The rationale for our study was to determine the pattern of ethanol drinking by the high alcohol-drinking (HAD) replicate lines of rats during adolescence and adulthood in both male and female rats. Rats were given 30 days of 24 h free-choice access to ethanol (15%, v/v) and water, with ad lib access to food, starting at the beginning of adolescence (PND 30) or adulthood (PND 90). Water and alcohol drinking patterns were monitored 22 h/day with a “lickometer” set-up. The results indicated that adolescent HAD-1 and HAD-2 males consumed the greatest levels of ethanol and had the most well defined ethanol licking binges among the age and sex groups with increasing levels of ethanol consumption throughout adolescence. In addition, following the first week of adolescence, male and female HAD-1 and HAD-2 rats differed in both ethanol consumption levels and ethanol licking behavior. Adult HAD-1 male and female rats did not differ from one another and their ethanol intake or licking behaviors did not change significantly over weeks. Adult HAD-2 male rats maintained a relatively constant level of ethanol consumption across weeks, whereas adult HAD-2 female rats increased ethanol consumption levels over weeks, peaking during the third week when they consumed more than their adult male counterparts. The results indicate that the HAD rat lines could be used as an effective animal model to examine the development of ethanol consumption and binge drinking in adolescent male and female rats providing information on the long-range consequences of adolescent alcohol drinking.

Three experiments used the intragastric alcohol consumption (IGAC) procedure to examine the effects of variations in passive ethanol exposure on withdrawal and voluntary ethanol intake in two inbred mouse strains, C57BL/6J (B6) and DBA/2J (D2). Experimental treatments were selected to induce quantitative differences in ethanol dependence and withdrawal severity by: (1) varying the periodicity of passive ethanol exposure (three, six or nine infusions/day); (2) varying the dose per infusion (low, medium or high); and (3) varying the duration of passive exposure (3, 5 or 10 days). All experiments included control groups passively exposed to water. B6 mice generally self-infused more ethanol than D2 mice, but passive ethanol exposure increased IGAC in both strains, with D2 mice showing larger relative increases during the first few days of ethanol access. Bout data supported the characterization of B6 mice as sippers and D2 mice as gulpers. Three larger infusions per day produced a stronger effect on IGAC than six or nine smaller infusions, especially in D2 mice. Increased IGAC was strongly predicted by cumulative ethanol dose and intoxication during passive exposure in both strains. Withdrawal during the passive exposure phase was also a strong predictor of increased IGAC in D2 mice. However, B6 mice showed little withdrawal, precluding analysis of its potential role. Overall, these data support the hypothesis that dependence-induced increases in IGAC are jointly determined by two processes that might vary across genotypes: (1) tolerance to aversive postabsorptive ethanol effects and (2) negative reinforcement (i.e. alleviation of withdrawal by self-administered ethanol).

Complex Mus musculus crosses, e.g., heterogeneous stock (HS), provide increased resolution for quantitative trait loci detection. However, increased genetic complexity challenges detection methods, with discordant results due to low data quality or complex genetic architecture. We quantified the impact of theses factors across three mouse crosses and two different detection methods, identifying procedures that greatly improve detection quality. Importantly, HS populations have complex genetic architectures not fully captured by the whole genome kinship matrix, calling for incorporating chromosome specific relatedness information. We analyze three increasingly complex crosses, using gene expression levels as quantitative traits. The three crosses were an F2 intercross, a HS formed by crossing four inbred strains (HS4), and a HS (HS-CC) derived from the eight lines found in the collaborative cross. Brain (striatum) gene expression and genotype data were obtained using the Illumina platform. We found large disparities between methods, with concordance varying as genetic complexity increased; this problem was more acute for probes with distant regulatory elements (trans). A suite of data filtering steps resulted in substantial increases in reproducibility. Genetic relatedness between samples generated overabundance of detected eQTLs; an adjustment procedure that includes the kinship matrix attenuates this problem. However, we find that relatedness between individuals is not evenly distributed across the genome; information from distinct chromosomes results in relatedness structure different from the whole genome kinship matrix. Shared polymorphisms from distinct chromosomes collectively affect expression levels, confounding eQTL detection. We suggest that considering chromosome specific relatedness can result in improved eQTL detection.

BACKGROUND: Alcohol abuse is frequently associated with nicotine (Nic) use. The current experiments were conducted to establish an oral operant ethanol + Nic (EtOH + Nic) co-use model and to characterize some aspects of EtOH + Nic co-use. METHODS: Rats were allowed to choose between EtOH alone or EtOH + Nic solutions. Additionally, alcohol-preferring (P) rats were allowed to concurrently self-administer 3 distinct EtOH solutions (10, 20, and 30%) with varying amounts of Nic (0.07, 0.14, or 0.21 mg/ml) under operant conditions. P rats were also allowed to concurrently self-administer 2 distinct amounts of Nic (0.07 and 0.14 mg/ml) added to saccharin (Sacc; 0.025%) solutions. RESULTS: During acquisition, P rats responded for the EtOH + Nic solutions at the same level as for EtOH alone, and responding for EtOH + Nic solutions was present throughout all drinking conditions. P rats also readily maintained stable self-administration behaviors for Nic + Sacc solutions. The results demonstrated that P rats readily acquired and maintained stable self-administration behaviors for EtOH + 0.07 and EtOH + 0.14 mg/ml Nic solutions. Self-administration of EtOH + 0.21 mg/ml Nic was established in only 50% of the subjects. P rats readily expressed seeking behaviors for the EtOH + Nic solutions and reacquired EtOH + Nic self-administration during relapse testing. In addition, tail blood samples indicated that EtOH + Nic co-use resulted in pharmacologically relevant levels of both EtOH and Nic in the blood. CONCLUSIONS: Overall, the results indicate that P rats readily consume EtOH + Nic solutions concurrently in the presence of EtOH alone, express drug-seeking behaviors, and will concurrently consume physiologically relevant levels of both drugs. These results support the idea that this oral operant EtOH + Nic co-use model would be suitable for studying the development of co-abuse and the consequences of long-term chronic co-abuse.

RATIONALE: One possible basis for the proclivity of ethanol and nicotine co-abuse is an interaction between the discriminative stimulus (S(D)) effects of each drug. OBJECTIVES: The current work sought to assess the discriminative control of ethanol and nicotine cues in mice trained with drug mixtures and to determine whether interactive mechanisms of overshadowing and potentiation occur. METHODS: Male C57BL/6J mice were trained to discriminate ethanol (1.5 g/kg) alone or ethanol plus nicotine (0.4, 0.8, or 1.2 mg/kg base) in experiment 1 and nicotine (0.8 mg/kg) alone or nicotine plus ethanol (0.5, 1.0, or 2.0 g/kg) in experiment 2. Stimulus generalizations of the training mixtures to ethanol, nicotine, and the drug combination were assessed. RESULTS: Ethanol (1.5 g/kg) retained discriminative control despite the inclusion of a progressively larger nicotine dose within the training mixtures in experiment 1. Although the nicotine S(D) was overshadowed by ethanol training doses \textgreater 0.5 g/kg in experiment 2, nicotine did potentiate the effects of low-dose ethanol. CONCLUSIONS: These findings are suggestive of dual mechanisms whereby ethanol (\textgreater0.5 g/kg) overshadows the S(D) effects of nicotine, and at lower doses (\textless1 g/kg) the salience of ethanol's S(D) effects is potentiated by nicotine. These mechanisms may contribute to the escalation of concurrent drinking and smoking in a binge-like fashion.

BACKGROUND: The High Drinking in the Dark (HDID) selected mouse line was bred for high blood ethanol (EtOH) concentration (BEC) following the limited access drinking in the dark (DID) test and is a genetic animal model of binge-like drinking. This study examines the microstructure of EtOH drinking in these mice and their control line during 3 versions of the DID test to determine how drinking structure differences might relate to overall intake and BEC. METHODS: Male mice from the HDID-1 replicate line and HS/Npt progenitor stock were tested in separate experiments on 2- and 4-day versions of the DID test, and on a 2-day 2-bottle choice DID test with 20% EtOH and water. Testing took place in home cages connected to a continuous fluid intake monitoring system, and drinking during the DID test was analyzed for drinking microstructure. RESULTS: HDID-1 mice had more drinking bouts, shorter interbout interval, larger bout size, greater total EtOH intake, and higher BECs than HS/Npt mice on the second day of the 2-day DID test. The 4-day DID test showed greater bout size, total EtOH intake, and BEC in the HDID-1 mice than the HS/Npt mice. Total EtOH intake and BECs for the HDID-1 mice in the DID tests averaged 2.6 to 3.0 g/kg and 0.4 to 0.5 mg/ml, respectively. The 2-bottle choice test showed no genotype differences in drinking microstructure or total consumption but did show greater preference for the EtOH solution in HDID-1 mice than HS/Npt. CONCLUSIONS: These results suggest that inherent differences in EtOH drinking structure between the HDID-1 and HS/Npt mice, especially the larger bout size in the HDID-1 mice, contribute to the difference in intake during the standard DID test.

Background Mouse lines are being selectively bred in replicate for high blood ethanol concentrations (BECs) achieved after a short period of ethanol (EtOH) drinking early in the circadian dark phase. High Drinking in the Dark-1 (HDID-1) mice were in selected generation S18, and the replicate HDID-2 line in generation S11. Methods To determine other traits genetically correlated with high DID, we compared naïve animals from both lines with the unselected, segregating progenitor stock, HS/Npt. Differences between HDID-1 and HS would imply commonality of genetic influences on DID and these traits. Results HDID-1 mice showed less basal activity, greater EtOH stimulated activity, and greater sensitivity to EtOH-induced foot slips than HS. They showed lesser sensitivity to acute EtOH hypothermia and longer duration loss of righting reflex than HS. HDID-1 and control HS lines did not differ in sensitivity on 2 measures of intoxication, the balance beam and the accelerating rotarod. None of the acute response results could be explained by differences in EtOH metabolism. HDID-2 differed from HS on some, but not all, of the above responses. Conclusions These results show that some EtOH responses share common genetic control with reaching high BECs after DID, a finding consistent with other data regarding genetic contributions to EtOH responses.

BACKGROUND: Mouse lines are being selectively bred in replicate for high blood ethanol concentrations (BECs) achieved after limited access of ethanol (EtOH) drinking early in the circadian dark phase. High Drinking in the Dark-1 (HDID-1) mice are in selected generation S21, and the replicate HDID-2 line in generation S14. Tolerance and withdrawal symptoms are 2 of the 7 diagnostic criteria for alcohol dependence. Withdrawal severity has been found in mouse studies to be negatively genetically correlated with EtOH preference drinking. METHODS: To determine other traits genetically correlated with high DID, we compared naïve animals from both lines with the unselected, segregating progenitor stock, HS/Npt. Differences between HDID-1 and HS would imply commonality of genetic influences on DID and these traits. RESULTS: Female HDID-1 and HDID-2 mice tended to develop less tolerance than HS to EtOH hypothermia after their third daily injection. A trend toward greater tolerance was seen in the HDID males. HDID-1, HDID-2, and control HS lines did not differ in the severity of acute or chronic withdrawal from EtOH as indexed by the handling-induced convulsion (HIC). Both HDID-1 and HDID-2 mice tended to have greater HIC scores than HS regardless of drug treatment. CONCLUSIONS: These results show that tolerance to EtOH's hypothermic effects may share some common genetic control with reaching high BECs after DID, a finding consistent with other data regarding genetic contributions to EtOH responses. Withdrawal severity was not negatively genetically correlated with DID, unlike its correlation with preference drinking, underscoring the genetic differences between preference drinking and DID. HDID lines showed greater basal HIC scores than HS, suggestive of greater central nervous system excitability.

Neuronal activity dependent pentraxin (Narp) interacts with α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) glutamate receptors to facilitate excitatory synapse formation by aggregating them at established synapses. Alcohol is well-characterized to influence central glutamatergic transmission, including AMPA receptor function. Herein, we examined the influence of injected and ingested alcohol upon Narp protein expression, as well as basal Narp expression in mouse lines selectively bred for high blood alcohol concentrations under limited access conditions. Alcohol up-regulated accumbens Narp levels, concomitant with increases in levels of the GluR1 AMPA receptor subunit. However, accumbens Narp or GluR1 levels did not vary as a function of selectively bred genotype. We next employed a Narp knock-out (KO) strategy to begin to understand the behavioral relevance of alcohol-induced changes in protein expression in several assays of alcohol reward. Compared to wild-type mice, Narp KO animals: fail to escalate daily intake of high alcohol concentrations under free-access conditions; shift their preference away from high alcohol concentrations with repeated alcohol experience; exhibit a conditioned place-aversion in response to the repeated pairing of 3 g/kg alcohol with a distinct environment and fail to exhibit alcohol-induced locomotor hyperactivity following repeated alcohol treatment. Narp deletion did not influence the daily intake of either food or water, nor did it alter any aspect of spontaneous or alcohol-induced motor activity, including the development of tolerance to its motor-impairing effects with repeated treatment. Taken together, these data indicate that Narp induction, and presumably subsequent aggregation of AMPA receptors, may be important for neuroplasticity within limbic subcircuits mediating or maintaining the rewarding properties of alcohol.

Individual mice differ in the dose of ethanol they will ingest voluntarily when it is offered during limited access periods in the circadian dark, a phenotype called drinking in the dark (DID). Substantial genetic variation in DID has been reported across a few standard inbred mouse strains, and a line of High Drinking in the Dark (HDID) mice has been established through selective breeding on the blood ethanol concentration (BEC) they attain at the end of a drinking session. Here, we report ethanol DID data for 23 inbred mouse strains, including 11 not previously reported, corroborating the genetic contributions to this trait. We also report data on a different ethanol drinking trait, the increased intake seen after multiple cycles of chronic intermittent exposure to ethanol vapor (CIE). Drinking escalated significantly during ethanol withdrawal. However, HDID mice and their HS controls showed equivalent escalation during withdrawal, demonstrating that withdrawal-associated drinking escalation is not a clear genetic correlate of selection on DID. Across inbred strains, DID is substantially genetically correlated with previously-published two-bottle ethanol preference drinking data assessed under conditions of continuous ethanol access. Although inbred strain data for withdrawal-associated drinking are not available, the current pattern of results suggests that withdrawal-associated drinking is genetically distinct from DID, while genetic contributions to DID and two-bottle preference drinking are substantially similar.

RATIONALE: Human ethanol withdrawal manifests as multiple behavioral deficits with distinct time courses. Most studies with mice index ethanol withdrawal severity with the handling-induced convulsion (HIC). Using the accelerating rotarod (ARR), we recently showed that ethanol withdrawal produced motor impairment. OBJECTIVES: This study aimed (a) to characterize further the ARR withdrawal trait, (b) to assess generalizability across additional behavioral assays, and (c) to test the genetic correlation between ethanol withdrawal ARR impairment and HICs. RESULTS: The severity of the ARR performance deficit depends on ethanol vapor dose and exposure duration, and lasts 1-4 days. Fatigue could not explain the deficits, which were also evident after intermittent exposure to ethanol vapor. Withdrawing mice were also impaired on a balance beam, but not on a static dowel or in foot slip errors per distance traveled in the parallel rod floor test, where they showed reduced locomotor activity. To assess genetic influences, we compared Withdrawal Seizure-Prone and -Resistant mice, genetically selected to express severe vs. mild withdrawal HICs, respectively. The ARR scores were approximately equivalent in all groups treated with ethanol vapor, though Withdrawal Seizure-Prone (WSP) mice may have displayed a slightly more severe deficit as control-treated WSP mice performed better than control-treated Withdrawal Seizure-Resistant mice. CONCLUSIONS: These studies show that ethanol withdrawal motor impairment is sensitive to a range of ethanol doses and lasts for several days. Multiple assays of behavioral impairment are affected, but the effects depend on the assay employed. Genetic contributions to withdrawal-induced ARR impairment appear largely distinct from those leading to severe or mild HICs.

Alcohol abuse causes widespread changes in gene expression in human brain, some of which contribute to alcohol dependence. Previous microarray studies identified individual genes as candidates for alcohol phenotypes, but efforts to generate an integrated view of molecular and cellular changes underlying alcohol addiction are lacking. Here, we applied a novel systems approach to transcriptome profiling in postmortem human brains and generated a systemic view of brain alterations associated with alcohol abuse. We identified critical cellular components and previously unrecognized epigenetic determinants of gene coexpression relationships and discovered novel markers of chromatin modifications in alcoholic brain. Higher expression levels of endogenous retroviruses and genes with high GC content in alcoholics were associated with DNA hypomethylation and increased histone H3K4 trimethylation, suggesting a critical role of epigenetic mechanisms in alcohol addiction. Analysis of cell-type-specific transcriptomes revealed remarkable consistency between molecular profiles and cellular abnormalities in alcoholic brain. Based on evidence from this study and others, we generated a systems hypothesis for the central role of chromatin modifications in alcohol dependence that integrates epigenetic regulation of gene expression with pathophysiological and neuroadaptive changes in alcoholic brain. Our results offer implications for epigenetic therapeutics in alcohol and drug addiction.

The objective of this study was to determine if there are common innate differences in gene expression or gene pathways in the ventral tegmental area (VTA) among 5 different pairs of rat lines selectively bred for high (HEC) or low (LEC) ethanol consumption: (a) alcohol-preferring (P) vs. alcohol-non-preferring (NP) rats; (b) high-alcohol-drinking (HAD) vs. low-alcohol-drinking (LAD) rats (replicate line pairs 1 and 2); (c) ALKO alcohol (AA) vs. nonalcohol (ANA) rats; and (d) Sardinian alcohol-preferring (sP) vs. alcohol-nonpreferring (sNP) rats. Microarray analysis revealed between 370 and 1340 unique named genes that significantly differed in expression between the individual line-pairs. Analysis using Gene Ontology (GO) and Ingenuity Pathways information indicated significant categories and networks in common for up to 3 line-pairs, but not for all 5 line-pairs; moreover, there were few genes in common in these categories and networks. ANOVA of the combined data for the 5 line-pairs indicated 1295 significant (p\textless0.01) differences in expression of named genes. Although no individual named gene was significant across all 5 line-pairs, there were 22 genes that overlapped in the same direction in 3 or 4 of the line-pairs. Overall, the findings suggest that (a) some biological categories or networks may be in common for subsets of line-pairs; and (b) regulation of different genes and/or combinations of multiple biological systems (e.g., transcription, synaptic function, intracellular signaling and protection against oxidative stress) within the VTA (possibly involving dopamine and glutamate) may be contributing to the disparate alcohol drinking behaviors of these line-pairs.

Sensitivity to ethanol intoxication, propensity to drink ethanol and vulnerability to develop alcoholism are all influenced by genetic factors. Conversely, exposure to ethanol or subsequent withdrawal produce gene expression changes, which, in combination with environmental variables, may participate in the emergence of compulsive drinking and relapse. The present review offers an integrated perspective on brain gene expression profiling in rodent models of predisposition to differential ethanol sensitivity or consumption, in rats and mice subjected to acute or chronic ethanol exposure, as well as in human alcoholics. The functional categories over-represented among differentially expressed genes suggest that the transcriptional effects of chronic ethanol consumption contribute to the neuroplasticity and neurotoxicity characteristic of alcoholism. Importantly, ethanol produces distinct transcriptional changes within the different brain regions involved in intoxication, reinforcement and addiction. Special emphasis is put on recent profiling studies that have provided some insights into the molecular mechanisms potentially mediating genome-wide regulation of gene expression by ethanol. In particular, current evidence for a role of transcription factors, chromatin remodeling and microRNAs in coordinating the expression of large sets of genes in animals predisposed to excessive ethanol drinking or exposed to protracted abstinence, as well as in human alcoholics, is presented. Finally, studies that have compared ethanol with other drugs of abuse have highlighted common gene expression patterns that may play a central role in drug addiction. The availability of novel technologies and a focus on mechanistic approaches are shaping the future of ethanol transcriptomics.

The effects of beverage alcohol (ethanol) on the body are determined largely by the rate at which it and its main breakdown product, acetaldehyde, are metabolized after consumption. The main metabolic pathway for ethanol involves the enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Seven different ADHs and three different ALDHs that metabolize ethanol have been identified. The genes encoding these enzymes exist in different variants (i.e., alleles), many of which differ by a single DNA building block (i.e., single nucleotide polymorphisms [SNPs]). Some of these SNPs result in enzymes with altered kinetic properties. For example, certain ADH1B and ADH1C variants that are commonly found in East Asian populations lead to more rapid ethanol breakdown and acetaldehyde accumulation in the body. Because acetaldehyde has harmful effects on the body, people carrying these alleles are less likely to drink and have a lower risk of alcohol dependence. Likewise, an ALDH2 variant with reduced activity results in acetaldehyde buildup and also has a protective effect against alcoholism. In addition to affecting drinking behaviors and risk for alcoholism, ADH and ALDH alleles impact the risk for esophageal cancer.

The INIA19 is a new, high-quality template for imaging-based studies of non-human primate brains created from high-resolution T1-weighted magnetic resonance (MR) images of 19 rhesus macaque (Macaca mulatta) animals. Combined with the comprehensive cortical and subcortical label map of the NeuroMaps atlas, the INIA19 is equally suitable for studies requiring both spatial normalization and atlas label propagation. Population-averaged template images are provided for both the brain and the whole head, to allow alignment of the atlas with both skull-stripped and unstripped data, and thus to facilitate its use for skull stripping of new images. This article describes the construction of the template using freely-available software tools, as well as the template itself, which is being made available to the scientific community (http://nitrc.org/projects/inia19/).

AIMS: Intermittent access (IA) to an alcohol (ethanol) solution can lead rats to higher ethanol intakes than continuous access, and a recent report showed increased drinking in C57BL/6J mice offered 20% ethanol vs. water 3X/week (Prior studies have offered ethanol during 24 h periods, either continuously or intermittently.). METHODS: We tested the high-preference C57BL/6J inbred mice: we also studied High Drinking in the Dark (HDID) mice, a line we have selectively bred to reach intoxicating blood ethanol levels after a short period of access to a single bottle of 20% ethanol. RESULTS: Neither HDID or C57BL/6J male mice offered ethanol every other day during only a 4-h access period showed greater daily intake than mice offered ethanol daily for 4 h. There was a small increase in drinking with 24 h IA in C57BL/6J mice. An experiment with HDID mice and their control heterogeneous stock stock modeled closely after a published study with C57BL/6J mice (Hwa, Chu, Levinson SA et al. Persistent escalation of alcohol drinking in C57BL/6J mice with intermittent access to 20% ethanol. Alcohol Clin Exp Res 2011;35:1938-1947) showed no significant elevation with 24 h IA exposure in either sex of any genotype. Finally, a near replication of the Hwa et al. study showed modestly greater intake in C57BL/6J mice, confirming the efficacy of 24 h IA. CONCLUSION: We conclude that 4 h of IA is likely insufficient to elevate drinking in mice. The lack of effect in HDID mice and their controls further suggests that not all genotypes respond to intermittency.

This paper introduces the Special Section: Pharmacotherapies for the Treatment of Alcohol Abuse and Dependence and provides a summary of patents targeting neurotransmitter systems not covered in the other four chapters. The World Health Organization notes that alcoholic-type drinking results in 2.5 million deaths per year, and these deaths occur to a disproportionately greater extent among adolescents and young adults. Developing a pharmacological treatment targeting alcohol abuse and dependence is complicated by (a) the heterogeneous nature of the disease(s), (b) alcohol affecting multiple neurotransmitter and neuromodulator systems, and (c) alcohol affecting multiple organ systems which in turn influence the function of the central nervous system. Presently, the USA Federal Drug Administration has approved three pharmacotherapies for alcoholism: disulfiram, naltrexone, and acamprosate. This chapter provides a summary of the following systems, which are not covered in the accompanying chapters; alcohol and acetaldehyde metabolism, opioid, glycinergic, GABA-A, neurosteroid, dopaminergic, serotonergic, and endocannabinoid, as well as patents targeting these systems for the treatment of alcoholism. Finally, an overview is presented on the use of pharmacogenetics and pharmacogenomics in tailoring treatments for certain subpopulations of alcoholics, which is expected to continue in the future.

Analysis of mouse brain gene expression, using strains that differ in alcohol consumption, provided a number of novel candidate genes that potentially regulate alcohol consumption. We selected six genes [beta-2-microglobulin (B2m), cathepsin S (Ctss), cathepsin F (Ctsf), interleukin 1 receptor antagonist (Il1rn), CD14 molecule (Cd14) and interleukin 6 (Il6)] for behavioral validation using null mutant mice. These genes are known to be important for immune responses but were not specifically linked to alcohol consumption by previous research. Null mutant mice were tested for ethanol intake in three tests: 24 hr two-bottle choice, limited access two-bottle choice and limited access to one bottle of ethanol. Ethanol consumption and preference were reduced in all the null mutant mice in the 24 hr two-bottle choice test, the test that was the basis for selection of these genes. No major differences were observed in consumption of saccharin in the null mutant mice. Deletion of B2m, Ctss, Il1rn, Cd14 and Il6 also reduced ethanol consumption in the limited access two bottle choice test for ethanol intake; with the Il1rn and Ctss null mutants showing reduced intake in all three tests (with some variation between males and females). These results provide the most compelling evidence to date that global gene expression analysis can identify novel genetic determinants of complex behavioral traits. Specifically, they suggest a novel role for neuroimmune signaling in regulation of alcohol consumption.