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Alcohol Consumption and Wheel Running in Adolescent Mice and Effects on Alcohol Consumption

Beth Chitel


Previous studies have shown an interaction between alcohol consumption and wheel running in C57BL/6 mice. We investigated adolescent alcohol consumption and subsequent adult alcohol consumption in C57BL/6 mice (N=26) using a 24-hour two-bottle choice paradigm, and determined whether any effects were dependent on wheel condition (no wheel, locked wheel, free wheel). Preliminary results were insignificant, but indicate possible trends which maybe confirmed in similar studies with a larger sample size.

Introduction: Alcohol & Humans

Alcohol Use & Dependence: Alcohol use is common in the United States and throughout many parts of the world (WHO). The Center for Disease Control (CDC) has recently reported that 52% of adults in the United States have had at least 12 drinks in the last year. In a 2003 National Survey on Drug Use and Health (NSDUH), 88% of the 14 million respondents aged 21 or older reported having used alcohol. While some evidence suggests moderate alcohol use (1 drink per day for women, two for men) offers health benefits, excessive alcohol use is accompanied by serious implications to health and society (American Heart Association, WHO, NSDUH, CDC, NIDA).

According to the WHO, there is increased risk of breast cancer and cirrhosis with increased alcohol consumption. Pregnant women who consume alcohol put the fetus at risk for Fetal Alcohol Syndrome, possibly causing facial abnormalities, stunted growth, and nervous system damage in the fetus (CDC). Alcohol dependence, defined by compulsive alcohol craving, increased tolerance, and abusive behavior (family problems, legal problems related to alcohol, impaired ability to function at school or work), can cause immunodeficiency, cancer, stroke, heart disease and liver disease (American Heart Association, CDC, WHO). After face-to-face interviews with 43,093 people, Hassin and colleagues (2007) reported a lifetime alcohol dependence risk of 12.5%. Alcohol use is related to 2.5 million deaths worldwide annually (WHO).

Society can bear the brunt of alcohol use. Social harms include unemployment, lost productivity by the drinker and the employer, violent and non-violent crimes, family problems, traffic accidents, and economic loss of productivity due to alcohol-related deaths (WHO). While some evidence may show that alcohol in moderation may have health benefits, the years of life lost to alcohol use are far more than the years of life saved (CDC, WHO).

Adolescent Alcohol Use: Adolescence is a period of neurological, physical, hormonal, and social behavior change. While the exact beginning and end of human adolescence is debated, the age range 12-18 years is typically considered to be adolescence, according to changes indicated above. Novelty-seeking, risk-taking, and conforming to peer groups are characteristic of adolescence, and may even be considered normative behavior (see Spear 2000, for review).

Evidence suggests that adolescents are less sensitive to perceived negative effects of alcohol consumption such as sedative effects and motor impairment (Silveri and Spear, 1998, ) and more sensitive to perceived pleasant effects of alcohol such as social facilitation, lowered inhibitions (Spear, 2000). 13 million of the 14 million respondents to the NSDUH initiated alcohol use prior to 21 years of age and this finding is not surprising givent the increased sensitivity of alcohol’s pleasant effects.

In addition to the implications of alcohol use by adults, evidence suggests that adolescents who use alcohol may become dependent much more quickly than adults. Adolescent alcohol use increases risk for alcohol and other drug abuse in adulthood, and is associated with detrimental behaviors in adulthood (Spear for review, 2000). The NSDUH reported that people who initiated alcohol use before age of 15 were five times more likely to report alcohol dependence in the last year than alcohol users who initiated use at age of 21 or older. Studies have shown that alcohol use in adolescence can adversely affect brain development, particularly areas involved in learning and memory (Spear, 2000; Pautassi et al., 2010).

Alcohol Studies in Animals

Physical and behavioral effects of alcohol consumption can also be seen in alcohol studies on mice and rats. And, like humans, alcohol consumption affects rodents differently, depending on age. Mouse and rat adolescents (usually from 21 to 59 days of age in mice and 28-42 days in rats, with some variability before and after that period) mirror some physical, neurological, and behavioral changes exhibited by human adolescents (Spear, 2000). For example, neural alterations appear to occur in the prefrontal cortex of both humans and rodents during adolescence; both human and rodent adolescents exhibit increased novelty-seeking and social interaction compared to human and rodent adults (Spear, 2000). The similar changes mice and rats undergo during the adolescent phase in addition to the large body of previous research on adult mice and rats, justifies the use of mice and rats to model human adolescent studies.

While alcohol studies using rodent models is a relatively new phenomenon, results of these studies have supported much of the preexisting research on adolescent humans. Adolescent rats are less sensitive to alcohol’s sedative and motor impairing effects. Rats and mice are able to drink more than their adult counterparts, and are more likely to develop a tolerance (Spear, 2000). Strong and colleagues found that mice that were exposed to alcohol during their adolescent years are likely to drink more alcohol as adults (2010).

In addition to age differences in alcohol consumption, studies with mice and rats have also shown that sex affects alcohol consumption. Several studies show greater alcohol intake in females. Strong and colleagues found that adult mice exposed to a binge drinking alcohol protocol as adolescents drank more as adults; this effect was exacerbated in the female mice (2010). Female C57BL/6 mice consistently drink more alcohol than C57BL/6 males (deVisser, L., 2007; Belknap et al., 1993). Ehringer et al., in 2009, found that C57BL/6 females drank less if they had access to a running wheel, whereas males drank less even if they had access to only a locked wheel. Results of the Ehringer et al. study suggest that exercise could decrease alcohol consumption.

Alcohol & Exercise: Mice have been shown to drink less alcohol if running wheels are available to them (McMillan et al., 1995; Ehringer et al., 2009). Mollenauer et al. (1991) found that wheel running mitigated some effects of alcohol consumption, suggesting cross-tolerance. Werme et al. found that rats with wheel access during alcohol deprivation increased alcohol consumption when alcohol was returned, although the wheels were still present (2002). Ozburn et al. found a similar deprivation effect, although the effect did not persist (2008). Studies also have shown that alcohol consumption is unaffected by access to running wheels (Crews et al., 2004; Moore et al., 2010; Ozburn et al., 2008).This multitude of results regarding exercise and alcohol use is very interesting, since, like alcohol, exercise stimulates the dopaminergic reward pathway. There is evidence that chronic exercise can be rewarding and addicting (Chan and Grossman, 1988; Chapman and De Castro, 1990; Furst and Germone, 1993; Pierce et al., 1997). Despite the popularity of alcohol, exercise, and adolescent research, this is the first study on exercise and alcohol use in adolescence, and subsequent effects on alcohol consumption in adulthood. Considering the consequences of adolescent alcohol use, the shared reward pathway of alcohol and exercise, and evidence supporting some relationship between alcohol use and exercise, the study of adolescent alcohol use and exercise and subsequent effects on adult consumption begs attention.

Dopaminergic Reward Pathway

Animals, including humans, repeat behaviors that are rewarding. We find our behaviors rewarding when they stimulate our brain’s reward pathways. One of those pathways is the dopaminergic reward pathway. Sex and food stimulate this pathway—an evolutionary trait that has reinforced behaviors necessary for human survival (NIDA). Exercise and alcohol consumption have similarly been found to stimulate the dopaminergic pathway and elicit pleasurable feelings in exercisers and/or drinkers, and are thus reinforcing. Hence, the possibility of addiction to exercise or alcohol exists for some people.

The dopaminergic reward pathway primarily consists of the ventral tegmental area (VTA), the prefrontal cortex (PFC), and the nucleus accumbens. The VTA extends axons to the nucleus accumbens and PFC—brain areas believed to play a role in motivation. When a rewarding stimulus is presented, the VTA increases release of the neurotransmitter dopamine in these projections, resulting in feelings of pleasure (Kandel, 1009).

Aim of Study

This study investigates whether or not exposure to alcohol in adolescence affects alcohol consumption in adulthood, and whether or not any effects are dependent on wheel condition.

Materials and Methods

Animals: The C57/BL/6 mouse strain is an inbred strain exhibiting high levels of alcohol consumption and wheel running (Belknap, Crabbe, and Young, 1993; Lerman et al., 2002; Lightfoot et al., 2004). The testing of 32 C57BL/6 IBG mice has been completed at this point, but only data for the 26 females is included in this study to control for sex differences (C57BL/6 females consistently drink more than males). Mice were bred at the Institute for Behavioral Genetics (IBG) at the University of Colorado, Boulder.

Housing: Mice were single-housed in 12 cm x 30.5 cm x 12.5cm polycarbonate cages with no wheel, a locked wheel, or free wheel, in a 12:12 light cycle room. Free running wheels were affixed with CatEye bike computer counters to measure distance. Food and water were available ad lib throughout the experiment. Each cage had enough bedding to cover the floor, and a bedding square.


Adolescence: Mice remained co-housed with their same-sex litter mates until PND (post-natal day) 30, or a couple days within the beginning of the adolescent stage, at which point they were weighed, ear-clipped for identification, and individually housed according to their randomly assigned condition for a 3-day acclimation period. Cage conditions were empty (no wheel), locked wheel (wheel is secured still with screws and washers), and free wheel (free-turning wheel). A locked wheel was used as a control to ensure that any difference in alcohol consumption between the free wheel and no-wheel (empty) groups would not be due merely to the novelty of the wheel, but to the exercise opportunity offered by a free-running wheel. After the 3-day acclimation period, on PND 33, 10% alcohol treatment began. Mice assigned to the alcohol condition had a two-bottle choice between alcohol and water, 24 hours a day. The positions (left or right) of water and alcohol bottles were switched daily to avoid place preference effects. Mice without alcohol had water only. Weights of alcohol and water bottles were recorded daily. Water, alcohol, and food were refilled as needed. Distance run, according to the CatEye counters, was recorded daily. On PND 43 (day 10 of the experiment), mice were weighed and rehoused with their same-sex litter mates. Food was weighed. Water and alcohol bottles were weighed for the last time in the adolescent phase.

Interim: Mice remained co-housed with their same-sex littermates in the IBG colony room for 17 nights. Bedding covered cage floors, and a bedding square was in each cage. Food and water were available ad lib. No running wheels or alcohol were available at this time.

Adulthood: In early adulthood, on PND 60, (after the 17-night interim), all mice were weighed and singly housed in polycarbonate cages with free wheels in the 12:12 light cycle room. Food was weighed, and was available with water ad lib. The acclimation period was again three days; no alcohol was available at this time. The protocol for adulthood testing, which began on PND 63, was identical to the adolescent protocol, except that all mice now had free-running wheels and a two-bottle choice between alcohol and water.

The final day of testing was PND 73. Alcohol and water bottles were weighed for the last time in the adult phase. Mice and food were weighed. Mice were euthanized using cervical dislocation, and brain tissue was collected for future examination of those tissues.

Statistical Analysis

SPSS 18.0 was used to conduct univariate ANOVAs for the following dependent variables:

  • Adolescent alcohol consumed (g/kg body weight)
  • Adult alcohol consumed (g/kg body weight)
  • Adolescent alcohol preference
  • Adult alcohol preference


Preliminary Results & Implications: Adolescents appeared to drink more alcohol in an empty cage than they did in a locked wheel or free wheel cage condition (Fig. 2). These results are consistent with Ehringer et al.’s study conclusions that mice with free wheels in their cages consumed less alcohol (only males in that study significantly decreased alcohol consumption with a locked wheel). That these results became inversed in adulthood was surprising. As adults, mice who had a free wheel in adolescence drank the most alcohol, mice who had locked wheels drank the second greatest amount, and mice who had had empty cages in adolescence now drank the least alcohol (Fig. 3). All mice had free wheels in the adult phase of the experiment. It is possible that mice that had a wheel or alcohol in adolescence experienced a deprivation effect in the 17-night interim, and drank more in adulthood to compensate for the alcohol and/or exercise reward they had been missing. Mice that had an empty cage and drank the most in adolescence now had a wheel. Their alcohol consumption may have decreased because of the new stimulus available in the cage. Overall, adult mice that had water only in adolescence appeared to drink slightly less than adult mice that had alcohol in adolescence.

In summary, results of this study were not significant, but suggest trends that might become significant in similar studies with a larger sample (N). These trends include increased initial alcohol consumption in adolescence when a running wheel is not available; increased alcohol consumption in adulthood if alcohol was available in adolescence; decreased alcohol consumption in adulthood when alcohol is initiated in adulthood and a running wheel is available; and any possible deprivation effects.

Future Directions

Further testing of adolescent running and alcohol consumption, and subsequent adult alcohol consumption, is warranted before any conclusions can be drawn. The sample was too small (N = 26) to be able to generalize the results to a greater population. Future modifications should be made if possible to prevent or reduce bottle fluid loss, cap chewing, squeaky wheels, and faulty distance measurements (not included in results). The possibility of effects of female cages and male cages side-by-side should be investigated before testing males and females in the same room.

It is common for activity levels in humans to decrease from adolescence to adulthood. If this situation were applied to mice, it would be similar to offering a wheel in adolescence and removing the wheel in adulthood. It would be interesting to investigate how this lifestyle shift would affect alcohol consumption in adult C57/BL6 mice.


The following people helped make this project possible: Marissa Ehringer, Todd Darlington, Nicole Hoft, Stacy Romero, and Jess Godfrey


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