Ethanol as a prodrug: brain metabolism of ethanol mediates its reinforcing effects--a commentary

Background: This commentary discusses a study by Karahanian and colleagues (2011) on the role of central nervous system acetaldehyde in the reinforcing effects of ethanol. The goal is to emphasize the importance of the study and to discuss future directions. Results: This important paper solidifies the idea that the levels of acetaldehyde in the central nervous system have profound effects in mediating the reinforcing actions of ethanol. This is accomplished by manipulating the brain levels of acetaldehyde produced from ethanol by the injection of lentivirus containing either an anti‐catalase shRNA construct or a rat liver alcohol dehydrogenase into the central nervous system and observing the effects on alcohol preference by high ethanol‐consuming rats. A factor not directly considered is that acetaldehyde is further metabolized to acetate, which also has some behavioral actions. Conclusions: The efficacy of lentivirus injections of enzyme inhibitors or enzymes themselves to alter a behavioral response to ethanol is clearly demonstrated here. The many other actions of ethanol that are postulated to be a result of the production of acetaldehyde in the brain remain to be investigated by similar techniques. Possible “therapeutic avenues to reduce chronic alcohol use” are envisioned.     

Effects of ethanol exposure on subsequent acquisition and extinction of ethanol self-administration and expression of alcohol-seeking behavior in adult alcohol-preferring (P) rats: I. Periadolescent exposure

Background The current study examined the effects of ethanol (EtOH) drinking during periadolescence on the subsequent acquisition and extinction of operant self‐administration of EtOH and expression of alcohol‐seeking behavior in adult alcohol‐preferring (P) rats to test the hypothesis that alcohol drinking during periadolescence produces enduring alterations that enhance the reinforcing properties of EtOH. Methods Periadolescent female P rats were given 24 hr free‐choice access to 15% (v/v) EtOH starting at postnatal day (PND) 30 and ending on PND 60 or were similarly housed and received water only. On PND 75, without any prior training, periadolescent alcohol‐drinking and periadolescent alcohol‐naïve rats were placed in standard two‐lever (15% EtOH and water) chambers to examine acquisition of EtOH self‐administration with a fixed ratio (FR) 1 schedule of reinforcement. After the acquisition phase and after stable responding was established on an FR5 for EtOH and FR1 for water, P rats underwent extinction training for both EtOH and water rewards. After extinction training and a 2 week home cage period, rats were returned to the operant chambers in the absence of reward for seven consecutive sessions (Pavlovian spontaneous recovery). After this testing period, animals were maintained in their home cage for a week before being returned to the operant chambers and allowed to respond for EtOH and water (reacquisition). Results Compared with periadolescent alcohol‐naïve rats, periadolescent alcohol‐drinking rats acquired EtOH responding sooner (i.e., in the first acquisition session), displayed a greater resistance to extinguish EtOH responding (i.e., higher levels of responding in sessions 4–6), had higher responding for more sessions on the EtOH lever in the absence of reward after a prolonged home cage rest period, and had a more prolonged elevated level of EtOH responding during reacquisition (four sessions versus one session). Conclusions Overall, the results suggest that periadolescent EtOH drinking by P rats produced long‐lasting alterations in the reinforcing effects of alcohol, which increased the likelihood that alcohol drinking would be initiated in adulthood, decreased the likelihood that once adult alcohol drinking began it could be extinguished easily, and increased the potential for relapse.     

Reward and relapse: complete gene-induced dissociation in an animal model of alcohol dependence

Background: In animal models of continuous alcohol self‐administration, in which physical dependence does not constitute the major factor of ethanol intake, 2 factors likely contribute to the perpetuation of alcohol self‐administration: (i) the rewarding effects of ethanol and (ii) the contextual conditioning cues that exist along with the process of self‐administration. Present studies are aimed at understanding the relative contribution of these factors on the perpetuation of heavy alcohol self‐administration, as an indication of relapse. Methods: Wistar‐derived UChB high ethanol drinker rats were allowed access to 10% ethanol and water on a 24‐hour basis. In initial studies, an anticatalase shRNA gene‐coding lentiviral vector aimed at inhibiting acetaldehyde generation was administered into the ventral tegmental area (VTA) of the animals prior to ethanol access. In subsequent studies, the lentiviral vector was administered to animals, which had consumed ethanol on a 24‐hour basis, or a 1‐hour basis, after the animals had reached high levels of ethanol intake for 60 to 80 days. In final studies, quinine (0.01%) was added to the ethanol solution to alter the conditioning taste/smell cues of alcohol that animals had chronically ingested. Results: Data indicate that the administration of an anticatalase vector into the VTA of naïve animals blocked reward and alcohol self‐administration, while it was, nevertheless, inactive in inhibiting alcohol self‐administration in rats that had been conditioned to ingest ethanol for over 2 months. The lack of inhibitory effect of the anticatalase vector on ethanol intake in animals that had chronically self‐administered ethanol was fully reversed when the contextual conditioning cues of the alcohol solution were changed. Conclusions: Data highlight the importance of conditioning factors in relapse and suggest that only abolishing or blunting it, along with long‐lasting pharmacological treatment to reduce ethanol reward, may have protracted effects in reducing alcohol self‐administration.     

Environmental Modulation of Alcohol Intake in Hamsters: Effects of Wheel Running and Constant Light Exposure

Background: Alcohol abuse leads to marked disruptions of circadian rhythms, and these disturbances in themselves can increase the drive to drink. Circadian clock timing is regulated by light, as well as by nonphotic influences such as food, social interactions, and wheel running. We previously reported that alcohol markedly disrupts photic and nonphotic modes of circadian rhythm regulation in Syrian hamsters. As an extension of this work, we characterize the hedonic interrelationship between wheel running and ethanol (EtOH) intake and the effects of environmental circadian disruption (long‐term exposure to constant light [LL]) on the drive to drink. Methods: First, we tested the effect of wheel running on chronic free‐choice consumption of a 20% (v/v) EtOH solution and water. Second, the effect of this alcohol drinking on wheel running in alcohol‐naive animals was investigated. Third, we assessed the influence of LL, known to suppress locomotor activity and cause circadian rhythm disruption, on EtOH consumption and wheel‐running behavior. Results: Inhibitory effects of wheel running on EtOH intake and vice versa were observed. Exposure to LL, while not affecting EtOH intake, induced rhythm splitting in 75% of the animals. Notably, the splitting phenotype was associated with lower levels of EtOH consumption and preference prior to, and throughout, the period of LL exposure. Conclusions: These results are evidence that exercise may offer an efficacious clinical approach to reducing EtOH intake. Also, predisposition for light‐induced (or other) forms of circadian disruption may modulate the drive to drink.     

Role of novelty and ethanol history in locomotor stimulation induced by binge-like ethanol intake

Background: The acute locomotor effects of voluntary ethanol (EtOH) intake in mice (stimulation/sedation) might be important behavioral indicators of an animals’ propensity to engage in EtOH consumption and/or EtOH seeking behaviors. Using a binge‐like EtOH intake model dubbed “Drinking‐in‐the‐Dark (DID),” we recently observed home cage locomotor stimulation in C57BL/6J mice during an acute EtOH intake session, but acute home cage locomotor sedation following repeated EtOH exposures. To determine the role of novelty and/or EtOH history on these previously described locomotor effects, and to determine the relationship between these variables on locomotor activity immediately following DID intake, we conducted 2 separate experiments. Methods: In experiment 1, mice were given access to either EtOH or water, and locomotor activity was monitored immediately afterwards. In experiment 2, mice were given 13 days access to EtOH or water solution while home cage locomotor activity was monitored. On the 14th day, half of the water consuming animals received EtOH access for the first time. On the 15th day, all animals received EtOH access, and locomotion was assessed afterwards in locomotor activity testing chambers. Results: In experiment 1, locomotor activity following DID was positively associated with EtOH intake and blood EtOH concentrations (BECs). In experiment 2, the group that received EtOH for the first time on the 14th day did not display locomotor stimulation. Locomotor activity following DID EtOH intake was positively associated with BECs in all groups regardless of EtOH history. Conclusions: These results suggest that (i) DID‐induced locomotor stimulation in the home cage may involve relative familiarity with the DID procedures, and (ii) locomotor stimulation immediately following DID is directly related to the relative concentration of EtOH in blood; an effect that is not altered by prior EtOH history. These data add new evidence of the pharmacological actions of binge‐like EtOH intake, and provide a basis by which we may explore the motivation and consequences of such binge consumption.     

Increase in Alcohol Intake,Reduced Flexibility of Alcohol Drinking,and Evidence of Signs of Alcohol Intoxication in Sardinian Alcohol‐Preferring Rats Exposed to Intermittent Access to 20% Alcohol

Background: This study assessed in Sardinian alcohol‐preferring (sP) rats a procedure known to promote alcohol drinking and based on the intermittent (once every other day) access to 2 bottles containing alcohol (20%, v/v) and water, respectively (Wise, 1973). Methods: To this end, sP rats were exposed – under the 2‐bottle choice regimen – to: (i) 10% (v/v) alcohol with continuous access (CA10%; i.e., the procedure under which sP rats had been selectively bred); (ii) 10% (v/v) alcohol with intermittent access (IA10%); (iii) 20% (v/v) alcohol with continuous access (CA20%); (iv) 20% (v/v) alcohol with intermittent access (IA20%; the “Wise” condition) (Experiment 1). Additional experiments assessed the influence of (i) adulteration with quinine of the alcohol solution (Experiment 2) and (ii) concurrent presentation of a saccharin solution (Experiment 3) on alcohol drinking under the CA10% and IA20% conditions. Finally, it was assessed whether alcohol drinking under the CA10% and IA20% conditions resulted in motor incoordination at the Rota‐Rod task, as a possible sign of alcohol intoxication (Experiment 4). Results: Daily alcohol intake markedly escalated in rats exposed to the IA20% condition, averaging 9.0 g/kg (in comparison with the average intake of 6.5 g/kg in the CA10% rat group). CA20% and IA10% rats displayed intermediate values of daily alcohol intake between those of CA10% and IA20% rats. Alcohol intake was virtually abolished by addition of quinine or by concurrent presentation of the saccharin solution in CA10% rats; conversely, alcohol intake in IA20% rats was only partially affected by gustatory aversion or concurrent presentation of an alternative reinforcer. Finally, alcohol intake in IA20%, but not in CA10%, rats resulted in clear motor‐incoordinating effects. Conclusions: These data suggest that the “Wise” procedure is effective in inducing marked increases in alcohol intake in sP rats. These increases are associated with a reduced flexibility of alcohol drinking (suggesting the development of “behavioral” dependence) and produce signs of alcohol intoxication that are not detected when sP rats are exposed to the more conventional CA10% condition.     

Impact of the Innate Immune Response in the Actions of Ethanol on the Central Nervous System

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

Low-dose stimulatory effects of ethanol during adolescence in rat lines selectively bred for high alcohol intake

Abstract : Background: The low‐dose stimulatory effect of ethanol (EtOH) in rats has been hypothesized to reflect its hedonic effects and to be associated with a genetic predisposition toward high alcohol preference. To test the hypothesis that phenotypes associated with high alcohol preference in adulthood are also present in adolescent rats at the time of onset of alcohol drinking, the current study examined the effects of EtOH on locomotor activity (LMA) during adolescence in lines of rats selectively bred for divergent alcohol intakes. Methods: Subjects were adolescent (31–40 days of age) rats from the alcohol‐preferring (P) and ‐nonpreferring (NP) lines and from the high–alcohol‐drinking (HAD) and low–alcohol‐drinking (LAD) replicate lines. On day 1, all subjects (n= 8–10/line/gender/dose) received intraperitoneal saline injections and were placed in the activity monitor for 30 min. On day 2, subjects received intraperitoneal saline or 0.25, 0.50, 0.75, 1.0, or 1.5 g EtOH/kg. Results: The LMA of male and female P rats was increased with low doses (0.25–0.75 g/kg) and decreased at the highest dose (1.5 g/kg) of EtOH. Similar effects were observed with low doses of EtOH on the LMA of HAD‐1 and HAD‐2 rats. None of the EtOH doses stimulated LMA in the NP, LAD‐1, or LAD‐2 rats, although all of the low–alcohol‐intake lines of rats showed decreased LMA at the highest dose of EtOH. Only the P rats among the high–alcohol‐consuming lines of rats showed decreased LMA at the highest dose of EtOH. Conclusion: Selective breeding for high alcohol consumption seems to be associated with increased sensitivity to the low‐dose stimulating effects of EtOH and reduced sensitivity to the high‐dose motor‐impairing effects of ethanol. The expression of these phenotypes emerges during adolescence by the age of onset of alcohol‐drinking behavior.     

Tolerance to ethanol's ataxic effects and alterations in ethanol-induced locomotion following repeated binge-like ethanol intake using the DID model

Background: Tolerance to the behavioral and subjective effects of alcohol (ethanol) is thought to be a major predictive factor for the development of alcoholism. Evidence from rodent models has supported this view with those animals most likely to develop tolerance generally drinking and preferring ethanol more so than those resistant to it. Despite this evidence, very little is known about the behavioral relationships between ethanol‐induced tolerance and consumption. The goal of this study was to evaluate the development of tolerance to the ataxic effects of ethanol using a mouse model of binge‐like intake dubbed “Drinking in the Dark” (DID; Physiol Behav 2005, 84:53–63). We hypothesized that mice would become tolerant to the ataxic effects of ethanol as this behavior is known to be altered at the blood ethanol concentrations reached using this model (≥80 mg/dl). Methods: To evaluate this, we gave daily DID ethanol or water access sessions to male C57BL/6J (B6) mice and monitored ataxia (and in some cases locomotion) at various time points. Results: In general, mice given 14 consecutive days of ethanol access displayed tolerance to the ataxic effects of ethanol compared to water‐drinking controls. These effects were coupled with alterations in locomotor behavior and in some cases differences in ethanol pharmacokinetics. Conclusions: Thus, we can conclude that tolerance to the behavioral effects of binge‐like ethanol intake might play a key role in the daily maintenance of this behavior and that these effects may be evidence of important neuroadaptations involved in the development of alcoholism.     

Commentary on Day and Colleagues : The Association Between Prenatal Alcohol Exposure and Behavior at 22 Years of Age—Adverse Effects of Risky Patterns of Drinking Among Low to Moderate Alcohol‐Using Pregnant Women

Day and colleagues have presented the first data showing that the behavioral effects of low to moderate prenatal alcohol exposure seen in childhood and adolescence persist into adulthood. Using the Achenbach Adult Self‐Report, they found dose‐dependent effects of prenatal exposure on internalizing, externalizing, and attention problems that persist in young adults and, thus, appear to be permanent. To date, few studies have attempted to identify thresholds at which prenatal alcohol exposure is harmful, although the animal literature suggests that even 1 to 2 binge episodes can result in adverse effects in the offspring. Four prospective longitudinal studies have reported adverse effects at what can be characterized as moderate exposure levels based on NIAAA criteria, but moderate drinking women often concentrate their alcohol use on 1 to 2 days per week, thereby engaging in binge drinking. In this study, binge drinking was not a strong predictor of adverse outcome when average daily dose was held constant, a conclusion that the authors note runs “counter to studies that have reported that binge drinking has a greater effect.” This inconsistency may be due to the difficulty of allocating variance that is shared (overlapping) between average daily dose and binge drinking (i.e., dose/occasion). Data from laboratory animal studies, in which dosage can be manipulated experimentally, demonstrate that a higher dose per occasion, the key feature of binge drinking, leads to more severe adverse effects. Day and colleagues' findings of adverse effects at low levels of exposure provides clear evidence that there is no safe level of drinking during pregnancy and that, even at low levels, drinking results in irreversible behavioral impairment. On the other hand, given the evidence from the animal and most human studies, it is important for all women who drink during pregnancy, even at light to moderate levels, to recognize that minimizing their intake per occasion and refraining from binge drinking can reduce risk to the fetus.     

A Metric of Maternal Prenatal Risk Drinking Predicts Neurobehavioral Outcomes in Preschool Children

Background: Fetal Alcohol Spectrum Disorders (FASDs), including Fetal Alcohol Syndrome, continue to be high‐incidence developmental disorders. Detection of patterns of maternal drinking that place fetuses at risk for these disorders is critical to diagnosis, treatment, and prevention, but is challenging and often insufficient during pregnancy. Various screens and measures have been used to identify maternal risk drinking but their ability to predict child outcome has been inconsistent. This study hypothesized that a metric of fetal “at‐risk” alcohol exposure (ARAE) derived from several indicators of maternal self‐reported drinking would predict alcohol‐related neurobehavioral dysfunctions in children better than individual measures of maternal alcohol consumption alone. Methods: Self‐reported peri‐conceptional and repeated maternal drinking during pregnancy were assessed with semi‐structured interviews and standard screens, i.e., the CAGE, T‐ACE, and MAST, in a prospective sample of 75 African‐American mothers. Drinking volumes per beverage type were converted to standard quantity and frequency measures. From these individual measures and screening instruments, a simple dichotomous index of prenatal ARAE was defined and used to predict neurobehavioral outcomes in the 4‐ to 5‐year‐old offspring of these women. Study outcomes included IQ, attention, memory, visual‐motor integration, fine motor skill, and behavior. Statistical analyses controlled for demographic and other potential confounders. Results: The current “at‐risk” drinking metric identified over 62% of the mothers as drinking at risk levels—23% more than the selection criterion identified—and outperformed all individual quantity and frequency consumption measures, including averages of weekly alcohol use and “binge” alcohol exposures (assessed as intake per drinking occasion), as well as an estimate of the Maternal Substance Abuse Checklist ( Coles et al., 2000 ), in predicting prenatal alcohol‐related cognitive and behavioral dysfunction in 4‐ to 5‐year‐old children. Conclusions: A metric reflecting multiple indices of “at‐risk” maternal alcohol drinking in pregnancy had greater utility in predicting various prenatal alcohol‐related neurobehavioral dysfunction and deficits in children compared to individual measures of maternal self‐reported alcohol consumption or a previous maternal substance abuse index. Assessing fetal risk drinking in pregnant women was improved by including multiple indicators of both alcohol consumption and alcohol‐related consequences and, if appropriate practical applications are devised, may facilitate intervention by health care workers during pregnancy and potentially reduce the incidence or severity of FASDs.     

Reverse microdialysis of a dopamine uptake inhibitor in the nucleus accumbens of alcohol-preferring rats: effects on dialysate dopamine levels and ethanol intake

Background: The mesolimbic dopamine (DA) system has been implicated in mediating the reinforcing actions of ethanol (EtOH). This study examines the effects of local perfusion of the DA uptake inhibitor GBR12909 (GBR) on (1) DA levels in the nucleus accumbens (NAc) and (2) EtOH drinking in alcohol‐preferring rats. Methods: Stable drinking of a 15% (v/v) EtOH solution (minimum of 0.75 g/kg body weight) was established in daily 1 hr limited access sessions. Rats were then implanted with bilateral guide cannulae aimed 4 mm above the NAc. After recovery from surgery, concentric microdialysis probes (2 mm dialysis membrane surface) were inserted into the NAc. Most placements were in the shell or overlapping both shell and core. Two days later, the probes were perfused at 1.0 μl/min with artificial cerebral spinal fluid (aCSF) for at least a 90 min washout period followed by collection of five basal samples over 150 min. Rats were then perfused with either aCSF alone or 10, 25, 100, or 200 μM of GBR for 240 min on the first day of microdialysis. During the last 60 min of the drug treatment phase, rats were given their scheduled access to 15% EtOH. All rats were then perfused with aCSF for the last 90 min of the experiment. The following day, the procedure was repeated, but animals that received aCSF on the first day were given a dose of GBR and rats given GBR on the first day received only aCSF. Results: GBR perfusion increased extracellular NAc DA levels dose dependently to more than 800% of basal levels at 100 to 200 μM but failed to alter EtOH intake (p > 0.05, paired t test) at any concentration tested. Moreover, after 100 μM of GBR perfusion had terminated, the extracellular levels of DA in the NAc remained elevated for approximately 24 hr (790% of day 1 basal;p < 0.05). The increase in dialysate DA levels observed during GBR perfusion with 100 μM was significantly greater for EtOH‐experienced rats than for EtOH‐naïve rats [F (7,59) = 14.85, p < 0.0001, analysis of variance, Student‐Newman‐Keuls post hoc test]. Conclusions: The results suggest (1) that EtOH drinking experience induces neuroadaptations that increase DA release in the NAc, and (2) that additional elevation in synaptic levels of DA in the NAc does not influence the maintenance of ongoing alcohol drinking under scheduled access conditions in alcohol‐preferring animals.     

Modulation of Brain Endocannabinoid Levels by Voluntary Alcohol Consumption in Alcohol-Preferring AA Rats

Background: The central nervous system cannabinoid CB1 receptors have been implicated in regulation of alcohol consumption. Less data are available on the role of the endogenous ligands for these receptors, anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG), in alcohol‐related behaviors. The purpose of this study was to assess the effects of voluntary alcohol consumption on the levels of these endocannabinoids in key brain areas mediating alcohol reinforcement. Methods: Female and male alcohol‐preferring AA (Alko, Alcohol) rats were trained to drink 10% (v/v) alcohol during 90‐min limited access sessions every second day. Following establishment of stable alcohol drinking, half of the subjects were killed immediately before the daily alcohol access (“pre‐session” group), while the other half was killed after the drinking session (“post‐session” group). A separate control group consisted of water‐drinking rats. AEA and 2‐AG levels were measured from prefrontal cortex (PFC), nucleus accumbens (NAc), caudate putamen (CPu), amygdala, and hippocampus using liquid chromatography–tandem mass spectrometry (LC/MS/MS). Results: Voluntary alcohol drinking caused widespread alterations in the levels of both AEA and 2‐AG. Compared to the water group, increased AEA levels were seen in the pre‐session group, but they were decreased immediately following limited access drinking in the female AA rats. Also 2‐AG levels were significantly elevated after long alcohol exposure, and an additional increase was found after limited access drinking in PFC. In males, however, the only alterations caused by alcohol drinking were significantly elevated AEA levels in NAc and CPu in the post‐session group. No changes were seen in the levels of 2‐AG. Conclusions: These results demonstrate that voluntary alcohol drinking modulates the levels of endocannabinoids in several brain areas implicated in alcohol reinforcement. AEA and 2‐AG were differentially affected, suggesting that they could have partially separate modulatory roles. Alterations were more widespread in females than males, possibly reflecting their higher alcohol intake. Taken together, alcohol‐induced release of endocannabinoids may have an important role in alcohol reinforcement and development of alcohol addiction.     

Fetal and infantile alcohol-mediated associative learning in the rat

BACKGROUND: Infant rats express conditioned responses to an odor experienced prenatally as a chemosensory cue associated with moderate alcohol intoxication. This study examined postnatal intake of a chemosensory cue (cineole) that had been paired with alcohol's unconditioned effects. It also tested the interaction between prenatal association and postnatal conditioning with cineole and alcohol. METHODS: Pregnant female rats intubated with cineole were given ethanol (EtOH).25 or 4.0 hr later. Other groups received only water or water paired with ethanol. During postnatal day 15 (PD15), infant consumption of cineole solution was assessed. After the cineole drinking test, pups were intubated with EtOH or water to assess infant conditioning. On PD16, all pups were tested for mouthing to milk alone or to a milk-cineole solution. RESULTS: Statistical analysis confirmed fetal associative conditioning attributable to the unconditioned effects of prenatal alcohol. Fetuses given explicit pairings of cineole and alcohol ingested less cineole on PD15 than control fetuses given a 4-hr interval between cineole and alcohol. On PD16, consumption of cineole was significantly increased by prenatal exposure to cineole. Teratogenic effects of this dose of prenatal alcohol did not affect postnatal associative or nonassociative behavior. CONCLUSIONS: Prenatal associative learning can be established through temporal contiguity between fetal chemosensory stimulation and alcohol's unconditioned properties. This associative memory survives to infancy and modulates intake patterns and behavioral reactivity to substances that were prenatally paired with alcohol intoxication.     

Effects of ethanol exposure on subsequent acquisition and extinction of ethanol self-administration and expression of alcohol-seeking behavior in adult alcohol-preferring (P) rats: II. Adult exposure

Background In a preceding study, we reported that ethanol (EtOH) consumption during periadolescence in alcohol‐preferring (P) rats produced significant effects on the acquisition, extinction, Pavlovian spontaneous recovery (PSR), and reacquisition of operant self‐administration of EtOH. The objective of the present study was to determine if EtOH consumption during adulthood produced similar effects on subsequent operant behaviors. Methods Adult female P rats (>135 days of age) were given 24 hr free‐choice access to 15% EtOH for 30 days or were similarly housed and received water only. After a 15 day period of no EtOH access and without any prior training, adult alcohol drinking and adult alcohol‐naïve rats were placed in standard two‐lever (15% EtOH and water) chambers to examine acquisition of EtOH self‐administration. After stable responding was established on a concurrent fixed ratio (FR) 5 FR1 schedule for EtOH versus water, the P rats underwent extinction training for nine sessions. After extinction and a 2 week home cage period (with no operant sessions or access to EtOH), rats were returned to the operant chambers in the absence of reward for seven consecutive sessions to test for PSR. After PSR testing, animals were maintained in their home cage for a week, before being reintroduced to the operant chambers and allowed to respond for EtOH and water. Results Both the adult alcohol‐drinking and adult alcohol‐naïve groups rapidly acquired EtOH self‐administration, expressed a pronounced PSR, which was augmented by EtOH priming and the presence of a discriminative stimulus (odor cue), and increased responding when EtOH was reinstated. Adult pre‐exposure to EtOH did not alter any of the operant measures. Conclusions The results of this study suggest that, unlike the results with EtOH pre‐exposure during periadolescence, chronic alcohol drinking by P rats in adulthood did not produce sufficient long‐lasting changes in neuronal function to alter subsequent operant acquisition of alcohol self‐administration, alcohol relapse, or alcohol‐seeking behavior.     

Effects of Ethyl Alcohol Administration to THA Rat Dams During Their Gestation Period on Learning Behavior and on Levels of Monoamines and Metabolites in the Brain of Pups after Birth

Children born from chronic alcoholic mothers have shown behavioral teratogenic effects more frequently than morphological malformations. To investigate the possible mechanisms and evaluate maternal alcohol dosage levels to induce behavioral dysfunctions, we gave pregnant Tokai High Avoider (THA) rats 0, 5,10, and 20% ethanol (EtOH) as drinking water during the gestation period. We evaluated the brain function of pups born of alcohol-administered dams. Sidman avoidance behavior test and the levels of monoamines (nor-adrenalin, dopamine, and serotonin) and metabolites (3,4-dihydroxy-phenyl acetic acid, homovanillic acid, and 5-hydroxyindole acetic acid) in whole brain were examined for neurobehavioral and neurochemical effects. EtOH-exposed THA offsprings showed high pre-and postnatal mortality, growth deficits, and brain weight reductions. Compared with the results of the same conditioning experiment using Wistar rats, the THA rat may have higher susceptibility to the effects of in utero EtOH exposure than Wistar rats. EtOH-exposed THA pups exhibited deficits in avoidance operant learning that were not shown in Wistar rats. We also observed the increased levels of all monoamines that were assumed to be related with the deficit of learning, the decreased levels of 3,4-dihydroxyphenyl acetic acid and homovanillic acid, and the unchanged levels of 5-hydroxyindole acetic acid in pups from dams administered 10 and 20% EtOH. However, contrary alteration of monoamines and their metabolites were shown in pups from 5% EtOH-administered dams.     

Suppression of Heavy Drinking and Alcohol Seeking by a Selective ALDH-2 Inhibitor

Background: Inherited human aldehyde dehydrogenase 2 (ALDH‐2) deficiency reduces the risk for alcoholism. Kudzu plants and extracts have been used for 1,000 years in traditional Chinese medicine to treat alcoholism. Kudzu contains daidzin, which inhibits ALDH‐2 and suppresses heavy drinking in rodents. Decreased drinking due to ALDH‐2 inhibition is attributed to aversive properties of acetaldehyde accumulated during alcohol consumption. However, daidzin can reduce drinking in some rodents without necessarily increasing acetaldehyde. Therefore, a selective ALDH‐2 inhibitor might affect other metabolic factors involved in regulating drinking. Methods: Aldehyde dehydrogenase 2 inhibitors were synthesized based on the co‐crystal structure of ALDH‐2 and daidzin. We tested the efficacy of a highly selective reversible ALDH‐2 inhibitor, CVT‐10216, in models of moderate and high alcohol drinking rats. We studied 2‐bottle choice and deprivation‐induced drinking paradigms in Fawn Hooded (FH) rats, operant self‐administration in Long Evans (LE), FH, and inbred P (iP) rats and in cue‐induced reinstatement in iP rats. We also assayed blood acetaldehyde levels as well as dopamine (DA) release in the nucleus accumbens (NAc) and tested possible rewarding/aversive effects of the inhibitor in a conditioned place preference (CPP) paradigm. Results: CVT‐10216 increases acetaldehyde after alcohol gavage and inhibits 2‐bottle choice alcohol intake in heavy drinking rodents, including deprivation‐induced drinking. Moreover, CVT‐10216 also prevents operant self‐administration and eliminates cue‐induced reinstatement of alcohol seeking even when alcohol is not available (i.e., no acetaldehyde). Alcohol stimulates DA release in the NAc, which is thought to contribute to increased drinking and relapse in alcoholism. CVT‐10216 prevents alcohol‐induced increases in NAc DA without changing basal levels. CVT‐10216 does not show rewarding or aversive properties in the CPP paradigm at therapeutic doses. Conclusion: Our findings suggest that selective reversible ALDH‐2 inhibitors may have therapeutic potential to reduce excessive drinking and to suppress relapse in abstinent alcoholics.     

Early Social Isolation in Male Long-Evans Rats Alters Both Appetitive and Consummatory Behaviors Expressed During Operant Ethanol Self-Administration

Background: Postweaning social isolation in rats produces profound and long‐lasting cognitive and behavioral deficits in adult animals. Importantly, this housing manipulation alters sensitivity to a number of drugs of abuse including ethanol. However, most studies with ethanol have utilized continuous or limited home‐cage access to examine interactions between juvenile social experience and drinking. More recently, social isolation was shown to increased ethanol responding in a “dipper” model of self‐administration ( Deehan et al., 2007 ). In the current study, we utilize a “sipper” operant self‐administration model to distinguish the effects of isolation rearing on ethanol seeking‐ and drinking‐related behaviors. Methods: Postweaning juvenile male Long‐Evans rats were placed into 2 housing groups for 6 weeks: one group consisted of individually housed animals; the second group was housed 4 animals per cage. Following the isolation period, anxiety‐like behavior was assessed to confirm the efficacy of the isolation procedure. In some animals, ethanol drinking in the home cage was assessed using a continuous access, 2‐bottle choice paradigm. All animals were then individually housed and trained to lever‐press for a sipper tube containing either an ethanol solution or a sucrose solution. Results: Postweaning social isolation increased the expression of anxiety‐like behavior in the elevated plus maze but not the light‐dark box. Ethanol consumption was also increased during continuous home‐cage access with the 2‐bottle choice paradigm. During operant self‐administration, isolation housing increased the response rate and increased ethanol consumption but did not alter responding for or consumption of sucrose. The housing manipulation did not change the total number of lever responses during extinction sessions. Paired‐pulse inhibition deficits that are characteristic of juvenile isolation remained intact after prolonged experience with sucrose self‐administration. Discussion: The effects of postweaning social isolation on ethanol drinking in the home cage are also manifest during operant self‐administration. Importantly, these alterations in adult operant self‐administration are ethanol‐specific.     

Involvement of arginine vasopressin and V1b receptor in alcohol drinking in Sardinian alcohol-preferring rats

Background: Recent animal studies have shown that the level of stress‐responsive arginine vasopressin (AVP) gene expression in the amygdala is increased during early withdrawal from long‐term heroin or cocaine administration. The selective AVP V1b receptor antagonist SSR149415 (capable of exerting antidepressant‐like and anxiolytic effects in animal models) also blocked stress‐induced reinstatement of drug‐seeking behavior. This study was undertaken to investigate the effects of alcohol and to determine whether (i) there are genetically determined differences in basal AVP mRNA levels in the medial/central amygdala (Me/CeA) and medial hypothalamus (MH) between selectively bred Sardinian alcohol‐preferring (sP) and alcohol‐nonpreferring (sNP) rats; (ii) the AVP mRNA levels are altered by long‐term alcohol drinking in sP rats; and (iii) the V1b receptor antagonist SSR149415 alters alcohol drinking in sP rats. Methods: In Experiment 1, AVP mRNA levels were measured in the Me/CeA and MH of alcohol‐naïve sP and sNP rats, and sP rats exposed to the standard, homecage 2‐bottle “alcohol versus water” choice regimen 24 h/d for 17 days. In Experiment 2, SSR149415 (0, 3, 10, or 30 mg/kg; intraperitoneal) was acutely administered 30 minutes before lights off to alcohol‐experienced sP rats. Alcohol, water, and food intake were monitored 6 and 24 hours later. Results: We found higher basal AVP mRNA levels in both Me/CeA and MH of alcohol‐naïve sP than sNP rats; alcohol consumption decreased AVP mRNA levels in both brain regions of sP rats, suggesting genetically determined differences between the 2 rat lines and in the effects of alcohol drinking in sP rats. Acute treatment with SSR149415 significantly reduced alcohol intake of sP rats. Conclusion: The stress‐responsive AVP/V1b receptor system is 1 component of the neural circuitry underlying high alcohol drinking in sP rats.     

Behavioral traits predicting alcohol drinking in outbred rats: an investigation of anxiety, novelty seeking, and cognitive flexibility

Background: Most adults in Western society consume alcohol regularly without negative consequences. For a small subpopulation, however, drinking can quickly progress to excessive and chronic intake. Given the dangers associated with alcohol abuse, it is critical to identify traits that may place an individual at risk for developing these behaviors. To that end, we used a rat model to determine whether anxiety‐related behaviors, novelty seeking, or cognitive flexibility predict excessive alcohol drinking under both limited and continuous access conditions. Methods: Adult male rats were assessed in a series of behavioral tasks (elevated plus maze [EPM], locomotor activity, and discrimination/reversal learning in a Y‐maze) followed by 6 weeks of daily, 1‐hour access to alcohol in a free‐choice, 2‐bottle paradigm (10% alcohol vs. tap water). Next, subjects were given the opportunity to consume alcohol for 72 hours in drinking chambers that permit separate measures of each drinking bout. Half of the animals experienced a 2‐week deprivation period between the limited and continuous access sessions. Results: Time spent on the open arms of the EPM, but not novelty seeking or discrimination/reversal learning, predicted alcohol consumption during limited, 1‐h/d access sessions to alcohol. Anxiety‐related behavior also predicted the escalation of intake when animals were given 72 hours of continuous access to alcohol. Bout size, but not frequency, was responsible for the increased consumption by high‐anxiety subjects during this period. Finally, intake during limited access sessions predicted intake during continuous access, but only in subjects with low intake during limited access. Conclusions: These findings confirm that preexisting anxiety‐related behavior predicts alcohol intake under several schedules of alcohol access. Moreover, when access is unlimited, the high‐anxiety‐related group exhibited an increase in bout size, but not frequency, of drinking. In addition, we show that modest intake when alcohol is restricted may or may not progress to excessive intake when the drug is freely available.