen only after 1 mg/kg morphine. Sensitization to the locomotor stimulating effects of morphine was revealed in the morphine-treated rats after a challenge with morphine (3 or 10 mg/kg, s.c.). The controls that had been drinking ethanol also showed a sensitized response after morphine (3 mg/kg). Conclusions: Ethanol did not interfere with the development of sensitization to morphine. Furthermore, the neuroadaptations induced by repeated exposure to ethanol were sufficient to cause behavioral cross-sensitization to morphine. Sensitization to the behavioral effects of morphine alone, however, neither enhances the reinforcing properties of voluntarily consumed ethanol nor contributes to increase in its intake. The increase in ethanol intake found after an acute dose of morphine was augmented in rats withdrawn from repeated treatment with morphine. The data suggest that the neuronal mechanisms underlying behavioral sensitization to morphine probably are distinct from those mediating reinforcement from ethanol and that the morphine-induced neuroadaptations contribute to the enhancement of increase in ethanol intake by morphine. [ABSTRACT FROM AUTHOR]

Background: Well-known reports suggest that the use of energy drinks might reduce the intensity of the depressant effects of alcohol. However, there is little scientific evidence to support this hypothesis. Objective and Methods: The present study aimed at evaluating the effects of the simultaneous ingestion of an alcohol (vodka37.5%v/v) and an energy drink (Red Bull®—3.57 mL/kg), compared with those presented after the ingestion of an alcohol or an energy drink alone. Twenty-six young healthy volunteers were randomly assigned to 2 groups that received 0.6 or 1.0 g/kg alcohol, respectively. They all completed 3 experimental sessions in random order, 7 days apart: alcohol alone, energy drink alone, or alcohol plus energy drink. We evaluated the volunteers' breath alcohol concentration, subjective sensations of intoxication, objective effects on their motor coordination, and visual reaction time. Results: When compared with the ingestion of alcohol alone, the ingestion of alcohol plus energy drink significantly reduced subjects' perception of headache, weakness, dry mouth, and impairment of motor coordination. However, the ingestion of the energy drink did not significantly reduce the deficits caused by alcohol on objective motor coordination and visual reaction time. The ingestion of the energy drink did not alter the breath alcohol concentration in either group. Conclusions: Even though the subjective perceptions of some symptoms of alcohol intoxication were less intense after the combined ingestion of the alcohol plus energy drink, these effects were not detected in objective measures of motor coordination and visual reaction time, as well as on the breath alcohol concentration. [ABSTRACT FROM AUTHOR]