Genetic and genomic signatures in ethanol withdrawal seizure-prone and seizure-resistant mice implicate genes involved in epilepsy and neuronal excitability.

Alcohol withdrawal is a clinically important consequence and potential driver of Alcohol Use Disorder. However, susceptibility to withdrawal symptoms, ranging from craving and anxiety to seizures and delirium, varies greatly. Selectively bred Withdrawal Seizure-Prone (WSP) and Seizure-Resistant (WSR) mice are an animal model of differential susceptibility to withdrawal and phenotypes with which withdrawal severity correlates. To identify innate drivers of alcohol withdrawal severity, we performed a multi-omic study of the WSP and WSR lines and F2 mice derived from them, using genomic, genetic, and transcriptomic analyses. Genes implicated in seizures and epilepsy were over-represented among those that segregated between WSP and WSR mice and that displayed differential expression in F2 mice high and low in withdrawal. Quantitative trait locus (QTL) analysis of ethanol withdrawal convulsions identified several genome-wide significant loci and pointed to genes that modulate potassium channel function and neural excitability. Perturbations of expression of genes involved in synaptic transmission, including GABAergic and glutamatergic genes, were prominent in prefrontal cortex transcriptome. Expression QTL (eQTL) analysis fine mapped genes within the peak ethanol withdrawal QTL regions. Genetic association analysis in human subjects provided converging evidence for the involvement of those genes in severity of alcohol withdrawal and dependence. Our results reveal a polygenic network and neural signaling pathways contributing to ethanol withdrawal seizures and related phenotypes that overlap with genes modulating epilepsy and neuronal excitability.

Midazolam, methamphetamine, morphine and nicotine intake in high-drinking-in-the-dark mice.

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.

Long-term alcohol drinking in High Drinking in the Dark mice is stable for many months and does not show alcohol deprivation effects.

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.

Effects of Pharmacologically Targeting Neuroimmune Pathways on Alcohol Drinking in Mice Selectively Bred to Drink to Intoxication.

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 2 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 (EtOH) binge-like alcohol drinking in female HDID-1 and HS/NPT mice. Tesaglitazar and fenofibrate did not alter 2-bottle choice (2BC) drinking in male HDID-1 or HS/NPT mice. However, tesaglitazar had no effect on DID EtOH intake but reduced blood alcohol levels (BAL), and fenofibrate increased DID intake with no effects on BAL. CAPE had no effect on EtOH 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 EtOH 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.

Targeting the Glucocorticoid Receptor Reduces Binge-Like Drinking in High Drinking in the Dark (HDID-1) Mice.

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 nondependent 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 (EtOH) 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. EtOH consumption and BECs were measured. EtOH 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 EtOH. 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 EtOH 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 EtOH intake or BECs in the HS/NPT mice. Although HDID-1 mice exhibit attenuated EtOH CTA, mifepristone administration did not enhance the aversive effects of EtOH in either a CTA or CPA task. CONCLUSION: These data suggest that the selection process increased sensitivity to GR antagonism on EtOH intake in the HDID-1 mice, and support a role for the GR as a genetic risk factor for high-risk alcohol intake.

What Is Addiction? How Can Animal and Human Research Be Used to Advance Research, Diagnosis, and Treatment of Alcohol and Other Substance Use Disorders?

The current article highlights key issues in defining, studying, and treating addiction, a concept related to but distinct from substance use disorders. The discussion is based upon a roundtable discussion at the 2017 annual meeting of the Research Society on Alcoholism where Warren K. Bickel and John C. Crabbe were charged with answering a range of questions posed by Kenneth J. Sher. All the presenters highlighted a number of central concerns for those interested in assessing and treating addiction as well as those seeking to conduct basic preclinical research that is amenable to meaningful translation to the human condition. In addition, the discussion illustrated both the power and limitations of using any single theory to explain multiple phenomena subsumed under the rubric of addiction. Among the major issues examined were the important differences between traditional diagnostic approaches and current concepts of addiction, the difficulty of modeling key aspects of human addiction in nonhuman animals, key aspects of addiction that have, to date, received little empirical attention, and the importance of thinking of recovery as a phenomenon that possibly involves processes distinct from those undergirding the development and maintenance of addiction.

Affective Behavior in Withdrawal Seizure-Prone and Withdrawal Seizure-Resistant Mice during Long-Term Alcohol Abstinence.

BACKGROUND: While the acute alcohol withdrawal syndrome has been well characterized both in human clinical studies and in experimental animals, much less is known regarding long-term affective disturbances that can sometimes persist during protracted abstinence. Nevertheless, since relapse often occurs long after acute detoxification and may be predicted by persistent affective disruption, a better understanding of the long-term behavioral consequences of prior alcohol dependence may lead to improved strategies for relapse prevention. METHODS: Male and female Withdrawal Seizure-Prone and Withdrawal Seizure-Resistant mice from the second selection replicate (WSP-2, WSR-2) were exposed to a 10-day chronic-intermittent ethanol vapor protocol (CIE) or plain air and then tested repeatedly on the sucrose preference test (SPT), marble burying test (MBT), and the light-dark box test (LDT) over 7 weeks of (forced) abstinence. RESULTS: While WSP and WSR mice differed significantly on tests of anxiety-like behavior (LDT, MBT), we found little evidence for long-term affective disruption following CIE in either line. The major exception was in the LDT, in that WSP but not WSR mice displayed longer latencies to enter the light compartment following CIE relative to air-controls. CONCLUSIONS: Selective breeding for acute withdrawal severity has resulted in differences in anxiety-like behavior between WSP and WSR mice. In contrast, however, genes contributing to the severity of acute withdrawal convulsions appear to have little overlap with those predisposing to affective disruption during long-term abstinence.

Gender-Specific Effects of Selection for Drinking in the Dark on the Network Roles of Coding and Noncoding RNAs.

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: Naïve 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.

Pharmacogenetic Manipulation of the Nucleus Accumbens Alters Binge-Like Alcohol Drinking in Mice.

BACKGROUND: Chronic alcohol intake leads to long-lasting changes in reward- and stress-related neuronal circuitry. The nucleus accumbens (NAc) is an integral component of this circuitry. Here, we investigate the effects of DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) on neuronal activity in the NAc and binge-like drinking. METHODS: C57BL/6J mice were stereotaxically injected with AAV2 hSyn-HA hM3Dq, -hM4Di, or -eGFP bilaterally into NAc [core + shell, core or shell]. We measured clozapine-n-oxide (CNO)-induced changes in NAc activity and assessed binge-like ethanol (EtOH) or tastant/fluid intake in a limited access Drinking in the Dark (DID) schedule. RESULTS: We found that CNO increased NAc firing in hM3Dq positive cells and decreased firing in hM4Di cells, confirming the efficacy of these channels to alter neuronal activity both spatially and temporally. Increasing NAc core + shell activity decreased binge-like drinking without altering intake of other tastants. Increasing activity specifically in the NAc core reduced binge-like drinking, and decreasing activity in the NAc core increased drinking. Manipulation of NAc shell activity did not alter DID. Thus, we find that increasing activity in the entire NAc, or just the NAc core is sufficient to decrease binge drinking. CONCLUSIONS: We conclude that the reduction in EtOH drinking is not due to general malaise, altered perception of taste, or reduced calorie-seeking. Furthermore, we provide the first evidence for bidirectional control of NAc core and binge-like drinking. These findings could have promising implications for treatment.

Commonalities and Distinctions Among Mechanisms of Addiction to Alcohol and Other Drugs.

BACKGROUND: Alcohol abuse is comorbid with abuse of many other drugs, some with similar pharmacology and others quite different. This leads to the hypothesis of an underlying, unitary dysfunctional neurobiological basis for substance abuse risk and consequences. METHODS: In this review, we discuss commonalities and distinctions of addiction to alcohol and other drugs. We focus on recent advances in preclinical studies using rodent models of drug self-administration. RESULTS: While there are specific behavioral and molecular manifestations common to alcohol, psychostimulant, opioid, and nicotine dependence, attempts to propose a unifying theory of the addictions inevitably face details where distinctions are found among classes of drugs. CONCLUSIONS: For alcohol, versus other drugs of abuse, we discuss and compare advances in: (i) neurocircuitry important for the different stages of drug dependence; (ii) transcriptomics and genetical genomics; and (iii) enduring effects, noting in particular the contributions of behavioral genetics and animal models.

Rewarding and aversive effects of ethanol in High Drinking in the Dark selectively bred mice.

Both rewarding and aversive effects contribute to alcohol consumption. Animals genetically predisposed to be high drinkers show reduced sensitivity to the aversive effects of alcohol, and in some instances, increased sensitivity to alcohol's rewarding effects. The present studies tested the high drinking in the dark (HDID) selected lines, a genetic model of drinking to intoxication, to determine whether intake in these mice was genetically related to sensitivity to alcohol aversion or reward. Male HDID mice from the first and second replicate lines (HDID-1 and HDID-2, respectively) and mice from the heterogeneous progenitor control population (HS/Npt, or HS) were conditioned for a taste aversion to a salt solution using two doses of alcohol, and lithium chloride (LiCl) and saline controls. In separate experiments, male and female HDID-1, HDID-2 and HS mice were conditioned for place preference using alcohol. HDID mice were found to have an attenuated sensitivity to alcohol at a moderate (2 g/kg) dose compared to HS mice, but did not differ on conditioned taste aversion to a high (4 g/kg) dose or LiCl or saline injections. HDID and HS mice showed comparable development of alcohol-induced conditioned place preference. These results indicate that high blood alcohol levels after drinking in the HDID mice is genetically related to attenuated aversion to alcohol, while sensitivity to alcohol reward is not altered in these mice. Thus, HDID mice may find a moderate dose of alcohol to be less aversive than control mice and consequently may drink more because of this reduced aversive sensitivity.

Genetic relationship between predisposition for binge alcohol consumption and blunted sensitivity to adverse effects of alcohol in mice.

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

Dual-trait selection for ethanol consumption and withdrawal: genetic and transcriptional network effects.

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.

Discovery and validation of genes driving drug-intake and related behavioral traits in mice.

Substance use disorders are heritable disorders characterized by compulsive drug use, the biological mechanisms for which remain largely unknown. Genetic correlations reveal that predisposing drug-naïve phenotypes, including anxiety, depression, novelty preference and sensation seeking, are predictive of drug-use phenotypes, thereby implicating shared genetic mechanisms. High-throughput behavioral screening in knockout (KO) mice allows efficient discovery of the function of genes. We used this strategy in two rounds of candidate prioritization in which we identified 33 drug-use candidate genes based upon predisposing drug-naïve phenotypes and ultimately validated the perturbation of 22 genes as causal drivers of substance intake. We selected 19/221 KO strains (8.5%) that had a difference from control on at least one drug-naïve predictive behavioral phenotype and determined that 15/19 (~80%) affected the consumption or preference for alcohol, methamphetamine or both. No mutant exhibited a difference in nicotine consumption or preference which was possibly confounded with saccharin. In the second round of prioritization, we employed a multivariate approach to identify outliers and performed validation using methamphetamine two-bottle choice and ethanol drinking-in-the-dark protocols. We identified 15/401 KO strains (3.7%, which included one gene from the first cohort) that differed most from controls for the predisposing phenotypes. 8 of 15 gene deletions (53%) affected intake or preference for alcohol, methamphetamine or both. Using multivariate and bioinformatic analyses, we observed multiple relations between predisposing behaviors and drug intake, revealing many distinct biobehavioral processes underlying these relationships. The set of mouse models identified in this study can be used to characterize these addiction-related processes further.

Selection for drinking in the dark alters brain gene coexpression networks.

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: Naïve 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.

Local changes in neurosteroid levels in the substantia nigra reticulata and the ventral tegmental area alter chronic ethanol withdrawal severity in male withdrawal seizure-prone mice.

BACKGROUND: Allopregnanolone (ALLO) is a potent positive modulator of γ-aminobutyric acidA receptors (GABAA Rs) that affects ethanol (EtOH) withdrawal. Finasteride (FIN), a 5α-reductase inhibitor that blocks the formation of ALLO and other GABAergic neurosteroids, alters EtOH sensitivity. Recently, we found that Withdrawal Seizure-Prone mice from the first genetic replicate (WSP-1) exhibited behavioral tolerance to the anticonvulsant effect of intrahippocampal ALLO during EtOH withdrawal and that intrahippocampal FIN significantly increased EtOH withdrawal severity. The purpose of this study was to determine whether neurosteroid manipulations in the substantia nigra reticulata (SNR) and ventral tegmental area (VTA) produced effects during EtOH withdrawal comparable to those seen with intrahippocampal ALLO and FIN. METHODS: Male WSP-1 mice were surgically implanted with bilateral guide cannulae aimed at the SNR or VTA at 2 weeks prior to EtOH vapor or air exposure for 72 hours. Initial studies examined the anticonvulsant effect of a single ALLO infusion (0, 100, or 400 ng/side) at a time corresponding to peak withdrawal in the air- and EtOH-exposed mice. Separate studies examined the effect of 4 FIN infusions (0 or 10 µg/side/d) during the development of physical dependence on the expression of EtOH withdrawal. RESULTS: ALLO infusion exerted a potent anticonvulsant effect in EtOH-naïve mice, but a diminished anticonvulsant effect during EtOH withdrawal. Administration of FIN into the SNR exerted a delayed proconvulsant effect in EtOH-naïve mice, whereas infusion into the VTA increased EtOH withdrawal duration. CONCLUSIONS: Activation of local GABAA Rs in the SNR and VTA via ALLO infusion is sufficient to exert an anticonvulsant effect in naïve mice and to produce behavioral tolerance to the anticonvulsant effect of ALLO infusion during EtOH withdrawal. Thus, EtOH withdrawal reduced sensitivity of GABAA Rs to GABAergic neurosteroids in 2 neuroanatomical substrates within the basal ganglia in WSP-1 male mice.

Escalation of intake under intermittent ethanol access in diverse mouse genotypes.

Experimental animals offered continuous 24-hour free choice access to ethanol rarely display voluntary ethanol consumption at levels sufficient to induce intoxication or to engender dependence. One of the simplest ways to increase voluntary ethanol intake is to impose temporal limitations on ethanol availability. Escalation of ethanol intake has been observed in both rats and mice under a variety of different schedules of alternating ethanol access and deprivation. Although such effects have been observed in a variety of rat and mouse genotypes, little is known concerning possible genetic correlations between responses to intermittent ethanol access and other ethanol-related phenotypes. In the present study, we examined the effects of intermittent ethanol access in mouse genotypes characterized by divergent responses to ethanol in other domains, including ethanol preference (C57BL/6J and C3H/HeJ mice), binge-like ethanol drinking (High Drinking in the Dark and HS/Npt mice) and ethanol withdrawal severity (Withdrawal Seizure-Prone and Withdrawal Seizure-Resistant mice). Although intermittent ethanol access resulted in escalated ethanol intake in all tested genotypes, the robustness of the effect varied across genotypes. On the other hand, we saw no evidence that the effects of intermittent access are correlated with either binge-like drinking or withdrawal severity, and only weak evidence for a genetic correlation with baseline ethanol preference. Thus, these different ethanol-related traits appear to depend on largely unique sets of genetic mediators.

The Collaborative Cross, a community resource for the genetic analysis of complex traits.

The goal of the Complex Trait Consortium is to promote the development of resources that can be used to understand, treat and ultimately prevent pervasive human diseases. Existing and proposed mouse resources that are optimized to study the actions of isolated genetic loci on a fixed background are less effective for studying intact polygenic networks and interactions among genes, environments, pathogens and other factors. The Collaborative Cross will provide a common reference panel specifically designed for the integrative analysis of complex systems and will change the way we approach human health and disease.

DISCOVERY AND VALIDATION OF GENES DRIVING DRUG-INTAKE AND RELATED BEHAVIORAL TRAITS IN MICE.

Substance use disorders (SUDs) are heritable disorders characterized by compulsive drug use, but the biological mechanisms driving addiction remain largely unknown. Genetic correlations reveal that predisposing drug-naïve phenotypes, including anxiety, depression, novelty preference, and sensation seeking, are predictive of drug-use phenotypes, implicating shared genetic mechanisms. Because of this relationship, high-throughput behavioral screening of predictive phenotypes in knockout (KO) mice allows efficient discovery of genes likely to be involved in drug use. We used this strategy in two rounds of screening in which we identified 33 drug-use candidate genes and ultimately validated the perturbation of 22 of these genes as causal drivers of substance intake. In our initial round of screening, we employed the two-bottle-choice paradigms to assess alcohol, methamphetamine, and nicotine intake. We identified 19 KO strains that were extreme responders on at least one predictive phenotype. Thirteen of the 19 gene deletions (68%) significantly affected alcohol use three methamphetamine use, and two both. In the second round of screening, we employed a multivariate approach to identify outliers and performed validation using methamphetamine two-bottle choice and ethanol drinking-in-the-dark protocols. We identified 15 KO strains that were extreme responders across the predisposing drug-naïve phenotypes. Eight of the 15 gene deletions (53%) significantly affected intake or preference for three alcohol, eight methamphetamine or three both (3). We observed multiple relations between predisposing behaviors and drug intake, revealing many distinct biobehavioral processes underlying these relationships. The set of mouse models identified in this study can be used to characterize these addiction-related processes further.

Preclinical and clinical evidence for suppression of alcohol intake by apremilast.

Treatment options for alcohol use disorders (AUDs) have minimally advanced since 2004, while the annual deaths and economic toll have increased alarmingly. Phosphodiesterase type 4 (PDE4) is associated with alcohol and nicotine dependence. PDE4 inhibitors were identified as a potential AUD treatment using a bioinformatics approach. We prioritized a newer PDE4 inhibitor, apremilast, as ideal for repurposing (i.e., FDA approved for psoriasis, low incidence of adverse events, excellent safety profile) and tested it using multiple animal strains and models, as well as in a human phase IIa study. We found that apremilast reduced binge-like alcohol intake and behavioral measures of alcohol motivation in mouse models of genetic risk for drinking to intoxication. Apremilast also reduced excessive alcohol drinking in models of stress-facilitated drinking and alcohol dependence. Using site-directed drug infusions and electrophysiology, we uncovered that apremilast may act to lessen drinking in mice by increasing neural activity in the nucleus accumbens, a key brain region in the regulation of alcohol intake. Importantly, apremilast (90 mg/d) reduced excessive drinking in non-treatment-seeking individuals with AUD in a double-blind, placebo-controlled study. These results demonstrate that apremilast suppresses excessive alcohol drinking across the spectrum of AUD severity.