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.

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.

Ethanol Conditioned Taste Aversion in High Drinking in the Dark Mice.

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.

An alcohol withdrawal test battery measuring multiple behavioral symptoms in mice.

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.

High Drinking in the Dark (HDID) mice are sensitive to the effects of some clinically relevant drugs to reduce binge-like drinking.

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 2h. 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 GABAB 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.

Fear conditioning in mouse lines genetically selected for binge-like ethanol drinking.

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.

Ethanol drinking in withdrawal seizure-prone and -resistant selected mouse lines.

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.

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.

Ethanol withdrawal-induced motor impairment in mice.

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

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 two-bottle ethanol preference drinking data assessed under conditions of continuous ethanol access. Although inbred strain data for withdrawal-associated drinking are not available, the current pattern of results suggests that withdrawal-associated drinking is genetically distinct from DID, while genetic contributions to DID and two-bottle preference drinking are substantially similar.

Genotypic differences in ethanol sensitivity in two tests of motor incoordination.

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.

Different data from different labs: lessons from studies of gene-environment interaction.

It is sometimes supposed that standardizing tests of mouse behavior will ensure similar results in different laboratories. We evaluated this supposition by conducting behavioral tests with identical apparatus and test protocols in independent laboratories. Eight genetic groups of mice, including equal numbers of males and females, were either bred locally or shipped from the supplier and then tested on six behaviors simultaneously in three laboratories (Albany, NY; Edmonton, AB; Portland, OR). The behaviors included locomotor activity in a small box, the elevated plus maze, accelerating rotarod, visible platform water escape, cocaine activation of locomotor activity, and ethanol preference in a two-bottle test. A preliminary report of this study presented a conventional analysis of conventional measures that revealed strong effects of both genotype and laboratory as well as noteworthy interactions between genotype and laboratory. We now report a more detailed analysis of additional measures and view the data for each test in different ways. Whether mice were shipped from a supplier or bred locally had negligible effects for almost every measure in the six tests, and sex differences were also absent or very small for most behaviors, whereas genetic effects were almost always large. For locomotor activity, cocaine activation, and elevated plus maze, the analysis demonstrated the strong dependence of genetic differences in behavior on the laboratory giving the tests. For ethanol preference and water escape learning, on the other hand, the three labs obtained essentially the same results for key indicators of behavior. Thus, it is clear that the strong dependence of results on the specific laboratory is itself dependent on the task in question. Our results suggest that there may be advantages of test standardization, but laboratory environments probably can never be made sufficiently similar to guarantee identical results on a wide range of tests in a wide range of labs. Interpretations of our results by colleagues in neuroscience as well as the mass media are reviewed. Pessimistic views, prevalent in the media but relatively uncommon among neuroscientists, of mouse behavioral tests as being highly unreliable are contradicted by our data. Despite the presence of noteworthy interactions between genotype and lab environment, most of the larger differences between inbred strains were replicated across the three labs. Strain differences of moderate effects size, on the other hand, often differed markedly among labs, especially those involving three 129-derived strains. Implications for behavioral screening of targeted and induced mutations in mice are discussed.