Ethanol self-administration and alterations in the livers of the cynomolgus monkey, Macaca fascicularis

Background: Most of the studies of alcoholic liver disease use models in which animals undergo involuntary administration of high amounts of ethanol and consume diets that are often high in polyunsaturated fatty acids. The objectives of this study were (1) to evaluate whether cynomolgus monkeys (Macaca fascicularis) drinking ethanol voluntarily and consuming a diet with moderate amounts of lipid would demonstrate any indices of alcoholic liver disease past the fatty liver stage and (2) to determine whether these alterations were accompanied by oxidative stress. Methods: Six adult male and 6 adult female cynomolgus monkeys were allowed to consume ethanol voluntarily for 18 to 19 months. Additional monkeys were maintained on the same consumption protocol, but were not provided with ethanol. During the course of the study, liver biopsy samples were monitored for lipid deposition and inflammation, serum for levels of liver enzymes, and urine for concentrations of the isoprostane (IsoP) metabolite, 2,3‐dinor‐5,6‐dihydro‐15‐F_2t‐IsoP, a biomarker for oxidative stress. Liver mitochondria were monitored for respiratory control and liver for concentrations of neutral lipids, adenine nucleotides, esterified F₂ isoprostanes, oxidized proteins, 4‐hydroxynonenal (HNE)‐protein adducts, and protein levels of cytochrome P‐450 2E1 and 3A4. Results: Ethanol consumption ranged from 0.9 to 4.05 g/kg/d over the period of the study. Serum levels of aspartate amino transferase were elevated in heavy‐consuming animals compared with those in ethanol‐naïve or moderate drinkers. Many of the ethanol consumers developed fatty liver and most showed loci of inflammation. Both hepatic energy charge and phosphorylation potential were decreased and NADH‐linked respiration was slightly, but significantly depressed in coupled mitochondria as a result of heavy ethanol consumption. The urinary concentrations of 2,3‐dinor‐5,6‐dihydro‐15‐F_2t‐IsoP increased as high as 33‐fold over that observed in ethanol‐abstinent animals. Liver cytochrome P‐450 2E1 concentrations increased in ethanol consumers, but there were no ethanol‐elicited increases in hepatic concentrations of the esterified F₂ isoprostanes, oxidized proteins, or HNE‐protein adducts. Conclusion: Our studies show that cynomolgus monkeys undergoing voluntary ethanol consumption for 1.5 years exhibit many of the features observed in the early stages of human alcoholic liver disease. Ethanol‐elicited fatty liver, inflammation, and elevated serum aspartate amino transferase were evident with a diet that contained modest amounts of polyunsaturated lipids. The dramatic increases in urinary IsoP demonstrated that the animals were being subjected to significant oxidative stress that correlated with their level of ethanol consumption.     

Comparison of ethanol metabolism in male and female cynomolgus macaques (Macaca fascicularis)

The physiological consequences of drinking ethanol differ among men and women; however, the biological basis of this gender difference is unknown. Our study characterized sex-related blood ethanol concentration (BEC) 60 min postethanol administration and ethanol elimination rates in male and female monkeys and across the phases of the menstrual cycle. Subjects were male (n = 4) and female (n = 4) cynomolgus monkeys (Macaca fascicularis) with a history of ethanol exposure and maintained at a lean body weight by food restriction. On three separate occasions, each monkey was administered 1.0 g/kg ethanol intragastrically and blood samples (20 microl) were collected every 60 min over a 5-hr period. For females, three phases of the menstrual cycle were determined by the presence of menses and plasma progesterone levels. There was no effect of menstrual cycle on mean 60 min BECs or mean rates of elimination. Mean BECs 60 min after 1.0 g/kg ethanol were: males = 86 mg/dl (+/- 2; n = 4) and females = 82 mg/dl (+/- 5; n = 4). There was no effect of sex on the highest BEC measured, which occurred at the 60 min time point in all subjects. Female monkeys did have faster average rates of ethanol elimination [34 +/- 2 (mg/dl)/hr] compared with males [23 +/- 1 (mg/dl)/hr]. The sex differences in metabolism of ethanol found with the macaque monkey model correlates well with human subject studies and suggests this is an appropriate model to further explore gender differences in response to ethanol.     

Pavlovian conditioning with ethanol: sign-tracking (autoshaping), conditioned incentive, and ethanol self-administration

BACKGROUND: Conditioned incentive theories of addictive behavior propose that cues signaling a drug's reinforcing effects activate a central motivational state. Incentive motivation enhances drug-taking and drug-seeking behavior. We investigated the behavioral response to cues associated with ethanol and their interaction with operant self-administration of ethanol. METHODS: In two experiments, rats received operant training to press a lever for a sweetened ethanol solution. After operant training, the animals were given Pavlovian pairings of a brief and localized cue light with the sweetened ethanol solution (no lever present). Lever pressing for ethanol was then re-established, and the behavioral effects of the cue light were tested during an ethanol self-administration session. RESULTS: The conditioned responses resulting from pairing cue lights with the opportunity to ingest ethanol had three main effects: (1) induction of operant behavior reinforced by ethanol, (2) stimulation of ethanol-seeking behavior (magazine entries), and (3) signal-directed behavior (i.e., autoshaping, or sign-tracking). Signal-directed behavior interacted with the other two effects in a manner predicted by the location of the cue light. These conditioned responses interact with operant responding for ethanol reinforcement. CONCLUSIONS: These findings demonstrate the importance of Pavlovian conditioning effects on ethanol self-administration and are consistent with conditioned incentive theories of addictive behavior.     

Age-dependent variation in behavior following acute ethanol administration in male and female adolescent rhesus macaques (Macaca mulatta)

Background: There has been considerable focus on the adolescent stage of development in the study of alcohol use and the etiology of alcohol‐related problems. Because adolescence is a process of dynamic change rather than a discrete or static stage of development, it is important to consider ontogenetic changes in the response to ethanol within the adolescent time period. In rodents, levels of ethanol‐induced motor impairment have been shown to increase from early to late adolescence. This study investigated associations between behavior following acute ethanol administration and age, rearing condition (mother‐reared vs nursery‐reared), and serotonin transporter (rh5‐HTTLPR) genotype in a sample of alcohol‐naïve adolescent rhesus macaques. Methods: Rhesus macaques (n=97; 41 males, 56 females), ranging in age from 28 to 48 months, were administered intravenous (IV) doses of ethanol (2.2 g/kg for males, 2.0 g/kg for females) twice in 2 separate testing sessions. A saline/ethanol group (n=16; 8 males, 6 females) was administered saline in 1 testing session and ethanol in the second session. Following each IV injection, subjects underwent a 30‐minute general motor behavioral assessment. Behavior in the saline/ethanol group was compared between the saline and ethanol‐testing sessions using analysis of variance. Behavioral data for the larger study sample were averaged between the 2 testing sessions and summarized using factor analysis. Rotated factor scores were used as dependent variables in multiple regression analyses to test for relationships between behavior and age, rearing condition, and rh5‐HTTLPR genotype. Results: During the ethanol‐testing session, behaviors indicative of motor impairment (stumbles, falls, sways, bumping the wall, and unsuccessful jumps) were frequently observed in the saline/ethanol group, while they did not occur under the saline‐testing session. Factor analysis of behavior following ethanol administration in the larger study sample yielded 3 factors: Ataxia, Impaired Jumping Ability, and Stimulation. Significant negative correlations between age and Ataxia were found for both males and females. Females also exhibited positive correlations between age and Impaired Jumping Ability and age and Stimulation. No significant correlations were found with either rearing condition or rh5‐HTTLPR genotype. Conclusions: These findings suggest that ontogenetic changes during adolescence in the behavioral response to ethanol differ between rodents and primates. Furthermore, sex differences in the behavioral response to ethanol appear to develop during adolescence.     

Neuropeptide-Y Y5 receptors modulate the onset and maintenance of operant ethanol self-administration

BACKGROUND: Neuropeptide Y (NPY) is the most abundant and widely distributed peptide in the mammalian central nervous system and increases feeding behavior at NPY Y1 or Y5 receptor subtypes. Recent pharmacological and mutant mouse data indicate that NPY activity at its receptors can influence ethanol self-administration, although the direction and strength of this influence are not clear. METHODS: Effects of the novel NPY Y5 receptor antagonist L-152,804 on the onset and maintenance of operant self-administration were examined in male C57BL/6J mice, which were trained to self-administer ethanol (10% v/v) versus water via the sucrose substitution method during 16 hr overnight sessions. After 4 months of baseline responding, mice were injected with L-152,804 (0, 10, 30, or 60 mg/kg, intraperitoneally) before operant sessions. Potential locomotor effects of L-152,804 and possible interaction with the sedative properties of ethanol also were examined. RESULTS: All three doses of L-152,804 significantly delayed the onset of ethanol-reinforced responding relative to vehicle injection. L-152,804 produced no effect on the total number of ethanol- or water-reinforced responses per 16 hr session. However, L-152,804 selectively modulated the temporal distribution of ethanol-reinforced responding depending on the dose (10 and 60 mg/kg) and time point measured in a manner consistent with blockade of ethanol reinforcement. Additional experiments determined that L-152,804 (10 or 60 mg/kg) did not alter spontaneous locomotor activity or influence the sedative effects of ethanol (4 g/kg). CONCLUSIONS: These results indicate that blockade NPY Y5 receptor activity modulates the onset and maintenance of ethanol self-administration. For this reason, NPY-Y5 receptor antagonists may be useful in medical management of alcohol abuse and alcoholism.     

Age and gender differences in response to neonatal ethanol withdrawal and polyamine challenge in organotypic hippocampal cultures

Background: Polyamines are synthesized and released in high concentrations during CNS development. These agents can potentiate N‐methyl‐D‐aspartate receptor (NMDAR) function and appear to play an important role in CNS development. Previous work has shown that polyamine release is increased during ethanol withdrawal (EWD). This likely promotes NMDAR overactivity and contributes to neurotoxicity during EWD, however, little is known regarding such effects in early neonatal brain. The present study compared the effects of EWD and polyamine exposure on toxicity in hippocampal slice cultures derived from postnatal day 2 (PND 2) or postnatal day 8 (PND 8) day‐old rats. Due to changes in NMDAR subtypes and response to polyamines, we predicted that slices taken from PND 2 pups would be more sensitive to EWD and polyamine challenge. Methods: Organotypic hippocampal slice cultures were obtained from neonatal rats either 2 or 8 days of age (PND 2 or PND 8). Five days after explantation, cultures were exposed to ETOH (50 mM‐ typically subthreshold for EWD induced cell death) for 10 days and then withdrawn from ETOH for 24‐hour in the presence of 100 μM of the polyamine spermidine and/or 100 μM ifenprodil, an NMDAR antagonist that blocks the NMDAR that is the most sensitive to polyamine modulation. Cytotoxicity was measured after 24‐hour by visualization of propidium iodide (PI) fluorescence. Results: There were clear age and gender‐dependent differences in response to EWD and to polyamines. EWD produced significant increases in PI uptake in all subregions (CA1, CA3 and DG) of cultures derived from PND 2 pups, but not PND 8 pups. Exposure of cultures to spermidine for 24‐hour also produced significant increases in cytotoxicity in all 3 regions of PND 2 cultures with no gender differences. In contrast, there were both gender and region‐specific differences in response to spermidine in cultures from PND 8. While the CA1 region of both sexes displayed increased cytotoxicity following spermidine exposure, only females showed increased cytotoxicity in the CA3 region while the DG appeared relatively insensitive to spermidine. Exposure to spermidine during EWD produced enhanced toxicity in all 3 hippocampal subregions in tissue from both PND 2 and PND 8 rats and this was reduced or prevented by co‐exposure to ifenprodil. Of interest, the PND 2 hippocampus was significantly more sensitive than the PND 8 hippocampus to the toxic effects of EWD and to spermidine during EWD in the DG and CA3 regions. Conclusions: Hippocampal slice cultures derived from PND 2 rats were more sensitive to the toxic effects of both EWD and EWD + spermidine exposure than were those derived from PND 8 rats. These findings are similar to recent behavioral data collected from our lab showing greater sensitivity to ETOH’s behavioral teratogenic effects when ETOH exposure in vivo occurred during the first postnatal week relative to the second postnatal week. Ifenprodil’s ability to block the toxic effects of spermidine during EWD suggests that excess activity of NR2B subunits of the NMDAR contributed to the excitatory and cytotoxic effects of EWD plus spermidine. While no sex differences in toxicity were observed in cultures taken from pups during the first postnatal week, these data do suggest that later in neonatal life (i.e., the second postnatal week), the female hippocampus may be more sensitive to polyamine‐induced neurotoxicity than males.     

Increased ethanol self-administration and anxiety-like behavior during acute ethanol withdrawal and protracted abstinence: regulation by corticotropin-releasing factor

BACKGROUND: Animal models of alcohol dependence suggest that long-term alterations in brain corticotropin-releasing factor (CRF) systems, key mediators of the behavioral stress response, may be involved in the development and reinstatement of dependence on drugs of abuse. The objective of the present study was to investigate the role of CRF in the regulation of ethanol self-administration and to examine the behavioral stress response during acute withdrawal and protracted abstinence. METHODS: Male Wistar rats were made dependent on ethanol via chronic exposure to ethanol vapor. Ethanol self-administration and exploratory behavior in the elevated plus maze were measured at 2 hr and 3 to 5 weeks after exposure. The role of CRF in ethanol self-administration was examined via central injection of the CRF receptor antagonist D-Phe-CRF(12-41). RESULTS: Rats showed increased responding for ethanol 2 hr and 3 to 5 weeks after chronic ethanol exposure, which was attenuated by central injection of D-Phe-CRF(12-41). In addition, rats displayed a decrease in open-arm exploration in the elevated plus maze when tested 2 hr and 4 weeks after exposure. CONCLUSIONS: These results indicate that chronic ethanol exposure leads to increased ethanol self-administration and decreased open-arm exploration in the elevated plus maze during acute withdrawal and protracted abstinence. Attenuation of ethanol self-administration via central injection of D-Phe-CRF(12-41) implicates CRF as an underlying mechanism regulating long-term motivational effects associated with alcohol dependence.     

Sex and strain differences in ethanol drinking: effects of gonadectomy

BACKGROUND: Alcohol drinking behavior in rats is known to be sexually dimorphic and strain-dependent. METHODS: To test whether the gonadal steroid milieu exerts activational effects on ethanol intake and can modulate individual sensitivity toward alcohol use and misuse, we examined the effects of gonadectomy on oral self-administration (OSA) of ethanol in male and female rats from different strains. After castration, animals were given continuous free choice between water and ethanol solutions. The ethanol concentration was progressively increased from 2% to 10% and maintained at 6% (the preferred concentration) for 24 days. Ethanol solutions were then withdrawn for 9 days. During the second phase of free-choice drinking, the ethanol concentration was gradually increased every 4 days by the following amounts, in order as listed: 6%, 12%, and 24%. RESULTS: Our results confirm both gender and strain differences in ethanol drinking: females exhibited higher ethanol intake than males, and the WKHA strain drank more than the WKY and SHR strains. However, except for a small decrease in ethanol drinking during the acquisition of ethanol OSA in males after castration, no clear-cut difference was found between gonadectomized and sham-operated animals during the maintenance of ethanol OSA behavior. CONCLUSIONS: These data suggest that gender and strain differences observed are insensitive to gonadal steroids during adulthood, and that different sensitivities to the effect of gonadal steroids do not explain the sex x strain interaction observed in ethanol drinking.     

Age differences in the expression of acute and chronic tolerance to ethanol in male and female rats

Background: Ontogenetic differences in response to ethanol (EtOH) challenge have been observed under a variety of circumstances, including varying reports of developmental differences in the expression of tolerance to EtOH. The purpose of the present experiment was to further explore potential differences in acute (AT) and chronic (CT) tolerance expression between adolescent and adult, male and female Sprague–Dawley rats, using the social interaction test. Methods: AT and CT to the social suppressing effects of a moderate dose of EtOH was assessed in adolescent and adult rats following intraperitoneal injections of 2.0 g/kg EtOH or saline daily for 10 days. At test, adults and adolescents were challenged with 1.0 or 1.25 g/kg EtOH, respectively, with AT and CT assessed at 5 and 25 minutes postinjection using ratios of impairment to brain ethanol concentrations (BrECs) at each time period (CT) and within‐session declines in impairment relative to BrECs (AT). Results: In adolescents, 10 days of EtOH pre‐exposure resulted in evidence of CT at 25 minutes postinjection, perhaps associated with an enhanced expression of AT. Among adults, signs of CT were seen at 5 minutes postinjection in adults, and may reflect neuroadaptations unassociated with AT, as with evidence of tolerance emerging only in adult control animals repeatedly exposed to saline injection prior to EtOH challenge on test day. Sex differences in tolerance expression were not observed at either age. Conclusions: Our results show ontogenetic differences between adolescents and adults in the short‐ and long‐term neuroadaptations that they express in response to repeated perturbations with EtOH. Together these findings add age of exposure and time of testing within the intoxication period as critical variables to be considered when exploring the complex relationship between AT and CT.     

The effect of naltrexone on alcohol's stimulant properties and self-administration behavior in social drinkers: influence of gender and genotype

Background: Few pharmacological treatments for alcohol dependence are available. Moreover, the best supported treatment, naltrexone hydrochloride, appears to work for only some. Methods: To investigate potential predictors of these differential responses, 40 social drinkers (20 women) were administered 6 days of treatment with naltrexone vs. placebo in a double‐blind, counterbalanced, crossover design. At the end of each treatment period, participants received a single dose of their preferred alcoholic beverage followed by the opportunity to work for additional alcohol units using a progressive ratio (PR) breakpoint paradigm. All subjects but one were genotyped for the A118G polymorphism of the mu opioid receptor gene (OPRM1). Results: Naltrexone decreased the ethanol‐induced ‘euphoria’ to a priming dose of alcohol in two subgroups: (i) in women, and (ii) in subjects with the A118G polymorphism of the mu opioid receptor gene (OPRM1). Naltrexone did not decrease motivation to work for additional alcoholic beverages on the PR task regardless of gender or genotype. Conclusions: The results add to the evidence that naltrexone decreases positive subjective effects of alcohol, with preferential effects in distinct subgroups. Similar effects in heavier drinkers might decrease alcohol use.     

Dopamine activity in the nucleus accumbens during consummatory phases of oral ethanol self-administration

BACKGROUND This present study was designed to clarify the role of dopamine in the nucleus accumbens during operant ethanol self-administration by separating bar pressing (ethanol seeking) from ethanol consumption. Furthermore, we sought to define the relationship between ethanol in the brain and the accumbal dopamine response after oral self-administration of ethanol. METHODS: Two separate groups of male Long-Evans rats were trained to bar press with 10% ethanol or water. Rats were trained to elicit an escalating number of bar presses across daily sessions before gaining access to the drinking solution for 20 min. Microdialysis was performed before (during a waiting period), during, and after bar pressing and drinking. A handling control group was included, but did not receive training. RESULTS: A significant increase in dopamine occurred during placement of the rats into the operant chamber in trained rats and handling controls. The lever-pressing period did not produce an increase in dialysate dopamine. Accumbal dopamine was increased in the first 5 min of ethanol, but not water, consumption. Ethanol appeared in the dialysate sample following ethanol availability, and peak concentrations were reached at 10 min. Most of the ethanol and water consumption occurred within 5 min of fluid access. The probes were distributed in the core (32%), shell (32%), and core plus shell (36%) regions of the nucleus accumbens. CONCLUSIONS: The enhancement of dopamine during transfer into the operant chamber does not depend on anticipation or operant training with ethanol or water reinforcement. Furthermore, the difference between the time course of accumbal dopamine and ethanol in dialysates suggests that the dopamine response is not solely due to pharmacological effects of ethanol. The dopamine response may be associated with the stimulus properties of ethanol presentation, which would be strongest during consumption.     

Gender differences in blood levels, but not brain levels, of ethanol in rats

Female rodents tend to drink more alcohol than males, a difference that emerges at puberty and appears to vary over the female estrous cycle. In addition, male and female rodents display different responses to alcohol; for example, female rats are reported to have faster elimination rates than males. We were interested in whether circulating ovarian hormones influence alcohol distribution to or elimination from the brain of rats, which might explain observed differences in drinking behavior. We administered 0.8 g/kg of ethanol via intraperitoneal injection to age-matched male and female Sprague-Dawley rats. Extracellular brain ethanol was sampled using microdialysis, and vascular ethanol concentrations were determined via tail blood collection, in two separate experiments. Ethanol pharmacokinetic parameters were calculated for both compartments. There were no differences in pharmacokinetic parameters due to gender or estrous cycle stage in brain ethanol concentration profiles. There were, however, differences in blood ethanol profiles: females showed faster elimination rates and a smaller area under the ethanol concentration versus time curve than males. In addition, the maximum concentration varied significantly across the estrous cycle. These results suggest that (1) circulating ovarian hormones do not influence alcohol distribution to the brain, but do influence distribution to more peripheral tissues such as the tail; and (2) apparent differences in tail blood alcohol levels may not reflect differences in brain levels.     

Effects of microinjection of the D2 dopamine antagonist raclopride into the ventral tegmental area on ethanol and sucrose self-administration

From previous microinjection studies, a reciprocal feedback between the nucleus accumbens and the ventral tegmental area (VTA) has been implicated in the reinforcing stimulus actions of ethanol and sucrose. In these studies, the effects of self administration of ethanol or sucrose solutions on maintained responding were similar when a dopamine antagonist was injected in the nucleus accumbens or a dopamine agonist was injected into the VTA. Our study was performed to determine if the effects on responding that had been observed when a dopamine agonist was injected into the nucleus accumbens would occur after an injection of a dopamine antagonist into the VTA. Male, Long-Evans rats were initially trained to lever press using either 10% ethanol or 75% sucrose solutions as the reinforcers. Bilateral guide cannulae were implanted to allow microinjection into the VTA of differing doses of the dopamine D2 antagonist, raclopride. Only at the highest dose tested (10 microg) was any effect observed on responding maintained by either reinforcer. The effect was minimal and different from that observed after the microinjection of a dopamine agonist into the nucleus accumbens. This suggests that either the actions of the nucleus accumbens agonist manipulation involved other processes or that the level of enhanced dopamine release in the nucleus accumbens from the VTA antagonist injection was not sufficient to mimic the effect of the nucleus accumbens agonist injections.     

Schedule-induced ethanol self-administration in DBA/2J and C57BL/6J mice

BACKGROUND: The purpose of these experiments was to provide an initial investigation into ethanol self-administration elicited in the schedule-induced polydipsia (SIP) paradigm. METHODS: Mature male mice were food deprived to between 80 and 85% of their baseline weight and received 20 daily 1 hr SIP test sessions in which a food pellet (20 mg) was delivered on a fixed-time 60 sec schedule. In different groups, the acquisition of drinking 5% (v/v) ethanol solution (experiment 1) or water (experiment 2) was recorded along with other behaviors that occurred in the test chambers. RESULTS: Results indicated that C57BL/6J mice drank significantly more ethanol than DBA/2J mice and that C57 mice achieved blood alcohol concentrations as high as 300 mg/dl. Blood alcohol concentrations were consistently correlated with g/kg ethanol intake. The groups did not differ in consumption of water. SIP test sessions using higher concentrations of ethanol (10-20% v/v, experiment 1) or sucrose solutions (0.1-2% w/v, experiment 2) then were performed. Group differences in ethanol consumption were maintained at all ethanol concentrations. Although DBAs drank more of a low concentration of sucrose (0.1%), when expressed as g/kg, sucrose intake was equivalent in the two strains at all concentrations. Analysis of the time course of drinking clearly showed that this behavior was adjunctive in nature. CONCLUSION: These results demonstrate the effectiveness of this procedure in inducing ethanol self-administration and its utility for investigating the genetic bases of vulnerability toward excessive ethanol consumption.     

Effects of SR141716A on ethanol and sucrose self-administration

BACKGROUND: Previous studies have demonstrated that administration of central cannabinoid receptor (CB1) ligands can produce marked effects on ingestive behaviors. However, the possible relationship to ethanol self-administration has not been fully examined. The present series of experiments was designed to characterize further the role of CB1 receptors in appetitive and consummatory behaviors related to sucrose and ethanol. METHODS: To determine the relative contribution of CB1 receptors to ethanol seeking and consumption, a series of experiments was designed using the sipper-tube model. In this paradigm, the appetitive and consummatory phases of ethanol and sucrose self-administration are separated. In the appetitive phase, animals are required to complete a response requirement (16 lever presses) within 20 min. If the requirement is successfully completed, access to a sipper tube containing either sucrose or ethanol (consummatory phase) is made available for 20 min. RESULTS: In the ethanol condition, the CB1 receptor antagonist SR141716A (0.3-3.0 mg/kg, ip) produced dose-related decreases in the probability of response requirement completion without significantly affecting latency to first lever press or overall lever press rate. In the sucrose condition, SR141716A (0.3-3.0 mg/kg, ip) increased first lever press latency without affecting lever press rate. In the consummatory phase, SR141716A (0.3-3.0 mg/kg, ip) administration markedly decreased total intake and the total number of licks for both ethanol and sucrose. CONCLUSIONS: These data indicate that CB1 receptors are involved in mediating both appetitive and consummatory aspects of ingestive behaviors related to sucrose and ethanol.     

Infection of owl monkeys (Aotus trivirgatus) and cynomolgus monkeys (Macaca fascicularis) with hepatitis E virus from Mexico.

Owl and cynomolgus monkeys were inoculated with hepatitis E virus (HEV) to compare disease models and produce antibody and virus. By immune electron microscopy (IEM), all six owl monkeys were shown to have serologic responses manifested by unusually high levels of anti-HEV at 6 months, but only three developed hepatitis. Virus-related antigen in liver (HEV Ag) was detected by immunofluorescence microscopy of biopsies from two of four owl monkeys; one with HEV Ag also had HEV in acute-phase bile (detected by IEM) and feces (detected by infecting another owl monkey). In contrast, cynomolgus monkeys propagated HEV to higher levels and all five had hepatitis. Moderate-to-high levels of HEV Ag correlated with detectable HEV in bile for both species. Thus, the value of using HEV-infected cynomolgus was confirmed. Owl monkeys were shown to be HEV-susceptible and sources of high-level anti-HEV; Sustained anti-HEV in these monkeys may also be useful for understanding immune responses.     

Operant self-administration of ethanol in Sardinian alcohol-preferring rats

BACKGROUND: "Work" for ethanol, that is, the ability of a laboratory animal to press a lever to gain access to ethanol, has been proposed as (a) a requirement for definition of an animal model of alcoholism and (b) a measure of ethanol-reinforcing properties. The present study evaluated oral self-administration of ethanol under an operant (lever pressing) procedure in selectively bred Sardinian alcohol-preferring (sP) and alcohol-nonpreferring (sNP) rats. METHODS: Rats from both lines were initiated to self-administer 10% ethanol, on a fixed ratio 1 schedule and in daily 30 min sessions, by using the Samson sucrose fading procedure. Subsequently, rats were exposed to increasing concentrations of ethanol up to 30% on a fixed ratio 4 schedule. Finally, the extinction responding for ethanol, defined as the maximal number of lever responses reached by each rat in the absence of ethanol reinforcement, was determined. RESULTS: The results indicated that sP rats acquired and maintained lever pressing for ethanol, self-administering mean amounts of ethanol in the range of 0.6 to 1.1 g/kg/session, which gave rise to mean blood ethanol levels in the 30 to 45 mg% range. Extinction responding for ethanol in sP rats averaged 73. In contrast, once sucrose was faded out, sNP rats displayed minimal levels of responding for ethanol, and extinction responding averaged 6. CONCLUSIONS: The results of the present study extend to the sP/sNP rat lines the finding that ethanol can be established as a reinforcer in selectively bred alcohol-preferring rats, whereas it has modest, if any, reinforcing properties in alcohol-nonpreferring rats.     

Individual differences in hyperlipidemia and vitamin E status in response to chronic alcohol self-administration in cynomolgus monkeys

Background: Chronic ethanol self‐administration induces oxidative stress and exacerbates lipid peroxidation. α‐Tocopherol is a potent lipid antioxidant and vitamin that is dependent upon lipoprotein transport for tissue delivery. Methods: To evaluate the extent to which vitamin E status is deranged by excessive alcohol consumption, monkeys voluntarily drinking ethanol (1.36 to 3.98 g/kg/d for 19 months, n = 11) were compared with nondrinkers (n = 5, control). Results: Three alcohol‐drinking animals developed hyperlipidemia with plasma triglyceride levels (1.8 ± 0.9 mM) double those of normolipidemic (NL) drinkers (0.6 ± 0.2) and controls (0.6 ± 0.3, p < 0.05); elevated plasma cholesterol (3.6 ± 0.5 mM) compared with NL drinkers (2.3 ± 0.2, p < 0.05) and controls (2.9 ± 0.3); and lower plasma α‐tocopherol per triglycerides (14 ± 6 mmol/mol) than controls (27 ± 8) and NL drinkers (23 ± 6, p < 0.05). Hyperlipidemic monkey liver α‐tocopherol (47 ± 15 nmol/g) was lower than NL drinkers (65 ± 13) and controls (70 ± 15, p = 0.080), as was adipose α‐tocopherol (84 ± 37 nmol/g) compared with controls (224 ± 118) and NL drinkers (285 ± 234, p < 0.05). Plasma apolipoprotein (apo) CIII increased compared to baseline at both 12 and 19 months in the normolipidemic (p = 0.0016 and p = 0.0028, respectively) and in the hyperlipidemic drinkers (p < 0.05 and p < 0.05, respectively). Plasma apo H concentrations at 19 months were elevated hyperlipidemics (p < 0.05) relative to concentrations in control animals. C‐reactive protein (CRP), a marker of inflammation, was increased compared to baseline at both the 12‐ and 19‐month time points in the normolipidemic (p = 0.005 and p = 0.0153, respectively) and hyperlipidemic drinkers (p = 0.016 and p = 0.0201, respectively). Conclusion: A subset of alcohol‐drinking monkeys showed a predisposition to alcohol‐induced hyperlipidemia. The defect in lipid metabolism resulted in lower plasma α‐tocopherol per triglycerides and depleted adipose tissue α‐tocopherol, and thus decreased vitamin E status.     

Noncontingent and response-contingent intravenous ethanol attenuates the effect of naltrexone on hypothalamic-pituitary-adrenal activity in rhesus monkeys

BACKGROUND: The mechanism by which the opioid antagonist naltrexone suppresses overconsumption of ethanol is unclear. Oral ethanol consumption in humans increases hypothalamic-pituitary-adrenal (HPA) activity, and recent studies suggest that naltrexone may reduce ethanol consumption by modifying the HPA-stimulating effects of ethanol. The purpose of this study was to measure in rhesus monkeys the effects of ethanol and naltrexone, alone and in combination, on plasma levels of adrenocorticotropin hormone (ACTH). METHODS: Nine adult male and female rhesus monkeys with chronic, indwelling intravenous catheters were maintained on tethers that allowed ethanol delivery and blood sampling. In one study, the monkeys received intramuscular injections of saline or 0.32 mg/kg naltrexone followed by noncontingent intravenous bolus infusions of saline or 0.3 to 1.8 g/kg ethanol. In a second study, other monkeys were given intramuscular injections of saline or 0.01 to 0.3 mg/kg naltrexone and subsequently responded on levers to receive intravenous saline or ethanol 0.03 g/kg per injection. RESULTS: Ethanol, delivered either response contingently or noncontingently, did not produce systematic changes in ACTH plasma levels. Naltrexone alone produced increases in plasma ACTH that were attenuated by the subsequent administration of noncontingent or response-contingent ethanol. Naltrexone also produced dose-dependent reductions in intravenous ethanol self-administration. Linear regression analysis indicated that ethanol intake was negatively correlated with the plasma levels of ACTH over time. CONCLUSIONS: The route of administration may modulate ethanol's effects on HPA activity. Ethanol may attenuate naltrexone's effect on the HPA axis by impairing HPA axis sensitivity to other stimuli. The negative correlation between ethanol intake and ACTH levels supports the notion that naltrexone's effect of increasing HPA axis activity may be related to its ability to suppress ethanol consumption.     

Controlled and behaviorally relevant levels of oral ethanol intake in rhesus macaques using a flavorant-fade procedure

BACKGROUND: Flavorant-fading procedures can initiate and maintain oral ethanol intake in rodents. The present study developed a similar procedure to achieve controlled and behaviorally relevant levels of ethanol intake in monkeys. METHODS: Male rhesus macaques (N = 13) were initially given the opportunity to consume 0.5 g/kg of a 1% (w/v) ethanol plus 4% (w/v) Tang solution in 1-hr limited-access sessions without the requirement of an operant response. Once consumption was stable at a particular concentration (%) and/or amount (g/kg), animals were given access to higher concentrations and/or amounts of ethanol. Animals were tested on a bimanual motor skill (BMS) task 20 and 90 min after consumption to assess behavioral impairment. Blood alcohol levels (BALs) were assessed after a session in which animals had the opportunity to consume up to 3.0 g/kg of 6% (w/v) ethanol. RESULTS: The gradual fading up of higher concentrations and amounts of ethanol resulted in controlled and robust levels (>2.0 g/kg) of ethanol intake in half of the subjects. Increasing the concentration of the sweetener from 4 to 6% (w/v) was effective in initiating consumption in three animals. Two monkeys required the additional step of presenting the increased-sweetener solutions after a meal (postprandial consumption) to initiate significant ethanol intake. Animals were significantly impaired on the BMS task after consumption of 2.0, 2.5, and 3.0 g/kg of ethanol. Individual consumption ranging from 0.8 to 3.0 g/kg of ethanol produced BALs of 18 to 269 mg/dl. CONCLUSIONS: The flavorant-fading procedure was effective in producing behaviorally relevant levels of ethanol consumption in rhesus macaques. This model facilitated a randomized-dose procedure to determine the behavioral effects of 0.5 to 3.0 g/kg of ethanol. This procedure therefore is of significant utility in determining behavioral or physiologic effects of specific doses of consumed ethanol in monkeys.