Amanda M. Barkley-Levenson

Ethanol Drinking Microstructure of a High Drinking in the Dark Selected Mouse Line

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

Progress in a replicated selection for elevated blood ethanol concentrations in HDID mice

Drinking in the dark (DID) is a limited access ethanol‐drinking phenotype in mice. High Drinking in the Dark (HDID‐1) mice have been bred for 27 selected generations (S27) for elevated blood ethanol concentrations (BECs) after a 4‐h period of access to 20% ethanol. A second replicate line (HDID‐2) was started later from the same founder population and is currently in S20. An initial report of response to selection in HDID‐1 was published after S11. This article reports genetic and behavioral characteristics of both lines in comparison with the HS controls. Heritability is low in both replicates (h2 = 0.09) but the lines have shown 4–5 fold increases in BEC since S0; 80% of HDID‐1 and 60% of HDID‐2 mice reach BECs greater than 1.0 mg/ml. Several hours after a DID test, HDID mice show mild signs of withdrawal. Although not considered during selection, intake of ethanol (g/kg) during the DID test increased by approximately 80% in HDID‐1 and 60% in HDID‐2. Common genetic influences were more important than environmental influences in determining the similarity between BEC and intake for HDID mice. Analysis of the partitioning of intake showed that 60% of intake is concentrated in the last 2 h of the 4 h session. However, this has not changed during selection. Hourly BECs during the DID test reach peak levels after 3 or 4 h of drinking. HDID mice do not differ from HS mice in their rate of elimination of an acute dose of alcohol.

High Drinking in the Dark Mice: A genetic model of drinking to intoxication

Drinking to intoxication is a critical component of risky drinking behaviors in humans, such as binge drinking. Previous rodent models of alcohol consumption largely failed to demonstrate that animals were patterning drinking in such a way as to experience intoxication. Therefore, few rodent models of binge-like drinking and no specifically genetic models were available to study possible predisposing genes. The High Drinking in the Dark (HDID) selective breeding project was started to help fill this void, with HDID mice selected for reaching high blood alcohol levels in a limited access procedure. HDID mice now represent a genetic model of drinking to intoxication and can be used to help answer questions regarding predisposition toward this trait as well as potential correlated responses. They should also prove useful for the eventual development of better therapeutic strategies.

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 alcohols 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.

Ethanol Withdrawal-Associated Drinking and Drinking in the Dark: Common and Discrete Genetic Contributions

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

Genotypic and sex differences in anxiety-like behavior and alcohol-induced anxiolysis in high drinking in the dark selected mice

Alcohol use disorders and anxiety disorders are highly comorbid in humans. In rodent lines selected for alcohol drinking, differences in anxiety-like behavior are also seen. The High Drinking in the Dark (HDID) lines of mice are selectively bred for drinking to intoxication during limited access to alcohol, and these mice represent a genetic model of risk for binge-like drinking. The present studies investigated whether these selected lines differ from control (HS) mice in basal anxiety behavior or in anxiolytic response to alcohol. We also assessed the genetic correlation between alcohol drinking in the dark (DID) and basal anxiety-like behavior using existing inbred strain data. Mice of both sexes and HDID replicates (HDID-1 and HDID-2) were tested on an elevated zero maze immediately following a DID test. In general, HDID mice showed more time spent in the open arms after drinking alcohol than HS mice, and open-arm time was significantly correlated with blood alcohol concentration. HDID-1 male mice also showed less anxiety-like behavior at baseline (water-drinking controls). In a separate experiment, HDID-1 and HS mice were tested for anxiolytic dose-response to acute alcohol injections. Both genotypes showed increasing time spent in the open arms with increasing alcohol doses, and HDID-1 and female mice had greater open-arm time across all doses. HDID-1 control males showed lower anxiety-like behavior than the HS control males. Inbred strain data analysis also showed no significant genetic relationship between alcohol DID and anxiety. These findings suggest that HDID selection has not produced systematic changes in anxiety-like behavior or sensitivity to alcohol-induced anxiolysis, though there is a tendency in the male mice of the first replicate toward reduced basal anxiety-like behavior. Therefore, anxiety state and sensitivity to alcohols anxiolytic effects do not appear to contribute significantly to the high drinking behavior of the HDID mice.

Methamphetamine drinking microstructure in mice bred to drink high or low amounts of methamphetamine

Genetic factors likely influence individual sensitivity to positive and negative effects of methamphetamine (MA) and risk for MA dependence. Genetic influence on MA consumption has been confirmed by selectively breeding mouse lines to consume high (MAHDR) or low (MALDR) amounts of MA, using a two-bottle choice MA drinking (MADR) procedure. Here, we employed a lickometer system to characterize the microstructure of MA (20, 40, and 80mg/l) and water intake in MAHDR and MALDR mice in 4-h limited access sessions, during the initial 4hours of the dark phase of their 12:12h light:dark cycle. Licks at one-minute intervals and total volume consumed were recorded, and bout analysis was performed. MAHDR and MALDR mice consumed similar amounts of MA in mg/kg on the first day of access, but MAHDR mice consumed significantly more MA than MALDR mice during all subsequent sessions. The higher MA intake of MAHDR mice was associated with a larger number of MA bouts, longer bout duration, shorter interbout interval, and shorter latency to the first bout. In a separate 4-h limited access MA drinking study, MALDR and MAHDR mice had similar blood MA levels on the first day MA was offered, but MAHDR mice had higher blood MA levels on all subsequent days, which corresponded with MA intake. These data provide insight into the microstructure of MA intake in an animal model of differential genetic risk for MA consumption, which may be pertinent to MA use patterns relevant to genetic risk for MA dependence.

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.

Identification of a novel, fast-acting GABAergic antidepressant

Current pharmacotherapies for depression exhibit slow onset, side effects and limited efficacy. Therefore, identification of novel fast-onset antidepressants is desirable. GLO1 is a ubiquitous cellular enzyme responsible for the detoxification of the glycolytic byproduct methylglyoxal (MG). We have previously shown that MG is a competitive partial agonist at GABA-A receptors. We examined the effects of genetic and pharmacological inhibition of GLO1 in two antidepressant assay models: the tail suspension test (TST) and the forced swim test (FST). We also examined the effects of GLO1 inhibition in three models of antidepressant onset: the chronic FST (cFST), chronic mild stress (CMS) paradigm and olfactory bulbectomy (OBX). Genetic knockdown of Glo1 or pharmacological inhibition using two structurally distinct GLO1 inhibitors (S-bromobenzylglutathione cyclopentyl diester (pBBG) or methyl-gerfelin (MeGFN)) reduced immobility in the TST and acute FST. Both GLO1 inhibitors also reduced immobility in the cFST after 5 days of treatment. In contrast, the serotonin reuptake inhibitor fluoxetine (FLX) reduced immobility after 14, but not 5 days of treatment. Furthermore, 5 days of treatment with either GLO1 inhibitor blocked the depression-like effects induced by CMS on the FST and coat state, and attenuated OBX-induced locomotor hyperactivity. Finally, 5 days of treatment with a GLO1 inhibitor (pBBG), but not FLX, induced molecular markers of the antidepressant response including brain-derived neurotrophic factor (BDNF) induction and increased phosphorylated cyclic-AMP response-binding protein (pCREB) to CREB ratio in the hippocampus and medial prefrontal cortex (mPFC). Our findings indicate that GLO1 inhibitors may provide a novel and fast-acting pharmacotherapy for depression.

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.