Unique behavioral and neurochemical effects induced by repeated adolescent consumption of caffeine-mixed alcohol in C57BL/6 mice.

The number of highly caffeinated products has increased dramatically in the past few years. Among these products, highly caffeinated energy drinks are the most heavily advertised and purchased, which has resulted in increased incidences of co-consumption of energy drinks with alcohol. Despite the growing number of adolescents and young adults reporting caffeine-mixed alcohol use, knowledge of the potential consequences associated with co-consumption has been limited to survey-based results and in-laboratory human behavioral testing. Here, we investigate the effect of repeated adolescent (post-natal days P35-61) exposure to caffeine-mixed alcohol in C57BL/6 mice on common drug-related behaviors such as locomotor sensitivity, drug reward and cross-sensitivity, and natural reward. To determine changes in neurological activity resulting from adolescent exposure, we monitored changes in expression of the transcription factor ΔFosB in the dopaminergic reward pathway as a sign of long-term increases in neuronal activity. Repeated adolescent exposure to caffeine-mixed alcohol exposure induced significant locomotor sensitization, desensitized cocaine conditioned place preference, decreased cocaine locomotor cross-sensitivity, and increased natural reward consumption. We also observed increased accumulation of ΔFosB in the nucleus accumbens following repeated adolescent caffeine-mixed alcohol exposure compared to alcohol or caffeine alone. Using our exposure model, we found that repeated exposure to caffeine-mixed alcohol during adolescence causes unique behavioral and neurochemical effects not observed in mice exposed to caffeine or alcohol alone. Based on similar findings for different substances of abuse, it is possible that repeated exposure to caffeine-mixed alcohol during adolescence could potentially alter or escalate future substance abuse as means to compensate for these behavioral and neurochemical alterations. © 2016 Robins et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Social Housing Leads to Increased Ethanol Intake in Male Mice Housed in Environmentally Enriched Cages

An individual's social environment affects alcohol intake. However, the complex interactions between social context and alcohol intake remain understudied in preclinical models. In the present study, we sought to characterize the effects of social housing on voluntary ethanol intake in male C567BL/6J mice using a continuous access two-bottle choice model. This was accomplished using HM2 cages, which allow for the continuous monitoring of individuals' fluid intake through radiofrequency tracking while they remain undisturbed in a group setting. These cages are moderately environmentally enriched compared to standard shoebox cages. By analyzing the levels of voluntary ethanol intake between socially- and individually-housed mice in HM2 cages, we were able to parse apart the effects of environmental enrichment vs. social enrichment. We found that while intake levels were overall lower than those observed when animals are singly housed in standard shoebox cages, socially-housed males consumed significantly more ethanol compared to individually-housed mice, suggesting that while environmental enrichment attenuates ethanol intake, social enrichment may, in fact, potentiate it. This effect was not specific for alcohol, however, in that ethanol preference did not differ as a product of social context. We also found that the total number of non-consummatory channel entries were consistently higher in individually-housed mice. Additionally, a single corticotropin releasing factor receptor 1 antagonist treatment significantly decreased both water and ethanol intake in socially- and individually-housed mice up to 3 h post-treatment, though the effect on water intake was longer lasting. This treatment also significantly decreased the number of non-consummatory channel entries in individually-housed mice, but not in socially-housed mice, suggesting that increased channel visits may be a stress-related behavior. Lastly, we examined blood ethanol concentrations and FosB immunoreactivity to characterize the physiological responses to ethanol intake in socially- and individually-housed mice. The number of FosB-positive cells in the centrally-projecting Edinger-Westphal nucleus and nucleus accumbens shell positively correlated with average baseline ethanol intake in individually-housed mice, but not in socially-housed mice. Overall, we found that social, but not environmental, enrichment can increase ethanol intake in male C57BL/6J mice. Future studies need to test this phenomenon in female mice and assess the generalizability of this finding

Repeated Use of the Psychoactive Substance Ethylphenidate Impacts Neurochemistry and Reward Learning in Adolescent Male and Female Mice

Schedule II prescription psychostimulants, such as methylphenidate (MPH), can be misused as nootropic drugs, i.e., drugs that enhance focus and cognition. When users are unable to obtain these prescribed medications, they may seek out novel psychoactive substances (NPSs) that are not yet scheduled. An example of a NPS reportedly being abused is ethylphenidate (EPH), a close analog of MPH but with a higher preference for the dopamine transporter compared with the norepinephrine transporter. Therefore, based upon this pharmacological profile and user self-reports, we hypothesized that repeated EPH exposure in adolescent mice may be rewarding and alter cognition. Here, we report that repeated exposure to 15 mg/kg EPH decreased spatial cognitive performance as assessed by the Barnes maze spatial learning task in adolescent male C57Bl/6 mice; however, male mice did not show alterations in the expression of mature BDNF – a protein associated with increased cognitive function – in key brain regions. Acute EPH exposure induced hyperlocomotion at a high dose (15 mg/kg, i.p.), but not a low dose (5 mg/kg, i.p.). Interestingly, mice exhibited significant conditioned place preference at the low EPH dose, suggesting that even non-stimulating doses of EPH are rewarding. In both males and females, repeated EPH exposure increased expression of deltaFosB – a marker associated with increased risk of drug abuse – in the dorsal striatum, nucleus accumbens, and prefrontal cortex. Overall, our results suggest that repeated EPH use in adolescence is psychostimulatory, rewarding, increases crucial brain markers of reward-related behaviors, and may negatively impact spatial performance

Critical Role for Gi/o-Protein Activity in the Dorsal Striatum in the Reduction of Voluntary Alcohol Intake in C57Bl/6 Mice

The transition from non-dependent alcohol use to alcohol dependence involves increased activity of the dorsal striatum. Interestingly, the dorsal striatum expresses a large number of inhibitory G-protein-coupled receptors (GPCRs), which when activated may inhibit alcohol-induced increased activity and can decrease alcohol consumption. Here, we explore the hypothesis that dorsal striatal Gi/o-protein activation is sufficient to reduce voluntary alcohol intake. Using a voluntary, limited-access, two-bottle choice, drink-in-the-dark model of alcohol (10%) consumption, we validated the importance of Gi/o signaling in this region by locally expressing neuron-specific, adeno-associated-virus encoded Gi/o-coupled muscarinic M4 designer receptors exclusively activated by designer drugs (DREADD) in the dorsal striatum and observed a decrease in alcohol intake upon DREADD activation. We validated our findings by activating Gi/o-coupled delta-opioid receptors (DORs), which are natively expressed in the dorsal striatum, using either a G-protein biased agonist or a β-arrestin-biased agonist. Local infusion of TAN-67, an in vitro-determined Gi/o-protein biased DOR agonist, decreased voluntary alcohol intake in wild-type and β-arrestin-2 knockout (KO) mice. SNC80, a β-arrestin-2 biased DOR agonist, increased alcohol intake in wild-type mice; however, SNC80 decreased alcohol intake in β-arrestin-2 KO mice, thus resulting in a behavioral outcome generally observed for Gi/o-biased agonists and suggesting that β-arrestin recruitment is required for SNC80-increased alcohol intake. Overall, these results suggest that activation Gi/o-coupled GPCRs expressed in the dorsal striatum, such as the DOR, by G-protein biased agonists may be a potential strategy to decrease voluntary alcohol consumption and β-arrestin recruitment is to be avoided

Exposure to caffeine-mixed alcohol attenuates caffeine-induced cocaine locomotor cross-sensitivity.

<p>Adolescent male animals exposured to water (H2O), 15 mg/kg caffeine (CAF), 1.5 g/kg alcohol (ALC), or caffeine-mixed alcohol (15 mg/kg caffeine, 1.5 g/kg alcohol, A+C) were challenged to 15 mg/kg cocaine in adulthood (n = 7–8 per group). Animals repeatedly exposed to caffeine alone exhibited increased cocaine locomotor cross-sensitization than animals exposed to water, alcohol, or caffeine-mixed alcohol. Significance by one-way ANOVA, *, p<0.05; data represented as mean ± SEM.</p

Caffeine-mixed alcohol exposure in adolescence increases natural reward consumption and preference.

<p>Adolescent exposure to caffeine-mixed alcohol (15 mg/kg caffeine, 1.5 g/kg alcohol, A+C) or water (H2O) altered natural reward consumption and preference in adulthood (n = 6–8 per group). An increase in saccharin preference was observed throughout testing between animals exposed to caffeine-mixed alcohol versus water controls (A). Animals exposed to caffeine-mixed alcohol consumed significantly more 2.0 mM saccharin solution (B) with a greater saccharin solution consumption overall compared to water controls (C). Significance by two-way ANOVA, *, p<0.05; **, p<0.01 or unpaired t-test, *, p<0.05; data represented as mean ± SEM.</p

Repeated caffeine-mixed alcohol exposure by oral gavage during adolescence sensitizes locomotor responses.

<p>Adolescent C57BL/6 mice were exposed to water (H2O), 1.5 g/kg alcohol (ALC), 15 mg/kg caffeine (CAF) or caffeine-mixed alcohol (A+C), exposure by daily oral gavage (n = 6 per group) for 4 weeks (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158189#pone.0158189.g002" target="_blank">Fig 2</a>). Locomotor activity was measured for 60 minutes directly following injection. Mice exposed to caffeine-mixed alcohol showed acute hyperlocomotion and significant locomotor sensitization over the course of four weeks (A). Differences in first and last exposure demonstrate the increase in locomotor activity over the locomotor testing sessions (B). Statistical significance was assessed by two-way, repeated measures ANOVA (time and treatment) followed by Bonferroni’s Multiple Comparison Test, *, p<0.05; **, p<0.01, ***, p<0.0005, ****, p<0.0001, #, p<0.05; data represented as mean ± SEM.</p

Adolescent exposure to caffeine-mixed alcohol during adolescence significantly increases ΔFosB expression levels in the nucleus accumbens similar to cocaine.

<p>Adolescent C57BL/6 mice (n = 6 per group) were repeatedly exposed by oral gavage to water (H2O), 15/mg/kg caffeine (CAF), 1.5 g/kg alcohol (ALC), caffeine-mixed alcohol (A+C), or 15 mg/kg cocaine (i.p., COC) for four weeks in adolescence, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158189#pone.0158189.g002" target="_blank">Fig 2</a>. Three days after the final locomotor session, animals were exposed once more to their respective treatment. Brains were removed 30 minutes after exposure to last treatment via transcardial perfusion. Coronal brain slices were immunohistochemically stained for ΔFosB expression in the dorsal striatum (A, D) and nucleus accumbens (B, E), as indicated in C. All treatments increased ΔFosB accumulation in the dorsal striatum compared to water controls (A, D). Increases in ΔFosB accumulation were observed in the nucleus accumbens in animals exposed to caffeine-mixed alcohol compared to alcohol or caffeine alone (B, E). Quantification was achieved by counting the number of ΔFosB for each treatment using ImageJ software. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s Multiple Comparison Test, *, p<0.05; **, p<0.01, ***, p<0.0005, #, p<0.05, ###, p<0.0005; data represented as mean ± SEM.</p

Behavioral Characterization of β-Arrestin 1 Knockout Mice in Anxiety-Like and Alcohol Behaviors

β-Arrestin 1 and 2 are highly expressed proteins involved in the desensitization of G protein-coupled receptor signaling which also regulate a variety of intracellular signaling pathways. Gene knockout (KO) studies suggest that the two isoforms are not homologous in their effects on baseline and drug-induced behavior; yet, the role of β-arrestin 1 in the central nervous system has been less investigated compared to β-arrestin 2. Here, we investigate how global β-arrestin 1 KO affects anxiety-like and alcohol-related behaviors in male and female C57BL/6 mice. We observed increased baseline locomotor activity in β-arrestin 1 KO animals compared with wild-type (WT) or heterozygous (HET) mice with a sex effect. KO male mice were less anxious in a light/dark transition test, although this effect may have been confounded by increased locomotor activity. No differences in sucrose intake were observed between genotypes or sexes. Female β-arrestin 1 KO mice consumed more 10% alcohol than HET females in a limited 4-h access, two-bottle choice, drinking-in-the-dark model. In a 20% alcohol binge-like access model, female KO animals consumed significantly more alcohol than HET and WT females. A significant sex effect was observed in both alcohol consumption models, with female mice consuming greater amounts of alcohol than males relative to body weight. Increased sensitivity to latency to loss of righting reflex (LORR) was observed in β-arrestin 1 KO mice although no differences were observed in duration of LORR. Overall, our efforts suggest that β-arrestin 1 may be protective against increased alcohol consumption in females and hyperactivity in both sexes