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.

Individual variation in the addiction liability of amphetamines has a heritable genetic component. We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene underlying decreased methamphetamine-induced locomotor activity in mice. Here, we showed that mice (both females and males) with a heterozygous mutation in the first coding exon of Hnrnph1 (H1 ϩ/Ϫ) showed reduced methamphetamine reinforcement and intake and dose-dependent changes in methamphetamine reward as measured via conditioned place preference. Furthermore, H1 ϩ/Ϫ mice showed a robust decrease in methamphetamine-induced dopamine release in the NAc with no change in baseline extracellular dopamine, striatal whole-tissue dopamine, dopamine transporter protein, dopamine uptake, or striatal methamphetamine and amphetamine metabolite levels. Immunohistochemical and immunoblot staining of midbrain dopaminergic neurons and their forebrain projections for TH did not reveal any major changes in staining intensity, cell number, or forebrain puncta counts. Surprisingly, there was a twofold increase in hnRNP H protein in the striatal synaptosome of H1 ϩ/Ϫ mice with no change in whole-tissue levels. To gain insight into the mechanisms linking increased synaptic hnRNP H with decreased methamphetamine-induced dopamine release and behaviors, synaptosomal proteomic analysis identified an increased baseline abundance of several mitochondrial complex I and V proteins that rapidly decreased at 30 min after methamphetamine administration in H1 ϩ/Ϫ mice. In contrast, the much lower level of basal synaptosomal mitochondrial proteins in WT mice showed a rapid increase. We conclude that H1 ϩ/Ϫ decreases methamphetamine-induced dopamine release, reward, and reinforcement and induces dynamic changes in basal and methamphetamine-induced synaptic mitochondrial function.

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.

We previously reported that commercially-sourced C57BL/6J (B6) male mice with a history of adult-onset binge-drinking exhibit anxiety-like behavior in early withdrawal, while the negative affective state incubates during protracted withdrawal in adolescent-onset binge-drinking males. As the results of such studies are potentially confounded by age-related differences in reactivity to environmental stress, we employed a 2-bottle-choice DID procedure (20 and 40% alcohol; 20 min habituation to the drinking cage) to examine the effects of binge-drinking on negative affect in male and female, adult and adolescent, B6 mice from our university colony. Unexpectedly, the mice in the initial experiment exhibited very low alcohol intake, with little sign of withdrawalinduced negative affect. This failure to replicate prompted us to examine how the duration of drinking cage habituation, the number of alcohol concentrations presented and the animal source might influence the propensity to binge-drink. Herein, we show that both male and female adult mice from our colony will binge-drink when allowed 45 min to habituate to the drinking cages, irrespective of whether mice are offered a choice between 2, 3 or 4 alcohol concentrations. Further, when drinking under 4-bottle-choice procedures (5, 10, 20 and 40% alcohol), adult-onset binge-drinking females exhibit robust negative affect in early withdrawal akin to that reported previously for adult males; however, the negative affective state persists for at least 30 days into withdrawal. Also unlike males, adolescent-onset binge-drinking females exhibit some signs of negative affect, as well as potentiated alcohol intake, in early withdrawal, which persist into later withdrawal. These latter data suggest that the age-related differences in the temporal patterning of the negative affective state produced by alcohol withdrawal may vary as a function of sex, which may have implications for understanding sex differences in the etiology of affective disorders and alcoholism co-morbidity.