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1.  Malondialdehyde-acetaldehyde (MAA) adducted proteins bind to scavenger receptor A in airway epithelial cells 
Alcohol (Fayetteville, N.Y.)  2014;48(5):493-500.
Co-exposure to cigarette smoke and ethanol generates malondialdehyde and acetaldehyde, which can subsequently lead to the formation of aldehyde-adducted proteins. We have previously shown that exposure of bronchial epithelial cells to malondialdehyde-acetaldehyde (MAA) adducted protein increases protein kinase C (PKC) activity and proinflammatory cytokine release. A specific ligand to scavenger receptor A (SRA), fucoidan, blocks this effect. We hypothesized that MAA-adducted protein binds to bronchial epithelial cells via SRA. Human bronchial epithelial cells (BEAS-2B) were exposed to MAA-adducted protein (either bovine serum albumin [BSA-MAA] or surfactant protein D [SPD-MAA]) and SRA examined using confocal microscopy, fluorescent activated cell sorting (FACS), and immunoprecipitation. Differentiated mouse tracheal epithelial cells (MTEC) cultured by air-liquid interface were assayed for MAA-stimulated PKC activity and keratinocyte-derived chemokine (KC) release. Specific cell surface membrane dye co-localized with upregulated SRA after exposure to MAA for 3–7 min and subsided by 20 min. Likewise, MAA-adducted protein co-localized to SRA from 3–7 min with a subsequent internalization of MAA by 10 min. These results were confirmed using FACS analysis and revealed a reduced mean fluorescence of SRA after 3 min. Furthermore, increased amounts of MAA-adducted protein could be detected by Western blot in immunoprecipitated SRA samples after 3 min treatment with MAA. MAA stimulated PKCε-mediated KC release in wild type, but not SRA knockout mice. These data demonstrate that aldehyde-adducted proteins in the lungs rapidly bind to SRA and internalize this receptor prior to the MAA-adducted protein stimulation of PKC-dependent inflammatory cytokine release in airway epithelium.
doi:10.1016/j.alcohol.2014.02.005
PMCID: PMC4128016  PMID: 24880893
lung; airway epithelial; scavenger receptor; malondialdehyde; acetaldehyde
2.  Intravenous Alcohol Self-Administration in the P Rat 
Alcohol (Fayetteville, N.Y.)  2014;48(5):419-425.
Alcohol consumption produces a complex array of effects that can be divided into two types: the explicit pharmacological effects of ethanol (which can be temporally separate from time of intake) and the more temporally “relevant” effects (primarily olfactory and taste) that bridge the time from intake to onset of the pharmacological effects. Intravenous (IV) self-administration of ethanol limits the confounding “non-pharmacological” effects associated with oral consumption, allows for controlled and precise dosing, and bypasses first order absorption kinetics, allowing for more direct and better-controlled assessment of alcohol’s effect on the brain. IV ethanol self-administration has been reliably demonstrated in mouse and human experimental models; however, models of IV self-administration have been historically problematic in the rat. An operant multiple-schedule study design was used to elucidate the role of each component of a compound IV-ethanol plus oral-sucrose reinforcer. Male alcohol-preferring P rats had free access to both food and water during all IV self-administration sessions. Animals were trained to press a lever for orally delivered 1% sucrose (1S) on a fixed ratio 4 schedule, and then surgically implanted with an indwelling jugular catheter. Animals were then trained to respond on a multiple FR4-FR4 schedule composed of alternating 2.5-min components across 30-min sessions. For the multiple schedule, two components were used: an oral 1S only and an oral 1S plus IV 20% ethanol (25 mg/kg/injection). Average total ethanol intake was 0.47 ± 0.04 g/kg. We found significantly higher earning of sucrose-only reinforcers and greater sucrose-lever error responding relative to the compound oral-sucrose plus IV-ethanol reinforcer. These response patterns suggest that sucrose, not ethanol, was responsible for driving overall responding. The work with a compound IV ethanol-oral sucrose reinforcer presented here suggests that the existing intravenous ethanol self-administration methodology cannot overcome the aversive properties of ethanol via this route in the rat.
doi:10.1016/j.alcohol.2013.12.007
PMCID: PMC4096581  PMID: 24835637
alcohol-preferring; multiple schedule; conflict
3.  Prenatal alcohol exposure alters expression of neurogenesis-related genes in an ex vivo cell culture model 
Alcohol (Fayetteville, N.Y.)  2014;48(5):483-492.
Prenatal alcohol exposure can lead to long-lasting changes in functional and genetic programs of the brain, which may underlie behavioral alterations seen in Fetal Alcohol Spectrum Disorder (FASD). Aberrant fetal programming during gestational alcohol exposure is a possible mechanism by which alcohol imparts teratogenic effects on the brain; however, current methods used to investigate the effects of alcohol on development often rely on either direct application of alcohol in vitro or acute high doses in vivo. In this study, we used our established moderate prenatal alcohol exposure (PAE) model, resulting in maternal blood alcohol content of approximately 20 mM, and subsequent ex vivo cell culture to assess expression of genes related to neurogenesis. Proliferating and differentiating neural progenitor cell culture conditions were established from telencephalic tissue derived from embryonic day (E) 15–17 tissue exposed to alcohol via maternal drinking throughout pregnancy. Gene expression analysis on mRNA derived in vitro was performed using a microarray, and quantitative PCR was conducted for genes to validate the microarray. Student's t tests were performed for statistical comparison of each exposure under each culture condition using a 95% confidence interval. Eleven percent of genes on the array had significantly altered mRNA expression in the prenatal alcohol-exposed neural progenitor culture under proliferating conditions. These include reduced expression of Adora2a, Cxcl1, Dlg4, Hes1, Nptx1, and Vegfa and increased expression of Fgf13, Ndn, and Sox3; bioinformatics analysis indicated that these genes are involved in cell growth and proliferation. Decreased levels of Dnmt1 and Dnmt3a were also found under proliferating conditions. Under differentiating conditions, 7.3% of genes had decreased mRNA expression; these include Cdk5rap3, Gdnf, Hey2, Heyl, Pard6b, and Ptn, which are associated with survival and differentiation as indicated by bioinformatics analysis. This study is the first to use chronic low to moderate PAE, to more accurately reflect maternal alcohol consumption, and subsequent neural progenitor cell culture to demonstrate that PAE throughout gestation alters expression of genes involved in neural development and embryonic neurogenesis.
doi:10.1016/j.alcohol.2014.06.001
PMCID: PMC4096774  PMID: 24954023
prenatal; alcohol; chronic; gene expression; neurogenesis; cell culture; development; epigenetic; DNA methylation
4.  Chronic intermittent ethanol exposure during adolescence: effects on social behavior and ethanol sensitivity in adulthood 
Alcohol (Fayetteville, N.Y.)  2014;48(5):433-444.
This study assessed long-lasting consequences of repeated ethanol exposure during two different periods of adolescence on 1) baseline levels of social investigation, play fighting, and social preference and 2) sensitivity to the social consequences of acute ethanol challenge. Adult male and female Sprague-Dawley rats were tested 25 days after repeated exposure to ethanol (3.5 g/kg intragastrically [i.g.], every other day for a total of 11 exposures) in a modified social interaction test. Early-mid adolescent intermittent exposure (e-AIE) occurred between postnatal days (P) 25–45, whereas late adolescent intermittent exposure (l-AIE) was conducted between P45–65. Significant decreases in social investigation and social preference were evident in adult male rats, but not their female counterparts following e-AIE, whereas neither males nor females demonstrated these alterations following l-AIE. In contrast, both e-AIE and l-AIE produced alterations in sensitivity to acute ethanol challenge in males tested 25 days after adolescent exposure. Ethanol-induced facilitation of social investigation and play fighting, reminiscent of that normally seen during adolescence, was evident in adult males after e-AIE, whereas control males showed an age-typical inhibition of social behavior. Males after l-AIE were found to be insensitive to the socially suppressing effects of acute ethanol challenge, suggesting the development of chronic tolerance in these animals. In contrast, females showed little evidence for alterations in sensitivity to acute ethanol challenge following either early or late AIE. The results of the present study demonstrate a particular vulnerability of young adolescent males to long-lasting detrimental effects of repeated ethanol. Retention of adolescent-typical sensitivity to the socially facilitating effects of ethanol could potentially make ethanol especially appealing to these males, therefore promoting relatively high levels of ethanol intake later in life.
doi:10.1016/j.alcohol.2014.01.012
PMCID: PMC4096834  PMID: 24928792
adolescence; ethanol exposure; social behavior; anxiety; social facilitation; sex differences
5.  Adolescent and adult rat cortical protein kinase A display divergent responses to acute ethanol exposure 
Alcohol (Fayetteville, N.Y.)  2014;48(5):463-470.
Adolescent rats display reduced sensitivity to many dysphoria-related effects of alcohol (ethanol) including motor ataxia and sedative hypnosis, but the underlying neurobiological factors that contribute to these differences remain unknown. The cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) pathway, particularly the type II regulatory subunit (RII), has been implicated in ethanol-induced molecular and behavioral responses in adults. Therefore, the current study examined cerebral cortical PKA in adolescent and adult ethanol responses. With the exception of early adolescence, PKA RIIα and RIIβ subunit levels largely did not differ from adult levels in either whole cell lysate or P2 synaptosomal expression. However, following acute ethanol exposure, PKA RIIβ P2 synaptosomal expression and activity were increased in adults, but not in adolescents. Behaviorally, intracerebroventricular administration of the PKA activator Sp-cAMP and inhibitor Rp-cAMP prior to ethanol administration increased adolescent sensitivity to the sedative-hypnotic effects of ethanol compared to controls. Sp-cAMP was ineffective in adults whereas Rp-cAMP suggestively reduced loss of righting reflex (LORR) with paralleled increases in blood ethanol concentrations. Overall, these data suggest that PKA activity modulates the sedative/hypnotic effects of ethanol and may potentially play a wider role in the differential ethanol responses observed between adolescents and adults.
doi:10.1016/j.alcohol.2014.01.011
PMCID: PMC4159958  PMID: 24874150
protein kinase A; cAMP; ethanol; adolescence
6.  Ethosuximide reduces electrographical and behavioral correlates of alcohol withdrawal seizure in DBA/2J mice 
Alcohol (Fayetteville, N.Y.)  2014;48(5):445-453.
Chronic alcohol abuse depresses the nervous system and, upon cessation, rebound hyperexcitability can result in withdrawal seizure. Withdrawal symptoms, including seizures, may drive individuals to relapse, thus representing a significant barrier to recovery. Our lab previously identified an upregulation of the thalamic T-type calcium (T channel) isoform CaV3.2 as a potential contributor to the generation and propagation of seizures in a model of withdrawal. In the present study, we examined whether ethosuximide (ETX), a T-channel antagonist, could decrease the severity of ethanol withdrawal seizures by evaluating electrographical and behavioral correlates of seizure activity. DBA/2J mice were exposed to an intermittent ethanol exposure paradigm. Mice were treated with saline or ETX in each withdrawal period, and cortical EEG activity was recorded to determine seizure severity. We observed a progression in seizure activity with each successive withdrawal period. Treatment with ETX reduced ethanol withdrawal-induced spike and wave discharges (SWDs), in terms of absolute number, duration of events, and contribution to EEG power reduction in the 6–10 Hz frequency range. We also evaluated the effects of ETX on handling-induced convulsions. Overall, we observed a decrease in handling-induced convulsion severity in mice treated with ETX. Our findings suggest that ETX may be a useful pharmacological agent for studies of alcohol withdrawal and treatment of resulting seizures.
doi:10.1016/j.alcohol.2014.01.010
PMCID: PMC4378959  PMID: 24933286
alcohol; withdrawal; seizure; ethosuximide; T-type calcium channel
7.  Effects of the Novel Cannabinoid CB1 Receptor Antagonist PF 514273 on the Acquisition and Expression of Ethanol Conditioned Place Preference 
Alcohol (Fayetteville, N.Y.)  2014;48(5):427-431.
The centrally expressed cannabinoid receptor (CB1) has been considered a potential therapeutic target in treating alcoholism. Though CB1 receptors have been shown to modulate primary and conditioned ethanol reward, much of this research employed animal models that require ethanol ingestion or oral routes of administration. This is problematic considering CB1 antagonist drugs have high anorectic liability and have been used clinically in the treatment of obesity. Therefore, the present study examined CB1 antagonism in DBA/2J mice using an unbiased ethanol-induced conditioned place preference (CPP) procedure, a paradigm that does not require ethanol ingestion. To evaluate the role of CB1 receptors in primary ethanol reward, the highly potent and selective novel CB1 antagonist 2-(2-chlorophenyl)-3-(4-chlorophenyl)-7-(2,2-difluoropropyl)-6,7-dihydro-2H-pyrazolo[3,4-f][1,4]oxazepin-8(5H)-one (PF 514273) was administered 30 min before place preference conditioning with a fixed dose of ethanol (acquisition). To evaluate the role of CB1 receptors in ethanol-conditioned reward, PF 514273 was administered 30 min before place preference testing (expression). Although PF 514273 reduced ethanol-stimulated and basal locomotor activity, it did not perturb the acquisition or expression of ethanol-induced CPP. Results from the present study appear inconsistent with other studies that have demonstrated a role for CB1 antagonism in ethanol reward using oral administration paradigms. Our findings suggest that CB1 antagonism may have greater involvement in consummatory behavior than ethanol reward.
doi:10.1016/j.alcohol.2014.01.013
PMCID: PMC4188509  PMID: 24954022
ethanol; reward; place preference; locomotor activity; CB1; PF 514273; rimonabant; SR141716; inbred mice; DBA/2J
8.  Serotonin-3 Receptors in the Posterior Ventral Tegmental Area Regulate Ethanol Self-Administration of Alcohol-Preferring (P) Rats 
Alcohol (Fayetteville, N.Y.)  2010;44(3):245-255.
Several studies indicated the involvement of serotonin-3 (5-HT3) receptors in regulating alcohol-drinking behavior. The objective of this study was to determine the involvement of 5-HT3 receptors within the ventral tegmental area (VTA) in regulating ethanol self-administration by alcohol-preferring (P) rats. Standard two-lever operant chambers were used to examine the effects of 7 consecutive bilateral micro-infusions of ICS205-930 (ICS), a 5-HT3 receptor antagonist, directly into the posterior VTA on the acquisition and maintenance of 15% (v/v) ethanol self-administration. P rats readily acquired ethanol self-administration by the 4th session. The three highest doses (0.125, 0.25 and 1.25 ug) of ICS prevented acquisition of ethanol self-administration. During the acquisition post-injection period, all rats treated with ICS demonstrated higher responding on the ethanol lever, with the highest dose producing the greatest effect. In contrast, during the maintenance phase, the 3 highest doses (0.75, 1.0 and 1.25 ug) of ICS significantly increased responding on the ethanol lever; following the 7-day dosing regimen, responding on the ethanol lever returned to control levels. Micro-infusion of ICS into the posterior VTA did not alter the low responding on the water lever, and did not alter saccharin (0.0125% w/v) self-administration.. Micro-infusion of ICS into the anterior VTA did not alter ethanol self-administration. Overall, the results of this study suggest that 5-HT3 receptors in the posterior VTA of the P rat may be involved in regulating ethanol self-administration. In addition, chronic operant ethanol self-administration, and/or repeated treatments with a 5-HT3 receptor antagonist may alter neuronal circuitry within the posterior VTA.
doi:10.1016/j.alcohol.2010.01.002
PMCID: PMC4516283  PMID: 20682192
alcohol-preferring rats; serotonin-3 receptor; ethanol self-administration; ventral tegmental area; ICS 205-930
9.  New evidence of ethanol's anxiolytic properties in the infant rat 
Alcohol (Fayetteville, N.Y.)  2014;48(4):367-374.
Ethanol induces appetitive, aversive, and anxiolytic effects that are involved in the development of ethanol use and dependence. Because early ethanol exposure produces later increased responsiveness to ethanol, considerable effort has been devoted to analysis of ethanol's appetitive and aversive properties during early ontogeny. Yet, there is a relative scarcity of research related to the anxiolytic effects of ethanol during early infancy, perhaps explained by a lack of age-appropriate tests. The main aim of this study was to validate a model for the assessment of ethanol's anxiolytic effects in the infant rat (postnatal days 13– 16). The potentially anxiolytic effects of ethanol tested included: i) amelioration of conditioned place aversion, ii) ethanol intake in the presence of an aversive conditioned stimulus, iii) the inhibitory behavioral effect in an anxiogenic environment, and iv) innate aversion to a brightly illuminated area in a modified light/dark paradigm. Ethanol doses employed across experiments were 0.0, 0.5, and 2.0 g/kg. Results indicated that a low ethanol dose (0.5 g/kg) was effective in attenuating expression of a conditioned aversion. Ethanol intake, however, was unaffected by simultaneous exposure to an aversive stimulus. An anxiogenic environment diminished ethanol-induced locomotor stimulation. Finally, animals given 0.5 g/kg ethanol and evaluated in a light/dark box showed increased time spent in the illuminated area and increased latency to escape from the brightly lit compartment than rats treated with a higher dose of ethanol or vehicle. These new results suggest that ethanol doses as low as 0.5 g/kg are effective in ameliorating an aversive and/or anxiogenic state in preweanling rats. These behavioral preparations can be used to assess ethanol's anxiolytic properties during early development.
doi:10.1016/j.alcohol.2014.01.007
PMCID: PMC4035812  PMID: 24776303
anxiolysis; ethanol motivational properties; conditioned aversion; light/dark test; infant rat
10.  Assessing behavioral control across reinforcer solutions on a fixed-ratio schedule of reinforcement in rats 
Alcohol (Fayetteville, N.Y.)  2014;48(4):337-344.
Instrumental behavior can shift from flexible, goal-directed actions to automatic, stimulus-response actions. The satiety-specific devaluation test assesses behavioral flexibility by evaluating reward seeking after temporary devaluation of the reinforcer via satiety; a decrease in responding compared to control conditions indicates goal-directed behavior. We have observed variability in the outcome of this test that may be dependent on the reinforcer. Another test of habit, contingency degradation, involves changing the action-outcome association over the course of retraining and determines whether reward seeking is sensitive to changing contingencies. We hypothesized that the outcome of the contingency-degradation test would remain consistent across reinforcers, while the satiety-specific devaluation test may vary across reinforcers because it depends on the ability of the reinforcer to induce satiety. Therefore, we trained rats to self-administer 1.5% sucrose, 10% sucrose, 10% ethanol, or 10 mM monosodium glutamate (MSG) on a fixed-ratio (FR5) schedule that has been shown to promote long-term, goal-directed responding. Next, behavioral flexibility was evaluated in three satiety-specific devaluation tests over 6 weeks. Finally, we investigated reward seeking after contingency-degradation training. All groups displayed sensitivity to satiety-specific devaluation in the first test, indicating goal-directed behavior. While the 10% sucrose and ethanol groups remained goal-directed, the 1.5% sucrose and MSG groups exhibited habit-like behavior in later tests. Nevertheless, all groups displayed decreased responding in an extinction session after contingency-degradation training, indicating goal-directed behavior. These results demonstrate that tests of behavioral flexibility can yield dissimilar results in the same rats. Next, rats from the 1.5% sucrose group underwent the entire experiment again, now self-administering 10% sucrose. These rats showed pronounced goal-directed behavior in satiety-specific and contingency-degradation tests under 10% sucrose conditions, further suggesting that the reinforcer solution affected the outcome of the satiety-specific devaluation test. We conclude that reinforcer characteristics should be considered when investigating habit-like behavior in alcohol research.
doi:10.1016/j.alcohol.2013.12.006
PMCID: PMC4031023  PMID: 24680666
ethanol; monosodium glutamate; sucrose; maltodextrin; satiety; operant
11.  Habit Formation: Implications for Alcoholism Research 
Alcohol (Fayetteville, N.Y.)  2014;48(4):327-335.
Characteristics of individuals with severe alcohol use disorders include heightened cue sensitivity, compulsive seeking, craving, and continued alcohol use in the face of negative consequences. Animal models are useful for understanding behavioral and neurological mechanisms underlying problematic alcohol use. Seeking of operant reinforcers including alcohol is processed by two mechanisms, commonly referred to as “goal-directed” (action-outcome) and “habitual” (stimulus-response). As substance use disorders are characterized by continued use regardless of unfavorable outcomes, it is plausible that drug use causes an unnatural disruption of these mechanisms. We present a critical analysis of literature pertaining to behavioral neuroscience alcoholism research involving habit formation.
Traditionally, when operant behavior is unaffected by a loss of subjective value of a reinforcer (devaluation), the behavior is considered habitual. Acquisition of instrumental behavior requires corticostriatal mechanisms that depend heavily on the prefrontal cortex and ventral striatum, whereas practiced behavior is more predominantly controlled by the dorsal striatum. Dopaminergic signaling is necessary for the neurological adaptations involved in stimulus-response action, and drugs of abuse appear to facilitate habitual behavior through high levels of dopamine release. Evidence suggests that the use of alcohol as a reinforcer expedites habit formation, and that a history of alcohol use produces alterations in striatal morphology, aids habit learning for non-psychoactive reinforcers, and promotes alcohol drinking despite aversive adulterants.
In this review, we suggest directions for future alcoholism research that seeks to measure action made despite a devalued outcome, including procedural modifications and genotypic, pharmacological, or neurological manipulations. Most alcoholism models currently in use fail to reach substantial blood ethanol concentrations, a shortcoming that may be alleviated through the use of high-drinking rodent lines. Additionally, satiety, one common mechanism of devaluing reinforcers, is not recommended for alcohol research because the psychoactive effects of alcohol depress response rates, mimicking devaluation effects. Overall, further research of habit formation and potentially related perseverative behaviors could be invaluable in discovering genetic variance, traits that correlate with persistent alcohol seeking, implicated neural structures and processes of alcohol use, and eventually novel pharmacological treatment for alcoholism.
doi:10.1016/j.alcohol.2014.02.004
PMCID: PMC4096986  PMID: 24835007
alcohol; habit learning; self-administration; goal-directed behavior; reinforcer devaluation; animal model
12.  Effect of Sub-Chronic Intermittent Ethanol Exposure on Spatial Learning and Ethanol Sensitivity in Adolescent and Adult Rats 
Alcohol (Fayetteville, N.Y.)  2014;48(4):353-360.
It has become clear that adolescence is a period of distinct responsiveness to the acute effects of ethanol on learning and other cognitive functions. However, the effects of repeated intermittent ethanol exposure during adolescence on learning and cognition are less well studied, and other effects of repeated ethanol exposure such as withdrawal and chronic tolerance complicate such experiments. Moreover, few studies have compared the effects of repeated ethanol exposure during adolescence and adulthood, and they have yielded mixed outcomes that may be related to methodological differences and/or secondary effects of ethanol on behavioral performance. One emerging question is whether relatively brief intermittent ethanol exposure (i.e., sub-chronic exposure) during adolescence or adulthood might alter learning at a time after exposure when chronic tolerance would be expected, and whether tolerance to the cognitive effects of ethanol might influence the effect of ethanol on learning at that time. To address this, male adolescent and adult rats were pre-treated with sub-chronic daily ethanol (five doses [4.0 g/kg, i.p.] or saline at 24-h intervals, across 5 days). Two days after the last pre-exposure, spatial learning was assessed on 4 consecutive days using the Morris water maze. Half of the animals from each treatment cell received ethanol (2.0 g/kg, i.p.) 30 min prior to each testing session and half of the animals received saline. Ethanol pre-exposure altered water maze performance in adolescent animals but not in adults, and acute ethanol exposure impaired learning in animals of both ages independent of pre-exposure condition. There was no evidence of cognitive tolerance in animals of either age group. These results indicate that a relatively short period of intermittent ethanol exposure during adolescence, but not adulthood, alters baseline water maze performance shortly after pre-exposure and does not induce cognitive tolerance to the effects of ethanol in either age group.
doi:10.1016/j.alcohol.2014.02.003
PMCID: PMC4084686  PMID: 24795209
adolescent; learning; water maze; tolerance; alcohol
13.  “Drinking in the Dark” (DID) Procedures: A Model of Binge-Like Ethanol Drinking in Non-Dependent Mice 
Alcohol (Fayetteville, N.Y.)  2013;48(3):235-241.
This review provides an overview of an animal model of binge-like ethanol drinking that has come to be called “drinking in the dark” (DID), a procedure that promotes high levels of ethanol drinking and pharmacologically relevant blood ethanol concentrations (BECs) in ethanol-preferring strains of mice. Originally described by Rhodes et al. (2005), the most common variation of the DID procedure, using singly housed mice, involves replacing the water bottle with a bottle containing 20% ethanol for 2 to 4 hours, beginning 3 hours into the dark cycle. Using this procedure, high ethanol drinking strains of mice (e.g., C57BL/6J) typically consume enough ethanol to achieve BECs greater than 100 mg/dL and to exhibit behavioral evidence of intoxication. This limited access procedure takes advantage of the time in the animal’s dark cycle in which the levels of ingestive behaviors are high, yet high ethanol intake does not appear to stem from caloric need. Mice have the choice of drinking or avoiding the ethanol solution, eliminating the stressful conditions that are inherent in other models of binge-like ethanol exposure in which ethanol is administered by the experimenter, and in some cases, potentially painful. The DID procedure is a high throughput approach that does not require extensive training or the inclusion of sweet compounds to motivate high levels of ethanol intake. The high throughput nature of the DID procedure makes it useful for rapid screening of pharmacological targets that are protective against binge-like drinking and for identifying strains of mice that exhibit binge-like drinking behavior. Additionally, the simplicity of DID procedures allows for easy integration into other paradigms, such as prenatal ethanol exposure and adolescent ethanol drinking. It is suggested that the DID model is a useful tool for studying the neurobiology and genetics underlying binge-like ethanol drinking, and may be useful for studying the transition to ethanol dependence.
doi:10.1016/j.alcohol.2013.08.005
PMCID: PMC4004717  PMID: 24275142
drinking-in-the-dark; binge-like; ethanol; intoxication; blood-ethanol; dependence
14.  The alcohol-preferring (P) and high-alcohol-drinking (HAD) rats – Animal Models of Alcoholism 
Alcohol (Fayetteville, N.Y.)  2013;48(3):209-215.
The objective of this article is to review the literature on the utility of using the selectively bred alcohol-preferring (P) and high-alcohol-drinking (HAD) lines of rats in studies examining high alcohol drinking in adults and adolescents, craving-like behavior, and the co-abuse of alcohol with other drugs. The P line of rats meets all of the originally proposed criteria for a suitable animal model of alcoholism. In addition, the P rat exhibits high alcohol-seeking behavior, demonstrates an alcohol deprivation effect (ADE) under relapse drinking conditions, consumes amounts of ethanol during adolescence equivalent to those consumed in adulthood, and co-abuses ethanol and nicotine. The P line also exhibits excessive binge-like alcohol drinking, attaining blood alcohol concentrations (BACs) of 200 mg% on a daily basis. The HAD replicate lines of rats have not been as extensively studied as the P rats. The HAD1,2 rats satisfy several of the criteria for an animal model of alcoholism, e.g., these rats will voluntarily consume ethanol in a free-choice situation to produce BACs between 50–200 mg%. The HAD1,2 rats also exhibit an ADE under repeated relapse conditions, and will demonstrate similar levels of ethanol intake during adolescence as seen in adults. Overall, the P and HAD1,2 rats have characteristics attributed to an early onset alcoholic, and can be used to study various aspects of alcohol use disorders.
doi:10.1016/j.alcohol.2013.09.044
PMCID: PMC4006324  PMID: 24268381
alcohol-preferring (P) rat; high-alcohol-drinking (HAD) rat; animal model of alcoholism; binge drinking; alcohol-seeking behavior
15.  Operant alcohol self-administration in dependent rats: focus on the vapor model 
Alcohol (Fayetteville, N.Y.)  2013;48(3):277-286.
Alcoholism (alcohol dependence) is characterized by a compulsion to seek and ingest alcohol (ethanol), loss of control over intake, and the emergence of a negative emotional state during withdrawal. Animal models are critical in promoting our knowledge of the neurobiological mechanisms underlying alcohol dependence. Here, we review the studies involving operant alcohol self-administration in rat models of alcohol dependence and withdrawal with the focus on the alcohol vapor model. In 1996, the first articles were published reporting that rats made dependent on alcohol by exposure to alcohol vapors displayed increased operant alcohol self-administration during acute withdrawal compared with nondependent rats (i.e., not exposed to alcohol vapors). Since then, it has been repeatedly demonstrated that this model reliably produces physical and motivational symptoms of alcohol dependence. The functional roles of various systems implicated in stress and reward, including opioids, dopamine, corticotropin-releasing factor (CRF), glucocorticoids, neuropeptide Y (NPY), γ-aminobutyric acid (GABA), norepinephrine, and cannabinoids, have been investigated in the context of alcohol dependence. The combination of models of alcohol withdrawal and dependence with operant self-administration constitutes an excellent tool to investigate the neurobiology of alcoholism. In fact, this work has helped lay the groundwork for several ongoing clinical trials for alcohol dependence. Advantages and limitations of this model are discussed, with an emphasis on what future directions of great importance could be.
doi:10.1016/j.alcohol.2013.08.006
PMCID: PMC4007394  PMID: 24290310
alcoholism; addiction; alcohol dependence; alcohol (ethanol) vapor; operant self-administration; compulsive behavior; rat; review
16.  Scheduled access alcohol drinking by alcohol-preferring (P) and high-alcohol-drinking (HAD) rats: modeling adolescent and adult binge-like drinking 
Alcohol (Fayetteville, N.Y.)  2013;48(3):225-234.
Binge alcohol drinking continues to be a public health concern among today’s youth and young adults. Moreover, an early onset of alcohol use, which usually takes the form of binge drinking, is associated with a greater risk for developing alcohol use disorders. Given this, it is important to examine this behavior in rat models of alcohol abuse and dependence. Toward that end, the objective of this article is to review findings on binge-like drinking by selectively bred alcohol-preferring (P) and high-alcohol-drinking (HAD) lines of rats. As reviewed elsewhere in this special issue, the P line meets all, and the HAD line meets most, of the proposed criteria for an animal model of alcoholism. One model of binge drinking is scheduled ethanol access during the dark cycle, which has been used by our laboratory for over 20 years. Our laboratory has also adopted a protocol involving the concurrent presentation of multiple ethanol concentrations. When this protocol is combined with limited access, ethanol intake is maximized yielding blood ethanol levels (BELs) in excess, sometimes greatly in excess, of 80 mg%. By extending these procedures to include multiple scheduled ethanol access sessions during the dark cycle for 5 consecutive days/week, P and HAD rats consume in 3 or 4 h as much as, if not more than, the amount usually consumed in a 24-h period. Under certain conditions, using the multiple scheduled access procedure, BELs exceeding 200 mg% can be achieved on a daily basis. An overview of findings from studies with other selectively bred, inbred, and outbred rats places these findings in the context of the existing literature. Overall, the findings support the use of P and HAD rats as animal models to study binge-like alcohol drinking and reveal that scheduled access procedures will significantly increase ethanol intake by other rat lines and strains as well.
doi:10.1016/j.alcohol.2013.10.004
PMCID: PMC4007416  PMID: 24290311
animal model of alcoholism; blood alcohol concentration; discrete bout; drinking-in-the-dark; excessive intake; extreme drinking; limited access; loss-of-control drinking; nocturnal drinking
17.  High Drinking in the Dark Mice: A genetic model of drinking to intoxication 
Alcohol (Fayetteville, N.Y.)  2013;48(3):217-223.
Drinking to intoxication is a critical component of risky drinking behaviors in humans, such as binge drinking. Previous rodent models of alcohol consumption largely failed to demonstrate that animals were patterning drinking in such a way as to experience intoxication. Therefore, few rodent models of binge-like drinking and no specifically genetic models were available to study possible predisposing genes. The High Drinking in the Dark (HDID) selective breeding project was started to help fill this void, with HDID mice selected for reaching high blood alcohol levels in a limited access procedure. HDID mice now represent a genetic model of drinking to intoxication and can be used to help answer questions regarding predisposition toward this trait as well as potential correlated responses. They should also prove useful for the eventual development of better therapeutic strategies.
doi:10.1016/j.alcohol.2013.10.007
PMCID: PMC4016107  PMID: 24360287
Binge; Drinking in the dark; Ethanol consumption; Genetics; Selective breeding
18.  Applications of schedule-induced polydipsia in rodents for the study of an excessive ethanol intake phenotype 
Alcohol (Fayetteville, N.Y.)  2014;48(3):265-276.
Schedule-induced polydipsia (SIP) is generated by subjecting a highly motivated animal to a sub-optimal rate of food reinforcement while also providing access to a fluid. SIP is one of several adjunctive (or displacement) behaviors that are expressed in an exaggerated form that is deemed ‘excessive’. This feature makes SIP an attractive model for studying an excessive ethanol drinking phenotype in rodents. Multiple experimental variables are crucial for the full manifestation of adjunctive drinking, including the degree of food deprivation, the inter-pellet interval selected, and the size of the food reward offered. Although these variables were extensively studied and optimized for water polydipsia in rats, a similarly customized approach to ethanol SIP and application of the procedure in mice have largely been curtailed in favor of the default variable values historically used for water SIP in rats. Further, ethanol SIP also requires careful consideration of variables such as taste and ethanol concentration. Investigation of the stress axis and neurochemical systems such as dopamine and serotonin in mediating adjunctive drinking stemmed from two leading hypotheses regarding the underlying mechanisms of SIP generation: 1) SIP as a coping strategy to mitigate stress associated with the aversive environmental condition, and 2) SIP as a displacement of reward in a highly motivated animal. Ethanol SIP is a powerful model of excessive intake because it can generate an ethanol-dependent state and sustain frequent and intoxicating levels of blood ethanol with voluntary oral consumption. The required food deprivation and the loss of the excessive drinking phenotype following removal of the generator schedule are the two main limitations of the model. Future utility of ethanol SIP will be enhanced by more fully dissecting the underlying hormonal and neurochemical mechanisms and optimizing experimental variables for ethanol SIP on a per species and strain basis.
doi:10.1016/j.alcohol.2014.01.005
PMCID: PMC4016177  PMID: 24680665
schedule-induced polydipsia; adjunctive drinking; stress; genetics; dependence; pharmacotherapy
19.  Operant Ethanol Self-Administration in Ethanol Dependent Mice 
Alcohol (Fayetteville, N.Y.)  2014;48(3):295-299.
While rats have been predominantly used to study operant ethanol self-administration behavior in the context of dependence, several studies have employed operant conditioning procedures to examine changes in ethanol self-administration behavior as a function of chronic ethanol exposure and withdrawal experience in mice. This review highlights some of the advantages of using operant conditioning procedures for examining the motivational effects of ethanol in animals with a history of dependence. As reported in rats, studies using various operant conditioning procedures in mice have demonstrated significant escalation of ethanol self-administration behavior in mice rendered dependent via forced chronic ethanol exposure in comparison to nondependent mice. This paper also presents a summary of these findings, as well as suggestions for future studies.
doi:10.1016/j.alcohol.2014.02.002
PMCID: PMC4020177  PMID: 24721194
operant conditioning; ethanol self-administration; ethanol dependence; mice
20.  Alcohol Dependence and Free-Choice Drinking in Mice 
Alcohol (Fayetteville, N.Y.)  2014;48(3):287-293.
Alcohol dependence continues to be an important health concern and animal models are critical to furthering our understanding of this complex disease. A hallmark feature of alcoholism is a significant increase in alcohol drinking over time. While several different animal models of excessive alcohol (ethanol) drinking exist for mice and rats, a growing number of laboratories are using a model that combines chronic ethanol exposure procedures with voluntary ethanol drinking with mice as experimental subjects. Primarily, these studies use a chronic intermittent ethanol (CIE) exposure pattern to render mice dependent and a 2-h limited access procedure to evaluate drinking behavior. Compared to non-dependent mice that also drink ethanol, the ethanol-dependent mice demonstrate significant increases in voluntary ethanol drinking. The increased drinking significantly elevates blood and brain ethanol concentrations compared to the non-dependent control mice. Studies report that the increased drinking by dependent mice is driven by neuroadaptations in glutamatergic and corticotropin-releasing factor signaling in different brain regions known to be involved in alcohol-related behaviors. The dysregulation of these systems parallels findings in human alcoholics and treatments that demonstrate efficacy in alcoholics can also reduce drinking in this model. Moreover, preclinical findings have informed the development of human clinical trials, further highlighting the translational potential of the model. As a result of these features, the CIE exposure and free-choice drinking model is becoming more widely used and promises to provide more insight into mechanisms of excessive drinking that may be important for developing treatments for human alcoholics. The salient features and possible future considerations for CIE exposure and free-choice drinking in mice are discussed.
doi:10.1016/j.alcohol.2013.11.006
PMCID: PMC4032174  PMID: 24530006
ethanol; dependence; chronic intermittent exposure; limited access; mouse; glutamate; CRF
21.  Binge drinking in alcohol-preferring sP rats at the end of the nocturnal period 
Alcohol (Fayetteville, N.Y.)  2014;48(3):301-311.
Sardinian alcohol-preferring (sP) rats have been selectively bred for high alcohol preference and consumption using the standard 2-bottle “alcohol (10%, v/v) vs. water” choice regimen with unlimited access; under this regimen, sP rats daily consume 6–7 g/kg alcohol. The present study assessed a new paradigm of alcohol intake in which sP rats were exposed to the 4-bottle “alcohol (10%, 20%, and 30%, v/v) vs. water” choice regimen during one of the 12 h of the dark phase of the daily light/dark cycle; the time of alcohol exposure was changed daily in a semi-random order and was unpredictable to rats. Alcohol intake was highly positively correlated with the time of the drinking session and averaged approximately 2 g/kg when the drinking session occurred during the 12th hour of the dark phase. Alcohol drinking during the 12th hour of the dark phase resulted in (a) blood alcohol levels averaging approximately 100 mg% and (b) severe signs of alcohol intoxication (e.g., impaired performance at a Rota-Rod task). The results of a series of additional experiments indicate that (a) both singular aspects of this paradigm (i.e., unpredictability of alcohol exposure and concurrent availability of multiple alcohol concentrations) contributed to this high alcohol intake, (b) alcohol intake followed a circadian rhythm, as it decreased progressively over the first 3 h of the light phase and then maintained constant levels until the beginning of the dark phase, and (c) sensitivity to time schedule was specific to alcohol, as it did not generalize to a highly palatable chocolate-flavored beverage. These results demonstrate that unpredictable, limited access to multiple alcohol concentrations may result in exceptionally high intakes of alcohol in sP rats, modeling – to some extent – human binge drinking. A progressively increasing emotional “distress” associated to rats’ expectation of alcohol might be the neurobehavioral basis of this drinking behavior.
doi:10.1016/j.alcohol.2014.02.001
PMCID: PMC4047429  PMID: 24680256
Time schedule of alcohol drinking; Limited and unpredictable access to alcohol; Experimental model of binge drinking; Sardinian alcohol-preferring (sP) rats
22.  Intermittent ethanol access schedule in rats as a preclinical model of alcohol abuse 
Alcohol (Fayetteville, N.Y.)  2014;48(3):243-252.
One of the major challenges in preclinical studies of alcohol abuse and dependence remains the development of paradigms that will elicit high ethanol intake and mimic the progressive transition from low or moderate social drinking to excessive alcohol consumption. Exposure of outbred rats to repeated cycles of free-choice ethanol intake and withdrawal with the use of intermittent access to 20% ethanol in a 2-bottle choice procedure (IA2BC) has been shown to induce a gradual escalation of voluntary ethanol intake and preference, eventually reaching ethanol consumption levels of 5–6 g/kg/24 h, and inducing pharmacologically relevant blood ethanol concentrations (BECs). This procedure has recently been gaining popularity due to its simplicity, high validity, and reliable outcomes. Here we review experimental and methodological data related to IA2BC, and discuss the usefulness and advantages of this procedure as a valuable pre-training method for initiating operant ethanol self-administration of high ethanol intake, as well as conditioned place preference (CPP). Despite some limitations, we provide evidence that IA2BC and related operant procedures provide the possibility to operationalize multiple aspects of alcohol abuse and addiction in a rat model, including transition from social-like drinking to excessive alcohol consumption, binge drinking, alcohol seeking, relapse, and neuroadaptations related to excessive alcohol intake. Hence, IA2BC appears to be a useful and relevant procedure for preclinical evaluation of potential therapeutic approaches against alcohol abuse disorders.
doi:10.1016/j.alcohol.2014.01.006
PMCID: PMC4102254  PMID: 24721195
ethanol; animal models; binge drinking; excessive drinking; intermittent access; operant self-administration; two-bottle choice; blood ethanol concentrations; neuroadaptations; relapse
23.  Environmental stressors influence limited-access ethanol consumption by C57BL/6J mice in a sex-dependent manner 
Alcohol (Fayetteville, N.Y.)  2014;48(8):741-754.
Environmental factors, including exposure to stress, are known to contribute to the propensity to consume ethanol. However, stress produces inconsistent effects on ethanol drinking in rodent models. Therefore, the present study examined the impact of different stressors on limited access ethanol consumption and determined whether there were sex-dependent differences in response to stress. To this end, male and female C57BL/6J mice had 2-hr access to a water and 10% ethanol solution, beginning 30 minutes before onset of the dark cycle. Once ethanol intake was stable, the effect of restraint, tail suspension, predator odor, foot shock, and tail pinch on subsequent intake was explored. Both plasma corticosterone (CORT) and allopregnanolone (ALLO) were assessed as indices of hypothalamic-pituitary-adrenal (HPA) axis activity and of endogenous neurosteroid levels respectively, following restraint, tail suspension and predator odor. Ethanol intake was decreased following restraint, tail suspension, foot shock, and tail pinch in both sexes, with stressor-related differences in the duration of the suppression. The effect of predator odor on ethanol intake was biphasic in females; ethanol consumption was significantly reduced on the day of stress but significantly increased on the following two days. In males, predator odor produced a delayed significant increase in ethanol intake on the second day after stress. All three stressors increased plasma CORT, with higher CORT levels in females when compared with males. Notably, there was a significant positive correlation between CORT levels immediately after predator odor stress and ethanol intake on the following day as well as a significant positive linear relationship between CORT levels immediately after restraint stress and ethanol intake on the following day in females. Furthermore, the three stressors produced a greater increase in ALLO levels in female versus male mice, but ALLO levels following predator odor were not correlated with subsequent ethanol intake. In summary, the type of stressor administered had a profound impact on subsequent ethanol consumption, with subtle sex differences in the magnitude and persistence of the effect. These findings are the first to demonstrate that a single, acute exposure to restraint, tail suspension, and predator odor stress increased plasma CORT and ALLO levels in animals with a history of ethanol consumption and that female mice were more responsive than males to the ability of stress to increase CORT levels as well as to the ability of predator odor stress to produce a delayed increase in ethanol intake. Because predator odor stress is a model of posttraumatic stress disorder, the present sex differences have important implications for future preclinical studies modeling the comorbidity of posttraumatic stress disorder and alcohol use disorders.
doi:10.1016/j.alcohol.2014.07.015
PMCID: PMC4371601  PMID: 25459519
environmental stress; corticosterone; allopregnanolone; predator odor; foot shock; tail pinch
24.  Oscillatory spatial profile of alcohol’s effects on the resting state: Anatomically-constrained MEG 
Alcohol (Fayetteville, N.Y.)  2014;48(2):89-97.
It has been firmly established that opening and closing the eyes strongly modulate the electro- and magnetoencephalography (EEG and MEG) signals acquired during wakeful rest. Certain features of the resting EEG are altered in chronic alcoholics and their offspring, and have been proposed as biomarkers for alcoholism. Spontaneous brain oscillations are also affected by pharmacological manipulations, but the spectral and spatial characteristics of these changes are not clear. This study examined effects of the eyes-open (EO) and eyes-closed (EC) resting paradigm and alcohol challenge on the spatial profile of spontaneous MEG and EEG oscillations. Whole-head MEG and scalp EEG signals were acquired simultaneously from healthy social drinkers (n = 17) who participated in both alcohol (0.6 g/kg ethanol for men, 0.55 g/kg for women) and placebo conditions in a counterbalanced design. Power of the signal was calculated with Fast Fourier Transform and was decomposed into its constituent theta (4–7 Hz), alpha (8–12 Hz), and beta (15–20 Hz) frequency bands. High-resolution structural MRI images were additionally obtained from all participants and used to constrain distributed minimum norm inverse source power estimates. The spatial estimates of the main generator nodes were in agreement with studies using a combined fMRI-EEG approach. Alpha band oscillations dominated the spectral profile and their source was estimated to the medial parieto-occipital area. Power in theta and beta bands was weaker overall and their sources were estimated to a more focal medial prefrontal area. EO and EC manipulation most strongly modulated power in the alpha band, but a wide-band power increase was observed during the EC condition. Alcohol intoxication increased alpha power, particularly during the EC condition. Application of this methodology to cohorts of chronic alcoholics or individuals at risk could potentially provide insight into the neural basis of oscillatory differences that may be predictive of the vulnerability to alcoholism.
doi:10.1016/j.alcohol.2013.12.004
PMCID: PMC3959272  PMID: 24530007
theta; alpha; beta; eyes-open; eyes-closed; alcohol
25.  Effects of stress, acute alcohol treatment, or both on pre-pulse inhibition in high- and low-alcohol preferring mice 
Alcohol (Fayetteville, N.Y.)  2014;48(2):113-122.
Pre-pulse inhibition of the acoustic startle reflex (PPI) is a measure of sensorimotor gating frequently used to assess information processing in both humans and rodents. Both alcohol and stress exposure can modulate PPI, making it possible to assess how stress and alcohol interact to influence information processing. Humans with an increased genetic risk for alcoholism are more reactive to stressful situations compared to those without a family history, and alcohol may have stress-dampening effects for those with high genetic risk. The purpose of the present study was to examine the effects of stress, acute alcohol exposure, or both on PPI in male and female mice selectively bred for high- (HAP2) and low- (LAP2) alcohol preference. Experiment 1 assessed the effects of various doses of acute alcohol on PPI. Experiments 2 and 3 assessed the effect of 10 days of restraint stress on subsequent PPI tested at 30 min (Experiment 2) or 24 h (Experiment 3) following the termination of stress exposure. Experiment 3 also examined the effects of acute alcohol treatment (0.75 g/kg) on PPI in mice previously exposed to stress or no stress. Results indicate that 0.75 and 1.0 g/kg doses of alcohol increased PPI in HAP2 but not LAP2 mice. When PPI was tested 30 min after stress exposure, stressed HAP2 mice showed a trend toward decreased PPI and stressed LAP2 mice showed a trend toward increased PPI. The combination of stress and alcohol treatment did not alter PPI in either line 24 h following the termination of stress exposure, suggesting that alcohol does not ameliorate the effect of stress on PPI. Stressed LAP2 mice had increased basal circulating corticosterone on the final stress exposure day compared to non-stressed LAP2 mice, and no difference was found between stressed and non-stressed HAP2 mice. The results suggest that high genetic risk for alcoholism may be related to increased sensitivity to alcohol and stress effects on PPI, and this sensitivity could signify an endophenotype for increased genetic risk to develop alcoholism.
doi:10.1016/j.alcohol.2013.12.002
PMCID: PMC3959585  PMID: 24507876
acoustic startle; pre-pulse inhibition; stress; alcohol; selectively bred mice; corticosterone

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