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.
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.
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.
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.
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.
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.
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 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.
•Doxycycline, minocycline, and tigecycline reduce binge alcohol drinking in high risk mice.•Female and male mice are responsive.•These data show that synthetic tetracycline derivatives are potential therapeutic agents for alcohol use.
Alcohol dependence (alcoholism) is accompanied by evidence of tolerance, withdrawal (physiological dependence), or compulsive behavior related to alcohol use. Studies of strain and individual ...differences using animal models for acute physiological dependence liability are useful means to identify potential genetic determinants of liability in humans. Behavioral and quantitative trait analyses were conducted using animal models for high risk versus resistance to acute physiological dependence. Using a two-step genetic mapping strategy, loci on mouse chromosomes 1, 4, and 11 were mapped that contain genes that influence alcohol withdrawal severity. In the aggregate, these three risk markers accounted for 68% of the genetic variability in alcohol withdrawal. Candidate genes in proximity to the chromosome 11 locus include genes encoding the alpha1, alpha6, and gamma2 subunits of type-A receptors for the inhibitory neurotransmitter, GABA. In addition, suggestive linkage is indicated for two loci on mouse chromosome 2, one near Gad1 encoding glutamic acid decarboxylase, and the other near the El2 locus which influences the seizure phenotype in the neurological mutant strain El. The present analyses detect and map some of the loci that increase risk to develop physiological dependence and may facilitate identification of genes related to the development of alcoholism. Syntenic conservation between human and mouse chromosomes suggests that human homologs of genes that increase risk for physiological dependence may localize to 1q21-q32, 2q24-q37/11p13, 9p21-p23/1p32-p22.1, and 5q32-q35.
Background
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.
Pre‐clinical studies of high alcohol drinking offer opportunities to better understand risk factors for Alcohol Use Disorders and test potential treatments. We report that several compounds shown to reduce ethanol drinking in other genotypes are not effective in our models of genetic risk for binge‐like drinking (High Drinking in the Dark mice). The most promising target we identified was the phosphodiesterase type 4 inhibitor, rolipram. These results highlight the importance of assessing generalizability when rigorously testing compounds for therapeutic development.
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.
Pharmacologically antagonizing glucocorticoid receptors with either mifepristone or CORT113176 reduces binge‐like ethanol consumption in the High Drinking in the Dark (HDID‐1) line of mice, but not their founder control line, the HS/Npt mice. These results suggest an important, previously unrecognized, role for the glucocorticoid receptor in the genetic risk for binge‐like ethanol intake, and may point to the glucocorticoid receptor as a potential biomarker target for identifying individuals at risk for excessive alcohol consumption.
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