Action Of Alcohol Research Articles

Sex-dependent differences in ethanol inhibition of mouse lateral orbitofrontal cortex neurons.

Biological differences between males and females likely influence responses to alcohol and the propensity to engage in excessive drinking. In both humans and rodents, females escalate alcohol use and develop addiction-like behaviors faster than males, while males exhibit more severe withdrawal symptoms during abstinence. The mechanisms underlying these differences are not yet known but may reflect fundamental differences in the ethanol sensitivity of neurons in reward and control areas of the brain. To address this question, we recorded current-evoked spiking of lateral orbitofrontal cortex (lOFC) neurons in male and female C57BL/6J mice following acute and chronic exposure to ethanol. Ethanol (11-66mM) reduced firing of lOFC neurons but produced less inhibition in neurons from female mice. As previously reported for male mice, the glycine receptor blocker strychnine blocked ethanol inhibition of spiking of lOFC neurons from female mice and prevented the ethanol-induced increase in tonic current. Following chronic intermittent ethanol (CIE) exposure, current-evoked spiking of lOFC neurons was significantly enhanced with a greater effect observed in males. After CIE treatment, acute ethanol had no effect on spiking in neurons from male mice, while it produced a slight but significant decrease in firing in females. Finally, like male mice, the inhibitory effect of the glycine transport inhibitor sarcosine was blunted in CIE-exposed female mice. Together, these results suggest that while lOFC neurons in male and female mice are similarly affected by ethanol, there are significant differences in sensitivity that may contribute to differences in alcohol actions between males and females.

Vasopressin and alcohol: a multifaceted relationship.

Arginine vasopressin (VP) has been implicated in a number of neuropsychiatric disorders with an emphasis on situations where stress increased the severity of the disorder. Based on this hypothesized role for VP in neuropsychiatric disorders, much research is currently being undertaken in humans and animals to test VP as a target for treatment of a number of these disorders including alcohol abuse. To provide a summary of the literature regarding the role of VP in alcohol- and stress-related behaviors including the use of drugs that target VP in clinical trials. Changes in various components of the VP system occur with alcohol and stress. Manipulating VP or its receptors can alter alcohol- and stress-related behaviors including tolerance to alcohol, alcohol drinking, and anxiety-like behavior. Finally, the hypothalamic-pituitary-adrenal axis response to alcohol is also altered by manipulating the VP system. However, clinical trials of VP antagonists have had mixed results. A review of VP's involvement in alcohol's actions demonstrates that there is much to be learned about brain regions involved in VP-mediated effects on behavior. Thus, future work should focus on elucidating relevant brain regions. By using previous knowledge of the actions of VP and determining the brain regions and/or systems involved in its different behavioral effects, it may be possible to identify a specific receptor subtype target, drug treatment combination, or specific clinical contexts that may point toward a more successful treatment.

Identification of alcohol-binding site(s) in proteins using diazirine-based photoaffinity labeling and mass spectrometry.

Defining molecular targets of alcohol and understanding the molecular mechanism of alcohol actions are necessary to develop effective therapeutics for alcohol use disorder (AUD). Here, we describe a detailed protocol for identifying alcohol-binding site(s) in proteins using diazirine-based azialcohol as photoaffinity labeling agents. Upon photoirradiation, azialcohol photoincorporates into alcohol-binding proteins. The stoichiometry and site of azialcohol photoincorporation can be determined using high-resolution mass spectrometry. Identification of the alcohol-binding residues in protein followed by measuring the biological significance of these residues in regulating alcohol action are important steps in characterizing the molecular targets of alcohol.

GMO safety assessment in the Russian Federation: the immunotoxicological studies

Ethanol (also called alcohol or ethyl alcohol) is the most commonly abused psychoactive drug. Its metabolism is the cause of a vast array of pathologic manifestations which affects almost all organs. The goal of this article is to describe and update the pathways responsible for the metabolism of ethanol and understand the factors which regulate its metabolism. Most alcohol is oxidized in the liver and general principles and overall mechanisms for alcohol oxidation will be summarized including the enzymatic pathways responsible for this metabolism. Rate-limiting steps in the overall metabolism of ethanol, including the activity of alcohol dehydrogenase isoforms, and the necessity to reoxidize NADH by substrate shuttle pathways and the mitochondrial respiratory chain will be discussed. Other ethanol-metabolizing pathways such as catalase and the microsomal ethanol-oxidizing system/CYP2E1 system will be characterized. The metabolism and role of acetaldehyde in the toxic actions of alcohol and ethanol drinking behavior will be discussed. Despite much knowledge of alcohol pharmacokinetics and metabolism, numerous questions which remain for further evaluation are presented and future research directions are suggested.

A Novel Peptide Restricts Ethanol Modulation of the BK Channel In Vitro and In Vivo.

Alcohol is a widely used and abused substance. A major unresolved issue in the alcohol research field is determining which of the many alcohol target proteins identified to date is responsible for shaping each specific alcohol-related behavior. The large-conductance, calcium- and voltage-activated potassium channel (BK channel) is a conserved target of ethanol. Genetic manipulation of the highly conserved BKα channel influences alcohol-related behaviors across phylogenetically diverse species that include worm, fly, mouse, and man. A pharmacological tool that prevents alcohol's action at a single target, like the BK channel, would complement genetic approaches in the quest to define the behavioral consequences of alcohol at each target. To identify agents that specifically modulate the action of ethanol at the BK channel, we executed a high-throughput phagemid-display screen in combination with a Caenorhabditis elegans behavioral genetics assay. This screen selected a novel nonapeptide, LS10, which moderated acute ethanol intoxication in a BK channel-humanized C. elegans strain without altering basal behavior. LS10's action in vivo was dependent upon BK channel functional activity. Single-channel electrophysiological recordings in vitro showed that preincubation with a submicromolar concentration of LS10 restricted ethanol-induced changes in human BKα channel gating. In contrast, no substantial changes in basal human BKα channel function were observed after LS10 application. The results obtained with the LS10 peptide provide proof-of-concept evidence that a combined phagemid-display/behavioral genetics screening approach can provide novel tools for understanding the action of alcohol at the BK channel and how this, in turn, exerts influence over central nervous system function.

Oral Cancer: An Evaluation of Knowledge and Awareness in Undergraduate Dental Students and the General Public.

To evaluate the knowledge of signs, symptoms and risk factors associated with oral cancer amongst undergraduate dental students and members of the general public. This study was open for a period of six months (Jan-June, 2013) to all undergraduate dental students in the 4th and 5th year of the dental science programme and dental patients attending the School of Dentistry, Griffith University, Australia. The survey evaluated the knowledge and awareness of clinical signs and symptoms and risk factors of oral cancers. A total of 100 undergraduate students and 150 patients provided informed consent and participated in this survey study. Both patients and dental students were aware of the importance of early detection of oral cancer. With the exception of smoking and persistent ulceration, this study indicated that the knowledge about oral cancer, its signs, symptoms and risk factors was limited amongst participants. This study highlights the need to raise awareness and knowledge pertaining to oral cancer, not only in the general community but also amongst those in the dental field. Specific points of concern were the common intraoral sites for oral cancer, erythroplakia as a risk factor, the synergistic action of smoking and alcohol, and HPV (human papilloma virus) as risk factors for oral cancer.

Issue Cover (July 2018)

Front cover: Background: Gestational alcohol exposure causes a range of neuropsychological disorders by modulating neurodevelopmental genes and proteins. The extent of damage depends on the stage of the embryo, as well as dosage, duration, and frequency of exposure. Main results: Employing hESC-derived neural cultures as an in vitro model we demonstrated that alcohol treatment perturbs MAPK/ERK signaling accompanied with deregulation of miR-145 by targeting the neural specifier Sox-2 causing compromised neuronal complexity which results in abnormal neurogenesis. Employing molecular endpoints in a humanized model we showed for the first time that acute alcohol exposure could lead to impaired brain development. Implication: These findings opens up an opportunity to further investigate the cell-autonomous and non-autonomous actions of alcohol on the central as well as peripheral nervous system. Image content: De novo generated neural progenitors stained for pan neuronal marker TUJ1 (green) by immunocytochemistry; nuclei was stained with DAPI (blue). Read the full article Transcriptional profiling of human neural precursors post alcohol exposure reveals impaired neurogenesis via dysregulation of ERK signaling and miR-145 by L. K. Louis, R. Gopurappilly, H. Surendran, S. Dutta, R. Pal (J. Neurochem. 2018, vol. 146 (1), pp. 47–62) on doi: 10.1111/jnc.14155

Does prenatal alcohol exposure cause a metabolic syndrome? (Non-)evidence from a mouse model of fetal alcohol spectrum disorder.

Although prenatal alcohol exposure (PAE) reduces offspring growth, it may increase obesity risk at adolescence. Animal models of PAE display glucose intolerance and increased adiposity, suggesting that PAE causes metabolic reprogramming. We tested this hypothesis in a mouse model of binge PAE, wherein pregnant C57Bl/6J females received 3 g/kg alcohol (ETOH) daily from gestational day 12.5 to 17.5; maltodextrin (MD) and medium chain triglycerides (MCT) served as isocaloric nutritional controls, and sham (H2O) treatment controlled for gavage stress. Our comprehensive assessment quantified body composition, energy expenditure, glucose tolerance, and cardiovascular function in offspring at age 17 weeks. Although ETOH pups were initially lighter than all other groups, they did not have a unique obesogenic phenotype. Instead, a similar obesogenic phenotype emerged in all three caloric groups (MCT, MD, ETOH), such that caloric groups had greater post-weaning weight gain (both sexes), reduced gonadal fat weight (males), and reduced glucose clearance (males) compared against H2O offspring. PAE did not affect body composition, respiratory exchange ratio, metabolic adaption to high-fat or low-fat diet, eating behavior, and blood pressure, and ETOH values did not differ from those obtained from isocaloric controls. Exposure to a higher alcohol dose (4.5 g/kg) or a high-fat (60%) diet did not exacerbate differences in body composition or glucose tolerance. “PAE-specific” effects on postnatal growth, glucose tolerance, adiposity, or hypertension only emerged when PAE offspring were compared just against H2O controls, or against MD controls. We conclude that prior reports of obesity and glucose intolerance in adult PAE offspring reflect the contribution of added gestational calories, and not alcohol’s pharmacologic action. Results suggest that the increased adiposity risk in FASD is not caused by metabolic reprogramming, and instead originates from behavioral, medication, and/or dietary practices. This study highlights the importance of appropriate dietary controls in nutritional studies of PAE.

Optogenetic investigation of neural mechanisms for alcohol-use disorder.

Optogenetic techniques have been widely used in the study of neuropsychiatric diseases such as anxiety, depression, and drug addiction. Cell-type specific targeting of optogenetic tools to neurons has contributed to a tremendous understanding of the function of neural circuits for future treatment of neuropsychiatric disorders. Though optogenetics has been widely used in many research areas, the use of optogenetic tools to uncover and elucidate neural circuit mechanisms of alcohol's actions in the brain are still developing. Here in this review article, we will provide a basic introduction to optogenetics and discuss how these optogenetic experimental approaches can be used in alcohol studies to reveal neural circuit mechanisms of alcohol's actions in regions implicated in the development of alcohol addiction.

Altered brain activity during withdrawal from chronic alcohol is associated with changes in IL-6 signal transduction and GABAergic mechanisms in transgenic mice with increased astrocyte expression of IL-6

Interleukin-6 (IL-6) is an important neuroimmune factor that is increased in the brain by alcohol exposure/withdrawal and is thought to play a role in the actions of alcohol on the brain. To gain insight into IL-6/alcohol/withdrawal interactions and how these interactions affect the brain, we are studying the effects of chronic binge alcohol exposure on transgenic mice that express elevated levels of IL-6 in the brain due to increased astrocyte expression (IL-6 tg) and their non-transgenic (non-tg) littermate controls. IL-6/alcohol/withdrawal interactions were identified by genotypic differences in spontaneous brain activity in electroencephalogram (EEG) recordings from the mice, and by Western blot analysis of protein activation or expression in hippocampus obtained from the mice after the final alcohol withdrawal period. Results from EEG studies showed frequency dependent genotypic differences in brain activity during withdrawal. For EEG frequencies that were affected by alcohol exposure/withdrawal in both genotypes, the nature of the effect was similar, but differed across withdrawal cycles. Differences between IL-6 tg and non-tg mice were also observed in Western blot studies of the activated form of STAT3 (phosphoSTAT3), a signal transduction partner of IL-6, and subunits of GABAA receptors (GABAAR). Regression analysis revealed that pSTAT3 played a more prominent role during withdrawal in the IL-6 tg mice than in the non-tg mice, and that the role of GABAAR alpha-5 and GABAAR alpha-1 in brain activity varied across genotype and withdrawal. Taken together, our results suggest that IL-6 can significantly impact mechanisms involved in alcohol withdrawal.

GABAA receptor polymorphisms in alcohol use disorder in the GWAS era.

Alcohol use disorder (AUD) is a chronic, relapsing, neuro-psychiatric illness of high prevalence and with a serious public health impact worldwide. It is complex and polygenic, with a heritability of about 50%, and influenced by environmental causal heterogeneity. Risk factors associated with its etiology have a genetic component. GABA (γ-aminobutyric acid) is a major inhibitory neurotransmitter in mammalian brain. GABAA receptors are believed to mediate some of the physiological and behavioral actions of alcohol. In this critical review, relevant genetic terms and type and methodology of the genetic studies are briefly explained. Postulated candidate genes that encode subunits of GABAA receptors, with all the reported SNPs, are presented. Genetic studies and meta-analyses examining polymorphisms of the GABAA receptor and their association with AUD predisposition are presented. The data are critically examined with reference to recent GWAS studies that failed to show relations between GABAA receptors and AUD. Restrictions and perspectives of the different findings are discussed.

Extra-endothelial TRPV1 channels participate in alcohol and caffeine actions on cerebral artery diameter.

Alcohol (ethyl alcohol; ethanol) and caffeine are the two most widely used psychoactive substances in the world. Caffeine and ethanol have both been reported to constrict cerebral arteries in several species, including humans. We have recently shown that application of 10-μM caffeine mixed with 50 mM ethanol to invitro pressurized cerebral arteries of rats reduced ethanol-induced constriction. This effect was dependent on the presence of nitric oxide (NO•) and could be observed in de-endothelialized arteries supplied with the NO donor sodium nitroprusside (SNP). The molecular target(s) of ethanol-caffeine interaction in cerebral arteries has remained unknown. In the present work, we used rat and mouse middle cerebral arteries (MCA) to identify the extra-endothelial effectors of NO-mediated, caffeine-induced protection against ethanol-evoked arterial constriction. Constriction of intact MCA of rat by either 50 mM ethanol or 10 μM caffeine was ablated in the presence of a selective TRPV1 pharmacological blocker. TRPV1 pharmacological block, but not block of TRPA1, PKG, or BK channels, removed caffeine-induced protection against ethanol-evoked rat MCA constriction, whether evaluated in arteries with intact endothelium or in SNP-supplemented, de-endothelialized arteries. In mouse arteries, caffeine-induced protection against ethanol-induced MCA constriction was significantly amplified, resulting in actual vasodilation, upon pharmacological block of TRPV1, and in TRPV1 knock-out arteries. Despite some species-specific differences, our study unequivocally demonstrates the presence of functional, extra-endothelial TRPV1 that participates in both endothelium-independent MCA constriction by separate exposure to ethanol or caffeine and caffeine-induced protection against ethanol-evoked MCA constriction.

Alcohol attenuates anti‐HCV function of IFN‐λ1 through up‐regulation of PLASy expression in human hepatic cells

Alcohol could compromise the anti-hepatitis C virus (HCV) function of interferon-alpha (IFN-α). However, little information is available about the effect of alcohol on interferon-lambda (IFN-λ, type III IFN), a novel candidate for development of therapy for HCV infection. Huh7 cells were infected with HCV JFH-1 virus, then treated with alcohol, and/or IFN-λ1. RT-PCR and Western blot were used to detect the levels of HCV and key cellular factors. Overexpression or silencing expression was performed to verify the role of key factors in alcohol-attenuated anti-HCV function of IFN-λ1. Alcohol treatment compromised anti-HCV effect of IFN-λ1 in HCV JFH-1-infected Huh7 cells, evidenced by the significantly increased levels of HCV RNA, and HCV core protein in alcohol-/IFN-λ1-treated cells compared to cells with IFN-λ1 treatment alone. Investigation of the mechanisms responsible for the alcohol action revealed that alcohol enhanced the expression of protein inhibitor of activated STAT (PIASy). Overexpression of PIASy compromised anti-HCV ability of IFN-λ1, whereas silencing expression of PIASy partly restored the alcohol-attenuated anti-HCV effect of IFN-λ1. More importantly, overexpression of PIASy significantly down-regulated the level of IFN-λ1-indcued phosphorylation of STAT1 (p-STAT1), an important adaptor in IFN-λ pathway, as well as reduced the expression of IFN-λ1-induced IFN-stimulated genes 56 (ISG56), and myxovirus resistance 1 (Mx1), two antiviral effectors in in IFN-λ pathway. These findings indicate that alcohol, through inducing the expression of negative regulator in IFN-λ pathway, inhibits IFN-λ-mediated anti-HCV action in human hepatic cells, which may lead to the poor efficacy of IFN-λ-based therapy against HCV infection.

Gene-edited CRISPy Critters for alcohol research.

Genetically engineered animals are powerful tools that have provided invaluable insights into mechanisms of alcohol action and alcohol-use disorder. Traditionally, production of gene-targeted animals was a tremendously expensive, time consuming, and technically demanding undertaking. However, the recent advent of facile methods for editing the genome at very high efficiency is revolutionizing how these animals are made. While pioneering approaches to create gene-edited animals first used zinc finger nucleases and subsequently used transcription activator-like effector nucleases, these approaches have been largely supplanted in an extremely short period of time with the recent discovery and precocious maturation of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system. CRISPR uses a short RNA sequence to guide a non-specific CRISPR-associated nuclease (Cas) to a precise, single location in the genome. Because the CRISPR/Cas system can be cheaply, rapidly, and easily reprogrammed to target nearly any genomic locus of interest simply by recoding the sequence of the guide RNA, this gene-editing system has been rapidly adopted by numerous labs around the world. With CRISPR/Cas, it is now possible to perform gene editing directly in early embryos from every species of animals that is of interest to the alcohol field. Techniques have been developed that enable the rapid production of animals in which a gene has been inactivated (knockout) or modified to harbor specific nucleotide changes (knockins). This system has also been used to insert specific DNA sequences such as reporter or recombinase genes into specific loci of interest. Genetically engineered animals created with the CRISPR/Cas system (CRISPy Critters) are being produced at an astounding pace. Animal production is no longer a significant bottleneck to new discoveries. CRISPy animal studies are just beginning to appear in the alcohol literature, but their use is expected to explode in the near future. CRISPy mice, rats, and other model organisms are sure to facilitate advances in our understanding of alcohol-use disorder.

To the question of laboratory diagnostics of fat droplets.

This study aims to compare of study of blood plasma for the presence of fat globules using the method of V.A. Cherkasov and the method what we developed in 60 patients with severe combined trauma. The use of the developed method increases the accuracy of qualitative and quantitative analysis by reducing the emulsifying action of the ethyl alcohol of Sudan IV, preventing uneven coloring and destruction of the preparation within 3 minutes after preparation. A classification of fat globules has been developed, capillary-hazardous globules of ≥ 8 μm in size and arterioles-hazardous globules ≥ 50 μm.

GABA and Glutamate Synaptic Coadaptations to Chronic Ethanol in the Striatum.

Alcohol (ethanol) is a widely used and abused drug with approximately 90% of adults over the age of 18 consuming alcohol at some point in their lifetime. Alcohol exerts its actions through multiple neurotransmitter systems within the brain, most notably the GABAergic and glutamatergic systems. Alcohol's actions on GABAergic and glutamatergic neurotransmission have been suggested to underlie the acute behavioral effects of ethanol. The striatum is the primary input nucleus of the basal ganglia that plays a role in motor and reward systems. The effect of ethanol on GABAergic and glutamatergic neurotransmission within striatal circuitry has been thought to underlie ethanol taking, seeking, withdrawal and relapse. This chapter reviews the effects of ethanol on GABAergic and glutamatergic transmission, highlighting the dynamic changes in striatal circuitry from acute to chronic exposure and withdrawal.

Effects of vitamin C and E on toxic action of alcohol on partial hepatectomy-induced liver regeneration in rats.

The purpose of this study was to investigate the influence of vitamins C and E on the toxic action of alcohol in rat liver regeneration. Male Sprague-Dawley rats subjected to 70% partial hepatectomy were divided into five groups (Groups 1 to 5). Rats in Groups 2 to 5 were only provided alcohol for drinking. Additionally, vitamin C, vitamin E, and vitamin C in combination with vitamin E were administered to Groups 3, 4, and 5, respectively. Alcohol inhibits liver regeneration, resulting in an increase in free radicals produced by alcohol metabolism and thus causing cellular damage and altering liver function. During liver regeneration, vitamins C and E significantly ameliorated liver injury from alcohol administration by reducing hepatic lipid peroxidation. Vitamins C and E protect against liver injury and dysfunction, attenuate lipid peroxidation, and thus may be more effective in combination than either vitamin alone against alcohol-mediated toxic effects during liver regeneration.

Regulation of Kisspeptin Synthesis and Release in the Preoptic/Anterior Hypothalamic Region of Prepubertal Female Rats: Actions of IGF-1 and Alcohol.

Alcohol (ALC) causes suppressed secretion of prepubertal luteinizing hormone-releasing hormone (LHRH). Insulin-like growth factor-1 (IGF-1) and kisspeptin (Kp) are major regulators of LHRH and are critical for puberty. IGF-1 may be an upstream mediator of Kp in the preoptic area and rostral hypothalamic area (POA/RHA) of the rat brain, a region containing both Kp and LHRH neurons. We investigated the ability of IGF-1 to stimulate prepubertal Kp synthesis and release in POA/RHA, and the potential inhibitory effects of ALC. Immature female rats were administered either ALC (3g/kg) or water via gastric gavage at 0730hours. At 0900hours, both groups were subdivided where half received either saline or IGF-1 into the brain third ventricle. A second dose of ALC (2g/kg) or water was administered at 1130hours. Rats were killed 6hours after injection and POA/RHA region collected. IGF-1 stimulated Kp, an action blocked by ALC. Upstream to Kp, IGF-1 receptor (IGF-1R) activation, as demonstrated by the increase in insulin receptor substrate 1, resulted in activation of Akt, tuberous sclerosis 2, ras homologue enriched in brain, and mammalian target of rapamycin (mTOR). ALC blocked the central action of IGF-1 to induce their respective phosphorylation. IGF-1 specificity and ALC specificity for the Akt-activated mTOR pathway were demonstrated by the absence of effects on PRAS40. Furthermore, IGF-1 stimulated Kp release from POA/RHA incubated invitro. IGF-1 stimulates prepubertal Kp synthesis and release following activation of a mTOR signaling pathway, and ALC blocks this pathway at the level of IGF-1R.

The Effects of ZnO Nanoparticles in Combination with Alcohol on Biosynthetic Potential of Saccharomyces cerevisiae

Abstract This paper reports about experimental results concerning the influence of 30 nm ZnO nanoparticles on biomass, carbohydrates, β-glucans, proteins accumulation and catalase enzyme activity at Saccharomyces cerevisiae CNMN-Y-20 yeast strain exposed to alcohol action. Alcohol in concentrations of 2%, 5% and 10% added to culture medium has been reported to stimulate β-glucans biosynthesis and to inhibit protein synthesis. Low biomass production, with 71% less that control, was detected in the experiments with 10% alcohol. ZnO nanoparticles in combination with alcohol do not offer sufficient protection for the proteins biosynthesis, but efficiently protect the carbohydrates and β-glucans biosynthetic processes, which contents in the biomass are with 16.6% and 19.9% higher than control, respectively. The maximum value of β-glucans content was established in case of cultivation of selected yeast strain on YPD medium supplemented with 5 mg/L nanoparticles ZnO and 2% alcohol. The obtained results allowed the elaboration of new procedure for directed synthesis of β-glucans that contributed to an increase of this component with 30.7%, compared to control.

Characterizing conditioned reactivity to sequential alcohol-predictive cues in well-trained rats.

Implicit learning about antecedent stimuli and the unconditional stimulus (US) properties of alcohol may facilitate the progressive loss of control over drinking. To model this learning, Cofresí etal. (2017) developed a procedure in which a discrete, visual conditional stimulus (houselight illumination; CS) predicted the availability of a retractable sipper that rats could lick to receive unsweetened alcohol [Alcoholism: Clinical and Experimental Research, 41, 608-617]. Here we investigated the possibility that houselight illumination, sipper presentation, and oral alcohol receipt might each exert control over alcohol seeking and drinking. We also determined the relationship between ingested dose and blood alcohol concentration, in order to validate the idea that the US is a post-ingestive action of alcohol. Finally, we tested a major prediction from the conditioning account of problematic drinking [Tomie, A., & Sharma, N. (2013). Current Drug Abuse Reviews, 6, 201-219], which is that once learned, responses elicited by a CS will promote drinking. We found that despite having constrained opportunities to drink alcohol during the conditioning procedure, ingested doses produced discriminable blood concentrations that supported cue conditioning. Based on our analysis of the dynamics of cue reactivity in well-trained rats, we found that houselight illumination triggered conditioned approach, sipper presentation evoked licking behavior, and alcohol receipt promoted drinking. Reactivity to these cues, which varied in terms of their temporal proximity to the alcohol US, persisted despite progressive intoxication or satiety. Additionally, rats with the greatest conditioned reactivity to the most distal alcohol cue were also the fastest to initiate drinking and drank the most. Our findings indicate that the post-ingestive effects of alcohol may condition multiple cues simultaneously in adult rats, and these multiple cues help to trigger alcohol seeking and drinking. Moreover, identification and characterization of these cues should be helpful for designing interventions that attenuate the power of these cues over behavior.