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.

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.

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.

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.

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.

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.

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.

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.

Ethanol tolerance and withdrawal severity in high drinking in the dark selectively bred mice.

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

Ethanol sensitivity in high drinking in the dark selectively bred mice.

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

Nucleus accumbens mGluR5-associated signaling regulates binge alcohol drinking under drinking-in-the-dark procedures.

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.

Intermittent availability of ethanol does not always lead to elevated drinking in mice.

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 impact of Drinking in the Dark (DID) procedural manipulations on ethanol intake in High Drinking in the Dark (HDID) mice.

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.

Ethanol-Related Behaviors in Mouse Lines Selectively Bred for Drinking to Intoxication.

Alcohol use disorder (AUD) is a devastating psychiatric disorder that has significant wide-reaching effects on individuals and society. Selectively bred mouse lines are an effective means of exploring the genetic and neuronal mechanisms underlying AUD and such studies are translationally important for identifying treatment options. Here, we report on behavioral characterization of two replicate lines of mice that drink to intoxication, the High Drinking in the Dark (HDID)-1 and -2 mice, which have been selectively bred (20+ generations) for the primary phenotype of reaching high blood alcohol levels (BALs) during the drinking in the dark (DID) task, a binge-like drinking assay. Along with their genetically heterogenous progenitor line, Hs/Npt, we tested these mice on: DID and drinking in the light (DIL); temporal drinking patterns; ethanol sensitivity, through loss of righting reflex (LORR); and operant self-administration, including fixed ratio (FR1), fixed ratio 3:1 (FR3), extinction/reinstatement, and progressive ratio (PR). All mice consumed more ethanol during the dark than the light and both HDID lines consumed more ethanol than Hs/Npt during DIL and DID. In the dark, we found that the HDID lines achieved high blood alcohol levels early into a drinking session, suggesting that they exhibit front loading like drinking behavior in the absence of the chronicity usually required for such behavior. Surprisingly, HDID-1 (female and male) and HDID-2 (male) mice were more sensitive to the intoxicating effects of ethanol during the dark (as determined by LORR), while Hs/Npt (female and male) and HDID-2 (female) mice appeared less sensitive. We observed lower HDID-1 ethanol intake compared to either HDID-2 or Hs/Npt during operant ethanol self-administration. There were no genotype differences for either progressive ratio responding, or cue-induced ethanol reinstatement, though the latter is complicated by a lack of extinguished responding behavior. Taken together, these findings suggest that genes affecting one AUD-related behavior do not necessarily affect other AUD-related behaviors. Moreover, these findings highlight that alcohol-related behaviors can also differ between lines selectively bred for the same phenotype, and even between sexes within those same line.

Binge drinking upregulates accumbens mGluR5-Homer2-PI3K signaling: functional implications for alcoholism.

The glutamate receptor-associated protein Homer2 regulates alcohol-induced neuroplasticity within the nucleus accumbens (NAC), but the precise intracellular signaling cascades involved are not known. This study examined the role for NAC metabotropic glutamate receptor (mGluR)-Homer2-phosphatidylinositol 3-kinase (PI3K) signaling in regulating excessive alcohol consumption within the context of the scheduled high alcohol consumption (SHAC) model of binge alcohol drinking. Repeated bouts of binge drinking ( approximately 1.5 g/kg per 30 min) elevated NAC Homer2a/b expression and increased PI3K activity in this region. Virus-mediated knockdown of NAC Homer2b expression attenuated alcohol intake, as did an intra-NAC infusion of the mGluR5 antagonist MPEP [2-methyl-6-(phenylethynyl)pyridine hydrochloride] (0.1-1 microg/side) and the PI3K antagonist wortmannin (50 ng/side), supporting necessary roles for mGluR5/Homer2/PI3K in binge alcohol drinking. Moreover, when compared with wild-type littermates, transgenic mice with an F1128R point mutation in mGluR5 that markedly reduces Homer binding exhibited a 50% reduction in binge alcohol drinking, which was related to reduced NAC basal PI3K activity. Consistent with the hypothesis that mGluR5-Homer-PI3K signaling may be a mechanism governing excessive alcohol intake, the "anti-binge" effects of MPEP and wortmannin were not additive, nor were they observed in the mGluR5(F1128R) transgenic mice. Finally, mice genetically selected for a high versus low SHAC phenotype differed in NAC mGluR, Homer2, and PI3K activity, consistent with the hypothesis that augmented NAC mGluR5-Homer2-PI3K signaling predisposes a high binge alcohol-drinking phenotype. Together, these data point to an important role for NAC mGluR5-Homer2-PI3K signaling in regulating binge-like alcohol consumption that has relevance for our understanding of the neurobiology of alcoholism and its pharmacotherapy.

Withdrawal severity after chronic intermittent ethanol in inbred mouse strains.

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.

Tetracycline derivatives reduce binge alcohol consumption in High Drinking in the Dark mice.

Alcohol use disorders (AUDs) are prevalent, and are characterized by binge-like drinking, defined by patterns of focused drinking where dosages ingested in 2-4 â€‹h reach intoxicating blood alcohol levels (BALs). Current medications are few and compliance with the relatively rare prescribed usage is low. Hence, novel and more effective medications are needed. We developed a mouse model of genetic risk for binge drinking (HDID: High Drinking in the Dark mice) by selectively breeding for high BALs after binge drinking. A transcriptional analysis of HDID brain tissue with RNA-Seq implicated neuroinflammatory mechanisms, and, more specifically extracellular matrix genes, including those encoding matrix metalloproteinases (MMPs). Prior experiments from other groups have shown that the tetracycline derivatives doxycycline, minocycline, and tigecycline, reduce binge drinking in inbred C57BL/6J mice. We tested these three compounds in female and male HDID mice and found that all three reduced DID and BAL. They had drug-specific effects on intake of water or saccharin in the DID assay. Thus, our results show that the effectiveness of synthetic tetracycline derivatives as potential therapeutic agents for AUDs is not limited to the single C57BL/6J genotype previously targeted, but extends to a mouse model of a population at high risk for AUDs.

Effects of Tacrolimus and Other Immune Targeting Compounds on Binge-Like Ethanol Drinking in High Drinking in the Dark Mice.

High Drinking in the Dark (HDID-1) mice represent a unique genetic risk model of binge-like drinking and a novel means of screening potential pharmacotherapies to treat alcohol use disorders (AUDs). We tested the effects of tacrolimus (0, 0.5, 1, and 2 mg/kg), sirolimus (0, 5, 10, and 20 mg/kg), palmitoylethanolamide (PEA; 0, 75, 150, and 225 mg/kg), and secukinumab (0, 5, 20, and 60 mg/kg) on binge-like ethanol intake (2-day, "Drinking in the Dark" [DID]) and blood alcohol levels (BALs) in HDID-1 mice. Tacrolimus reduced ethanol intake and BALs. Tacrolimus had no effect on water intake, but reduced saccharin intake. There was no effect of sirolimus, PEA, or secukinumab on ethanol intake or BALs. These results compare and contrast with previous work addressing these compounds or their targeted mechanisms of action on ethanol drinking, highlighting the importance of screening a wide range of models and genotypes to inform the role of neuroimmune signaling in AUDs.