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The High Drinking in the Dark mouse lines (HDID-1 and HDID-2) were selectively bred to achieve high blood ethanol concentrations (BECs) in the Drinking in the Dark (DID) task, a widely used model of binge-like intake of 20% ethanol. There are several components that differentiate DID from other animal models of ethanol intake: time of day of testing, length of ethanol access, single-bottle access, and individual housing. Here, we sought to determine how some of these individual factors contribute to the high ethanol intake observed in HDID mice. HDID-1, HDID-2, and non-selected HS/NPT mice were tested in a series of DID experiments where one of the following factors was manipulated: length of ethanol access, fluid choice, number of ethanol bottles, and housing condition. We observed that 1) HDID mice achieve intoxicating BECs in DID, even when they are group-housed; 2) HDID mice continue to show elevated ethanol intake relative to HS/NPT mice during an extended access session, but this is most apparent during the first 4 h of access; and 3) offering a water choice during DID prevents elevated intake in the HDID-1 mice, but not necessarily in HDID-2 mice. Together, these results suggest that the lack of choice in the DID paradigm, together with the length of ethanol access, are important factors contributing to elevated ethanol intake in the HDID mice. These results further suggest important differences between the HDID lines in response to procedural manipulations of housing condition and ethanol bottle number in the DID paradigm, highlighting the distinct characteristics that each of these lines possess, despite being selectively bred for the same phenotype.

The high-drinking-in-the-dark (HDID) lines of mice were selectively bred for achieving high blood alcohol levels in the drinking-in-the-dark (DID) task and have served as a unique genetic risk model for binge-like alcohol intake. However, little is known about their willingness to consume other addictive drugs. Here, we examined (a) whether the HDID-1 and HDID-2 lines of mice would voluntarily consume midazolam, methamphetamine, morphine and nicotine in a DID test and (b) whether the HDID lines differ from their founders, heterogeneous stock/Northport (HS/NPT), in consumption levels of these drugs at the concentrations tested. Separate groups of HDID-1, HDID-2 and HS/NPT mice were given 4 days of access to each drug, using the single-bottle, limited-access DID paradigm. Male and female mice of both HDID lines consumed all four offered drugs. We observed no genotype differences in 40 μg/ml methamphetamine intake, but significant differences in nicotine, midazolam and morphine intake. Both HDID lines drank significantly more (150 μg/ml) midazolam than their founders, providing strong support for a shared genetic contribution to binge ethanol and midazolam intake. HDID-2 mice, but not HDID-1 mice, consumed more morphine (700 μg/ml) and more nicotine across a range of concentrations than HS/NPT mice. These results demonstrate that the HDID mice can be utilized for tests of voluntary drug consumption other than ethanol and highlight potentially important differences between HDID lines in risk for elevated drug intake.

BACKGROUND: Chronic alcohol exposure can alter glucocorticoid receptor (GR) function in some brain areas that promotes escalated and compulsive-like alcohol intake. GR antagonism can prevent dependence-induced escalation in drinking, but very little is known about the role of GR in regulating high-risk non-dependent alcohol intake. Here, we investigate the role of GR in regulating binge-like drinking and aversive responses to alcohol in the High Drinking in the Dark (HDID-1) mice, which have been selectively bred for high blood ethanol concentrations (BECs) in the Drinking in the Dark (DID) test, and in their founder line, the HS/NPT. METHODS: In separate experiments, male and female HDID-1 mice were administered one of several compounds that inhibited GR or its negative regulator, FKBP51 (mifepristone [12.5, 25, 50, 100 mg/kg], CORT113176 [20, 40, 80 mg/kg], and SAFit2 [10, 20, 40 mg/kg]) during a 2-day DID task. Ethanol consumption and BECs were measured. Ethanol conditioned taste and place aversion (CTA and CPA, respectively) were measured in separate HDID-1 mice after mifepristone administration to assess GR’s role in regulating the conditioned aversive effects of ethanol. Lastly, HS/NPT mice were administered CORT113176 during DID to assess whether dissimilar effects from those of HDID-1 would be observed, which could suggest that selective breeding had altered sensitivity to the effects of GR antagonism on binge-like drinking. RESULTS: GR antagonism (with both mifepristone and CORT113176) selectively reduced binge-like ethanol intake and BECs in the HDID-1 mice, while inhibition of FKBP51 did not alter intake or BECs. In contrast, GR antagonism had no effect on ethanol intake or BECs in the HS/NPT mice. Although HDID-1 mice exhibit attenuated ethanol CTA, mifepristone administration did not enhance the aversive effects of ethanol in either a CTA or CPA task. CONCLUSION: These data suggest that the selection process increased sensitivity to GR antagonism on ethanol intake in the HDID-1 mice, and support a role for the GR as a genetic risk factor for high-risk alcohol intake.

BACKGROUND: Rodent models of high alcohol drinking offer opportunities to better understand factors for Alcohol Use Disorders (AUD) and test potential treatments. Selective breeding was carried out to create two unique High Drinking in the Dark (HDID-1, HDID-2) mouse lines that represent models of genetic risk for binge-like drinking. A number of studies have indicated that neuroimmune genes are important for regulation of alcohol drinking. We tested whether compounds shown to reduce drinking in other models also reduce alcohol intake in these unique genetic lines. METHODS: We report tests of gabapentin, tesaglitazar, fenofibrate, caffeic acid phenethyl ester (CAPE), ibrutinib, and rolipram. Although these compounds have different mechanisms of action, they have all been shown to reduce inflammatory responses. We evaluated effects of these compounds on alcohol intake. In order to facilitate comparison with previously published findings for some compounds, we employed similar schedules that were previously used for that compound. RESULTS: Gabapentin increased ethanol binge-like alcohol drinking in female HDID-1 and HS/NPT mice. Tesaglitazar and fenofibrate did not alter two bottle choice (2BC) drinking in male HDID-1 or HS/NPT mice. However, tesaglitazar had no effect on DID ethanol intake but reduced blood alcohol levels (BAL), and fenofibrate increased DID intake with no effects on BAL. CAPE had no effect on ethanol intake. Ibrutinib reduced intake in female HDID-1 in initial testing, but did not reduce intake in a second week of testing. Rolipram reduced DID intake and BALs in male and female HDID-1, HDID-2, and HS/NPT mice. CONCLUSIONS: A number of compounds shown to reduce ethanol drinking in other models and genotypes are not effective in HDID mice, or their genetically heterogeneous founders, HS/NPT. The most promising compound was the PDE4 inhibitor, rolipram. These results highlight the importance of assessing generalizability when rigorously testing compounds for therapeutic development.

We have modelled genetic risk for binge-like drinking by selectively breeding High Drinking in the Dark-1 and −2 (HDID-1 and HDID-2) mice for their propensity to reach intoxicating blood alcohol levels (BALs) after binge-like drinking in a single bottle, limited access paradigm. Interestingly, in standard two-bottle choice (2BC) tests for continuously available alcohol versus water, HDID mice show modest levels of preference. This indicates some degree of independence of the genetic contributions to risk for binge-like and sustained, continuous access drinking. We had few data where the drinking in the dark (DID) tests of binge-like drinking had been repeatedly performed, so we serially offered multiple DID tests to see whether binge-like drinking escalated. It did not. We also asked whether HDID mice would escalate their voluntary intake with prolonged exposure to alcohol 2BC. They did not. Lastly, we assessed whether an alcohol deprivation effect (ADE) developed. ADE is a temporary elevation in drinking typically observed after a period of abstinence from sustained access to alcohol choice. With repetition, these periods of ADE sometimes have led to more sustained elevations in drinking. We therefore asked whether repeated ADE episodes would elevate choice drinking in HDID mice. They did not. After nearly 500 days of alcohol access, the intake of HDID mice remained stable. We conclude that a genetically-enhanced high risk for binge-like drinking is not sufficient to yield alterations in long-term alcohol intake.

Treatment options for Alcohol Use Disorders (AUD) have minimally advanced since 2004, while the annual deaths and economic toll have become alarmingly high. Bringing potential therapeutics beyond the bench and into the clinic for AUD requires rigorous pharmacological screening across molecular, behavioral, pre-clinical, and clinical studies in neuroscience. The repurposing of FDA approved compounds is an effective and expedited means of screening pharmacotherapies for AUD. Here, we demonstrate that apremilast, a phosphodiesterase type 4 inhibitor that is FDA approved for psoriasis and psoriatic arthritis, reduces binge-like alcohol intake and behavioral measures of motivation in unique, preclinical genetic risk models for drinking to intoxication and reduces excessive alcohol drinking in models of stress facilitated drinking and alcohol dependence. In a double blind, placebo-controlled human laboratory study in non-treatment seeking individuals with AUD, apremilast significantly reduced the number of drinks per day. Lastly, using site-directed drug infusions and electrophysiology we determined that apremilast may act by increasing neural activity in the nucleus accumbens, an important alcohol-related brain region, to reduce alcohol intake in mice. These results demonstrate that apremilast reduces excessive alcohol drinking across a spectrum of AUD severity and support its importance as a potential therapeutic for AUD.

BACKGROUND Transcriptional differences between heterogeneous stock mice and high drinking-in-the-dark selected mouse lines have previously been described based on microarray technology coupled with network-based analysis. The network changes were reproducible in 2 independent selections and largely confined to 2 distinct network modules; in contrast, differential expression appeared more specific to each selected line. This study extends these results by utilizing RNA-Seq technology, allowing evaluation of the relationship between genetic risk and transcription of noncoding RNA (ncRNA); we additionally evaluate sex-specific transcriptional effects of selection. METHODS Naive mice (N = 24/group and sex) were utilized for gene expression analysis in the ventral striatum; the transcriptome was sequenced with the Illumina HiSeq platform. Differential gene expression and the weighted gene co-expression network analysis were implemented largely as described elsewhere, resulting in the identification of genes that change expression level or (co)variance structure. RESULTS Across both sexes, we detect selection effects on the extracellular matrix and synaptic signaling, although the identity of individual genes varies. A majority of nc RNAs cluster in a single module of relatively low density in both the male and female network. The most strongly differentially expressed transcript in both sexes was Gm22513, a small nuclear RNA with unknown function. Associated with selection, we also found a number of network hubs that change edge strength and connectivity. At the individual gene level, there are many sex-specific effects; however, at the annotation level, results are more concordant. CONCLUSIONS In addition to demonstrating sex-specific effects of selection on the transcriptome, the data point to the involvement of extracellular matrix genes as being associated with the binge drinking phenotype.

Transcriptome-based drug discovery has identified new treatments for some complex diseases, but has not been applied to alcohol use disorder (AUD) or other psychiatric diseases, where there is a critical need for improved pharmacotherapies. High Drinking in the Dark (HDID-1) mice are a genetic model of AUD risk that have been selectively bred (from the HS/Npt line) to achieve intoxicating blood alcohol levels (BALs) after binge-like drinking. We compared brain gene expression of HDID-1 and HS/Npt mice, to determine a molecular signature for genetic risk for high intensity, binge-like drinking. Using multiple computational methods, we queried LINCS-L1000 (Library of Integrated Network-Based Cellular Signatures), a database containing gene expression signatures of thousands of compounds, to predict candidate drugs with the greatest potential to decrease alcohol consumption. Our analyses predicted novel compounds for testing, many with anti-inflammatory properties, providing further support for a neuroimmune mechanism of excessive alcohol drinking. We validated the top 2 candidates in vivo as a proof-of-concept. Terreic acid (a Bruton's tyrosine kinase inhibitor) and pergolide (a dopamine and serotonin receptor agonist) robustly reduced alcohol intake and BALs in HDID-1 mice, providing the first evidence for transcriptome-based drug discovery to target an addiction trait. Effective drug treatments for many psychiatric diseases are lacking, and the emerging tools and approaches outlined here offer researchers studying complex diseases renewed opportunities to discover new or repurpose existing compounds and expedite treatment options.

The comorbidity of substance- and alcohol-use disorders (AUD) with other psychiatric conditions, especially those related to stress such as post-traumatic stress disorder (PTSD), is well-established. Binge-like intoxication is thought to be a crucial stage in the development of the chronic relapsing nature of the addictions, and self-medication through binge-like drinking is commonly seen in PTSD patients. We have selectively bred two separate High Drinking in the Dark (HDID-1 and HDID-2) mouse lines to reach high blood ethanol concentrations (BECs) after a 4-h period of access to 20% ethanol starting shortly after the onset of circadian dark. As an initial step toward the eventual goal of employing binge-prone HDID mice to study PTSD-like behavior including alcohol binge drinking, we sought first to determine their ability to acquire conditioned fear. We asked whether these mice acquired, generalized, or extinguished conditioned freezing to a greater or lesser extent than unselected control HS/Npt mice. In two experiments, we trained groups of 16 adult male mice in a standard conditioned fear protocol. Mice were tested for context-elicited freezing, and then, in a novel context, for cue-induced freezing. After extinction tests, renewal of conditioned fear was tested in the original context. Mice of all three genotypes showed typical fear responding. Context paired with shock elicited freezing behavior in a control experiment, but cue unpaired with shock did not. These studies indicate that fear learning per se does not appear to be influenced by genes causing predisposition to binge drinking, suggesting distinct neural mechanisms. However, HDID mice are shown to be a suitable model for studying the role of conditioned fear specifically in binge-like drinking.

Two independent lines of High Drinking in the Dark (HDID-1, HDID-2) mice have been bred to reach high blood alcohol levels after a short period of binge-like ethanol drinking. Male mice of both lines were shown to have reduced sensitivity to develop a taste aversion to a novel flavor conditioned by ethanol injections as compared with their unselected HS/NPT founder stock. We have subsequently developed inbred variants of each line. The current experiments established that reduced ethanol-conditioned taste aversion is also seen in the inbred variants, in both males and females. In other experiments, we asked whether HDID mice would ingest sufficient doses of ethanol to lead to a conditioned taste aversion upon retest. Different manipulations were used to elevate consumption of ethanol on initial exposure. Access to increased ethanol concentrations, to multiple tubes of ethanol, and fluid restriction to increase thirst motivation all enhanced initial drinking of ethanol. Each condition led to reduced intake the next day, consistent with a mild conditioned taste aversion. These experiments support the conclusion that one reason contributing to the willingness of HDID mice to drink to the point of intoxication is a genetic insensitivity to the aversive effects of ethanol.

Despite acceptance that risk for alcohol-use disorder (AUD) has a large genetic component, the identification of genes underlying various components of risk for AUD has been hampered in humans, in part by the heterogeneity of expression of the phenotype. One aspect of AUD is physical dependence. Alcohol withdrawal is a serious consequence of alcohol dependence with multiple symptoms, many of which are seen in multiple species, and can be experienced over a wide-ranging time course. In the present three studies, we developed a battery of withdrawal tests in mice, examining behavioral symptoms from multiple domains that could be measured over time. To permit eventual use of the battery in different strains of mice, we used male and female mice of a genetically heterogeneous stock developed from intercrossing eight inbred strains. Withdrawal symptoms were assessed using commonly used tests after administration of ethanol in vapor for 72 continuous hours. We found significant effects of ethanol withdrawal versus air-breathing controls on nearly all symptoms, spanning 4 days following ethanol vapor inhalation. Withdrawal produced hypothermia, greater neurohyperexcitability (seizures and tremor), anxiety-like behaviors using an apparatus (such as reduced transitions between light and dark compartments), anhedonia (reduced sucrose preference), Straub tail, backward walking, and reductions in activity; however, there were no changes in thermal pain sensitivity, hyper-reactivity to handling, or anxiety-like emergence behaviors in other apparatus. Using these data, we constructed a refined battery of withdrawal tests. Individual differences in severity of withdrawal among different tests were weakly correlated at best. This battery should be useful for identifying genetic influences on particular withdrawal behaviors, which should reflect the influences of different constellations of genes.

Background There is a serious public health need for better understanding of alcohol use disorder disease mechanisms and for improved treatments. At this writing, only three drugs are approved by the Food and Drug Administration as medications to treat alcohol use disorders – disulfiram, naltrexone, and acamprosate. Binge drinking is a form of abusive alcohol drinking defined by the NIAAA as a drinking to blood alcohol levels (BALs) > 0.08% during a period of approximately 2 h. To model genetic risk for binge-like drinking, we have used selective breeding to create a unique animal model, High Drinking in the Dark (HDID) mice. Behavioral characterization of HDID mice has revealed that HDID mice exhibit behavioral impairment after drinking, withdrawal after a single binge-drinking session, and escalate their intake in response to induction of successive cycles of dependence. Notably, HDID mice do not exhibit altered tastant preference or alcohol clearance rates. We therefore asked whether drugs of known clinical relevance could modulate binge-like ethanol drinking in HDID mice, reasoning that this characterization of HDID responses should inform future use of this genetic animal model for screening and development of novel potential therapeutics. Methods We tested the efficacy of acamprosate and naltrexone to reduce binge-like drinking in HDID mice. Additionally, we tested the GABA B receptor agonist, baclofen, based on recent pre-clinical and clinical studies demonstrating that it reduces alcohol drinking. We elected not to include disulfiram due to its more limited clinical usage. Mice were tested after acute doses of drugs in the limited-access Drinking in the Dark (DID) paradigm. Results HDID mice were sensitive to the effects of acamprosate and baclofen, but not naltrexone. Both drugs reduced binge-like drinking. However, naltrexone failed to reduce drinking in HDID mice. Thus, HDID mice may represent a useful model for screening novel compounds.

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 >60,000 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.

Drinking in the dark (DID) is a limited access ethanol-drinking phenotype in mice. High Drinking in the Dark (HDID-1) mice have been bred for 27 selected generations (S27) for elevated blood ethanol concentrations (BECs) after a 4-h period of access to 20% ethanol. A second replicate line (HDID-2) was started later from the same founder population and is currently in S20. An initial report of response to selection in HDID-1 was published after S11. This article reports genetic and behavioral characteristics of both lines in comparison with the HS controls. Heritability is low in both replicates (h(2) = 0.09) but the lines have shown 4-5 fold increases in BEC since S0; 80% of HDID-1 and 60% of HDID-2 mice reach BECs greater than 1.0 mg/ml. Several hours after a DID test, HDID mice show mild signs of withdrawal. Although not considered during selection, intake of ethanol (g/kg) during the DID test increased by approximately 80% in HDID-1 and 60% in HDID-2. Common genetic influences were more important than environmental influences in determining the similarity between BEC and intake for HDID mice. Analysis of the partitioning of intake showed that 60% of intake is concentrated in the last 2 h of the 4 h session. However, this has not changed during selection. Hourly BECs during the DID test reach peak levels after 3 or 4 h of drinking. HDID mice do not differ from HS mice in their rate of elimination of an acute dose of alcohol.

Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mouse lines were bidirectionally selectively bred, respectively, to have severe or mild ethanol withdrawal handling-induced convulsions (HICs) after cessation of 3 days of ethanol vapor inhalation. Murine genotypes with severe withdrawal have been found to show low ethanol consumption, and high consumers show low withdrawal. An early drinking study with WSP and WSR mice showed modest evidence consistent with this genetic correlation, but there were several limitations to that experiment. We therefore conducted a thorough assessment of two bottle ethanol preference drinking in both replicate pairs of WSP/WSR selected lines in mice of both sexes. Greater preference drinking of WSR-2 than WSP-2 female mice confirmed the earlier report. However, in the parallel set of selected lines, the WSP-1 mice drank more than the WSR-1s. Naive mice tested for preference for sucrose, saccharin and quinine did not differ markedly for any tastant. Finally, in a test of binge-like drinking, Drinking in the Dark (DID), WSP mice drank more than WSR mice and attained significantly higher (but still modest) blood ethanol concentrations. Tests of acute withdrawal after DID showed a mild, but significant elevation in handling-induced convulsions in the WSP line. These results provide further evidence that 2-bottle ethanol preference and DID are genetically distinguishable traits.

Among animals at risk for excessive ethanol consumption such as the HDID selected mouse lines, there is considerable individual variation in the amount of ethanol consumed and the associated blood ethanol concentrations (BECs). For the HDID lines, this variation occurs even though the residual genetic variation associated with the DID phenotype has been largely exhausted and thus is most likely associated with epigenetic factors. Here we focus on the question of whether the genes associated with individual variation in HDID-1 mice are different from those associated with selection (risk) (Iancu et al., 2013). Thirty-three HDID-1 mice were phenotyped for their BECs at the end of a standard DID trial, were sacrificed 3 weeks later, and RNA-Seq was used to analyze the striatal transcriptome. The data obtained illustrate that there is considerable overlap of the risk and variation gene sets, both focused on the fine-tuning of synaptic plasticity.

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.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.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.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.

Background: To study withdrawal, ethanol is usually administered chronically without interruption. However, interest has recurred in models of episodic exposure. Increasing evidence suggests that chronic intermittent exposure to ethanol leads to a sensitization effect in both withdrawal severity and ethanol consumption. The goal of the present study was to examine mouse inbred strain differences in withdrawal severity following chronic intermittent exposure using the handling-induced convulsion as the behavioral endpoint. We also sought to compare the withdrawal responses of inbred strains across acute, chronic continuous, and chronic intermittent exposure regimens. Methods: Male mice from 15 standard inbred strains were exposed to ethanol vapor for 16 hours each day for 3 days and removed to an air chamber during the intervening 8 hours. Mice in the control groups were handled the same, except that they were exposed only to air. Daily blood ethanol concentrations were averaged for each mouse to estimate total dose of ethanol experienced. Results: Across strains, mice had an average daily blood ethanol concentration (BEC) of 1.45 ± 0.02 mg/ml and we restricted the range of this value to 1.00–2.00 mg/ml. To evaluate strain differences, we divided data into two dose groups based on BEC, low dose (1.29 ± 0.1 mg/ml) and high dose (1.71 ± 0.02 mg/ml). After the third inhalation exposure, ethanol-exposed and air-exposed groups were tested hourly for handling-induced convulsions for 10 hour and at hour 24 and 25. Strains differed markedly in the severity of withdrawal (after subtraction of air control values) in both dose groups. Conclusion: The chronic intermittent exposure paradigm is sufficient to elicit differential withdrawal responses across nearly all strains. Data from the high-dose groups correlated well with withdrawal data derived from prior acute (single high dose) and chronic continuous (for 72 hours) ethanol withdrawal studies, supporting the influence of common genes on all three responses.

The latency of mice and rats to fall from the accelerating rotarod can differ markedly between laboratories using the same brand of rod as well as between studies using different kinds of rods. These discrepancies can arise from different rod diameters, surface textures, test protocols, or laboratory environmental factors beyond the test itself, but it is also possible that the actual acceleration rates of the different rods do not correspond to the nominal rates set on the devices. This paper describes a simple method to measure acceleration rate of the rotarod and to set the rate to a desired value for any brand of rod.

Withdrawal after chronic ethanol (EtOH) affects body temperature, goal-directed behavior and motor function in mice and increases general central nervous system excitability. Nest-building tests have been used to assay these states but to this point have not been employed as measures of EtOH withdrawal severity. We first refined nest-scoring methods using a genetically heterogeneous stock of mice (HS/Npt). Mice were then made physically dependent following three days of chronic EtOH vapor inhalation to produce average blood EtOH concentrations (BECs) of 1.89 mg/mL. EtOH withdrawal affected the progression of nest building over time when mice were tested 2–4 days after removal from three days of chronic exposure to EtOH. In a separate group of mice, chronic EtOH vapor inhalation (BECs 1.84 mg/mL) suppressed nest building over days 1–2 but not days 2–3 of withdrawal. In a following experiment, EtOH withdrawal dose-dependently slowed recovery of nest building for up to 32 h. Finally, we determined that long-lasting nest-building deficits extend to mice undergoing withdrawal from a high dose (4 g/kg) of acute EtOH. Sex differences for nest building were absent following EtOH exposure. In mice naive to EtOH treatments, male mice had lower pre-test body temperatures and increased nest scores across a two-day testing period compared to females. These results suggest that nest building can be used to assess chronic and acute EtOH withdrawal severity in mice.

Withdrawal from benzodiazepines in physically dependent rodents often requires that the drug be dislodged from its receptor with a competitive antagonist. Withdrawal Seizure-Prone (WSP) mice were selectively bred for their susceptibility to handling-induced withdrawal convulsions following chronic treatment with ethanol. Reflecting pleiotropic genetic influences, they also experience more severe withdrawal from other sedative-hypnotics including the benzodiazepine, diazepam. We used this susceptible genotype to test whether other benzodiazepine receptor (BZR) agonists also produce physical dependence following acute administration, comparing studies of spontaneous withdrawal with those where convulsions were precipitated by a BZR antagonist (flumazenil). Separate groups of mice were tested following a single injection of one of eight BZR agonists. Several doses of each drug were tested for spontaneous withdrawal, and a single dose of each drug was tested for precipitated withdrawal. Withdrawal convulsions were seen after all of the drugs by at least one method, suggesting that BZR agonists as a class elicit acute physical dependence in this susceptible genotype.

UNLIKE WITH HUMAN subjects, assessment of the reinforcing effects of ethanol in rodents is necessarily a matter of inference. The two basic approaches are to allow the animal to self-administer ethanol or to use principles of Pavlovian conditioning to pair ethanol's effects with a specific cue and assess approach or avoidance of that cue in a subsequent drug-free test (Cunningham and Phillips, 2003). The earliest studies of voluntary self-administration (Richter and Campbell, 1940) were adapted from the nutrition field and offered rats two bottles, one containing an alcohol solution in tap water and the other tap water alone. These studies reported that self-administration was dependent on concentration of ethanol offered. Individual differences in ethanol preference among rats also were noted. This basic procedure is usually termed a “two-bottle ethanol preference” test, which has many variants. The earliest attempts to document a genetic contribution to individual differences in preference drinking were successful. Mardones and Segovia–Requelme (1983) successfully bred rats to prefer or avoid ethanol-containing solutions, and McClearn and Rodgers (1959) demonstrated that C57BL/6 (B6) inbred mice had nearly absolute preference for 10% ethanol over tap water, that DBA/2 mice were near-teetotalers, and that other inbred strains showed intermediate preference. Fairly large surveys of 14 (Rodgers, 1972) or 15 (Belknap et al., 1993) inbred strains reinforced the primacy of B6 but revealed no other strains with higher preference. The genetic contribution to the trait was further evidenced by the relatively high preference of other strains (C57 L/J, C57BR/cdJ) from the C57BL lineage (Belknap et al., 1993). Subsequent analyses of even larger panels of inbred mouse strains (28 strains: Bachmanov et al., personal communication; 22 strains: Finn et al., unpublished) also have failed to reveal a more extreme preferrer of alcohol solutions than B6. Ethanol intake is often reported using two indices. One is the “preference ratio,” or the proportion of total fluid intake that is taken from the ethanol bottle in a two-bottle ethanol preference test. As already noted, preference ratio depends on the concentration offered, and the usual finding is that preference increases as concentration increases, up to a point, but thereafter, preference ratios decline. That is, the relationship is an inverted-U shaped curve. The second index, g of ethanol consumed per kg body weight, gives a direct measure of the amount of ethanol consumed and relates directly to blood ethanol concentration. In the studies reported below, we report both indices but base our interpretations of the data only on g/kg intake. From the strain surveys reported above, and from the data reported in many (>100) other published studies of 24-hr two-bottle ethanol preference, it has been well-documented that male B6 mice will self administer ethanol in the range of 10–14 g/kg/day, while female B6 mice will self-administer in the range of 12–18 g/kg/day, when the choice is between a 10% ethanol solution and water. Many null mutants and transgenics have been tested for ethanol preference. Drinking in these studies necessarily compares intake of the null mutant versus the wild-type onto which the mutation has been bred. While some of the wild-types in these studies are B6, many are not, and often the wild type is itself a hybrid of two or more inbred strains, a segregating intercross, or a partial backcross to an inbred strain. A survey of many such studies reported that about 1/3 found increased ethanol preference versus wild-type in the null mutants, 1/3 found a reduction, and about 1/3 no effect (Cunningham and Phillips, 2003). However, there are no reports of which we are aware where substantially greater g/kg doses of ethanol than those self-administered by B6 are reported. In one of our laboratories (Texas), we have been screening null mutants for ethanol preference. The background strain on which the mutant α1 S267Q glycine receptor transgenic was held was the F1 hybrid of B6 and FVB/NJ (FVB). As reported in Experiment 1, we noted a very high intake of ethanol in the B6×FVB F1 hybrids. To substantiate this finding, and to test the hypothesis of an effect of maternal genotype, we attempted to repeat the finding in another laboratory (Oregon). As we are also exploring other methods for inducing high levels of ethanol self-administration (Finn et al., 2005; Rhodes et al., 2005), we also tested B6×FVB F1 mice in these newer, limited access procedures. We found that B6×FVB F1 mice indeed self administer extremely high levels of ethanol in two-bottle preference tests. They appeared to drink more than B6 mice in the standard two-bottle preference test, and resembled B6 in the newer assays that lead to higher brain concentrations of ethanol.

We have established that there is a considerable amount of common genetic influence on physiological dependence and associated withdrawal from sedative-hypnotic drugs including alcohol, benzodiazepines, barbiturates and inhalants. We previously mapped two loci responsible for 12 and 9% of the genetic variance in acute alcohol and pentobarbital withdrawal convulsion liability in mice, respectively, to an approximately 28-cM interval of proximal chromosome 11. Here, we narrow the position of these two loci to a 3-cM interval (8.8 Mb, containing 34 known and predicted genes) using haplotype analysis. These include genes encoding four subunits of the GABA(A) receptor, which is implicated as a pivotal component in sedative-hypnotic dependence and withdrawal. We report that the DBA/2J mouse strain, which exhibits severe withdrawal from sedative-hypnotic drugs, encodes a unique GABA(A) receptor gamma2 subunit variant compared with other standard inbred strains including the genetically similar DBA/1J strain. We also demonstrate that withdrawal from zolpidem, a benzodiazepine receptor agonist selective for alpha1 subunit containing GABA(A) receptors, is influenced by a chromosome 11 locus, suggesting that the same locus (gene) influences risk of alcohol, benzodiazepine and barbiturate withdrawal. Our results, together with recent knockout studies, point to the GABA(A) receptor gamma2 subunit gene (Gabrg2) as a promising candidate gene to underlie phenotypic differences in sedative-hypnotic physiological dependence and associated withdrawal episodes.

We previously mapped quantitative trait loci (QTL) responsible for approximately 26% of the genetic variance in acute alcohol and barbiturate (i.e., pentobarbital) withdrawal convulsion liability to a < 1 cM (1.8 Mb) interval of mouse chromosome 4. To date, Mpdz, which encodes the multiple PSD95/DLG/ZO-1 (PDZ) domain protein (MPDZ), is the only gene within the interval shown to have allelic variants that differ in coding sequence and/or expression, making it a strong candidate gene for the QTL. Previous work indicates that Mpdz haplotypes in standard mouse strains encode distinct protein variants (MPDZ1-3), and that MPDZ status is genetically correlated with severity of withdrawal from alcohol and pentobarbital. Here, we report that MPDZ status cosegregates with withdrawal convulsion severity in lines of mice selectively bred for phenotypic differences in severity of acute withdrawal from alcohol [i.e., High Alcohol Withdrawal (HAW) and Low Alcohol Withdrawal (LAW) lines] or pentobarbital [High Pentobarbital Withdrawal (HPW) and Low Pentobarbital Withdrawal (LPW) lines]. These analyses confirm that MPDZ status is associated with severity of alcohol and pentobarbital withdrawal convulsions. Using a panel of standard inbred strains of mice, we assessed the association between MPDZ status with seizures induced by nine chemiconvulsants. Our results show that MPDZ status is genetically correlated with seizure sensitivity to pentylenetetrazol, kainate and other chemiconvulsants. Our results provide evidence that Mpdz may have pleiotropic effects on multiple seizure phenotypes, including seizures associated with withdrawal from two classes of central nervous system (CNS) depressants and sensitivity to specific chemiconvulsants that affect glutaminergic and GABAergic neurotransmission.

Motor incoordination is frequently used as a behavioral index of intoxication by drugs that depress the central nervous system. Two tasks that have been used to assay incoordination in mice, the balance beam and the grid test, were evaluated to optimize aspects of apparatus and testing procedures for studying genetic differences. Mice of eight inbred strains were given one of several doses of ethanol or saline and tested for intoxication. Strains differed in sensitivity to ethanol in both tests, indicating a significant influence of genotype on ethanol sensitivity. For the balance beam, the width of the beam affected the strain sensitivity pattern, and only the widest beam worked well at all doses. For the grid test, both ethanol dose and the time after drug injection affected strains differentially. Although the behavioral sign of intoxication recorded for both tests was a foot-slip error, the correlations of strain means for ethanol sensitivity across the two tasks were generally not significant. This suggests that the genes influencing ethanol sensitivity in the two tasks are mostly different. These results make clear that a single set of task parameters is insufficient to characterize genetic influences on behavior. Several other issues affect the interpretation of data using these tests.

Mice from 8 to 21 inbred strains were tested for sensitivity to ethanol intoxication using a range of doses and three different measures: the screen test, the dowel test and a test of grip strength. Strains differed under nearly all conditions. For the dowel test, two dowel widths were employed, and mice were tested immediately or 30 min after ethanol. For the dowel and screen tests, low doses failed to affect some strains, and the highest doses failed to discriminate among mice, maximally affecting nearly all. For grip strength, a single ethanol dose was used, and mice of all strains were affected. Pharmacokinetic differences among strains were significant, but these could not account for strain differences in intoxication. For doses and test conditions in the middle range, there were only modest correlations among strain means within a test. In addition, genotypic correlations across tests were modest to quite low. These results suggest that different specific versions of a test reflect the influence of different genes, and that genetic influences on different tests were also distinct.

Ethanol (alcohol) withdrawal-induced convulsions are a key index of physical dependence on ethanol and a clinically important consequence of alcohol abuse in humans. In rodent models, severity of withdrawal is strongly influenced by genotype. For example, many studies have reported marked differences in withdrawal severity between the WSR (Withdrawal Seizure Resistant) and WSP (Withdrawal Seizure Prone) mouse strains selectively bred for over 25 generations to differ in chronic withdrawal severity. Therefore, we used an F(2) intercross between the inbred WSP and WSR strains for a genome-wide search for quantitative trait loci (QTLs), which are chromosomal sites containing genes influencing the magnitude of withdrawal. We also used the recently developed HW, RHW (high withdrawal) and LW, RLW (low withdrawal) lines selectively bred for the same trait and in the same manner as the WSP, WSR lines. QTL analysis was then used to dissect the continuous trait distribution of withdrawal severity into component loci, and to map them to broad chromosomal regions by using the Pseudomarker 0.9 and Map Manager QT29b programs. This genome-wide search identified five significant QTLs influencing chronic withdrawal severity on Chromosomes (Chrs) 1 (proximal), 4 (mid), 8 (mid), 11 (proximal), and 14 (mid), plus significant interactions (epistasis) between loci on Chr 11 with 13, 4 with 8, and 8 with 14.

Rating scales for difficulty in capturing and holding mice were devised that proved to be easy to use and highly sensitive to differences among mouse strains on the A and B priority lists of the Mouse Phenome Project. The simplicity of the scales makes it feasible to rate wildness during behavioral test sessions without adding much to testing time or distracting the technician from the principal task at hand. Overall wildness and placidity ratings obtained by combining capture and hold ratings provide a good impression of the difficulties encountered while working with lab mice in the course of complex experiments. Ratings of 21 inbred strains during the course of 15 behavioral tests in two laboratories demonstrated that the SPRET/Ei, PERA/Ei, CAST/Ei and SWR/J strains were particularly difficult to handle. The NOD/LtJ strain posed no special challenge in the Edmonton laboratory but was very difficult to handle in the Portland lab. The rating scales should be useful for judging the difficulties in working with novel targeted or induced mutations in mice as well as effects of a variety of environmental treatments or drugs.

To elucidate pathways from specific genes to complex behaviors, assays of mouse behavior need to be valid, reliable and replicable across laboratories. Behavioral assays are proving to be as complex as the intricate cellular and molecular pathways that are the main interest of many mouse users. There is no perfect behavioral test, but we propose some aphorisms to stimulate discussion that is necessary for continued progress in task development. For maximal utility, a behavioral test should yield valid data for most of the commonly used inbred mouse strains. Tests of simple, ubiquitous behaviors usually yield meaningful data for most mice, especially when based on automated scoring or on simple physical measures that are likely to be replicable across laboratories. Extreme test scores resulting from non-performance on a task can inflate the apparent reliability of a test, and devious adaptations to a task can undermine its validity. The optimal apparatus configuration for certain genetic or pharmacological analyses might depend on the particular laboratory environment. Despite our best efforts, the mice will continue to win some innings.

Rationale. We postulated that genetic determinants of different responses to pentobarbital (PB) in mice would differ from response to response. Objectives. Mice from 14 standard inbred strains were tested for sensitivity to several effects of acute PB. Methods. Strains were tested for sensitivity to PB-induced low-dose stimulation and high-dose depression of locomotor activity, reduced rearing, hypothermia, and ataxia assessed on a rotarod, using four doses of PB or saline. Results. Strains differed in sensitivity to PB for all responses. Correlations among strain means indicated that strain sensitivity to a particular effect of PB generalized rather well across doses. Sensitivities to some of the different behavioral responses were also significantly correlated. For example, strains less sensitive to PB-induced enhanced locomotor activity were also significantly more sensitive to the drug's hypothermic effects. Some responses were genetically independent. Brain PB concentrations were also determined, and appeared to be unrelated to inbred strain drug sensitivities. Conclusions. Overall, these results suggest that there are multiple genetic determinants of behavioral sensitivity to PB effects. That is, genetically influenced sensitivity to PB is not monolithic, but is somewhat specific to the particular response variable studied, a result that also characterizes genetic control of responses to other drugs.

Background: Genetic factors are well known to play an important role in determining individual differences in the metabolism of ethanol (EtOH), and several specific polymorphic loci have been identified that significantly contribute to the variability of EtOH metabolism in humans. However, these variant genes are either alcohol or aldehyde dehydrogenases, and the identification of new gene products that contribute to variation in alcohol metabolism would be useful. Methods: To identify quantitative trait loci (QTLs), we correlated variation in polymorphic markers with blood EtOH concentration and the rate of EtOH metabolism (β) in C57BL/6J and DBA/2J strains and in 25 of their recombinant inbred strains after 2 and 3 g/kg of EtOH intraperitoneally. Results: A QTL associated with β values for both doses was definitively mapped to the proximal region of chromosome 17, syntenic with human chromosome 6q25–27. Seven to 12 chromosomal regions were provisionally identified for each phenotype; several were associated with 2 or more phenotypes. Each QTL suggests the location of a gene or genes affecting EtOH pharmacokinetics. Candidate genes suggested by these analyses included several whose gene products are known to be induced by EtOH (e.g., superoxide dismutase, glutathione transferase, and cytochrome P450 2E1), as well as several whose gene products have signaling functions likely to contribute to this induction. Conclusions: These studies provide evidence for the existence of genes affecting EtOH metabolism in multiple chromosomal regions. Future studies will be required to identify the chromosome 17 gene product. Use of other genetic populations, such as B6D2F2 crosses, will be required to determine which of the provisional loci represent true and which represent false-positive associations.

Rationale: Alcoholism is associated with withdrawal (physical dependence), tolerance, or a maladaptive pattern of alcohol (ethanol) use. The well-documented difference in susceptibility to withdrawal after chronic ethanol exposure between the C57BL/6J and DBA/2J mouse strains provides an excellent starting point for dissecting genetic influences involved in physical dependence on ethanol. A quantitative trait locus (QTL) identifies the genomic location of a gene (or genes) affecting a trait of interest. Objectives: A genome-wide QTL mapping study was carried out to dissect the multifactorial nature of withdrawal after chronic ethanol exposure using 400 B6D2F2 mice. Methods: To induce physical dependence, we used a standard paradigm in which mice were exposed to ethanol vapor for 72 h. The mice were then tested hourly for handling-induced convulsions (HICs) for 10 h and at hours 24 and 25. Ethanol withdrawal severity was first computed as the area under the 25-h HIC curve. Separate regression residuals were then calculated that corrected for individual differences in blood ethanol concentration at the time of withdrawal and baseline HIC severity (i.e. before ethanol exposure). Results: Statistical mapping yielded significant evidence (P

Potentially life-threatening seizures can occur following withdrawal from benzodiazepines, ethanol, or barbiturates. In animals, withdrawal severity has been shown to be partially genetically determined for each drug class. Susceptibility to these drugs is partially determined by common genetic factors, but the evidence is conflicting. We tested the hypothesis that acute benzodiazepine withdrawal convulsions are influenced by at least some genes that also affect withdrawal from ethanol and pentobarbital. Results in inbred mouse strains demonstrate that strain susceptibility is genetically correlated with susceptibility to ethanol and pentobarbital. The proportion of variance accounted for by genetic factors common to diazepam and ethanol was estimated at 69%. Results contrast with previous data obtained in mice that were serially tested for withdrawal severity from ethanol, pentobarbital, and then diazepam, because serial testing of mice significantly affected the previous results for some strains. Diazepam withdrawal severity was also genetically correlated with pentobarbital withdrawal. Together, these results suggest that some genes influence severity of withdrawal from several types of depressant drugs.

Alcohol dependence (alcoholism) is accompanied by evidence of tolerance, withdrawal (physiological dependence), or compulsive behavior related to alcohol use. Studies of strain and individual differences using animal models for acute physiological dependence liability are useful means to identify potential genetic determinants of liability in humans. Behavioral and quantitative trait analyses were conducted using animal models for high risk versus resistance to acute physiological dependence. Using a two-step genetic mapping strategy, loci on mouse chromosomes 1, 4, and 11 were mapped that contain genes that influence alcohol withdrawal severity. In the aggregate, these three risk markers accounted for 68% of the genetic variability in alcohol withdrawal. Candidate genes in proximity to the chromosome 11 locus include genes encoding the α1, α6, and γ2subunits of type-A receptors for the inhibitory neurotransmitter, GABA. In addition, suggestive linkage is indicated for two loci on mouse chromosome 2, one nearGad1encoding glutamic acid decarboxylase, and the other near theEl2locus which influences the seizure phenotype in the neurological mutant strain El. The present analyses detect and map some of the loci that increase risk to develop physiological dependence and may facilitate identification of genes related to the development of alcoholism. Syntenic conservation between human and mouse chromosomes suggests that human homologs of genes that increase risk for physiological dependence may localize to 1q21–q32, 2q24–q37/11p13, 9p21–p23/1p32–p22.1, and 5q32–q35.

Background Data from C57BL/6J (B6) × DBA/2J (D2) F2 intercrosses (B6xD2 F2), standard and recombinant inbred strains, and heterogeneous stock mice indicate that a reciprocal (or inverse) genetic relationship exists between alcohol consumption and withdrawal severity. Furthermore, some genetic studies have detected reciprocal quantitative trait loci (QTLs) for these traits. We used a novel mouse model developed by simultaneous selection for both high alcohol consumption/low withdrawal and low alcohol consumption/high withdrawal and analyzed the gene expression and genome-wide genotypic differences. Methods Randomly chosen third selected generation (S3) mice (N = 24/sex/line), bred from a B6xD2 F2, were genotyped using the Mouse Universal Genotyping Array, which provided 2,760 informative markers. QTL analysis used a marker-by-marker strategy with the threshold for a significant log of the odds (LOD) set at 10. Gene expression in the ventral striatum was measured using the Illumina Mouse 8.2 array. Differential gene expression and the weighted gene co-expression network analysis (WGCNA) were implemented. Results Significant QTLs for consumption/withdrawal were detected on chromosomes (Chr) 2, 4, 9, and 12. A suggestive QTL mapped to Chr 6. Some of the QTLs overlapped with known QTLs mapped for 1 of the traits individually. One thousand seven hundred and forty-five transcripts were detected as being differentially expressed between the lines; there was some overlap with known withdrawal genes (e.g., Mpdz) located within QTL regions. WGCNA revealed several modules of co-expressed genes showing significant effects in both differential expression and intramodular connectivity; a module richly annotated with kinase-related annotations was most affected. Conclusions Marked effects of selection on expression and network structure were detected. QTLs overlapping with differentially expressed genes on Chr 2 (distal) and 4 suggest that these are cis-eQTLs (Chr 2: Kif3b, Kcnq2; Chr 4: Mpdz, Snapc3). Other QTLs identified were on Chr 2 (proximal), 9, and 12. Network results point to involvement of kinase-related mechanisms and outline the need for further efforts such as interrogation of noncoding RNAs.

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 Naive 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

Background Heterogeneous stock (HS/NPT) mice have been used to create lines selectively bred in replicate for elevated drinking in the dark (DID). Both selected lines routinely reach a blood ethanol (EtOH) concentration (BEC) of 1.00 mg/ml or greater at the end of the 4-hour period of access in Day 2. The mechanisms through which genetic differences influence DID are currently unclear. Therefore, the current study examines the transcriptome, the first stage at which genetic variability affects neurobiology. Rather than focusing solely on differential expression (DE), we also examine changes in the ways that gene transcripts collectively interact with each other, as revealed by changes in coexpression patterns. Methods Naive mice (N = 48/group) were genotyped using the Mouse Universal Genotyping Array, which provided 3,683 informative markers. Quantitative trait locus (QTL) analysis used a marker-by-marker strategy with the threshold for a significant logarithm of odds (LOD) set at 10.6. Gene expression in the ventral striatum was measured using the Illumina Mouse 8.2 array. Differential gene expression and the weighted gene coexpression network analysis (WGCNA) were implemented largely as described elsewhere. Results Significant QTLs for elevated BECs after DID were detected on chromosomes 4, 14, and 16; the latter 2 were associated with gene-poor regions. None of the QTLs overlapped with known QTLs for EtOH preference drinking. Ninety-four transcripts were detected as being differentially expressed in both selected lines versus HS controls; there was no overlap with known preference genes. The WGCNA revealed 2 modules as showing significant effects of both selections on intramodular connectivity. A number of genes known to be associated with EtOH phenotypes (e.g., Gabrg1, Glra2, Grik1, Npy2r, and Nts) showed significant changes in connectivity. Conclusions We found marked and consistent effects of selection on coexpression patterns; DE changes were more modest and less concordant. The QTLs and differentially expressed genes detected here are distinct from the preference phenotype. This is consistent with behavioral data and suggests that the DID and preference phenotypes are markedly different genetically.

Ethanol Withdrawal-Associated Drinking and Drinking in the Dark: Common and Discrete Genetic Contributions 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 twobottle ethanol preference drinking data assessed under conditions of continuous ethanol access. Although inbred strain data for withdrawalassociated 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.

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.

The genomic map locations of specific genes controlling behaviors can be identified by studying a panel of recombinant inbred (RI) mouse strains. The progenitor C57BL/6J (B6) and DBA/2J (D2) strains, and 19 of the BXD RI strains derived from an F2 cross of these progenitors, were tested for 3% and 10% ethanol (EtOH) intake. The test sequence began with two-bottle free choice between tap water and unsweetened ethanol, and ended with free choice between water and saccharin-sweetened ethanol. Saccharin preference was also measured. Correlational analyses indicated that 59% of the genetic variance in 10% ethanol and sweetened 10% ethanol consumption was held in common, 24% of the genetic variance in saccharin and sweetened 10% ethanol consumption was held in common, and only 7% of the genetic variance in saccharin and unsweetened 10% ethanol consumption was held in common. These percentages for 3% ethanol solutions were 21%, 36%, and 14%. In addition, the severity of handling-induced convulsions during ethanol withdrawal was found to be significantly associated with the amount of ethanol consumed from the sweetened ethanol drinking tubes, suggesting that genetic differences in avidity for ethanol could lead to the development of physical dependence. Quantitative trait loci (QTL) analyses revealed that several genetic markers were associated with ethanol consumption levels, including markers for the D2 dopamine receptor. QTL analyses of saccharin and sweetened ethanol consumption identified the sac locus, thought to determine the ability to detect saccharin. In general, our results suggest that saccharin and ethanol consumption are determined by the actions of multiple genes (QTL), some in common, and suggest specific map locations of several such QTL on the mouse genome.

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 naive 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 Alcohol increases the expression of Group 1 metabotropic glutamate receptors (mGluRs) and their associated scaffolding protein Homer2 and stimulates phosphatidylinositol 3-kinase (PI3K) within the nucleus accumbens (NAC). Moreover, functional studies suggest that NAC Group 1 mGluR/Homer2/PI3K signaling may be a potential target for pharmacotherapeutic intervention in alcoholism. Methods Immunoblotting was conducted to examine the effects of alcohol consumption under drinking-in-the-dark (DID) procedures on Group 1 mGluR-associated proteins in C57BL/6J (B6) mice. Follow-up behavioral studies examined the importance of Group 1 mGluR/Homer2/PI3K signaling within the NAC shell for limited-access alcohol drinking. Finally, immunoblotting examined whether the NAC expression of Group 1 mGluR-associated proteins is a genetic correlate of high alcohol drinking using a selectively bred high DID (HDID-1) mouse line. Results Limited-access alcohol drinking under DID procedures up-regulated NAC shell Homer2 levels, concomitant with increases in mGluR5 and NR2B. Intra-NAC shell blockade of mGluR5, Homer2, or PI3K signaling, as well as transgenic disruption of the Homer binding site on mGluR5, decreased alcohol consumption in B6 mice. Moreover, transgenic disruption of the Homer binding site on mGluR5 and Homer2 deletion both prevented the attenuating effect of mGluR5 and PI3K blockade upon intake. Finally, the basal NAC shell protein expression of mGluR1 and Homer2 was increased in offspring of HDID-1 animals. Conclusions Taken together, these data further implicate Group 1 mGluR signaling through Homer2 within the NAC in excessive alcohol consumption.

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.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.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.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.