GABAA receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR), and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABAA receptor subunits. (S)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ1” antagonist), when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests), but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ2” antagonist) did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting) ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABAA receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo.

BACKGROUND: Initial sensitivity to ethanol (EtOH) and the capacity to develop acute functional tolerance (AFT) to its adverse effects may influence the amount of alcohol consumed and may also predict future alcohol use patterns. The current study assessed sensitivity and AFT to the ataxic and hypnotic effects of EtOH in the first replicate of mice (HDID-1) selectively bred for high blood EtOH concentrations (BECs) following limited access to EtOH in the Drinking in the Dark (DID) paradigm. METHODS: Naïve male and female HDID-1 and HS/Npt mice from the progenitor stock were evaluated in 3 separate experiments. In Experiments 1 and 2, EtOH-induced ataxia was assessed using the static dowel task. In Experiment 3, EtOH-induced hypnosis was assessed by using modified restraint tubes to measure the loss of righting reflex (LORR). RESULTS: HDID-1 mice exhibited reduced initial sensitivity to both EtOH-induced ataxia (p \textless 0.001) and hypnosis (p \textless 0.05) relative to HS/Npt mice. AFT was calculated by subtracting the BEC at loss of function from the BEC at recovery (Experiments 1 and 3) or by subtracting BEC at an initial recovery from the BEC at a second recovery following an additional alcohol dose (Experiment 2). The dowel test yielded no line differences in AFT, but HS/Npt mice developed slightly greater AFT to EtOH-induced LORR than HDID-1 (p \textless 0.05). CONCLUSIONS: These results suggest that HDID-1 mice exhibit aspects of blunted ataxic and hypnotic sensitivity to EtOH which may influence their high EtOH intake via DID, but do not display widely different development of AFT. These findings differ from previous findings with the high alcohol-preferring (HAP) selected mouse lines, suggesting that genetic predisposition for binge, versus other forms of excessive alcohol consumption, is associated with unique responses to EtOH-induced motor incoordination.

Numerous selective breeding experiments have been performed with rodents, in an attempt to understand the genetic basis for innate differences in preference for alcohol consumption. Quantitative trait locus (QTL) analysis has been used to determine regions of the genome that are associated with the behavioral difference in alcohol preference/consumption. Recent work suggests that differences in gene expression represent a major genetic basis for complex traits. Therefore, the QTLs are likely to harbor regulatory regions (eQTLs) for the differentially expressed genes that are associated with the trait. In this study, we examined brain gene expression differences over generations of selection of the third replicate lines of high and low alcohol-preferring (HAP3 and LAP3) mice, and determined regions of the genome that control the expression of these differentially expressed genes (de eQTLs). We also determined eQTL regions (rv eQTLs) for genes that showed a decrease in variance of expression levels over the course of selection. We postulated that de eQTLs that overlap with rv eQTLs, and also with phenotypic QTLs, represent genomic regions that are affected by the process of selection. These overlapping regions controlled the expression of candidate genes (that displayed differential expression and reduced variance of expression) for the predisposition to differences in alcohol consumption by the HAP3/LAP3 mice.

Complex Mus musculus crosses provide increased resolution to examine the relationships between gene expression and behavior. While the advantages are clear, there are numerous analytical and technological concerns that arise from the increased genetic complexity that must be considered. Each of these issues is discussed, providing an initial framework for complex cross study design and planning.

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.

Extensive genetic and genomic studies of the relationship between alcohol drinking preference and withdrawal severity have been performed using animal models. Data from multiple such publications and public data resources have been incorporated in the GeneWeaver database with \textgreater60,000 gene sets including 285 alcohol withdrawal and preference-related gene sets. Among these are evidence for positional candidates regulating these behaviors in overlapping quantitative trait loci (QTL) mapped in distinct mouse populations. Combinatorial integration of functional genomics experimental results revealed a single QTL positional candidate gene in one of the loci common to both preference and withdrawal. Functional validation studies in Ap3m2 knockout mice confirmed these relationships. Genetic validation involves confirming the existence of segregating polymorphisms that could account for the phenotypic effect. By exploiting recent advances in mouse genotyping, sequence, epigenetics, and phylogeny resources, we confirmed that Ap3m2 resides in an appropriately segregating genomic region. We have demonstrated genetic and alcohol-induced regulation of Ap3m2 expression. Although sequence analysis revealed no polymorphisms in the Ap3m2-coding region that could account for all phenotypic differences, there are several upstream SNPs that could. We have identified one of these to be an H3K4me3 site that exhibits strain differences in methylation. Thus, by making cross-species functional genomics readily computable we identified a common QTL candidate for two related bio-behavioral processes via functional evidence and demonstrate sufficiency of the genetic locus as a source of variation underlying two traits.

Neuroinflammatory mechanisms contribute to the brain pathology resulting from human immunodeficiency virus (HIV) infection. Magnetic resonance spectroscopy (MRS) has been touted as a suitable method for discriminating in vivo markers of neuroinflammation. The present MRS study was conducted in four groups: alcohol dependent (A, n = 37), HIV-infected (H, n = 33), alcohol dependent + HIV infected (HA, n = 38) and healthy control (C, n = 62) individuals to determine whether metabolites would change in a pattern reflecting neuroinflammation. Significant four-group comparisons were evident only for striatal choline-containing compounds (Cho) and myo-inositol (mI), which follow-up analysis demonstrated were due to higher levels in HA compared with C individuals. To explore the potential relevance of elevated Cho and mI, correlations between blood markers, medication status and alcohol consumption were evaluated in H + HA subjects. Having an acquired immune deficiency syndrome (AIDS)-defining event or hepatitis C was associated with higher Cho; lower Cho levels, however, were associated with low thiamine levels and with highly active antiretroviral HIV treatment (HAART). Higher levels of mI were related to greater lifetime alcohol consumed, whereas HAART was associated with lower mI levels. The current results suggest that competing mechanisms can influence in vivo Cho and mI levels, and that elevations in these metabolites cannot necessarily be interpreted as reflecting a single underlying mechanism, including neuroinflammation.

LIM-domain-only 3 (LMO3) is a transcriptional regulator involved in central nervous system development and neuroblastoma. Our previous studies implicated a potential role for LMO3 in regulating ethanol sensitivity and consumption. Here, we examined behavioral responses to ethanol in a line of Lmo3 null (Lmo3(Z) ) mice, utilizing the ethanol-induced loss-of-righting-reflex (LORR) test, two-bottle choice ethanol consumption and the drinking in the dark (DID) test, which models binge-like ethanol consumption. We found that Lmo3(Z) mice exhibited increased sedation time in response to ethanol in the LORR test and drank significantly more ethanol in the DID test compared with their wild-type counterparts, but showed no differences in two-bottle choice ethanol consumption. To explore where LMO3 may be acting in the brain to produce an ethanol phenotype, we also examined reporter gene (β-galactosidase) expression in heterozygous Lmo3(Z) mice and found strong expression in subcortical areas, particularly in those areas implicated in drug abuse, including the nucleus accumbens (Acb), cortex, hippocampus and amygdala. We also examined Lmo3 expression in the brains of wild-type mice who had undergone the DID test and found a negative correlation between Lmo3 expression in the Acb and the amount of ethanol consumed, consistent with the increased binge-like drinking observed in Lmo3(Z) mice. These results support a role for LMO3 in regulating behavioral responses to ethanol, potentially through its actions in the Acb.

Abstract: The problem of computing logical network models to account for temporal dependencies among interacting genes and environmental stimuli from high-throughput transcriptomic data is addressed. A logical network reconstruction algorithm was developed that uses the statistical significance as a criterion for network selection to avoid false interactions arising from pure chance. Using temporal gene expression data collected from the brains of alcohol-treated mice in an analysis of the molecular response to alcohol, this algorithm identified several genes from a major neuronal pathway as putative components of the alcohol response mechanism. Three of these genes have known specific associations with alcohol response as reported in the literature. Several other potentially relevant genes were also highlighted, in agreement with independent results from literature mining. These genes may play a role in the response to alcohol. Additional, previously-unknown interactions were discovered in the logical network that, subject to biological verification, may offer new clues in the search for the elusive molecular mechanisms of alcoholism.

Some anti-inflammatory medications reduce alcohol consumption in rodent models. Inhibition of phosphodiesterases (PDE) increases cAMP and reduces inflammatory signaling. Rolipram, an inhibitor of PDE4, markedly reduced ethanol intake and preference in mice and reduced ethanol seeking and consumption in alcohol-preferring fawn-hooded rats (Hu et al., ; Wen et al., ). To determine if these effects were specific for PDE4, we compared nine PDE inhibitors with different subtype selectivity: propentofylline (nonspecific), vinpocetine (PDE1), olprinone, milrinone (PDE3), zaprinast (PDE5), rolipram, mesopram, piclamilast, and CDP840 (PDE4). Alcohol intake was measured in C57BL/6J male mice using 24-h two-bottle choice and two-bottle choice with limited (3-h) access to alcohol. Only the selective PDE4 inhibitors reduced ethanol intake and preference in the 24-h two-bottle choice test. For rolipram, piclamilast, and CDP840, this effect was observed after the first 6 h but not after the next 18 h. Mesopram, however, produced a long-lasting reduction of ethanol intake and preference. In the limited access test, rolipram, piclamilast, and mesopram reduced ethanol consumption and total fluid intake and did not change preference for ethanol, whereas CDP840 reduced both consumption and preference without altering total fluid intake. Our results provide novel evidence for a selective role of PDE4 in regulating ethanol drinking in mice. We suggest that inhibition of PDE4 may be an unexplored target for medication development to reduce excessive alcohol consumption.

Excessive ethanol drinking in rodent models may involve activation of the innate immune system, especially toll-like receptor 4 (TLR4) signaling pathways. We used intracellular recording of evoked GABAergic inhibitory postsynaptic potentials (eIPSPs) in central amygdala (CeA) neurons to examine the role of TLR4 activation by lipopolysaccharide (LPS) and deletion of its adapter protein CD14 in acute ethanol effects on the GABAergic system. Ethanol (44, 66 or 100mM) and LPS (25 and 50μg/ml) both augmented eIPSPs in CeA of wild type (WT) mice. Ethanol (44mM) decreased paired-pulse facilitation (PPF), suggesting a presynaptic mechanism of action. Acute LPS (25μg/ml) had no effect on PPF and significantly increased the mean miniature IPSC amplitude, indicating a postsynaptic mechanism of action. Acute LPS pre-treatment potentiated ethanol (44mM) effects on eIPSPs in WT mice and restored ethanol's augmenting effects on the eIPSP amplitude in CD14 knockout (CD14 KO) mice. Both the LPS and ethanol (44-66mM) augmentation of eIPSPs was diminished significantly in most CeA neurons of CD14 KO mice; however, ethanol at the highest concentration tested (100mM) still increased eIPSP amplitudes. By contrast, ethanol pre-treatment occluded LPS augmentation of eIPSPs in WT mice and had no significant effect in CD14 KO mice. Furthermore, (+)-naloxone, a TLR4-MD-2 complex inhibitor, blocked LPS effects on eIPSPs in WT mice and delayed the ethanol-induced potentiation of GABAergic transmission. In CeA neurons of CD14 KO mice, (+)-naloxone alone diminished eIPSPs, and subsequent co-application of 100mM ethanol restored the eIPSPs to baseline levels. In summary, our results indicate that TLR4 and CD14 signaling play an important role in the acute ethanol effects on GABAergic transmission in the CeA and support the idea that CD14 and TLR4 may be therapeutic targets for treatment of alcohol abuse.

High-throughput next-generation sequencing is now entering its second decade. However, it was not until 2008 that the first report of sequencing the brain transcriptome appeared (Mortazavi, Williams, Mccue, Schaeffer, & Wold, 2008). These authors compared short-read RNA-Seq data for mouse whole brain with microarray results for the same sample and noted both the advantages and disadvantages of the RNA-Seq approach. While RNA-Seq provided exon level resolution, the majority of the reads were provided by a small proportion of highly expressed genes and the data analysis was exceedingly complex. Over the past 6 years, there have been substantial improvements in both RNA-Seq technology and data analysis. This volume contains 11 chapters that detail various aspects of sequencing the brain transcriptome. Some of the chapters are very methods driven, while others focus on the use of RNA-Seq to study such diverse areas as development, schizophrenia, and drug abuse. This chapter briefly reviews the transition from microarrays to RNA-Seq as the preferred method for analyzing the brain transcriptome. Compared with microarrays, RNA-Seq has a greater dynamic range, detects both coding and noncoding RNAs, is superior for gene network construction, detects alternative spliced transcripts, and can be used to extract genotype information, e.g., nonsynonymous coding single nucleotide polymorphisms. RNA-Seq embraces the complexity of the brain transcriptome and provides a mechanism to understand the underlying regulatory code; the potential to inform the brain-behavior-disease relationships is substantial.

We have investigated the expression of chromatin-regulating genes in the prefrontal cortex and in the shell subdivision of the nucleus accumbens during protracted withdrawal in mice with increased ethanol drinking after chronic intermittent ethanol (CIE) vapor exposure and in mice with a history of non-dependent drinking. We observed that the methyl-CpG binding protein 2 (MeCP2) was one of the few chromatin-regulating genes to be differentially regulated by a history of dependence. As MeCP2 has the potential of acting as a broad gene regulator, we investigated sensitivity to ethanol and ethanol drinking in MeCP2(308/) (Y) mice, which harbor a truncated MeCP2 allele but have a milder phenotype than MeCP2 null mice. We observed that MeCP2(308/) (Y) mice were more sensitive to ethanol's stimulatory and sedative effects than wild-type (WT) mice, drank less ethanol in a limited access 2 bottle choice paradigm and did not show increased drinking after induction of dependence with exposure to CIE vapors. Alcohol metabolism did not differ in MeCP2(308/) (Y) and WT mice. Additionally, MeCP2(308/) (Y) mice did not differ from WT mice in ethanol preference in a 24-hour paradigm nor in their intake of graded solutions of saccharin or quinine, suggesting that the MeCP2(308/) (Y) mutation did not alter taste function. Lastly, using the Gene Set Enrichment Analysis algorithm, we found a significant overlap in the genes regulated by alcohol and by MeCP2. Together, these results suggest that MeCP2 contributes to the regulation of ethanol sensitivity and drinking.

Acute alcohol intoxication causes cellular changes in the brain that last for hours, while chronic alcohol use induces widespread neuroadaptations in the nervous system that can last a lifetime. Chronic alcohol use and the progression into dependence involve the remodeling of synapses caused by changes in gene expression produced by alcohol. The progression of alcohol use, abuse, and dependence can be divided into stages, which include intoxication, withdrawal, and craving. Each stage is associated with specific changes in gene expression, cellular function, brain circuits, and ultimately behavior. What are the molecular mechanisms underlying the transition from recreational use (acute) to dependence (chronic)? What cellular adaptations result in drug memory retention, leading to the persistence of addictive behaviors, even after prolonged drug abstinence? Research into the neurobiology of alcoholism aims to answer these questions. This chapter will describe the molecular adaptations caused by alcohol use and dependence, and will outline key neurochemical participants in alcoholism at the molecular level, which are also potential targets for therapy.

Neuroimaging has consistently documented reductions in the brain tissue of alcoholics. Inability to control comorbidity, environmental insult, and nutritional deficiency, however, confound the ability to assess whether ethanol itself is neurotoxic. Here we report monkey oral ethanol self-administration combined with MR imaging to characterize brain changes over 15 months in 18 well-nourished rhesus macaques. Significant brain volume shrinkage occurred in the cerebral cortices of monkeys drinking ≥ 3 g/kg ethanol/day (12 alcoholic drinks) at 6 months, and this persisted throughout the period of continuous access to ethanol. Correlation analyses revealed a cerebral cortical volumetric loss of \textasciitilde0.11% of the intracranial vault for each daily drink (0.25 g/kg), and selective vulnerability of cortical and non-cortical brain regions. These results demonstrate for the first time a direct relation between oral ethanol intake and measures of decreased brain gray matter volume in vivo in primates. Notably, greater volume shrinkage occurred in monkeys with younger drinking onset that ultimately became heavier drinkers than monkeys with older drinking onset. The pattern of volumetric changes observed in nonhuman primates following 15 months of drinking suggests that cerebral cortical gray matter changes are the first macroscopic manifestation of chronic ethanol exposure in the brain.

Ethanol exposure and withdrawal alter the generation of new neurons in the adult hippocampus. The endogenous opioid system, in particular the μ opioid receptor (MOR), can modulate neural progenitors and also plays a critical role in ethanol drinking and dependence. In the present study, we sought to determine whether MOR contributes to the effects of ethanol on the dentate gyrus (DG) neurogenic niche. MOR wild-type (WT), heterozygous (Het) and knockout (KO) littermates were subjected to voluntary ethanol drinking in repeated limited-access two-bottle choice (2BC) sessions. MOR deficiency did not alter progenitor proliferation, neuronal differentiation and maturation, apoptosis or microglia in ethanol-naïve mice. When exposed to five consecutive weeks of 2BC, MOR mutant mice exhibited a gene-dosage dependent reduction of ethanol consumption compared to WT mice. Introducing a week of ethanol deprivation between each week of 2BC increased ethanol consumption in all genotypes and produced equivalent intakes in WT, Het and KO mice. Under the latter paradigm, ethanol drinking decreased progenitor proliferation and neuronal differentiation in the DG of WT mice. Interestingly, WT mice exhibited a strong negative correlation between ethanol intake and proliferation, which was disrupted in Het and KO mice. Moreover, MOR deficiency blocked the effect of ethanol on neuronal differentiation. MOR deficiency also protected against the neuroimmune response to ethanol drinking. Finally, chronic binge drinking induced a paradoxical decrease in apoptosis, which was independent of MOR. Altogether our data suggest that MOR is implicated in some of the neuroplastic changes produced by chronic ethanol exposure in the DG.

Alcohol and other drugs of abuse have significant impacts on the neuroimmune system. Studies have demonstrated that drugs of abuse interact with the neuroimmune system and alter neuroimmune gene expression and signaling, which in turn contribute to various aspects of addiction. As the key component of the CNS immune system, neuroimmune factors mediate neuroinflammation and modulate a wide range of brain function including neuronal activity, endocrine function, and CNS development. These neuromodulatory properties of immune factors, together with their essential role in neuroinflammation, provide a new framework to understand neuroimmune mechanisms mediating brain functional and behavioral changes contributing to addiction. This chapter highlights recent advances in understanding neuroimmune changes associated with exposure to alcohol and other drugs of abuse, including opiates, marijuana, methamphetamine, and cocaine. It provides a brief overview on what we know about neuroimmune signaling and its role in drug action and addiction.

Immune or brain proinflammatory signaling has been linked to some of the behavioral effects of alcohol. Immune signaling appears to regulate voluntary ethanol intake in rodent models, and ethanol intake activates the immune system in multiple models. This bidirectional link raises the possibility that consumption increases immune signaling, which in turn further increases consumption in a feed-forward cycle. Data from animal and human studies provide overlapping support for the involvement of immune-related genes and proteins in alcohol action, and combining animal and human data is a promising approach to systematically evaluate and nominate relevant pathways. Based on rodent models, neuroimmune pathways may represent unexplored, nontraditional targets for medication development to reduce alcohol consumption and prevent relapse. Peroxisome proliferator-activated receptor agonists are one class of anti-inflammatory medications that demonstrate antiaddictive properties for alcohol and other drugs of abuse. Expression of immune-related genes is altered in animals and humans following chronic alcohol exposure, and the regulatory influences of specific mRNAs, microRNAs, and activated cell types are areas of intense study. Ultimately, the use of multiple datasets combined with behavioral validation will be needed to link specific neuroimmune pathways to addiction vulnerability.

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.

Alcohol use disorders (AUDs) have a staggering socioeconomic impact. Few therapeutic options are available, and they are largely inadequate. These shortcomings highlight the urgent need to develop effective medications to prevent and/or treat AUDs. A critical barrier is the lack of information regarding the molecular target(s) by which ethanol (EtOH) exerts its pharmacological activity. This review highlights findings implicating P2X4 receptors (P2X4Rs) as a target for the development of therapeutics to treat AUDs and discusses the use of ivermectin (IVM) as a potential clinical tool for treatment of AUDs. P2XRs are a family of ligand-gated ion channels (LGICs) activated by extracellular ATP. Of the P2XR subtypes, P2X4Rs are expressed the most abundantly in the CNS. Converging evidence suggests that P2X4Rs are involved in the development and progression of AUDs. First, in vitro studies report that pharmacologically relevant EtOH concentrations can negatively modulate ATP-activated currents. Second, P2X4Rs in the mesocorticolimbic dopamine system are thought to play a role in synaptic plasticity and are located ideally to modulate brain reward systems. Third, alcohol-preferring (P) rats have lower functional expression of the p2rx4 gene than alcohol-non-preferring (NP) rats suggesting an inverse relationship between alcohol intake and P2X4R expression. Similarly, whole brain p2rx4 expression has been shown to relate inversely to innate 24 h alcohol preference across 28 strains of rats. Fourth, mice lacking the p2rx4 gene drink more EtOH than wildtype controls. Fifth, IVM, a positive modulator of P2X4Rs, antagonizes EtOH-mediated inhibition of P2X4Rs in vitro and reduces EtOH intake and preference in vivo. These findings suggest that P2X4Rs contribute to EtOH intake. The present review summarizes recent findings focusing on the P2X4R as a molecular target of EtOH action, its role in EtOH drinking behavior and modulation of its activity by IVM as a potential therapy for AUDs.