Differential importance of nucleus accumbens Ox1Rs and AMPARs for female and male mouse binge alcohol drinking.

Alcohol use disorder exhausts substantial social and economic costs, with recent dramatic increases in female problem drinking. Thus, it is critically important to understand signaling differences underlying alcohol consumption across the sexes. Orexin-1 receptors (Ox1Rs) can strongly promote motivated behavior, and we previously identified Ox1Rs within nucleus accumbens shell (shell) as crucial for driving binge intake in higher-drinking male mice. Here, shell Ox1R inhibition did not alter female mouse alcohol drinking, unlike in males. Also, lower dose systemic Ox1R inhibition reduced compulsion-like alcohol intake in both sexes, indicating that female Ox1Rs can drive some aspects of pathological consumption, and higher doses of systemic Ox1R inhibition (which might have more off-target effects) reduced binge drinking in both sexes. In contrast to shell Ox1Rs, inhibiting shell calcium-permeable AMPA receptors (CP-AMPARs) strongly reduced alcohol drinking in both sexes, which was specific to alcohol since this did not reduce saccharin intake in either sex. Our results together suggest that the shell critically regulates binge drinking in both sexes, with shell CP-AMPARs supporting intake in both sexes, while shell Ox1Rs drove drinking only in males. Our findings provide important new information about sex-specific and -general mechanisms that promote binge alcohol intake and possible targeted therapeutic interventions.

A novel NMDA receptor-based intervention to suppress compulsion-like alcohol drinking.

Compulsive drives for alcohol, where intake persists despite adverse consequences, are substantial obstacles to treating Alcohol Use Disorder (AUD). However, there are limited treatment options and thus considerable interest in identifying new, potent and safe pharmacotherapies. We found that non-canonical N-methyl-d-aspartate receptors (NMDARs), active at hyperpolarized potentials, drive compulsion-like alcohol drinking in rats without affecting regular, alcohol-only intake. Congruent human studies suggest that NMDAR inhibition reduces alcohol drinking in treatment-seekers but not non-treatment-seekers and suppresses craving. These cross-species studies of consumption under conflict indicate that inhibiting non-canonical NMDARs could be of clinical value for AUD. d-serine activates NMDARs overall, but actually inhibits non-canonical NMDARs. Also, d-serine has been widely tested in humans as a moderate NMDAR modulator, but some nephrotoxicity concerns remain, and thus any strategy that reduces d-serine exposure could be of broad utility. Here, co-administration of sodium benzoate (NaBenz), which reduces d-serine breakdown, allowed subthreshold d-serine levels to suppress compulsion-like alcohol drinking without altering normal alcohol-only consumption, providing a novel intervention for AUD and underscoring the importance of non-canonical NMDARs for compulsion-like intake. Low NaBenz doses alone had no average effect on intake. NaBenz/d-serine reduced compulsion-like intake in nearly all animals, while higher d-serine alone decreased compulsion-like intake with less of an effect in lower-drinking subjects. Thus, combining subthreshold NaBenz and d-serine suppressed compulsion-like intake, helping both to alleviate some d-serine concerns, and, importantly, to reduce consequence-resistant consumption across nearly all individuals. Therefore, NaBenz/d-serine likely represents an FDA-approved and immediately-accessible pharmacotherapy to help counteract compulsion-like drives and treat AUD.

Drinking despite adversity: behavioral evidence for a head down and push strategy of conflict-resistant alcohol drinking in rats.

Compulsive alcohol drinking, where intake persists regardless of adverse consequences, plays a major role in the substantial costs of alcohol use disorder. However, the processes that promote aversion-resistant drinking remain poorly understood. Compulsion-like responding has been considered automatic and reflexive and also to involve higher motivation, since drinking persists despite adversity. Thus, we used lickometry, where microstructural behavioral changes can reflect altered motivation, to test whether conflict-resistant intake [quinine-alcohol (QuiA)] reflected greater automaticity or motivation relative to alcohol-only drinking (Alc). Front-loading during QuiA and Alc suggested incentive to drink in both. However, the relationship between total licking and intake was less variable during QuiA, as was lick volume, without changes in average responding. QuiA bout organization was also less variable, with fewer licks outside of bouts (stray licks) and fewer gaps within bouts. Interestingly, QuiA avoidance of stray licking continued into short bouts, with fewer short and more medium-length bouts, which was striking given their minor impact on intake. Instead, more effort at bout onset could allow short bouts to persist longer. Indeed, while QuiA licking was overall faster, QuiA bouts were especially fast at bout initiation. However, few QuiA changes individually predicted greater intake, perhaps suggesting an overarching strategy during aversion-resistant responding. Thus, our results indicate that aversion-resistant intake exhibited less variability, where increased automaticity could decrease need for awareness, and stronger bout initiation, which might prolong responding despite adversity. This may reflect a collective strategy, which we call Head Down and Push responding that facilitates conflict-resistant, compulsion-like intake.

Limited Excessive Voluntary Alcohol Drinking Leads to Liver Dysfunction in Mice.

BACKGROUND: Liver damage is a serious and sometimes fatal consequence of long-term alcohol intake, which progresses from early-stage fatty liver (steatosis) to later-stage steatohepatitis with inflammation and fibrosis/necrosis. However, very little is known about earlier stages of liver disruption that may occur in problem drinkers, those who drink excessively but are not dependent on alcohol. METHODS: We examined how repeated binge-like alcohol drinking in C57BL/6 mice altered liver function, as compared with a single binge-intake session and with repeated moderate alcohol consumption. We measured a number of markers associated with early- and later-stage liver disruption, including liver steatosis, measures of liver cytochrome P4502E1 (CYP2E1) and alcohol dehydrogenase (ADH), alcohol metabolism, expression of cytokine mRNA, accumulation of 4-hydroxynonenal (4-HNE) as an indicator of oxidative stress, and alanine transaminase/aspartate transaminase as a measure of hepatocyte injury. RESULTS: Importantly, repeated binge-like alcohol drinking increased triglyceride levels in the liver and plasma, and increased lipid droplets in the liver, indicators of steatosis. In contrast, a single binge-intake session or repeated moderate alcohol consumption did not alter triglyceride levels. In addition, alcohol exposure can increase rates of alcohol metabolism through CYP2E1 and ADH, which can potentially increase oxidative stress and liver dysfunction. Intermittent, excessive alcohol intake increased liver CYP2E1 mRNA, protein, and activity, as well as ADH mRNA and activity. Furthermore, repeated, binge-like drinking, but not a single binge or moderate drinking, increased alcohol metabolism. Finally, repeated, excessive intake transiently elevated mRNA for the proinflammatory cytokine IL-1B and 4-HNE levels, but did not alter markers of later-stage liver hepatocyte injury. CONCLUSIONS: Together, we provide data suggesting that even relatively limited binge-like alcohol drinking can lead to disruptions in liver function, which might facilitate the transition to more severe forms of liver damage.

Ethanol drives aversive conditioning through dopamine 1 receptor and glutamate receptor-mediated activation of lateral habenula neurons.

There has been increasing interest in the lateral habenula (LHb) given its potent regulatory role in many aversion-related behaviors. Interestingly, ethanol can be rewarding as well as aversive; we therefore investigated whether ethanol exposure alters pacemaker firing or glutamate receptor signaling in LHb neurons in vitro and also whether LHb activity in vivo might contribute to the acquisition of conditioned place aversion to ethanol. Surprisingly, in epithalamic slices, low doses of ethanol (1.4 mM) strongly accelerated LHb neuron firing (by ~60%), and ethanol's effects were much reduced by blocking glutamate receptors. Ethanol increased presynaptic glutamate release, and about half of this effect was mediated by dopamine subtype 1 receptors (D1Rs) and cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. In agreement with these findings, c-Fos immunoreactivity in LHb regions was enhanced after a single administration of a low dose of ethanol (0.25 g/kg i.p.). Importantly, the same dose of ethanol in vivo also produced strong conditioned place aversion, and this was prevented by inhibiting D1Rs or neuronal activity within the LHb. By contrast, a higher dose (2 g/kg) led to ethanol conditioned place preference, which was enhanced by inhibiting neuronal activity or D1Rs within the LHb and suppressed by infusing aminomethylphosphonic acid or the D1R agonist SKF38393 within the LHb. Our in vitro and in vivo observations show, for the first time, that ethanol increases LHb excitation, mediated by D1R and glutamate receptors, and may underlie a LHb aversive signal that contributes to ethanol-related aversion.

Binge ethanol-drinking potentiates corticotropin releasing factor R1 receptor activity in the ventral tegmental area.

BACKGROUND: Corticotropin releasing factor (CRF) and urocortin play an important role in many stress responses and also can regulate ethanol (EtOH) intake. Adaptations in CRF signaling in the central amygdala promote EtOH consumption after long-term EtOH intake in dependent animals and also after brief periods of binge EtOH intake. Thus, even brief episodes of EtOH consumption can alter the function of the CRF system, allowing CRF to regulate EtOH intake. Here, we examined whether brief binge EtOH consumption leads to CRF receptor adaptations within the ventral tegmental area (VTA), a structure involved in signaling rewarding and aversive events and important in the development and expression of drug and alcohol addiction. METHODS: We utilized a mouse model of binge drinking known as drinking in the dark (DID), where C57BL/6J mice drink approximately 6 g/kg in 4 hours and achieve blood EtOH concentrations of approximately 100 mg/dl, which is equivalent to binge drinking in humans. We used ex vivo whole-cell recordings from putative VTA dopamine (DA) neurons to examine CRF regulation of NMDA receptor (NMDAR) currents. We also examined the impact of CRF receptor antagonist injection in the VTA on binge EtOH intake. RESULTS: Ex vivo whole-cell recordings from putative VTA DA neurons showed enhanced CRF-mediated potentiation of NMDAR currents in juvenile mice that consumed EtOH in the DID procedure. CRF-induced potentiation of NMDAR currents in EtOH-drinking mice was blocked by administration of CP-154,526 (3 µM), a selective CRF1 receptor antagonist. Furthermore, intra-VTA infusion of CP-154,526 (1 µg) significantly reduced binge EtOH consumption in adult mice. These results were not due to alterations of VTA NMDAR number or function, suggesting that binge drinking may enhance signaling through VTA CRF1 receptors onto NMDARs. CONCLUSIONS: Altered CRF1 receptor-mediated signaling in the VTA promotes binge-like EtOH consumption in mice, which supports the idea that CRF1 receptors may therefore be a promising pharmacological target for reducing binge drinking in humans.

The dual orexin/hypocretin receptor antagonist, almorexant, in the ventral tegmental area attenuates ethanol self-administration.

Recent studies have implicated the hypocretin/orexinergic system in reward-seeking behavior. Almorexant, a dual orexin/hypocretin R(1) and R(2) receptor antagonist, has proven effective in preclinical studies in promoting sleep in animal models and was in Phase III clinical trials for sleep disorders. The present study combines behavioral assays with in vitro biochemical and electrophysiological techniques to elucidate the role of almorexant in ethanol and sucrose intake. Using an operant self-administration paradigm, we demonstrate that systemic administration of almorexant decreased operant self-administration of both 20% ethanol and 5% sucrose. We further demonstrate that intra-ventral tegmental area (VTA) infusions, but not intra-substantia nigra infusions, of almorexant reduced ethanol self-administration. Extracellular recordings performed in VTA neurons revealed that orexin-A increased firing and this enhancement of firing was blocked by almorexant. The results demonstrate that orexin/hypocretin receptors in distinct brain regions regulate ethanol and sucrose mediated behaviors.

Motivation for alcohol becomes resistant to quinine adulteration after 3 to 4 months of intermittent alcohol self-administration.

BACKGROUND: Continued consumption of alcohol despite deleterious consequences is a hallmark of alcoholism and represents a critical challenge to therapeutic intervention. Previous rat studies showed that enduring alcohol self-administration despite pairing alcohol with normally aversive stimuli was only observed after very long-term intake (>8 months). Aversion-resistant alcohol intake has been previously interpreted to indicate pathological or compulsive motivation to consume alcohol. However, given the time required to model compulsive alcohol seeking in previous studies, there is considerable interest in developing more efficient and quantitative rodent models of aversion-resistant alcohol self-administration. METHODS: Outbred Wistar rats underwent 3 to 4 months or approximately 1.5 months of intermittent, home-cage, two-bottle access (IAA) to 20% alcohol (v/v) or water. Then, after brief operant training, the effect of the bitter-tasting quinine (0.1 g/l) on the motivation to seek alcohol was quantified via progressive ratio (PR). Motivation for quinine-adulterated 2% sucrose under PR was assayed in a separate cohort of 3 to 4 months IAA rats. The effects of quinine on home-cage alcohol consumption in IAA rats and rats with continuous access to alcohol were also examined. Finally, a dose-response for quinine taste preference in IAA and continuous-access animals was determined. RESULTS: Motivation for alcohol after 3 to 4 months IAA, measured using an operant PR procedure, was not altered by adulteration of alcohol with 0.1 g/l quinine. In contrast, after 3 to 4 months of IAA, motivation for sucrose under PR was significantly reduced by adulteration of sucrose with 0.1 g/l quinine. In addition, motivation for alcohol after only approximately 1.5 months IAA was significantly reduced by adulteration of alcohol with 0.1 g/l quinine. Furthermore, home-cage alcohol intake by IAA rats was insensitive to quinine at concentrations (0.01, 0.03 g/l) that significantly reduced alcohol drinking in animals with continuous access to alcohol. Finally, no changes in quinine taste preference after 3 to 4 months IAA or continuous access to alcohol were observed. CONCLUSIONS: We have developed a novel and technically simple hybrid operant/IAA model in which quinine-resistant motivation for alcohol is evident after an experimentally tractable period of time (3 to 4 months vs. 8 months). Quinine dramatically reduced sucrose and water intake by IAA rats, indicating that continued responding for alcohol in IAA rats despite adulteration with the normally aversive quinine might reflect maladaptive or compulsive motivation for alcohol. This model could facilitate identification of novel therapeutic interventions for pathological alcohol seeking in humans.