Cessation from chronic alcohol abuse often produces a dysphoric state that can persist into protracted withdrawal. This dysphoric state is theorized to function as a negative reinforcer that maintains excessive alcohol consumption and/or precipitates relapse in those struggling to abstain from alcohol. However, we know relatively little regarding the impact of cessation from binge drinking on behavioral measures of negative affect and related neurobiology. Male C57BL/6J mice were given access to unsweetened 20% alcohol for 6 weeks under modified Drinking-in-the-Dark procedures, followed by behavioral testing beginning either 1 or 21 days into withdrawal. Mice were administered a behavioral test battery consisting of: the elevated plus maze, light/dark box, novel object test, marble burying test, Porsolt forced swim test and sucrose preference test to assess anxiogenic and depressive signs. Egr1 immunostaining was used to quantify cellular activity within the central nucleus of the amygdala (CEA), basolateral amygdala (BLA), bed nucleus of the stria terminalis (BNST), and the nucleus accumbens (Acb) shell (AcbSh) and core (AcbC). Compared to water controls, alcohol-drinking mice exhibited higher indices of emotionality in the majority of behavioral assays. The hyper-emotionality exhibited by binge drinking mice was apparent at both withdrawal time-points and correlated with higher Egr1+ cell counts in the CEA and BNST, compared to controls. These data show that affective symptoms emerge very early after cessation of binge drinking and persist into protracted withdrawal. A history of binge drinking is capable of producing enduring neuroadaptations within brain circuits mediating emotional arousal.

Binge-drinking is the most prevalent form of alcohol abuse and while an early life history of binge-drinking is a significant risk factor for subsequent alcoholism and co-morbid affective disorders, relatively little is known regarding the biobehavioral impact of binge-drinking during the sensitive neurodevelopmental period of adolescence. In adult mice, a month-long history of binge-drinking elicits a hyper-glutamatergic state within the nucleus accumbens (Acb), coinciding with hyper-anxiety. Herein, we employed a murine model of binge-drinking to determine whether or not: (1) withdrawal-induced changes in brain and behavior differ between adult and adolescent bingers; and (2) increased behavioral signs of negative affect and changes in Acb expression of glutamate-related proteins would be apparent in adult mice with less chronic binge-drinking experience (14 days, approximating the duration of mouse adolescence). Adult and adolescent male C57BL/6J mice were subjected to a 14-day binge-drinking protocol (5, 10, 20 and 40% alcohol (v/v) for 2 h/day), while age-matched controls received water. At 24 h withdrawal, half of the animals from each group were assayed for negative affect, while tissue was sampled from the shell (AcbSh) and core (AcbC) subregions of the remaining mice for immunoblotting analyses. Adult bingers exhibited hyper-anxiety when tested for defensive marble burying. Additionally, adult bingers showed increased mGlu1, mGlu5, and GluN2b expression in the AcbSh and PKCε and CAMKII in the AcbC. Compared to adults, adolescent mice exhibited higher alcohol intake and blood alcohol concentrations (BACs); however, adolescent bingers did not show increased anxiety in the marble-burying test. Furthermore, adolescent bingers also failed to exhibit the same alcohol-induced changes in mGlu and kinase protein expression seen in the adult bingers. Irrespective of age, bingers exhibited behavioral hyperactivity in the forced swim test (FST) compared to water drinkers, which was paralleled by an increase in AcbC levels of GluN2b. Thus, a 2-week period of binge-drinking is sufficient to produce a hyper-anxious state and related increases in protein indices of Acb glutamate function. In contrast, adolescents were resilient to many of the effects of early alcohol withdrawal and this attenuated sensitivity to the negative consequences of binge drinking may facilitate greater alcohol intake in adolescent drinkers.

Binge-drinking is common in underage alcohol users, yet we know little regarding the biopsychological impact of binge-drinking during early periods of development. Prior work indicated that adolescent male C57BL6/J mice with a 2-week history of binge-drinking (PND28-41) are resilient to the anxiogenic effects of early alcohol withdrawal. Herein, we employed a comparable Drinking-in-the-Dark model to determine how a prior history of binge-drinking during adolescence (EtOHadolescents) influences emotionality (assayed with the light-dark box, marble burying test, and the forced swim test) and the propensity to consume alcohol in later life, compared to animals without prior drinking experience. For additional comparison, adult mice (EtOHadults) with comparable drinking history (PND56-69) were subdivided into groups tested for anxiety/drinking either on PND70 (24 h withdrawal) or PND98 (28 days withdrawal). Tissue from the nucleus accumbens shell (AcbSh) and central nucleus of the amygdala (CeA) was examined by immunoblotting for changes in the expression of glutamate-related proteins. EtOHadults exhibited some signs of hyperanxiety during early withdrawal (PND70), but not during protracted withdrawal (PND98). In contrast, EtOHadolescents exhibited robust signs of anxiety-l and depressive-like behaviors when tested as adults on PND70. While all alcohol-experienced animals subsequently consumed more alcohol than mice drinking for the first time, alcohol intake was greatest in EtOHadolescents. Independent of drinking age, the manifestation of withdrawal-induced hyperanxiety was accompanied by reduced Homer2b expression within the CeA and increased Group1 mGlu receptor expression within the AcbSh. The present data provide novel evidence that binge-drinking during adolescence produces a state characterized by profound negative affect and excessive alcohol consumption that incubates with the passage of time in withdrawal. These data extend our prior studies on the effects of subchronic binge-drinking during adulthood by demonstrating that the increase in alcoholism-related behaviors and glutamate-related proteins observed in early withdrawal dissipate with the passage of time. Our results to date highlight a critical interaction between the age of binge-drinking onset and the duration of alcohol withdrawal in glutamate-related neuroplasticity within the extended amygdala of relevance to the etiology of psychopathology, including pathological drinking, in later life.

Binge alcohol-drinking elicits symptoms of negative affect such as anxiety upon cessation, which is a source of negative reinforcement for perpetuating this pattern of alcohol abuse. Binge-induced anxiety during early (24 h) withdrawal is associated with increased expression of metabotropic glutamate receptor 5 (mGlu5) within the nucleus accumbens shell (AcbSh) of adult male mice, but was unchanged in anxiety-resilient adolescents. Herein, we determined the role of mGlu5 signaling in withdrawal-induced anxiety via pharmacological manipulation using the mGlu5 negative allosteric modulator MTEP and the positive allosteric modulator CDPPB. Adult (PND 56) and adolescent (PND 28) male C57BL/6J mice binge-drank for 14 days under 3-bottle-choice procedures for 2 h/day; control animals drank water only. Approximately 24 h following the final alcohol presentation, animals were treated with 30 mg/kg IP MTEP, CDPPB, or vehicle and then tested, thirty minutes later, for behavioral signs of anxiety. Vehicle-treated binge-drinking adults exhibited hyperanxiety in all paradigms, while vehicle-treated binge-drinking adolescents did not exhibit withdrawal-induced anxiety. In adults, 30 mg/kg MTEP decreased alcohol-induced anxiety across paradigms, while 3 mg/kg MTEP was anxiolytic in adult water controls. CDPPB was modestly anxiogenic in both alcohol- and water-drinking mice. Adolescent animals showed minimal response to either CDPPB or MTEP, suggesting that anxiety in adolescence may be mGlu5-independent. These results demonstrate a causal role for mGlu5 in withdrawal-induced anxiety in adults and suggest age-related differences in the behavioral pharmacology of the negative reinforcing properties of alcohol.

Traditionally, a reduction in floating behavior or immobility in the Porsolt forced swim test (FST) is employed as a predictor of antidepressant efficacy. However, over the past several years, our studies of alcohol withdrawal-induced negative affect consistently indicate the coincidence of increased anxiety-related behaviors on various behavioral tests with reduced immobility in the FST. Further, this behavioral profile correlates with increased mGlu5 protein expression within limbic brain regions. As the role for mGlu5 in anxiety is well established, we hypothesized that the reduced immobility exhibited by alcohol-withdrawn mice when tested in the FST might reflect anxiety, possibly a hyper-reactivity to the acute swim stressor. Herein, we evaluated whether or not the decreased FST immobility during alcohol withdrawal responds to systemic treatment with a behaviorally-effective dose of the prototypical anxiolytic, buspirone (5 mg/kg). We also determined the functional relevance of the withdrawal-induced increase in mGlu5 expression for FST behavior by comparing the effects of buspirone to a behaviorally effective dose of the mGlu5 negative allosteric modulator MTEP (3 mg/kg). Adult male C57BL/6J mice were subjected to a 14-day, multi-bottle, binge-drinking protocol that elicits hyper-anxiety and increases glutamate-related protein expression during early withdrawal. Control animals received only water. At 24hr withdrawal, animals from each drinking condition were subdivided into groups and treated with an IP injection of buspirone, MTEP, or vehicle, 30min prior to the FST. Drug effects on general locomotor activity were also assessed. As we reported previously, alcohol-withdrawn animals exhibited significantly reduced immobility in the FST compared to water controls. Both buspirone and MTEP significantly increased immobility in alcohol-withdrawn animals, with a modest increase also seen in water controls. No significant group differences were observed for locomotor activity, indicating that neither anxiolytic was sedating. These results provide predictive validity for increased swimming/reduced immobility in the FST as a model of anxiety and provide novel evidence in favor of mGlu5 inhibition as an effective therapeutic strategy for treating hyperanxiety during alcohol withdrawal.