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The cAMP signaling pathway has emerged as an important modulator of the pharmacological effects of ethanol. In this respect, the cAMP-dependent protein kinase has been shown to play an important role in the modulation of several ethanol-induced behavioral actions. Cellular levels of cAMP are maintained by the activity of adenylyl cyclases and phosphodiesterases. In the present work we have focused on ascertaining the role of PDE4 in mediating the neurobehavioral effects of ethanol. For this purpose, we have used the selective PDE4 inhibitor Ro 20-1724. This compound has been proven to enhance cellular cAMP response by PDE4 blockade and can be administered systemically. Swiss mice were injected intraperitoneally (i.p.) with Ro 20-1724 (0-5 mg/kg; i.p.) at different time intervals before ethanol (0-4 g/kg; i.p.) administration. Immediately after the ethanol injection, locomotor activity, loss of righting reflex, PKA footprint and enzymatic activity were assessed. Pretreatment with Ro 20-1724 increased ethanol-induced locomotor stimulation in a dose-dependent manner. Doses that increased locomotor stimulation did not modify basal locomotion or the suppression of motor activity produced by high doses of this alcohol. Ro 20-1724 did not alter the locomotor activation produced by amphetamine or cocaine. The time of loss of righting reflex evoked by ethanol was increased after pretreatment with Ro 20-1724. This effect was selective for the narcotic effects of ethanol since Ro 20-1724 did not affect pentobarbital-induced narcotic effects. Moreover, Ro 20-1724 administration increased the PKA footprint and enzymatic activity response elicited by ethanol. These data provide further evidence of the key role of the cAMP signaling pathway in the central effects of ethanol., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Rationale: The cAMP-dependent protein kinase A (PKA) signaling transduction pathway has been shown to play an important role in the modulation of several ethanol-induced behaviors. Different studies have demonstrated intracellular calcium (Ca(2+))-dependent activation of the PKA cascade after ethanol administration. Thus, the cAMP cascade mediator Ca(2+)-dependent calmodulin (CaM) has been strongly implicated in the central effects of ethanol., Objectives: In this study, we assessed the role of the CaM inhibitor W7 on ethanol-induced stimulation, ethanol intake, and ethanol-induced activation of PKA., Methods: Swiss mice were pretreated with W7 (0-10 mg/kg) 30 min before ethanol (0-3.75 g/kg) administration. Immediately, animals were placed during 20 min in an open-field chamber. Ethanol (10 %, v/v) intake in 2 h was assessed using a limited access paradigm. Experiments with caffeine (0-15 mg/kg), cocaine (0-4 mg/kg), and saccharine (0.1 %, w/v) were designed to compare their results to those obtained with ethanol. Western blot was assayed 45 min after ethanol administration., Results: Results showed that pretreatment with W7, reduced selectively in a dose-dependent fashion ethanol-induced locomotor stimulation and ethanol intake. The ethanol-induced activation of PKA was also prevented by W7 administration., Conclusions: These results demonstrate that CaM inhibition resulted in a selective reduction of ethanol-stimulating effects and ethanol intake. The PKA activation induced by ethanol was blocked after the CaM blockade with W7. These results provide further evidence of the key role of cellular Ca(2+)-dependent pathways on the central effects of ethanol.

In the central nervous system ethanol (EtOH) is metabolized into acetaldehyde by different enzymes. Brain catalase accounts for 60% of the total production of EtOH-derived acetaldehyde, whereas cerebral cytochrome P450 2E1 (CYP 2E1) produces 20% of this metabolite. Acetaldehyde formed by the activity of central catalase has been implicated in some of the neurobehavioral properties of EtOH, yet the contribution of CYP 2E1 to the pharmacological actions of this drug has not been investigated. Here we assessed the possible participation of CYP 2E1 in the behavioral effects of EtOH. Thus, we induced CYP 2E1 activity and expression by exposing mice to chronic acetone intake (1% v/v for 10 days) and examined its consequences on the stimulating and uncoordinating effects of EtOH (0-3.2 g/kg) injected intraperitoneally. Our data showed that 24 h after withdrawal of acetone brain expression and activity of CYP 2E1 was induced. Furthermore, the locomotion produced by EtOH was boosted over the same interval of time. Locomotor stimulation produced by amphetamine or tert-butanol was unchanged by previous treatment with acetone. EtOH-induced motor impairment as evaluated in a Rota-Rod apparatus was unaffected by the preceding exposure to acetone. These results indicate that cerebral CYP 2E1 activity could contribute to the locomotor-stimulating effects of EtOH, and therefore we suggest that centrally produced acetaldehyde might be a possible mediator of some EtOH-induced pharmacological effects., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Centrally formed acetaldehyde has proven to be responsible for several psychopharmacological effects induced by ethanol. In addition, it has been suggested that the cAMP-PKA signaling transduction pathway plays an important role in the modulation of several ethanol-induced behaviors. Therefore, we hypothesized that acetaldehyde might be ultimately responsible for the activation of this intracellular pathway. We used three pharmacological agents that modify acetaldehyde activity (α-lipoic acid, aminotriazole, and d-penicillamine) to study the role of this metabolite on EtOH-induced PKA activation in mice. Our results show that the injection of α-lipoic acid, aminotriazole and d-penicillamine prior to acute EtOH administration effectively blocks the PKA-enhanced response to EtOH in the brain. These results strongly support the hypothesis of a selective release of acetaldehyde-dependent Ca(2+) as the mechanism involved in the neurobehavioral effects elicited by EtOH., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

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 Ca(2+) -dependent cAMP-PKA cascade mediators play a critical role in the neurobehavioral response to EtOH, being of special relevance to the Ca(2+) -dependent adenylyl cyclases 1 and 8. We hypothesize an intracellular PKA activation elicited by EtOH administration, which may be regulated by a Ca(2+) -dependent mechanism as an early cellular response. Thus, the present work aims to explore the role of Ca(2+) (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 Ca(2+) 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 Ca(2+) release from the endoplasmic reticulum resulted in a decreased EtOH-induced activation of PKA. On the contrary, extracellular-to-cytoplasm Ca(2+) manipulations did not prevent the PKA activation by EtOH., Conclusions: Altogether, these results show the critical role of stored Ca(2+) 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., (Copyright © 2013 by the Research Society on Alcoholism.)

Hydrogen peroxide is the co-substrate 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 acetaldehyde has been involved in many of the effects of EtOH. Previous research demonstrated that treatments that change the levels of cerebral H2O2 available to catalase modulate the locomotor-stimulating effects of EtOH and its volitional intake in rodents. However, the source of H2O2 which is used by catalase to form Compound I and mediates the psychoactive actions of EtOH is unknown. One cause of the generation of H2O2 in the brain comes from the deamination of biogenic amines by the activity of MAO-A. Here we explore the consequences of the administration of the MAO-A inhibitor clorgyline on EtOH-induced locomotion and voluntary EtOH drinking. For the locomotor activity tests, we injected Swiss (RjOrl) mice intraperitoneally (IP) with clorgyline (0-10mg/kg) and later (0.5-8h) with EtOH (0-3.75 g/kg; IP). Following these treatments, mice were placed in locomotor activity chambers to measure their locomotion. For the drinking experiments, mice of the C57BL/6J strain were injected IP with clorgyline prior to offering them an EtOH (20%) solution following a drinking-in-the-dark procedure. Additional experiments were performed to assess the selectivity of this compound in altering EtOH-stimulated locomotion and EtOH intake. Moreover, we indirectly tested the ability of clorgyline to reduce brain H2O2 levels. We showed that this treatment selectively reduced EtOH-induced locomotion and its self-administration. Moreover, this compound decreased central H2O2 levels available to catalase. We suggest that H2O2 derived from the deamination of biogenic amines by the activity of MAO-A could determine the formation of brain EtOH-derived acetaldehyde. This centrally-formed acetaldehyde within the neurons of the aminergic system could play a role in the neurobehavioral properties of EtOH., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Previous studies have shown that both 3-amino-1,2,4-triazole (AT), which inhibits metabolism of ethanol (EtOH) to acetaldehyde by inhibiting catalase, and D-penicillamine (D-P), an acetaldehyde-sequestering agent, modulate EtOH-conditioned place preference (CPP) in male albino Swiss (IOPS Orl) mice. These studies followed a reference-dose-like procedure, which involves comparing cues that have both been paired with EtOH. However, the role of EtOH-derived acetaldehyde has not been examined using a standard CPP method, and efficacy of these treatments could be different under the two circumstances. In the present investigation, we manipulated the strength of CPP across five separate studies and evaluated the effect of D-P and AT on EtOH-induced CPP following a standard unbiased CPP procedure. Mice received pairings with vehicle-saline injections with one cue and, alternatively, with AT- and D-P-EtOH with another cue. Our studies indicate that AT and D-P only disrupt CPP induced by EtOH in mice when the number of conditioning sessions and the dose of EtOH are low. These findings suggest that acquisition of EtOH-induced CPP may depend on the levels of acetaldehyde available during memory acquisition and the strength of the memory. Therefore, we propose that, at least when the memory processes are labile, brain acetaldehyde could participate in the formation of Pavlovian learning elicited by EtOH.

Rationale: Hydrogen peroxide (H2O2) is the co-substrate used by catalase to metabolize ethanol to acetaldehyde in the brain. This centrally formed acetaldehyde has been involved in several ethanol-related behaviors., Objectives: The present research evaluated the effect of the H2O2 scavenger, alpha lipoic acid (LA), on the acquisition and reconditioning of ethanol-induced conditioned place preference (CPP)., Methods: Mice received pairings of a distinctive floor stimulus (CS+) associated with intraperitoneal injections of ethanol (2.5 g/kg). On alternate days, animals received pairings of a different floor stimulus (CS-) associated with saline injections. A different group of animals received pairings with the (CS-) associated with saline injections, and on alternate days they received LA (100 mg/kg) injected 30 min prior to ethanol (2.5 g/kg) administration paired with the (CS+). A preference test assessed the effect of LA on the acquisition of ethanol-induced CPP. A similar procedure was followed to study the effect of LA on the acquisition of cocaine- and morphine-induced CPP. A separate experiment evaluated the effect of LA on the reconditioning of ethanol-induced CPP. In addition, we investigated the consequence of LA administration on central H2O2 levels., Results: LA selectively blocked the acquisition of ethanol-induced CPP. Moreover, this compound impaired the reconditioning of ethanol-induced CPP. Additionally, we found that LA diminished H2O2 levels in the brain., Conclusions: These data suggest that a decline in H2O2 availability by LA might impede the formation of brain ethanol-derived acetaldehyde by catalase, which results in an impairment of the rewarding properties of ethanol.

Calcium has been characterized as one of the most ubiquitous, universal and versatile intracellular signals. Among other substances with the ability to alter intracellular calcium levels, ethanol has been described as particularly relevant because of its social and economic impact. Ethanol effects on calcium distribution and flux in vitro have been widely studied, showing that acute ethanol administration can modulate intracellular calcium concentrations in a dose dependent manner. Intracellular calcium released from the endoplasmic reticulum plays a determinant role in several cellular processes. In this study, we aim to assess the effect of dantrolene, a ryanodine receptor antagonist, on three different ethanol-elicited behaviors: locomotor activity, loss of righting reflex and ethanol intake. Mice were challenged with an injection of dantrolene (0-5 mg/kg, i.p.) 30 min before ethanol (0-4 g/kg, i.p.) administration. Animals were immediately placed in an open field cylinder to monitor distance travelled horizontally or in a V-shaped trough to measure righting reflex recovery time. For ethanol intake, dantrolene (0-5mg/kg, i.p.) was administered 30 min before ethanol (20%, v/v) exposure, following a drinking in the dark paradigm. Our results showed that dantrolene selectively reduces ethanol-induced stimulation, loss of righting reflex, and ethanol intake in a dose dependent manner. Together, these data suggest that intracellular calcium released from the endoplasmic reticulum may play a critical role in behavioral effects caused by ethanol, and point to a calcium-dependent pathway as a possible cellular mechanism of action for ethanol., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: In the brain, the enzyme catalase by reacting with H(2)O(2) forms Compound I (catalase-H(2)O(2) system), which is the main system of central ethanol metabolism to acetaldehyde. Previous research has demonstrated that acetaldehyde derived from central-ethanol metabolism mediates some of the psychopharmacological effects produced by ethanol. Manipulations that modulate central catalase activity or sequester acetaldehyde after ethanol administration modify the stimulant effects induced by ethanol in mice. However, the role of H(2)O(2) in the behavioral effects caused by ethanol has not been clearly addressed. The present study investigated the effects of ebselen, an H(2)O(2) scavenger, on ethanol-induced locomotion., Methods: Swiss RjOrl mice were pre-treated with ebselen (0-50mg/kg) intraperitoneally (IP) prior to administration of ethanol (0-3.75g/kg; IP). In another experiment, animals were pre-treated with ebselen (0 or 25mg/kg; IP) before caffeine (15mg/kg; IP), amphetamine (2mg/kg; IP) or cocaine (10mg/kg; IP) administration. Following these treatments, animals were placed in an open field to measure their locomotor activity. Additionally, we evaluated the effect of ebselen on the H(2)O(2)-mediated inactivation of brain catalase activity by 3-amino-1,2,4-triazole (AT)., Results: Ebselen selectively prevented ethanol-induced locomotor stimulation without altering the baseline activity or the locomotor stimulating effects caused by caffeine, amphetamine and cocaine. Ebselen reduced the ability of AT to inhibit brain catalase activity., Conclusions: Taken together, these data suggest that a decline in H(2)O(2) levels might result in a reduction of the ethanol locomotor-stimulating effects, indicating a possible role for H(2)O(2) in some of the psychopharmacological effects produced by ethanol., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Calcium (Ca(2+)) has been characterized as one of the most ubiquitous, universal and versatile intracellular signaling molecules responsible for controlling numerous cellular processes. Ethanol-induced effects on Ca(2+) distribution and flux have been widely studied in vitro, showing that acute ethanol administration can modulate intracellular Ca(2+) concentrations in a dose dependent manner. In vivo, the relationship between Ca(2+) manipulation and the corresponding ethanol-induced behavioral effects have focused on Ca(2+) flux through voltage-gated Ca(2+) channels. The present study investigated the role of inward Ca(2+) currents in ethanol-induced psychomotor effects (stimulation and sedation) and ethanol intake. We studied the effects of the fast Ca(2+) chelator, BAPTA-AM, on ethanol-induced locomotor activity and the sedative effects of ethanol. Swiss (RjOrl) mice were pretreated with BAPTA-AM (0-10 mg/kg) 30 min before an ethanol (0-4 g/kg) challenge. Our results revealed that pretreatment with BAPTA-AM prevented locomotor stimulation produced by ethanol without altering basal locomotion. In contrast, BAPTA-AM reversed ethanol-induced hypnotic effects. In a second set of experiments, we investigated the effects of intracellular Ca(2+) chelation on ethanol intake. Following a drinking-in-the-dark methodology, male C57BL/6J mice were offered 20% v/v ethanol, tap water, or 0.1% sweetened water. The results of these experiments revealed that BAPTA-AM pretreatment (0-5 mg/kg) reduced ethanol consumption in a dose-dependent manner while leaving water and sweetened water intake unaffected. Our findings support the role of inward Ca(2+) currents in mediating different behavioral responses induced by ethanol. Our results are discussed together with data indicating that ethanol appears to be more sensitive to intracellular Ca(2+) manipulations than other psychoactive drugs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Neuroplasticity associated with drug-induced behavioral sensitization has been associated with excessive drug pursuit and consumption characteristic of addiction. Repeated intraperitoneal (ip) injections of ethanol (EtOH) can induce psychomotor sensitization in mice. In terms of its clinical relevance, however, it is important to determine whether this phenomenon can also be produced by voluntary EtOH consumption., Methods: The present investigation used a drinking-in-the-dark (DID) methodology to induce high levels of EtOH drinking in mice; EtOH replaces water for 2 or 4h, starting 3h after the beginning of the dark cycle. Animals followed a 3-week DID protocol prior to an evaluation of EtOH-induced locomotor activity (acute and repeated EtOH). For the first week, animals had access to 20% EtOH. On weeks 2 and 3, different concentrations of EtOH (10, 20 or 30%) were used. Three different inbred strains of mice were used: C57BL/6J (B6), BALB/cByJ (BALB), and CXBK. The CXBK mouse line was used because of its reduced expression and functioning of brain mu-opioid receptors, which have been suggested to participate in the development of EtOH-induced sensitization. B6 and BALB mice were used as controls., Results: B6 and CXBK mice presented comparable levels of EtOH drinking (approx. 3g/kg in 2h), that were higher than those showed by BALB. All animals, regardless of genotype, adjusted volume of EtOH intake to obtain stable g/kg of EtOH across concentrations. Previous EtOH DID produced (B6) or potentiated (BALB) sensitization to EtOH; this effect was not seen in CXBK. Western blot analysis showed a reduced number of mu-opioid receptors in several brain regions of CXBK as compared to that of B6 and BALB mice., Conclusions: In summary, here we show that the DID methodology can be used to trigger EtOH-induced neuroplasticity supporting psychomotor sensitization, a process that might require participation of mu-opioid receptors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Many people begin to experiment with alcohol during adolescence, an important developmental period during which sex differences in the effects of ethanol appear. In the present study we evaluated the effect of ethanol (0, 0.625, 1.25 or 2.5 g/kg) on the acquisition of a conditioned place preference (CPP) in early and late adolescent male and female mice. In addition, we assessed the capacity of ethanol to induce reinstatement of the CPP after its extinction. CPP was induced in early and late adolescent females with 2.5 g/kg, and in early adolescent males with 1.25 or 2.5 g/kg of ethanol. No CPP was observed in late adolescent males. Priming with ethanol reinstated the CPP induced by the highest dose in early adolescent male and early and late adolescent female mice. Our data suggest that early adolescents of both sex and late adolescent females are particularly vulnerable to the effects of ethanol., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Rationale: The main system of central ethanol oxidation is mediated by the enzyme catalase. By reacting with H(2)O(2), brain catalase forms compound I (the catalase-H(2)O(2) system), which is able to oxidize ethanol to acetaldehyde in the brain. Previous studies have demonstrated that pharmacological manipulations of brain catalase activity modulate the stimulant effects of ethanol in mice. However, the role of H(2)O(2) in the behavioral effects of ethanol has not yet been clearly addressed., Objectives: In the present study, we investigated the effects of alpha-lipoic acid (LA), a scavenging agent for H(2)O(2), on ethanol-induced locomotor stimulation., Methods: CD-1 mice were pretreated with LA [0-100 mg/kg, intraperitoneally (IP)] 0-60 min prior to administration of ethanol (0-3.75 g/kg, IP). In another experiment, animals were pretreated with LA (0, 25, or 50 mg/kg, IP) 30 min before cocaine (10 mg/kg, IP), amphetamine (2 mg/kg, IP), or caffeine (25 mg/kg, IP). After these treatments the animals were placed in an open-field chamber and their locomotor activity was measured for 20 min., Results: LA 25, 50, and 100 mg/kg IP prevented ethanol-induced locomotor stimulation. LA did not affect the locomotor-stimulating effects of cocaine, amphetamine, and caffeine. Additionally, we demonstrated that LA prevents the inactivation of brain catalase by 3-amino-1,2,4-triazole, thus indicating that H(2)O(2) levels are reduced by LA., Conclusions: These data support the idea that a decrease in cerebral H(2)O(2) production by LA administration inhibits ethanol-stimulated locomotion. This study suggests that the brain catalase-H(2)O(2) system, and by implication centrally formed acetaldehyde, plays a key role in the psychopharmacological effects of ethanol.

Background: Increasing evidence indicates that mu- and delta-opioid receptors are decisively involved in the retrieval of memories underlying conditioned effects of ethanol. The precise mechanism by which these receptors participate in such effects remains unclear. Given the important role of the proopiomelanocortin (POMc)-derived opioid peptide beta-endorphin, an endogenous mu- and delta-opioid receptor agonist, in some of the behavioral effects of ethanol, we hypothesized that beta-endorphin would also be involved in ethanol conditioning., Methods: In this study, we treated female Swiss mice with estradiol valerate (EV), which induces a neurotoxic lesion of the beta-endorphin neurons of the hypothalamic arcuate nucleus (ArcN). These mice were compared to saline-treated controls to investigate the role of beta-endorphin in the acquisition, extinction, and reinstatement of ethanol (0 or 2 g/kg; intraperitoneally)-induced conditioned place preference (CPP)., Results: Immunohistochemical analyses confirmed a decreased number of POMc-containing neurons of the ArcN with EV treatment. EV did not affect the acquisition or reinstatement of ethanol-induced CPP, but facilitated its extinction. Behavioral sensitization to ethanol, seen during the conditioning days, was not present in EV-treated animals., Conclusions: The present data suggest that ArcN beta-endorphins are involved in the retrieval of conditioned memories of ethanol and are implicated in the processes that underlie extinction of ethanol-cue associations. Results also reveal a dissociated neurobiology supporting behavioral sensitization to ethanol and its conditioning properties, as a beta-endorphin deficit affected sensitization to ethanol, while leaving acquisition and reinstatement of ethanol-induced CPP unaffected., (Copyright © 2011 by the Research Society on Alcoholism.)

Calcium flux through voltage gate calcium channels (VGCC) is involved in many neuronal processes such as membrane depolarization, gene expression, hormone secretion, and neurotransmitter release. Several studies have shown that either acute or chronic exposure to ethanol modifies calcium influx through high voltage activated channels. Of special relevance is the L-type VGCC. Pharmacological manipulation of L-type calcium channels affects ethanol intake, ethanol discrimination and manifestations of withdrawal syndrome. The present study investigates the role of L-type channels on the psychomotor effects (stimulation and sedation/ataxia) of ethanol by testing the effects of different L-type calcium channel blockers (CCB) on such behaviors. Mice were pretreated intraperitoneally with the CCB, diltiazem (0-40 mg/kg) or verapamil (0-30 mg/kg) 30 min before ethanol (0-3.5 g/kg). Locomotion was measured in an open field chamber for 20 min immediately after ethanol. The two CCB tested prevented locomotor stimulation, but not locomotor suppression produced by ethanol. Doses of the two CCB which reduced ethanol stimulation, did not alter spontaneous locomotion. The ataxic effects of ethanol (1.25 g/kg), measured with an accelerating rotarod task, were not affected by diltiazem (20mg/kg) or verapamil (15 mg/kg). In addition, our results indicated that ethanol is more sensitive to the antagonism of L-type calcium channels than other drugs with stimulant properties; doses of the two CCB that reduced ethanol stimulation did not reduce the psychomotor effects of amphetamine, caffeine or cocaine. In conclusion, these data provide further evidence of the important involvement of L-type calcium channels in the behavioral effects produced by ethanol., (Copyright 2010 Elsevier B.V. All rights reserved.)

Objectives: It has been shown that acetaldehyde is an active metabolite of ethanol with central actions that modulate behavior. Catalase has been proposed as the main enzyme responsible for the synthesis of acetaldehyde from ethanol in the brain. Recent studies, however, suggest that cytochrome, in particular the isoform P450 2E1, can also contribute to the central metabolism of ethanol., Methods: Cytochrome P4502E1 knockout (KO) mice were used to assess the involvement of this isoenzyme in some of the acute and chronic behavioral effects of ethanol. Ethanol-induced locomotion, locomotor sensitization, and voluntary ethanol intake were evaluated in cytochrome P4502E1 KO mice and their wild-type (WT) counterparts., Results: Spontaneous locomotion in KO mice was lower than that seen in the WT mice. Acute administration of ethanol (1.5 g/kg, intraperitoneally) increased locomotion to a similar extent in both strains of mice. Repeated intermittent administration of ethanol produced sensitization in both strains, but it was very subtle in the KO mice compared with the effect in the WT mice. KO mice showed a reduction in preference for ethanol intake at low concentrations (4-8% v/v). Interestingly, western blot for catalase in the brain and liver showed that KO mice had higher levels of catalase expression compared with WT mice., Conclusion: These results show some impact of the mutation on ethanol-induced sensitization and on voluntary ethanol preference. The lack of a substantial impact of the mutation can be explained by the fact that the KO animals have a compensatory increase in catalase expression compared with WT mice, therefore possibly showing alterations in the formation of acetaldehyde after ethanol administration.

Rationale: Considerable evidence indicates that brain ethanol metabolism mediated by catalase is involved in modulating some of the behavioral and physiological effects of this drug, which suggests that the first metabolite of ethanol, acetaldehyde, may have central actions. Previous results have shown that acetaldehyde administered into the lateral ventricles produced anxiolysis in a novel open arena in rats., Objectives: The present studies investigate the effects of centrally formed acetaldehyde on ethanol-induced anxiolysis., Materials and Methods: The effects of the catalase inhibitor sodium azide (SA; 0 or 10 mg/kg, IP) on ethanol-induced anxiolysis (0.0, 0.5, or 1.0 g/kg, IP) were evaluated in CD1 mice in two anxiety paradigms, the elevated plus maze and the dark/light box. Additional studies assessed the effect of the noncompetitive catalase inhibitor 3-amino-1,2,4-triazole (AT; 0.5 g/kg, IP) and the acetaldehyde inactivation agent D: -penicillamine (50 mg/kg, IP) on the plus maze., Results: SA reduced the anxiolytic effects of ethanol on several parameters evaluated in the elevated plus maze and in the dark/light box. In the plus maze, AT completely blocked and D-penicillamine significantly reduced the anxiolytic properties of ethanol., Conclusions: Thus, when cerebral metabolism of ethanol into acetaldehyde is blocked by catalase inhibitors, or acetaldehyde is inactivated, there is a suppressive effect on the anxiolytic actions of ethanol. These data provide further support for the idea that centrally formed or administered acetaldehyde can contribute to some of the psychopharmacological actions of ethanol, including its anxiolytic properties.

It is suggested that some of the behavioral effects of ethanol, including its psychomotor properties, are mediated by beta-endorphin and opioid receptors. Ethanol-induced increases in the release of hypothalamic beta-endorphin depend on the catalasemic conversion of ethanol to acetaldehyde. Here, we evaluated the locomotor activity in rats microinjected with ethanol directly into the hypothalamic arcuate nucleus (ArcN), the main site of beta-endorphin synthesis in the brain and a region with high levels of catalase expression. Intra-ArcN ethanol-induced changes in motor activity were also investigated in rats pretreated with the opioid receptor antagonist, naltrexone (0-2 mg/kg) or the catalase inhibitor 3-amino-1,2,4-triazole (AT; 0-1 g/kg). We found that ethanol microinjections of 64 or 128, but not 256 microg, produced locomotor stimulation. Intra-ArcN ethanol (128 microg)-induced activation was prevented by naltrexone and AT, whereas these compounds did not affect spontaneous activity. The present results support earlier evidence indicating that the ArcN and the beta-endorphinic neurons of this nucleus are necessary for ethanol to induce stimulation. In addition, our data suggest that brain structures that, as the ArcN, are rich in catalase may support the formation of ethanol-derived pharmacologically relevant concentrations of acetaldehyde and, thus be of particular importance for the behavioral effects of ethanol.

It has been suggested that some of the behavioral effects produced by ethanol are mediated by its first metabolite, acetaldehyde. The present research addressed the hypothesis that catalase-dependent metabolism of ethanol to acetaldehyde in the brain is an important step in the production of ethanol-related affective properties. Firstly, we investigated the contribution of brain catalase in the acquisition of ethanol-induced conditioned place preference (CPP). Secondly, the specificity of the catalase inhibitor 3-amino-1,2,4-triazole (AT) was evaluated with morphine- and cocaine-induced CPP. Finally, to investigate the role of catalase in the process of relapse to ethanol seeking caused by re-exposure to ethanol, after an initial conditioning and extinction, mice were primed with saline and ethanol or AT and ethanol and tested for reinstatement of CPP. Conditioned place preference was blocked in animals treated with AT and ethanol. Morphine and cocaine CPP were unaffected by AT treatment. However, the reinstatement of place preference was not modified by catalase inhibition. Taken together, the results of the present study indicate that the brain catalase-H(2)O(2) system contributes to the acquisition of affective-dependent learning induced by ethanol, and support the involvement of centrally-formed acetaldehyde in the formation of positive affective memories produced by ethanol.

Acetaldehyde, the main biological metabolite of ethanol, is nowadays considered to mediate some ethanol-induced effects. Previous studies on alcohol effect attenuation have shown that D-(-)-penicillamine (3-mercapto-D-valine), a thiol amino acid, acts as an effective agent for the inactivation of acetaldehyde. In the study reported here, laboratory rats were treated with ethanol and D-(-)-penicillamine at different doses looking for the interaction (in vivo) of D-(-)-penicillamine with metabolically formed acetaldehyde following a condensation reaction to form the stable adduct (4S,2RS)-2,5,5-trimethylthiazolidine-4-carboxylic acid (TMTCA). A novel and rapid procedure based on liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) was developed for quantification and reliable identification of TMTCA in different rat tissues, including plasma, liver and brain. Firstly, plasma was obtained from whole blood. Then, proteins were precipitated from plasma, brain and liver extracts with acetonitrile and the clarified extracts diluted 10-fold. A 20 microL aliquot of the final extracts was then analyzed using an Atlantis C18 5 microm, 100x2 mm column which was connected to the electrospray source of a LC/triple quadrupole mass spectrometer. The analyte was detected in positive ion mode acquiring four MS/MS transitions in selected reaction monitoring (SRM) mode. The method has been validated and it has proved to be fast, reliable and sensitive. The accuracy and precision were evaluated by means of recovery experiments from plasma, liver and brain samples fortified at two concentration levels obtaining satisfactory recoveries in all cases: 95 and 105% in plasma (at 10 and 100 ng/mL, respectively), 79 and 89% in brain (100 and 1000 ng/g), 85 and 99% in liver (100 and 1000 ng/g). Precision, expressed as repeatability, was in all tissues analyzed lower than 17% at the two concentrations tested. The estimated detection limits were 1 ng/mL in plasma, 4 ng/g in brain and 5 ng/g in liver. The limit of quantitation objective (the lowest concentration that was validated with acceptable results) was set up at 10 ng/mL for plasma and 100 ng/g for brain and liver tissue. The reliable identification of the analyte was ensured by the acquisition of four transitions and by their ion abundance ratio measurement. Due to its excellent selectivity and sensitivity, the method developed in this work provides an excellent tool for the specific determination of this cyclic amino acid in biological samples., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

The antioxidant enzyme catalase by reacting with H(2)O(2), forms the compound known as compound I (catalase-H(2)O(2)). This compound is able to oxidise ethanol to acetaldehyde in the CNS. It has been demonstrated that 3-nitropropionic acid (3-NPA) induces the activity of the brain catalase-H(2)O(2) system. In this study, we tested the effect of 3-NPA on both the brain catalase-H(2)O(2) system and on the acute locomotor effect of ethanol. To find the optimal interval for the 3-NPA-ethanol interaction mice were treated with 3-NPA 0, 45, 90 and 135min before an ethanol injection (2.4mg/kg). In a second study, 3-NPA (0, 15, 30 or 45mg/kg) was administered SC to animals 90min before saline or several doses of ethanol (1.6 or 2.4g/kg), and the open-field behaviour was registered. The specificity of the effect of 3-NPA (45mg/kg) was evaluated on caffeine (10mg/kg IP) and cocaine (4mg/kg)-induced locomotion. The prevention of 3-NPA effects on both ethanol-induced locomotion and brain catalase activity by L-carnitine, a potent antioxidant, was also studied. Nitropropionic acid boosted ethanol-induced locomotion and brain catalase activity after 90min. The effect of 3-NPA was prevented by l-carnitine administration. These results indicate that 3-NPA enhanced ethanol-induced locomotion by increasing the activity of the brain catalase system.

Acetaldehyde, the first metabolite of ethanol, may mediate some ethanol-induced effects. Previous research in our laboratory has shown that D-penicillamine, an inactivation agent for acetaldehyde, is effective in decreasing locomotor stimulation and conditioned place preference induced by ethanol in mice. In the present study, the effects of D-penicillamine on the voluntary consumption of ethanol were assessed. Male rats were offered ethanol under restricted access, without food or water deprivation. Daily availability of ethanol was limited to a 15-min period in the home cages. When the response for 10% ethanol was stable, rats received an intraperitoneal (IP) injection of D-penicillamine (0, 25, 50 or 75 mg/kg) over a 5-day period, given 30 min before exposure to ethanol. In a second study we determined the specificity of D-penicillamine effects (50 mg/kg) on voluntary sucrose consumption (3%). Another study was conducted to evaluate whether IP D-penicillamine (50 mg/kg) alters taste reactivity responses. In the final experiment, rats were treated with intracerobroventricular (ICV) infusions of D-penicillamine (75 microg) for 5 days before drinking ethanol or sucrose. D-Penicillamine was found to reduce ethanol intake in a dose-dependent manner. Sucrose consumption was also affected by this thiol amino acid. We also demonstrated that D-penicillamine produced changes in the ingestive and flavor properties of sucrose and ethanol, measured by means of a taste reactivity test. When D-penicillamine was administered ICV, only voluntary ethanol consumption was modified. These findings indicate that the central inactivation of acetaldehyde blocks ethanol intake in rats, and suggest that acetaldehyde plays a key role in the motivational properties of ethanol.

Nonspecific blockade of opioid receptors has been found to prevent development of behavioral sensitization to ethanol. Whether this effect is achieved through a specific opioid receptor subtype, however, is not clear. The present study investigated, for the first time, the role of specific opioid receptor subtypes in the development of ethanol-(2.5 g/kg/day; six sessions) induced locomotor sensitization in mice. We confirmed previous results showing that the nonspecific antagonism of opioid receptors (naltrexone; 0-2 mg/kg) prevented the development of behavioral sensitization to ethanol, an effect attained at doses presumed to occupy only mu opioid receptors. This was confirmed by using the selective mu opioid receptor antagonist CTOP (0-1.5 mg/kg), which also blocked sensitization to ethanol. The selective delta receptor antagonist, naltrindole (0-10 mg/kg), however, did not alter sensitization. We further assessed the role of mu opioid receptors in sensitization to ethanol by exploring the involvement of mu(1), mu(1+2), and mu(3) opioid receptor subtypes. Results of these experiments revealed that the blockade of mu(1) (naloxonazine; 0-30 mg/kg) or mu(3) opioid receptors (3-methoxynaltrexone; 0-6 mg/kg) did not prevent locomotor sensitization to ethanol. Using naloxonazine under treatment conditions that block mu(1+2) opioid receptor subtypes we observed a retarded sensitization. The present data suggest that the concurrent inactivation of all mu opioid receptor subtypes may be required to prevent the neural adaptations underlying the development of behavioral sensitization to ethanol. In addition, these results support previous data suggesting a putative role for the mu opioid receptor endogenous ligand, beta-endorphin, and the hypothalamic arcuate nucleus in ethanol sensitization.

A series of experiments was conducted to investigate the locomotor effects of local injections of ethanol and the ethanol metabolite, acetaldehyde, into substantia nigra pars reticulata (SNr). Infusions of ethanol into SNr resulted in a dose-related increase in locomotor activity, with maximal effects at a dose of 1.4 micromol. Ethanol injected into a control site dorsal to substantia nigra failed to stimulate locomotion, and another inactive site was identified in brainstem areas posterior to substantia nigra. The locomotor effects of intranigral ethanol (1.4 micromol) were reduced by coadministration of 10 mg/kg sodium azide, a catalase inhibitor that acts to reduce the metabolism of ethanol into acetaldehyde in the brain. SNr infusions of acetaldehyde, which is the first metabolite of ethanol, also increased locomotion. Taken together, these results indicate that SNr is one of the sites at which ethanol and acetaldehyde may be acting to induce locomotor activity. These results are consistent with the hypothesis that acetaldehyde is a centrally active metabolite of ethanol, and provide further support for the idea that catalase activity is a critical step in the regulation of ethanol-induced motor activity. These studies have implications for understanding the brain mechanisms involved in mediating the ascending limb of the biphasic dose-response curve for the effect of ethanol on locomotor activity.

Rationale: There is evidence to suggest that acetaldehyde is involved in the control of ethanol-seeking behavior and reward. D -penicillamine, a thiol amino acid, is a highly selective agent for the inactivation of acetaldehyde. Previous studies from our laboratory have demonstrated that D -penicillamine prevents both behavioral stimulation induced by ethanol and acetaldehyde-produced locomotor depression in mice., Objectives: The contribution of ethanol-derived acetaldehyde to the affective effects of ethanol (preference and aversion) was assessed using an unbiased place conditioning design., Methods: Male mice received four pairings of a distinctive floor stimulus (CS+: GRID+ or HOLE+) with injections of saline and ethanol (2 g/kg) given before (preference) or after (aversion) the 5-min exposure to the place conditioning apparatus. A different floor stimulus (CS-: GRID- or HOLE-), associated with saline-saline injections on alternate days, was presented. For a different group of animals, the pairings with the CS+ were associated with saline and ethanol injections, but on alternate days, they received D -penicillamine (50 or 75 mg/kg) and ethanol injections paired with the CS-floor stimulus. A 60-min preference test was carried out 24 h after the last conditioning trial. A similar procedure was followed to test the effect of D -penicillamine on morphine (16 mg/kg) and cocaine-induced (20 mg/kg) conditioned place preference (CPP)., Results: CPP and conditioned place aversion (CPA) were observed for ethanol, but D -penicillamine only blocked CPP. D -penicillamine, by itself, did not produce either rewarding or aversive effects. CPP observed for morphine and cocaine was unaffected by D -penicillamine pretreatment., Conclusions: The results of the present study suggest that the selective inactivation of acetaldehyde blocked the rewarding, but not aversive, effects of ethanol and support the role of this ethanol metabolite in the affective properties of ethanol.

Rationale: Novelty associated with behavioral testing has been shown to enhance psychostimulant- and morphine-induced locomotor stimulation. Evidence has demonstrated that novelty increases dopamine (DA) activity, and habituation to a novel environment reduces such activation. However, it is not clear whether novelty modulates ethanol-induced behavioral stimulation and whether DA plays a role in this effect., Objectives: The present work sought to demonstrate a role of habituation to test procedure as a factor that could modulate the involvement of DA in ethanol-induced locomotor stimulation., Methods: Non-habituated (NH) and habituated (H) Swiss mice pretreated with DA D1- (SCH23390; 0-0.045 mg/kg) or D2-receptor (sulpiride; 0-50 mg/kg) antagonists were tested for ethanol (0-2.5 g/kg)-induced locomotor stimulation. Experiments with amphetamine (0-4 mg/kg), morphine (0-5 mg/kg) and caffeine (0-15 mg/kg)were designed to compare their results to those obtained with ethanol. The effect of the non-selective opioid receptor antagonist naltrexone (0-1.5 mg/kg) was also tested on ethanol-induced locomotor stimulation., Results: NH and H animals did not differ in their locomotor response to ethanol or caffeine; however, amphetamine- and morphine-induced stimulation was greater in NH than in H mice. SCH23390 only reduced ethanol-induced stimulation at doses that also reduced spontaneous activity in both NH and H mice. Sulpiride decreased ethanol-stimulated behavior only in the NH condition. Habituation did not modify the effect of sulpiride on amphetamine-, morphine- or caffeine-induced activation. Naltrexone (0-1.5 mg/kg) reduced ethanol-induced stimulation regardless of habituation., Conclusions: The present data suggest that the participation of DA D2-receptors in ethanol-induced behavioral stimulation requires the presence of novelty. Results also support the involvement of neurotransmitter systems other than DA (i.e., endogenous opioid system) as important substrates mediating ethanol-induced locomotor activation.

The involvement of catalase in ethanol-induced locomotion has been clearly proven. However, studies addressing the role of this enzyme in the effects that ethanol exerts on memory are lacking. In the present study, the social recognition test (SRT) was used to evaluate ethanol effects on memory. In this test, the reduction in investigation time of a juvenile conspecific, when this social stimulus is presented for the second time, is considered a reliable index of memory. Exploration ratios (ER) were calculated to evaluate the recognition capacity of mice. Ethanol (0.0, 0.5, 1.0 or 1.5g/kg, i.p.) was administered immediately after the first juvenile presentation, and 2h later the juvenile was re-exposed to the adult. Additionally, adult mice received aminotriazole (AT) or sodium azide (two catalase inhibitors) 5h or 30 min before juvenile presentation, respectively. Ethanol (1.0 and 1.5g/kg) was able to reduce ER, indicating an improving effect on memory. This improvement was prevented by either AT or sodium azide pre-treatment. However, neither AT nor sodium azide attenuated the memory-enhancing capacity of NMDA or nicotine, suggesting a specific interaction between catalase inhibitors and ethanol in their effects on memory. The present results suggest that brain catalase activity could mediate the memory-enhancing capacity of ethanol and add further support to the idea that this enzyme mediates some of the psychopharmacological effects produced by ethanol.

Background: D-Penicillamine, a sulfhydryl amino acid derived from penicillin, is a highly selective agent for sequestering in vivo acetaldehyde, the first metabolic product of ethanol. A substantial amount of research supports the idea that brain acetaldehyde, produced by central ethanol metabolism, plays a key role in determining some of the behavioral effects of ethanol administration. This study addressed two questions. First, we tested if D-penicillamine was able to modify the depressant effects of acetaldehyde on behavior. Second, we studied the effect of D-penicillamine on ethanol-induced behavioral stimulation., Methods: Mice were pretreated with 75.00 mg/kg of D-penicillamine, and 30 min later, they received acetaldehyde at 0, 100, 200, or 300 mg/kg intraperitoneally. Different groups of animals were treated with 0.0, 37.5, 75, 150, or 300 mg/kg of D-penicillamine simultaneously 30, 90, 150, or 210 min before the intraperitoneal administration of saline or 1.2, 1.8, 2.4, 3.0, or 3.6 g/kg of ethanol, respectively. The specificity of D-penicillamine effects was addressed using two drugs: cocaine (4 mg/kg) and caffeine (15 mg/kg)., Results: Our results revealed that behavioral depression caused by acetaldehyde (200 and 300 mg/kg) could be attenuated by D-penicillamine treatment. In addition, D-penicillamine was also effective in lowering behavioral locomotion induced by ethanol (1.8 and 2.4 g/kg), without altering spontaneous locomotor activity. This sulfhydryl amino acid specifically modified the effect of ethanol on locomotion because cocaine- or caffeine-induced locomotion was unaffected. In addition, blood ethanol levels were not different between D-penicillamine- and saline-pretreated mice., Conclusions: Behavioral effects produced by acetaldehyde and ethanol are blocked when animals are treated with D-penicillamine, an effective sequestration agent for acetaldehyde. These results suggest that some of the psychopharmacological effects, classically attributed to ethanol, could be mediated by its first metabolite, acetaldehyde.

Previous studies have demonstrated that administration of nonspecific opioid antagonists such as naltrexone or naloxone reduces ethanol-induced locomotor activity in mice. However, because of their broad pharmacological profile, it remains unclear through which opioid receptor this antagonism is achieved. Therefore, the present study was aimed at further investigating the role of the different opioid receptors in ethanol-induced (2.5 g/kg) locomotion in mice. First, we compared the effect of naltrexone (0-2 mg/kg) on ethanol-induced locomotion with that of the selective delta-opioid receptor antagonist, naltrindole (0-10 mg/kg). Results of this first set of data revealed that naltrexone completely blocked this effect of ethanol at doses suggested to occupy only mu-opioid receptors, and naltrindole did not modify ethanol-induced locomotion. In a second set of experiments, we further investigated the involvement of mu-opioid receptors in ethanol-stimulated motor activity by assessing the implication of mu(1)-, mu(1/2)-, and mu(3)-opioid receptor subtypes. Results revealed that mu(1/2)-, and to a lesser extent mu(3)-, but not mu(1)-opioid receptor subtypes are involved in the psychomotor actions of ethanol. Data are discussed together with previous results which have emphasized the critical dependence of ethanol-induced motor behaviors on opioid receptors, as well as, of the integrity of beta-endorphin synthesizing neurons from the hypothalamic Arcuate Nucleus.

Previous studies have demonstrated that there is a bidirectional modulation of ethanol-induced locomotion produced by drugs that regulate brain catalase activity. In the present study we have assessed the effect in rats of intraperitoneal, intraventricular or intracraneal administration of the catalase inhibitor sodium azide in the locomotor changes observed after ethanol (1 g/kg) administration. Our results show that sodium azide prevents the effects of ethanol in rats locomotion not only when sodium azide was systemically administered but also when it was intraventricularly injected, then confirming that the interaction between catalase and ethanol takes place in Central Nervous System (CNS). Even more interestingly, the same results were observed when sodium azide administration was restricted to the hypothalamic Arcuate nucleus (ARC), a brain region which has one of the highest levels of expression of catalase. Therefore, the results of the present study not only confirm a role for brain catalase in the mediation of ethanol-induced locomotor changes in rodents but also point to the ARC as a major neuroanatomical location for this interaction. These results are in agreement with our reports showing that ethanol-induced locomotor changes are clearly dependent of the ARC integrity and, especially of the POMc-synthesising neurons of this nucleus. According to these data we propose a model in which ethanol oxidation via catalase could produce acetaldehyde into the ARC and to promote a release of beta-endorphins that would activate opioid receptors to produce locomotion and other ethanol-induced neurobehavioural changes.

Background: Growing evidence indicates that brain catalase activity is involved in the psychopharmacological actions of ethanol. Recent data suggest that participation of this enzymatic system in some ethanol effects could be mediated by the endogenous opioid system. The present study assessed whether brain catalase has a role in ethanol-induced activation of the HPA axis, a neuroendocrine system modulated by the endogenous opioid neurotransmission., Methods: Swiss male mice received an intraperitoneal injection of the catalase inhibitor 3-amino-1,2,4-triazole (AT; 0-1 g/kg), and 0 to 20 hr after this administration, animals received an ethanol (0-4 g/kg; intraperitoneally) challenge. Thirty, 60, or 120 min after ethanol administration, plasma corticosterone levels were determined immunoenzymatically. In addition, we tested the effects of 45 mg/kg of cyanamide (another catalase inhibitor) and 0 to 2 mg/kg of naltrexone (nonselective opioid receptor antagonist) on ethanol-induced enhancement in plasma corticosterone values., Results: The present study revealed that AT boosts ethanol-induced increase in plasma corticosterone levels in a dose- and time-dependent manner. However, it did not affect corticosterone values when measured after administration of saline, cocaine (4 mg/kg, intraperitoneally), or morphine (30 mg/kg, intraperitoneally). The catalase inhibitor cyanamide (45 mg/kg, intraperitoneally) also increased ethanol-related plasma corticosterone levels. These effects of AT and cyanamide on ethanol-induced corticosterone values were observed under treatment conditions that decreased significantly brain catalase activity. Indeed, a significant correlation between effects of catalase manipulations on both variables was found. Finally, we found that the administration of naltrexone enhanced the levels of plasma corticosterone after the administration of saline or ethanol., Conclusions: This study shows that the inhibition of brain catalase increases ethanol-induced plasma corticosterone levels. Results are discussed together with previous findings suggesting a putative linkage between brain ethanol metabolism and the endogenous opioid system to explain some of the neuroendocrine effects of ethanol.

The C57BL/6J strain of inbred mice shows a characteristic pattern of ethanol-induced behaviors: very weak acute locomotor stimulation, a lack of locomotor-sensitizing effect of ethanol, and a high level of ethanol intake. This strain has relatively low levels of activity of the ethanol metabolizing enzyme catalase, and it has been proposed that brain catalase plays a role in the modulation of some behavioral effects of ethanol. In the first study of the present paper, we investigated the effects of pharmacological manipulations of brain catalase activity on C57BL/6J mice in acute ethanol-induced locomotion and ethanol intake. Results indicated that the reduction in motor activity produced by ethanol was reversed by pretreatment with catalase potentiators and it was enhanced by catalase inhibitors. In addition, ethanol intake was highly correlated with brain catalase activity in mice treated with a catalase potentiator. In the second study, F1 hybrid mice (SWXB6) from the outbred Swiss-Webster mice and the inbred C57BL/6J mice were used. Basal brain catalase activity levels of F1 mice were intermediate between to those of the two progenitor genotypes. That profile of catalase activity was parallel to the acute-ethanol-induced locomotion and to repeated-ethanol-induced motor sensitization effects observed across the three types of mice. These data suggest that brain catalase activity modifications in the C57BL/6J strain change the pattern of several ethanol-related behaviors in this inbred mouse.

Several studies have pointed out that the mu opioid receptor (MOR) can play a key role in some of the behavioural effects of ethanol. In the present study, the implication of the MOR in ethanol-induced locomotion in mice was assessed. First, the effects of the administration of different naltrexone doses (0.001-1.000 mg/kg) on the locomotor changes produced by ethanol (2.5 g/kg) were evaluated. In a second set of experiments, the ability of repeated naltrexone (6 mg/kg) administrations to modify the effects of ethanol was also assessed on mice locomotion. The results of the present study revealed that an acute naltrexone administration reduced dose-dependently ethanol-induced locomotion. Conversely, after repeated naltrexone injections, a transient boost of ethanol induced locomotor activity was observed. Thus, the results of the present study revealed that the effects of these naltrexone pretreatments on ethanol-induced locomotion are similar to the previously described changes on MOR activity. Moreover, the same (acute and chronic) naltrexone pretreatments produced similar changes on the locomotion of mice after a challenge with morphine (a MOR agonist), but not after tert-butanol (an alcohol which does not release beta-endorphins) administration. Therefore, our results are discussed in terms of the proved ability of ethanol to promote the release of beta-endorphins and, consequently, to activate the MOR.

Lesions of the arcuate nucleus by monosodium glutamate, goldthioglucose and oestradiol valerate treatments are known to prevent the acute stimulating effect of ethanol in mice. On the basis of these results, the current study analysed whether a lesion of the arcuate nucleus by monosodium glutamate was able to block ethanol-induced locomotor sensitization. To produce the arcuate nucleus lesions, pups were injected with saline or monosodium glutamate (4 mg/g body weight) subcutaneously on 5 alternate days, starting on postnatal day one. Sensitization treatments began 10 weeks after the initial lesions. Sensitization training consisted of six trials on alternate days, in which groups of mice were treated with ethanol (2 g/kg) or saline, and then tested in an open-field for the induction of locomotor activity. The present study demonstrated that animals with monosodium glutamate-induced lesions did not develop locomotor sensitization to ethanol. Different groups of mice were used to assay blood ethanol levels and to evaluate the effect of arcuate nucleus lesions on psychostimulant-induced locomotor sensitization. Sensitization to cocaine or amphetamine was spared in monosodium glutamate-pre-treated animals, although the lesion of arcuate nucleus reduced the sensitivity of mice to cocaine. Our findings therefore suggest that the arcuate nucleus may be critical for the neuroadaptations that underlie the behavioural sensitization to ethanol, in contrast to those mediating psychostimulant-induced sensitization.

To investigate how diethyldithiocarbamate (DDTC) affects the stimulating effect of ethanol on open-field locomotion, mice were pretreated with different doses of DDTC 8 hours prior to ethanol. The effect of DDTC and saline on different ethanol doses was analyzed. DDTC reduces ethanol-induced locomotor activity in a dose-dependent manner, not the spontaneous locomotor activity. Aldehyde dehydrogenase might influence psychopharmacological effects of ethanol.

Rationale: Over the last few years, a role for the brain catalase-H(2)O(2) enzymatic system has been suggested in the behavioural effects observed in rodents after ethanol administration. This role seems to be related to the ability of cerebral catalase to metabolise ethanol to acetaldehyde using H(2)O(2)as a co-substrate. On the other hand, it has been shown that normobaric hyperoxia increases the rate of cerebral H(2)O(2) production in rodents in vivo. Thus, substrate-level changes could regulate brain catalase activity, thereby modulating the behavioural effects of ethanol., Objectives: The aim of the present study was to assess if the enhancement of cerebral H(2)O(2) production after hyperoxia exposure results in a boost of ethanol-induced locomotion in mice., Methods: CD-1 mice were exposed to air or 99.5% O(2) inhalation (for 15, 30, or 45 min) and 0, 30, 60 or 120 min after this treatment, ethanol-induced locomotion was measured. The H(2)O(2)-mediated inactivation of endogenous brain catalase activity following an injection of 3-amino-1,2,4-triazole was used as a measure of the rate of cerebral H(2)O(2) production., Results: Hyperoxia exposure (30 or 45 min) potentiated the locomotor-stimulating effects of ethanol (2.5 or 3.0 g/kg), whereas cocaine (4 mg/kg) or caffeine (15 mg/kg)-induced locomotion and blood ethanol levels were unaffected. Moreover, the results also confirmed brain H(2)O(2) overproduction in mice., Conclusions: The present results suggest that an increase in brain H(2)O(2) production potentiates ethanol-induced locomotion. Therefore, this study provides further support for the notion that the brain catalase-H(2)O(2) system, and by implication centrally formed acetaldehyde, plays a key role in the mediation of ethanol's psychopharmacological effects.

It has been suggested that the endogenous opioid system, especially beta-endorphins, may play an important role in the behavioral effects of ethanol. The main site of beta-endorphin synthesis in the brain is the hypothalamic arcuate nucleus (ARC). In the present study, we used the neurotoxins monosodium glutamate (MSG) or goldthioglucose (GTG) to produce a selective ARC lesion and to assess its effects on the locomotion observed after ethanol administration. The results show that MSG or GTG pre-treatment produces a blockade of the increased locomotion produced by the injection of low and moderate doses of ethanol (0.5 and 1.5 g/kg, respectively). These effects were observed in the absence of any change in blood ethanol levels. On the other hand, MSG (but not GTG) pre-treatment enhanced the locomotor depression produced by higher doses of this alcohol (2.5 g/kg). Finally, caffeine (10 mg/kg)-induced locomotion was unaffected by the aforementioned neurotoxic agents. Thus, taken together, the present results suggest that MSG and GTG administration produce a blockade of the stimulating effects of ethanol on locomotion in mice and thus provides further support for a role of the ARC in the behavioral effects observed after ethanol administration.

Background: It has been demonstrated that ethanol decreases blood luteinizing hormone (LH) levels in rodents. This effect seems to be produced by the capacity of ethanol to release beta-endorphins from the hypothalamic arcuate nucleus and, in a second step, by a mu-receptor-mediated inhibitory effect of these peptides on hypothalamic LH-releasing hormone-synthesizing neurons. However, it has been reported that, in primary hypothalamic cultures, the ethanol-produced beta-endorphin release is mediated by the enzyme catalase. Therefore, the aim of this study was to assess whether catalase inhibition modifies ethanol effects on blood LH levels., Methods: Swiss albino mice were pretreated with the catalase inhibitor 3-amino-1,2,4-triazole (AT; 0.0-0.5 g/kg) and, 3.5 hr later, saline, ethanol (2.5 g/kg), or morphine (30 mg/kg) was administered. Blood samples were collected 2 hr after ethanol administration, and LH levels were immunoenzymatically assayed., Results: The catalase inhibitor AT dose-dependently blocked the ethanol-produced decrease in blood LH levels without altering those observed after saline or morphine administration. This effect was highly correlated with the decrease in brain catalase activity produced by AT., Conclusions: These results show an antagonistic effect between AT and ethanol on blood LH levels and suggest a role of brain catalase activity on this effect of ethanol. Data are discussed in terms of a possible functional relationship between brain catalase and beta-endorphins in the mediation of some of the psychopharmacological consequences observed after ethanol administration.

To assess the role of cyanamide and 4-methylpyrazole (4-MP) in mediating ethanol-induced locomotor activity in mice, they were pretreated with cyanamide (12.5, 25, or 50 g/kg) prior to one ethanol injection (2.4 g/kg) and showed significantly depressed locomotor activity compared with control groups. Cyanamide (25 mg/kg) also cancelled out the biphasic action of ethanol (0, 0.8, 1.6, 2.4, 3.2, or 4 g/kg) on locomotor activity. The action of cyanamide and 4-MP in combined administration was also tested. Our data show that pretreatment with 4-MP alone does not change the spontaneous or ethanol-induced locomotor activity. Conversely, when mice were pretreated with cyanamide and 4-MP, the depressive effect of cyanamide on the locomotor activity induced by ethanol disappeared, and the locomotor activity rose to levels similar to those of the control group, recovering the biphasic ethanol effect. These effects cannot be attributed to peripheral elevated blood acetaldehyde levels, as pretreatment with 4-MP prevents accumulation of acetaldehyde. These data might suggest some influence of brain catalase and aldehyde dehydrogenase (ALDH) on the effects of ethanol.

Rationale: It has been proposed that brain aldehyde dehydrogenase (ALDH) plays a role in the modulation of some psychopharmacological effects of ethanol. Diethyldithiocarbamate (DDTC), an ALDH inhibitor, elevates blood acetaldehyde levels in the presence of ethanol. Concurrent administration with 4-methylpyrazole (4-MP), an alcohol dehydrogenase inhibitor, prevents peripheral accumulation of acetaldehyde by DDTC., Objective: To investigate the effects of concurrent DDTC and 4-MP administration on ethanol-induced locomotor activity in mice., Methods: Mice were pretreated IP with saline (S+S) or 4-MP (10 mg/kg) (S+4-MP), then received IP injections of ethanol (0, 0.8, 1.6, 2.4, 3.2 and 4 g/kg) prior to testing in the open field., Results: Pretreatment with 4-MP does not modify the spontaneous or ethanol-induced locomotor activity. In the second experiment, the DDTC (114, 228 and 456 mg/kg) and 4-MP (DDTC+4-MP) were administered 8 h prior to testing locomotor activity in the open field. Animals were then treated with ethanol (0, 0.8, 1.6, 2.4, 3.2 and 4 g/kg), and placed in open field chambers. The locomotor activity of animals pretreated with DDTC and 4-MP was significantly enhanced here compared to groups S+S and S+4-MP. These effects cannot be attributed to elevated blood acetaldehyde levels, as pretreatment with 4-MP prevented peripheral accumulation of acetaldehyde., Conclusions: These data suggest that brain ALDH may contribute to the effects of ethanol on locomotor activity. This role of the enzyme ALDH in some of the psychopharmacological effects of ethanol may be a result of its ability to regulate levels of acetaldehyde in brain.

The effect of lead acetate and 3-amino-1, 2, 4-triazole (AT) on ethanol-induced loss of righting reflex (LORR) and brain catalase activity was studied in an attempt to confirm earlier observations on the involvement of catalase in ethanol-induced effects. Lead acetate (0 or 100 mg/kg) or AT (0 or 500 mg/kg) was injected (acutely) into mice 7 days or 5 h before testing. Other mice were exposed to drinking fluid containing 500 ppm lead acetate for 60 days. On the test day, mice received an intraperitoneal injection of ethanol (4.0 or 4.5 g/kg) and the duration of LORR was recorded. Acute lead-treated animals demonstrated a reduction in the duration of the LORR. However, both chronic administration of lead acetate and AT treatment increased the duration of ethanol-produced LORR. Furthermore, brain catalase activity in acute lead pretreated animals showed a significant induction, whereas it was reduced in chronic lead and AT treated mice. These results suggest that brain catalase activity, and by implication centrally formed acetaldehyde, may modulate ethanol-induced LORR.

In order to evaluate the effect of hypotaurine on ethanol-induced locomotion, different groups of mice received an injection of saline or 5.62, 8.45, 11.25, 16.87 or 33.75 mg/kg of hypotaurine 30 min prior to administering ethanol (2.4 g/kg). The duration of the effect of hypotaurine was explored by treating animals with ethanol 0, 30, 60 and 90 min after hypotaurine pretreatment. The effect of hypotaurine on acute stimulating ethanol locomotion was evaluated by pretreating animals with saline or 11.25 mg/kg of hypotaurine 30 or 60 min before ethanol (1.6, 2.4, 3.2 g/kg) or saline injections. Hypotaurine (11.25 mg/kg) required 30 min to boost, specifically ethanol-stimulated locomotion (2.4 g/kg). These results suggest a central locus for the interaction, firstly, because blood ethanol levels were not different between hypotaurine and saline pretreated mice, and, secondly, because a cotreatment with beta-alanine (22 mg/kg), a beta-amino acid that counteracts the transfer of hypotaurine across the blood-brain barrier (BBB), prevented the enhancement in ethanol-induced locomotion produced by hypotaurine.

The present study deals with the effect of L-cysteine on the acute stimulating effects of ethanol. Swiss albino mice were pretreated with 0.0, 18.75, 37.50, or 75.00 mg/kg of cysteine simultaneously, 30 or 60 min, before the administration of saline or 0.8, 1.6, 2.4, or 3.2 g/kg of ethanol at 20% vol./vol. After these treatments, mice were left in the open-field chamber for 20 min, and locomotor activity was evaluated for the last 10 min. The specificity of the effects of L-cysteine was analyzed with the use of two drugs that also induce locomotor activity--d-amphetamine and methanol. Mice received L-cysteine (37.5 mg/kg), and 30 min after this treatment d-amphetamine (2 mg/kg) or methanol (2.4 g/kg) was injected. Data showed that L-cysteine at 37.5 mg/kg was able to increase locomotion induced by 2.4 and 3.2 g/kg of ethanol when it was administered 30 min before ethanol injection. The effects of L-cysteine are specific for the stimulation of ethanol on locomotion, because L-cysteine does not alter d-amphetamine-induced locomotor activity or methanol-induced locomotion. Moreover, blood ethanol levels were not affected by L-cysteine pretreatment. Therefore, the present findings demonstrated that ethanol-induced locomotor effects are enhanced by L-cysteine, in a manner similar to that of other sulfur amino acids.

It has been demonstrated that acute administration of lead to mice enhances brain catalase activity and ethanol-induced locomotion. These effects of lead seem to be related, since they show similar time courses and occur at similar doses. In the present study, in an attempt to further evaluate the relation between brain catalase activity and lead-induced changes in ethanol-stimulated locomotion, the interaction between lead acetate and 3-amino-1H,2,4-triazole (AT), a well-known catalase inhibitor, was assessed. In this study, lead acetate or saline was acutely injected intraperitoneally to Swiss mice at doses of 50 or 100 mg/kg 7 days before testing. On the test day, animals received an intraperitoneal injection of AT (0, 10, or 500 mg/kg). Five hours following AT treatment, ethanol (0.0 or 2.5 g/kg, ip) was injected and the animals were placed in open-field chambers, in which locomotion was measured for 10 min. Neither lead exposure nor AT administration, either alone or in combination, had any effect on spontaneous locomotor activity. AT treatment reduced ethanol-induced locomotion as well as brain catalase activity. On the other hand, ambulation and brain catalase activity were significantly increased by both doses of lead. Furthermore, AT significantly reduced the potentiation produced by lead acetate on brain catalase and on ethanol-induced locomotor activity in a dose-dependent manner. A significant correlation was found between locomotion and catalase activity across all test conditions. The results show that brain catalase activity is involved in the effects of lead acetate on ethanol-induced locomotion in mice. Thus, this study confirms the notion that brain catalase provides the molecular basis for understanding some of the mechanisms of the action of ethanol in the central nervous system.

It has been suggested that the endogenous opioid system, especially b-endorphins (b-ep), can play a key role in the behavioral effects of ethanol. A single injection of estradiol valerate (EV) produces a neurotoxic effect on the b-endorphin cell population of the hypothalamic arcuate nucleus. In the present study we questioned whether mice pretreated with EV, exhibit any alterations in ethanol-induced behavioral effects. Female Swiss mice were pretreated with EV (2 mg/0.2 ml per mice) or vehicle and, 8 weeks later, these animals were challenged with ethanol (0.0-3.2 g/kg). Immediately after ethanol injection, mice were placed in the open field chambers and locomotor activity was assessed. EV administration did not produce any change in spontaneous locomotor activity but, conversely, blocked the locomotor activity induced by low (0.8 g/kg) and moderate (1.6 or 2.4 g/kg) doses of ethanol. Interestingly, the behavioral effects of higher doses of ethanol on locomotor activity as well as on the duration of the loss of righting reflex were unaffected by EV. Moreover, neither rota-rod performance or blood ethanol levels were affected by EV. In a second study, the effects of EV pre-treatment on caffeine- and 1-propanol-induced locomotor activity was tested. No differences were observed between groups in caffeine- or 1-propanol-induced locomotion. The results of the present study indicate that EV blocks ethanol-induced locomotor activity and that this effect can not be related with any difference in ethanol levels or nonspecific motor impairment. Furthermore, they suggest that b-ep containing neurons of the hypothalamic arcuate nucleus may play a role in some, but not all, behavioral effects of ethanol.

Objective: The present experiments analyze the effects of the brain catalase inhibitor 3-amino-1,2,4-triazole (AT) on the locomotor activity induced by ethanol., Method: In the first experiment, mice received injections of either AT (0.5 g/kg) or saline (S) 5 hours prior to an ethanol injection (0, 0.8, 1.6, 2.4, 3.2 or 4 g/kg). In the second experiment, five different groups of mice received injections of AT (0, 0.010, 0.030, 0.060, 0.125, 0.250 or 0.500 g/kg) 5 hours prior to being injected with 1.6 g/kg of ethanol. In the third experiment, six groups of mice were treated with AT (0.5 g/kg), simultaneously, 2.5, 5, 10 or 20 hours before the administration of 1.6 g/kg of ethanol. Immediately after ethanol injection, mice were placed individually in the open-field apparatus for 20 minutes. In another set of experiments, the effects of AT on brain catalase activity were studied. Animals were injected with AT at 0, 0.010, 0.030, 0.060, 0.125, 0.250 or 0.500 g/kg, and 5, 10 or 20 hours following AT treatment mice were perfused and the brain was removed., Results: Pretreating mice with AT reduces ethanol-induced locomotor activity (1.6, 2.4 and 3.2 g/kg) without altering spontaneous locomotion. Pretreatment with AT (from 0.125 g/kg to 0.5 g/kg) produced a clear dose-dependent decrease of ethanol locomotion and brain catalase activity. The effect of AT was observed 5 and 10 hours after the injection of this drug, and it disappeared 20 hours following AT treatment., Conclusions: Current data showed a parallel property of AT in producing a remarkable dose- and time-dependent decrease in catalase activity and ethanol locomotion.

Several reports have demonstrated that acute lead acetate administration enhances brain catalase activity in animals. Other reports have shown a role of brain catalase in ethanol-induced behaviors. In the present study we investigated the effect of acute lead acetate on brain catalase activity and on ethanol-induced locomotion, as well as whether mice treated with different doses of lead acetate, and therefore, with enhanced brain catalase activity, exhibit an increased ethanol-induced locomotor activity. Lead acetate or saline was injected IP in Swiss mice at doses of 50, 100, 150, or 200 mg/kg. At 7 days following this treatment, ethanol (0.0, 1.5, 2.0, 2.5, or 3.0 g/kg) was injected IP, and the animals were placed in the open-field chambers. Results indicated that the locomotor activity induced by ethanol was significantly increased in the groups treated with lead acetate. Maximum ethanol-induced locomotor activity increase was found in animals treated with 100 mg/kg of lead acetate and 2.5 g/kg of ethanol. Total brain catalase activity in lead-pretreated animals also showed a significant induction, which was maximum at 100 mg/kg of lead acetate treatment. No differences in blood ethanol levels were observed among treatment groups. The fact that brain catalase and ethanol-induced locomotor activity followed a similar pattern could suggest a relationship between both lead acetate effects and also a role for brain catalase in ethanol-induced behaviors.

A role for brain catalase in the mediation of some psychopharmacological effects of ethanol has been proposed. In the present study, we investigated the effects of repeated cyanamide injections on the activity of brain catalase, as well as on the ethanol-induced locomotion of mice. Male Swiss mice were pre-treated with cyanamide (10 mg/kg; three times per day, 5 days) or saline. At different times (2, 3, 6 or 9 days) following this treatment, animals were injected with ethanol. Immediately following this ethanol challenge, animals were placed in the open field chambers and locomotor activity was assessed for 10 min. Results indicated an increase in ethanol-induced locomotion of mice pre-treated with cyanamide 2, 3 or 6 days before the ethanol challenge. Brain catalase activity showed an enhancement at the same time period and the two variables showed a significant correlation. No differences between pre-treatment groups on ethanol blood levels were observed at time of testing. In a second study, the effects of these cyanamide treatment conditions on d-amphetamine-induced locomotor activity were assessed. Results indicated no differences between pre-treatment groups in d-amphetamine-induced locomotion. Thus, these data suggest that repeated daily injections of cyanamide can simultaneously induce both brain catalase and locomotor activity, and that these effects may be strongly related. Furthermore, the present study provides further support for the notion that brain catalase activity may be a factor mediating some of the psychopharmacological effects of ethanol.