- Email: [email protected]
Reduced alcohol consumption in mice with access to a running wheel
Alcohol 43 (2009) 443e452
Reduced alcohol consumption in mice with access to a running wheel Marissa A. Ehringera,b,*, Nicole R. Hofta, Matthias Zunhammerc a
University of Colorado, Institute for Behavioral Genetics, 1480 30th Street, 447 UCB, Boulder, CO 80309-0447, USA b Department of Integrative Physiology, 354 UCB, Boulder, CO 80309-0354, USA c University of Regensburg, Department of Psychiatry, Psychosomatics, and Psychotherapy, 93053 Regensburg, Germany Received 11 August 2008; received in revised form 8 May 2009; accepted 20 June 2009
Abstract Studies of the behavioral effects of alcohol in humans and rodent models have implicated a number of neurological pathways and genes. Separate studies have shown that certain regions of the brain are involved in behavioral responses to exercise. The aim of this study was to determine whether mice which normally voluntarily consume high amounts of alcohol (C57BL/6 strain) would exhibit reduced alcohol consumption when given access to a running wheel under two different models of voluntary consumption: unlimited access two-bottle choice and limited access drinking in the dark (DID). Under the two-bottle choice model, the animals voluntarily consumed less alcohol when a wheel was present in their cage. However, sex-specific differences emerged because female mice voluntarily consumed less alcohol when they have the opportunity to exercise on a running wheel, whereas male mice consumed less alcohol even if the running wheel was locked. There were no significant differences observed in alcohol metabolism or food consumption. Under the DID protocol, no differences in alcohol consumption were observed in the presence of a running wheel. These results suggest that exercise may be a useful approach to consider for treatment for some types of chronic human alcohol problem behaviors, but may be less applicable to human binge drinking. Ó Published by Elsevier Inc. Keywords: Alcohol preference; Exercise; Wheel running; Mouse model
Introduction In recent years, it has become apparent that common neuronal pathways may be involved in a wide range of addictive behaviors, including drugs of abuse, overeating, gambling, and sexual addictions, and that these behaviors are often comorbid with other psychiatric disorders such as depression, stress, and anxiety (Goldman et al., 2005; Nestler, 2005; Volkow and Wise, 2005). Although other neurobiological pathways are likely to be involved, the mesolimbic dopaminergic system has become the focus of study for understanding reward responses. Dopaminergic cell bodies in the ventral tegmental area project to forebrain nuclei, including the nucleus accumbens, and it has been shown that these neurons can be activated by reward stimuli (Carelli et al., 2000).
This study was supported by University of Colorado Council on Research and Creative Work, Junior Faculty Development Award to M.A.E. * Corresponding author. University of Colorado, Institute for Behavioral Genetics, 1480 30th Street, 447 UCB, Boulder, CO 80309-0447, USA. Tel.: þ1-303-492-1463; fax: þ1-303-492-8063. E-mail address: [email protected] (M.A. Ehringer). 0741-8329/09/$ e see front matter Ó Published by Elsevier Inc. doi: 10.1016/j.alcohol.2009.06.003
A number of studies have shown that exercise has addictive properties among people who intensely exercise on a regular basis (Chan and Grossman, 1988; Chapman and De Castro, 1990; Furst and Germone, 1993; Pierce et al., 1997). These studies have shown that exercise can become a chronic activity, that the need to continue to exercise persists despite physical and psychological symptoms, and that ‘‘dependency’’ is associated with quantitative factors, including frequency, duration, and intensity. Recently, Bamber et al. have begun developing specific diagnostic criteria for exercise dependence that includes two main components: impaired function and withdrawal (Bamber et al., 2003). Gutgesell et al. (1996) have found that although runners typically report more occasions of drinking than nonrunners, among those who have a history of problem drinking, runners report drinking less alcohol than nonrunners. Similarly, intense exercise has been shown to lead to a significant decrease in alcohol cravings in recovering alcoholics (Ussher et al., 2004). These effects suggest that running and alcohol may be activating shared neural pathways. Wheel running in rodents has been used in connection with models of hyperactivity (Rhodes et al., 2003), stress response (Greenwood et al., 2003, 2005), response to
444
M.A. Ehringer et al. / Alcohol 43 (2009) 443e452
novelty (Ferreira et al., 2006), food deprivation, and drugs of abuse (Larson and Carroll, 2005; Mathes and Kanarek, 2001). A landmark study by Freed and Yamamoto (1985) demonstrated that dopamine signaling could be selectively activated by voluntary motor behavior, but not by passive movement of the head. Similarly, studies of mice selectively bred for hyperactive wheel running will decrease their running when administered dopamine reuptake blockers (Rhodes and Garland, 2003). Wheel running has been used as a model of physical activity dependence (Ferreira et al., 2006) and shown to confer conditioned place preference after only 2 h of access (Lett et al., 2000). The role of exercise in modulating neuronal plasticity and regeneration has become a topic of great interest (Dishman et al., 2006; Vaynman and Gomez-Pinilla, 2006), and is in contrast to the neurodegenerative effects of alcohol (Nixon, 2006). A recent report by Crews et al. (Crews et al., 2004) demonstrated that access to a running wheel could reverse alcohol’s inhibitory effect on neural stem-cell proliferation. The authors suggest ‘‘the opposing effects of ethanol consumption and exercise on neurogenesis could contribute to the CNS pathology and health benefits, respectively, of these two behaviors.’’ It has been shown that wheel running can prime adult sensory neurons for enhanced axonal regeneration (Molteni et al., 2004) and that activity-dependent plasticity is retained into adulthood (Zito and Svoboda, 2002). At least some of the positive effects observed with running, including increased neurogenesis, learning, and long-term potentiation appear to be mediated via hippocampal mechanisms (van Praag et al., 1999a). In animal research, several studies have been conducted to examine possible effects of exercise on voluntary alcohol consumption. Rats bred to accept ethanol (P rats) show decreased ethanol intake and increased water intake when given access to a running wheel (McMillan et al., 1995), with no effect on food consumption. Werme et al. (2002) showed that Lewis rats, which ran during a withdrawal period following alcohol exposure showed increased alcohol consumption compared with those that did not have access to wheels during the withdrawal period, concluding that running increases ethanol preference following withdrawal. The same group also showed that Nurr1 deficient mice (heterozygous for the Nurr1 gene deletion) displayed lower alcohol consumption and less running behavior than their littermate wild-type controls (Werme et al., 2003). The Nurr1 protein is a member of the NGFI-B family of transcription factors and plays an important role in signaling pathways involved in differentiation and phenotypic expression of dopaminergic neurons (Sacchetti et al., 2006). However, Crews et al. (2004) reported no difference in voluntary alcohol consumption in male C57BL/6 mice with access to a running wheel. This is in agreement with Ozburn et al. (Ozburn et al., 2008), who found that voluntary ethanol consumption of female C57BL/6J mice remained stable under a protocol involving alternating periods of access to running wheels.
In addition to alcohol, a few studies have examined selfadministration of other drugs, including amphetamine and cocaine. Kanarek et al. (Kanarek et al., 1995) reported that male Sprague-Dawley rats consumed significantly less amphetamine when the animals had access to running wheels compared with no access to a wheel. Likewise, Smith et al. (Smith et al., 2008) found that wheel running was associated with decreased positive-reinforcing effects of cocaine in female Long-Evans rats, as measured using a progressive ratio schedule. This collection of studies supports the possibility that hedonic substitution of alcohol by exercise might be a useful approach for preventing or treating alcohol and drug dependence. However, variation in study designs makes it difficult to draw overarching conclusions regarding the interaction of these two behaviors. Most of the studies have used rats, but four different strains have been used and in most cases only one sex was tested. Among the three mouse studies using different behavioral paradigms, only females were used in two studies (Ozburn et al., 2008; Werme et al., 2003), whereas only males were tested in the third study (Crews et al., 2004). The purpose of the present study was to examine whether providing C57BL/6 mice access to a running wheel would lead to decreased alcohol intake using two common measures of voluntary consumption. The C57BL/6 strain was selected for these pilot studies because among commonly used inbred mouse strains they show high levels of alcohol consumption and high levels of voluntary wheel running (Belknap et al., 1993; Lerman et al., 2002; Lightfoot et al., 2004). An unlimited access two-bottle choice (water or ethanol) protocol was selected because it has been well characterized as a model of alcohol behavior for over 30 years (Belknap et al., 1993; Fuller, 1964; Yoneyama et al., 2008). In addition, we also used the relatively new drinking-in-the-dark (DID) protocol, believed to be a model of binge drinking in which the animals have limited access to alcohol during the dark cycle (Boehm et al., 2008; Kamdar et al., 2007; Rhodes et al., 2007). Experimental procedures Animals and housing The C57BL/6IBG mice were bred at the Institute for Behavioral Genetics at the University of Colorado, Boulder. On collection, mice were in group housing on a 12-h light:12-h dark cycle (lights on at 0700 h) and provided with ad libitum access to food and water. When they were 60e90 days old, the animals were placed in individual cages, with or without running wheels (as noted in the following) and allowed to acclimate for 3e7 days. The cages without wheel were standard 30 13 17-cm polycarbonate cages, whereas the cages with wheel were slightly larger to accommodate the wheels (30.3 20.6 26 cm with wheels 242 mm in diameter), available from Respironics Mini Mitter (Bend, OR, USA). The cages were
M.A. Ehringer et al. / Alcohol 43 (2009) 443e452
checked for food and water daily, and animals were weighed every 4 days. The local Institutional Animal Care and Use Committee approved all procedures. Ethanol preference testing The same animals were used for Ethanol preference testing and Combined ethanol preference and voluntary wheel running. The mice were given a choice between tap water and different concentrations of ethanol diluted in tap water in two 15-mL tubes with a metal sipper tube. Three percent ethanol solution was provided in the ethanol tube on days 1 and 2, 7% solution was provided on days 3 and 4, and then the solution remained at 10% for days 5 through 13. A diagram of the behavioral testing procedure is shown in Fig. 1A. The placement of the tubes was reversed daily during weighing and refilling to prevent the mice from developing placement preference. To account for evaporation, calibration tubes containing water and ethanol were placed in empty cages in the testing room, measured daily, and these amounts were subtracted from all data for individual mice. All together, 37 animals (19 males and 18 females) were tested. A Student’s two-tailed t-test was used to compare drinking levels (g alcohol/kg body weight) in males and females (SPSS 16.0, Chicago, IL). Voluntary wheel running Mice were placed in cages containing free wheels for 13 days with free access to food and water. The distance run by the mice was recorded daily using a magnet system attached to the wheel for which number of revolutions was collected by a counter, next to the cage. All together, 31 animals (16 males and 15 females) were used. A Student’s two-tailed t-test was used to compare running distances in males and females. Combined ethanol preference and voluntary wheel running The ethanol paradigm described as previously mentioned, introducing 3%, 7%, and 10% ethanol, was conducted with mice in cages that contained movable running wheels and locked running wheels (Fig. 1A). Daily ethanol consumption and distance run data were collected. SPSS 16.0 was used to conduct a one-way analysis of variance (ANOVA) within each sex to compare mean alcohol consumption using two measures: grams of alcohol per kilogram of body weight and ethanol preference ratio (PR), the ratio of volume of alcohol consumed divided by the total volume of liquid consumed. Tukey’s post hoc tests were used to determine significant differences between the three cage condition groups (sedentary, locked wheel, or movable wheel). One-way ANOVAs were also used to examine whether the total volume of liquid consumed differed by cage condition within each sex. Finally, a Student’s two-tailed t-test was used to compare running distances in the presence or absence of alcohol within each
445
sex. All together, 108 animals (59 males and 49 females) were used, including the 37 baseline-drinking animals used for the sex comparison of baseline ethanol drinking (as previously mentioned). A similar two-bottle choice procedure was repeated with a different set of animals whereby the alcohol concentration was increased gradually to 20% ethanol (Fig. 1B). Animals had access to water and ethanol each day, where the ethanol bottle concentrations were 3% on days 1e2, 7% on days 3e4, 10% on days 5e6, 13% on days 7e8, 17% on days 9e10, and 20% on days 11e14. Data from days 11e14 were analyzed using a Student’s t-test to compare ethanol consumption between sedentary and running animals.
Alcohol metabolism Mice were individually housed in cages with or without running wheels for 2 weeks to determine if animals that experienced a ‘‘pretreatment’’ of running showed faster rates of alcohol metabolism. At the end of the 2-week period, animals were injected with ethanol into the intraperitoneal cavity (3 g/kg). Retro-orbital sinus blood was collected nine times following the injection to determine blood ethanol concentrations (BECs) (10, 20, 30, 45, 60, 90, 120, 180, and 240 min). The BEC was measured using a modified enzymatic assay, which couples the conversion of ethanol to acetaldehyde and the conversion of nicotinamide adenine dinucleotide (NAD) to NAD with hydrogen (NADH) by the addition of alcohol dehydrogenase (ADH from yeast). The amount of NADH produced is measured at 340 nm using a spectrophotometer, and ethanol concentrations can be calculated in reference to a standard curve (Smolen et al., 1986). All together, 16 animals (8 males and 8 females) were used. Linear regression curves were fit to estimate a slope of alcohol decay for each animal. A Student’s two-tailed t-test was used to test for differences in these slopes by sex and cage condition (sedentary or running).
Food consumption The standard 13-day 10% alcohol protocol was administered to an additional set of mice with and without access to running wheels. This experiment was conducted twice. In the first batch, food pellets were glued into small petri dishes (60 15 mm) and attached to the side of the cage using Velcro. The petri dishes were removed, weighed, and replenished daily (10 males, 5 sedentary and 5 running; 20 females, 10 sedentary and 10 running). In the second experiment, food pellets were placed in the hopper next to the liquid bottles. These pellets were removed, weighed, and replenished daily. In this second batch, there were 15 males (8 sedentary, 7 running) and 15 females (8 sedentary, 7 running).
446
M.A. Ehringer et al. / Alcohol 43 (2009) 443e452
A 3-7 days single housing
Days 1-2 3% EtOH
Days 3-4 7%EtOH
Days 5-13 10%EtOH
15 days with free access to wheel or locked wheel
B 3-7 days single housing
Days 1-2 3% EtOH
Days 3-4 7%EtOH
Days 5-6 10% EtOH
Days 7-8 13%EtOH
Days 11-14 20%EtOH
Days 9-10 17%EtOH
14 days free access to wheel
C Days 1-7 group housing Reverse light cycle
Days 8-14 single housing Reverse light cycle
Days 15-18 2 hour limited access to 20% alcohol
11 days with free access to wheel
Fig. 1. Schematic of behavioral testing protocols. (A) Two-bottle choice unlimited access to 10% ethanol. Animals were allowed to acclimate in their individual cages with or without running wheels for 48 h prior to introduction of alcohol. (B) Two-bottle choice unlimited access to 20% ethanol. (C) Drinking in the dark.
DID and voluntary wheel running Animal breeding, housing, and cage conditions were as previously mentioned, except animals for this experiment were acclimated to a reverse light cycle (lights OFF at 0700 h, lights ON at 1900 h) for 7 days in group housing (5 mice per cage), following by 7 additional acclimation days in their individual cages. Mice had unlimited access to water and food at all times up, until the 4-day DID protocol began. Three hours after the lights were off (1000 h), water bottles were replaced with bottles containing 20% (vol/vol) ethanol for 2 h, at which time the amount of ethanol consumed was recorded and the water bottle replaced. A diagram of the behavioral testing protocol is shown in Fig. 1C. For those animals in cages with moveable wheels, distance run was recorded daily. A Student’s t-test was used to compare amount of alcohol consumed during the limited access period within each sex by cage condition (sedentary or running). Results All reported values are mean 6 standard error for the 10% ethanol testing days, unless otherwise noted. Baseline ethanol consumption and wheel running As has been shown previously (Middaugh et al., 1999), females consumed significantly more ethanol than males (P 5 .01, t 5 2.725). The females drank 20.77 6 1.48 g/kg (n 5 18) ethanol daily, whereas the males drank 15.75 6 1.12 g/kg (n 5 19) ethanol (Table 1). These consumption levels are slightly higher than reports ranging
from approximately 9 g/kg daily in males to 13 g/kg in females (Bachmanov et al., 1996; Belknap et al., 1993; Meliska et al., 1995; Middaugh et al., 1999), but they have remained consistent over approximately 3 years of testing. Likewise, female C57BL/6IBG mice ran significantly farther than males (P 5 .017, t 5 2.522). Females ran 5722.37 6 806.37 m/day (n 5 16), whereas males ran only 3527.91 6 373.54 m/day (n 5 16). All subsequent analyses were performed in males and females separately. Similar results were found for 3% and 7% ethanol testing days as shown in Fig. 2. Ethanol consumption in presence of running wheel One-way ANOVA revealed a significant difference in total liquid consumption among females that was dependent on cage condition (F 5 4.68, P 5 .014), where Tukey’s post hoc test indicated the mice with a movable wheel drank more total liquid (6.28 6 0.33 g) than either of the other two groups (5.34 6 0.22 g and 5.36 6 0.77 g; P ! .05). This increase in overall drinking was associated with a decrease in ethanol intake because a significant difference in alcohol consumption between animals for the three cage conditions was also present (F 5 4.64, P 5 .015 for g/kg and F 5 4.75, P 5 .013 for PR). Tukey’s post hoc test revealed that female mice with access to a running wheel consumed significantly less alcohol (13.7 6 2.31 g/kg or 0.51 6 0.07 PR) than sedentary mice (20.7 6 1.5 g/kg or 0.78 6 0.05 PR) (P ! .05), or mice with a locked wheel (20.3 6 1.6 g/kg or 0.73 6 0.05 PR) (P ! .05) (Table 1). There was no difference in the mean distance (m) run per day in mice with or without access
M.A. Ehringer et al. / Alcohol 43 (2009) 443e452 Table 1 Summary of all experiments Experiment and variable
Males
N
Two-bottle choice ethanol and wheel running Mean g/kg alcohol at 10% Sedentary 15.75 6 1.12 19 Locked wheel 11.84 6 1.44 16 Free wheel 9.11 6 1.02 24
Females
N
20.77 6 1.48 20.32 6 1.64 13.74 6 2.13
18 18 13
Mean preference ratio (PR) at 10% Sedentary 0.66 6 0.05 Locked wheel 0.62 6 0.07 Free wheel 0.40 6 0.05
19 16 24
0.78 6 0.05 0.73 6 0.05 0.51 6 0.07
18 18 13
Total liquid consumption at 10% (g) Sedentary 5.63 6 0.21 Locked wheel 4.85 6 0.27 Free wheel 6.06 6 0.18
19 16 24
5.34 6 0.22 5.36 6 0.18 6.28 6 0.33
18 18 13
Mean g/kg alcohol at 20% Sedentary 10.16 6 2.67 Free wheel 7.74 6 1.92
17 12
19.15 6 3.22 8.35 6 2.37
13 11
Mean PR at 20% Sedentary Free wheel
0.25 6 0.06 0.18 6 0.05
17 12
0.40 6 0.07 0.15 6 0.04
13 11
Total liquid consumption at 20% (g) Sedentary 5.02 6 0.17 Free wheel 6.18 6 0.37
17 12
5.33 6 0.77 6.54 6 0.32
13 11
Alcohol metabolism Slope of regression line for alcohol clearance Sedentary 0.63 6 0.09 8 8 Free wheel 0.86 6 0.09
0.69 6 0.09 0.86 6 0.09
8 8
Food consumption Mean g food consumed daily (10% alcohol) Sedentary 3.67 6 0.12 13 Free wheel 4.27 6 0.20 12
3.39 6 0.14 3.86 6 0.11
18 17
Mean g food consumed daily (7% alcohol) Sedentary 4.10 6 0.23 13 Free wheel 3.60 6 0.36 12
3.44 6 0.24 3.67 6 0.18
18 17
Mean g food consumed daily (3% alcohol) Sedentary 3.60 6 0.11 13 Free wheel 4.22 6 0.15 12
3.30 6 0.11 4.08 6 0.16
18 17
5.68 6 0.48 5.38 6 0.32
12 10
Drinking in the dark Mean g/kg alcohol (2-h limited access) Sedentary 5.39 6 0.26 Free wheel 5.47 6 0.37
10 10
to alcohol (data not shown). Fig. 2A, B shows daily alcohol consumption means by cage condition group for the 3%, 7%, and 10% ethanol days. One-way ANOVA showed a significant difference in alcohol consumption among males between animals for the three cage conditions (F 5 8.723, P 5 .001 for g/kg and F 5 7.37, P 5 .001 for PR) (Fig. 2C, D). Results differed slightly for the g/kg consumption measure compared with the PR measure. Tukey’s post hoc test revealed a significant difference between the sedentary group and the movable wheel group (P ! .001), a trend between sedentary mice and the locked wheel group (P 5 .07), and no difference
447
between the locked wheel and movable wheel (P 5 .241). The mean alcohol consumption values were 9.1 6 1.0 g/kg (free wheel), 11.8 6 1.4 g/kg (locked wheel), and 15.7 6 1.1 g/kg (sedentary) (Table 1). However, there was a statistically significant difference in the total amount of liquid consumed dependent on cage type (F 5 7.89, P 5 .001). Tukey’s post hoc analysis revealed that the mice with a locked wheel drank less total liquid than either of the other two groups. Those with a locked wheel drank 4.85 6 0.27 g compared with 5.6 6 0.21 g for the sedentary animals and 6.06 6 0.18 g for the running mice (P 5 .047 and P 5 .001, respectively). Using PR as the alcohol consumption measure revealed significant differences between the sedentary group (0.66 6 0.05 PR) and the free running group (0.40 6 0.05 PR) where P 5 .03, and between the locked wheel group (0.62 6 0.07 PR) and the free running group (P 5 .014). There was no difference in the mean distance (m) run per day in mice with and without access to alcohol (data not shown). Fig. 2C, D shows daily alcohol consumption means by cage condition group for the 3%, 7%, and 10% ethanol days. Repeated-measure ANOVA showed that for days 5e13 (when the concentration of ethanol is 10%), the variables alcohol g/kg, PR, and distance run do not differ significantly within subject (all P O .05), suggesting that these behaviors are fairly consistent across this time period for an individual animal. In addition, there were no significant differences in body weight changes over the course of the experiment (F 5 0.864, P 5 .427). Results for the two-bottle choice at 20% ethanol were similar to those for the 10% concentration. Fig. 3 shows the daily alcohol consumption means for females (A and B) and males (C and D) at all concentrations of ethanol (3%, 7%, 10%, 13%, 17%, and 20%) in the sedentary and running mice. There was a significant difference in g/kg of ethanol consumed (t 5 2.62, P 5 .016) and PR (t 5 3.08, P 5 .005), but no difference in total amount of liquid consumed (t 5 1.36, P 5 .19) among the females. Sedentary females consumed 19.15 6 3.22 g/kg of 20% ethanol daily, whereas running mice consumed 8.35 6 2.37 g/kg. Their respective PRs for ethanol were 0.40 6 0.07 and 0.15 6 0.04 (Table 1). In the males, there were no significant differences in g/kg alcohol consumed or in PR (both P O .40). However, as can be seen from the mean values in Table 1 and Fig. 3, the trend toward decreased alcohol consumption with access to a running wheel could be seen. In addition, there was a significant difference in total amount of liquid consumed (t 5 2.84, P 5 .012). The sedentary males drank a total volume of 5.02 6 0.17 g, whereas the running mice drank 6.18 6 0.37 g. Alcohol metabolism Two groups of mice were individually housed with water and food, half with access to a running wheel and half in a standard cage, which were subjected to eight retro-orbital
448
M.A. Ehringer et al. / Alcohol 43 (2009) 443e452
B
Females
Female 1.0
no wheel wheel free wheel w/ brake
no wheel wheel free wheel w/ brake
20
0.8
Mean volume ethanol / total volume consumed
Mean g/kg Ethanol Consumed
A 25
15
10
5
0.6
0.4
0.2
0.0
0 2
4
6
8
10
2
4
8
D
Males
10
Males
1.0
no wheel wheel free wheel w/ brake
20
no wheel wheel free wheel w/ brake
0.8
Mean volume ethanol / total volume consumed
Mean g/kg Ethanol Consumed
C 25
6
% ethanol (v/v)
% ethanol (v/v)
15
10
5
0
0.6
0.4
0.2
0.0 2
4
6
8
10
% ethanol (v/v)
2
4
6
8
10
% ethanol (v/v)
Fig. 2. (A) Mean daily g/kg of 3%, 7%, and 10% ethanol consumed in female C57BL/6 mice in a standard cage (black circle), with access to a locked wheel (black triangle) or a running wheel (open circle). (B) Mean daily ethanol preference ratio (PR) (vol/vol) consumed in female C57BL/6 mice in a standard cage (black circle), with access to a locked wheel (black triangle) or a running wheel (open circle). (C) Mean daily g/kg of 3%, 7%, and 10% ethanol consumed in male C57BL/6 mice in a standard cage (black circle), with access to a locked wheel (black triangle) or a running wheel (open circle). (D) Mean daily ethanol PR (vol/vol) consumed in male C57BL/6 mice in a standard cage (black circle), with access to a locked wheel (black triangle) or a running wheel (open circle). Error bars show 61 standard error.
sinus blood collections at the end of the 2 weeks of ‘‘treatment.’’ This experiment was repeated twice, first with eight mice (four male and four female) and again 2 years later with eight additional mice. There was no significant difference in the metabolism of ethanol by sex (F 5 2.29, P 5 .16) nor by treatment (F 5 0.20, P 5 .67). Fig. 4 shows the mean BEC levels for each time point for each group, with female runners and nonrunners in panel A and males in panel B. Food consumption To determine if food consumption played a role in the drinking behavior between mice with or without a running wheel, food consumption (g) was measured daily (Table 1). As described in the Experimental procedures, this experiment was conducted twice. In the first batch, there was no significant difference in the amount of alcohol consumed between sedentary and running mice. We hypothesized that perhaps the process of opening the cage twice daily to remove and replace the petri dish with food might have led to additional stress that affected their drinking. Therefore, we repeated the experiment but placed the food in the
hopper next to their liquid bottles. However, we noted that the removal and replacement of food from the hopper did create some ‘‘loud’’ metallic noises, which often awakened and visibly agitated the mice. Results from both batches were comparable, so the data were combined. There was no significant difference in mean g/kg of 10% alcohol consumed, and no difference in alcohol PR (all P O .50). Among the females, the runners (17) consumed 16.8 6 2.3 g/kg or a ratio of 0.57 6 0.09 ethanol, whereas the sedentary mice (18) consumed 18.6 6 2.1 g/kg or a ratio of 0.61 6 0.08. Although not significant, there is a directional trend for the runners to drink less alcohol than the sedentary mice. However, among the males, the runners (12) consumed 12.5 6 2.0 g/kg or 0.52 6 0.10 PR of alcohol, and the sedentary animals (13) drank only 11.7 6 2.1 g/kg or 0.51 6 0.10 PR. Examining the food data, there were no significant differences in the amount of food eaten between runners and sedentary mice, although there was a trend for the runners to eat more at all alcohol concentrations except in the males at 7% alcohol. These data are presented in Table 1.
M.A. Ehringer et al. / Alcohol 43 (2009) 443e452
B
Females
Female 1.0
no wheel wheel free
no wheel wheel free
20
Mean volume ethanol / total volume consumed
Mean g/kg Ethanol Consumed
A 25
449
15
10
5
0.8
0.6
0.4
0.2
0 0.0
5
10
15
5
20
D
Males no wheel wheel free
15
20
Males 1.0
Mean volume ethanol / total volume consumed
Mean g/kg Ethanol Consumed
C 25
10
% ethanol (v/v)
% ethanol (v/v)
20
15
10
no wheel wheel free 0.8
0.6
0.4
0.2
5
0.0
0
5
10
15
20
% ethanol (v/v)
5
10
15
20
% ethanol (v/v)
Fig. 3. (A) Mean daily g/kg of 3%, 7%, 10%, 13%, 17%, and 20% ethanol consumed in female C57BL/6 mice in a standard cage (black circle) or with a running wheel (open circle). (B) Mean daily ethanol preference ratio (PR) (vol/vol) consumed in female C57BL/6 mice in a standard cage (black circle) or with a running wheel (open circle). (C) Mean daily g/kg of 3%, 7%, 10%, 13%, 17%, and 20% ethanol consumed in male C57BL/6 mice in a standard cage (black circle) or with a running wheel (open circle). (D) Mean daily ethanol PR (vol/vol) consumed in male C57BL/6 mice in a standard cage (black circle) or with a running wheel (open circle). Error bars show 61 standard error.
DID and voluntary wheel running Under standard cage conditions (no wheel), there was no difference between males and females in the amount of alcohol consumed during the 2-h limited access period (females, 5.68 6 0.48 g/kg; males, 5.39 6 0.26 g/kg; t 5 0.51, P 5 .62), so both sexes were analyzed together. There were no differences in the daily running distances between animals with and without access to alcohol (data not shown). Sedentary mice consumed 5.55 6 0.28 g/kg of alcohol, whereas the mice with access to a running wheel drank 5.43 6 0.24 g/kg (t 5 0.34, P 5 .74). Data for each sex separately are presented in Table 1. A total of 20 mice (10 males and 10 females) were tested with the running wheels and 22 sedentary mice (10 males and 12 females), for a total of 42 mice.
Discussion Here, we have shown that C57BL/6IBG mice alter their alcohol consumption patterns when given unlimited access to a running wheel over several days, and that these behavioral responses differ in males and females. C57BL/6IBG
females drank significantly less alcohol when a running wheel was provided and movable, but not when the wheel was locked, suggesting that the decreased consumption is likely to be (at least in part) due to exercise and not simply a novel environment. In C57BL/6IBG males, the mice drank significantly less alcohol (g/kg of body weight) in the presence of a wheel, including showing a trend to drink less when it was locked, suggesting that a novel environment may be a key factor. However, it was noted that the males also drank less total liquid when the brake was engaged on the wheel. This led to the idea that perhaps the males were using the locked wheel as a ‘‘jungle gym’’ and perhaps increased activity simply limited the time they spent drinking in general. However, when the data were analyzed on the basis of comparing ethanol PR, males showed a similar pattern as the females. They drank a lower ratio of alcohol when they could run, but not when the wheel was locked, providing support to the notion that exercise is a key component. To further characterize these behaviors, we examined other aspects of the behavioral testing procedure, including increasing the alcohol concentration to 20%, and possible differences in alcohol metabolism and food consumption.
450
M.A. Ehringer et al. / Alcohol 43 (2009) 443e452
A 400
Females no wheel wheel free
mg % ethanol
300
200
100
0 0
20
40
60
80
100
120
140
160
180
200
Minutes post injection
B
Males
400
no wheel wheel free
mg % ethanol
300
200
100
0 0
20
40
60
80
100
120
140
160
180
200
Minutes post injection Fig. 4. Pharmacokinetic profile of blood ethanol concentrations in female (A) and male (B) mice with or without access to a running wheel prior to an intraperitoneal injection of alcohol.
The female mice behaved similarly when presented with 20% ethanol, showing significant decreases in alcohol consumption when they had access to a running wheel. Results were not significant for the male mice drinking 20% ethanol, but such as the 10% ethanol experiments, the data were confounded by the fact that the running mice drank significantly more total liquid than did the sedentary males. Our data suggest that these differences in alcohol consumption are not likely to reflect differences in alcohol metabolism between sedentary and running mice. Animals given 2 weeks of ‘‘pretreatment’’ of voluntary running prior to an intraperitoneal injection of alcohol did not differ in their rate of metabolism compared with ‘‘untreated’’ sedentary animals in a cage with no wheel. Furthermore, we did not find a significant difference between sedentary and running mice in the amount of food consumed. However, the two experiments examining food intake revealed an unexpected result because the process of weighing the food each day appeared to affect the drinking results, whereby there was no significant difference in alcohol consumption between sedentary and running mice. In our ongoing work using this behavioral model, we continue to observe a significant decrease in alcohol consumption among females in the presence of a running
wheel, using a protocol where the food is weighed every 4 days, corresponding to the same days the mice are weighed. Furthermore, the experiments testing a range of concentrations up to 20% ethanol (Fig. 3) consistently show decreased ethanol intake in the presence of a wheel. This finding highlights the challenges and complexities of studying animal behavior, but it is an interesting observation because it is in agreement with work showing connections between alcohol and stress (Chester et al., 2006), and exercise and stress (Greenwood and Fleshner, 2008). In our standard experiment, we provide an excess of food pellets in the bottom of the cage, so it is only necessary to replenish them every few days. One explanation for differing results for the food experiments might be related to a stress response induced by daily contact with their food, leading to high-alcohol consumption even in the presence of a running wheel. These observations support the hypothesis that common neuronal pathways may be activated by alcohol, exercise, and stress, and that access to a running wheel may provide an alternative neurological reward to those rewards normally stimulated by alcohol consumption. Results from testing using the DID protocol, believed to be a model of binge drinking, revealed an interesting difference. Using this limited access model, neither male nor female C57BL/6IBG mice showed any differences in alcohol consumption. This finding is important because it reflects potential important differences in the underlying neurobiology related to chronic alcohol intake over long periods of time compared with binge drinking. Our findings suggest that although regular exercise may have the potential to be a useful treatment component for some alcohol problem behaviors, it may be less effective for others, such as binge drinking (Vogel, 2002). These results are in agreement with several of the previous studies supporting the idea that the rewarding properties of exercise may substitute in part for the rewarding properties of alcohol and other substances. Wheel running has been shown to lead to decreased intake of amphetamine (Kanarek et al., 1995) and decreased positive-reinforcing effects of cocaine (Smith et al., 2008) in rats. The C57BL/6IBG mice are similar to the selectively bred alcohol-preferring P rats in that they voluntary drink high levels of alcohol, and they both show decreased intake of alcohol in the presence of a running wheel (McMillan et al., 1995). As access to a running wheel had no effect on alcohol intake in the nonpreferring rats, it would be interesting to test other strains of mice with different baseline voluntary drinking and wheel-running behaviors to determine whether individual genetic differences may contribute to this response. There is support for this idea from the work conducted in the Nurr1 deficient mice, which show lower alcohol intake and less running than their wild-type counterparts (Werme et al., 2003). In contrast to our work, Crews et al. (Crews et al., 2004) did not observe any difference in alcohol intake in male C57BL/6 mice, but this analysis was not the main focus of their study and very few animals were tested. As the effect
M.A. Ehringer et al. / Alcohol 43 (2009) 443e452
in our study is less pronounced in males, this discrepancy may be due in part to differences in testing procedure and perhaps modest genetic differences between the two different sources of the C57BL/6 mice. Likewise, it has been shown that within an individual animal, alternating access periods to running wheels does not lead to differences in alcohol intake (Ozburn et al., 2008). In a small number of animals, we examined this phenomenon also by locking the wheels for 5 days at the end of our standard 13-day 10% two-bottle choice experiment and did not see any evidence for increased drinking (unpublished data). However, it is possible that there may be important temporal considerations for these types of study designs, because we cannot predict whether 5 or 7 days (as used by Ozburn et al., 2008) would constitute sufficient length of exposure to a different environment to lead to meaningful differences in alcohol consumption. It is difficult to make a direct comparison to the findings of Werme et al. (Werme et al., 2002) because they were examining ethanol preference in rats following an ethanol withdrawal period. The experimental design used here differs dramatically because there was no attempt to induce alcohol dependency in the mice, given the availability of water at all times. Although one explanation for the connection between voluntary alcohol consumption and wheel running might be common neuronal reward pathway activation by both behaviors, alternative reasons for this decreased alcohol consumption exist. For example, ethanol is a diuretic, and wheel running may result in additional loss of water. Alcohol consumption may have decreased to maintain water balance. Future studies should attempt to address these possibilities. A major limitation of this work is the relative inability to disentangle the effect of exercise from a possible effect of environmental enrichment, particularly for the male animals. In the female C57BL/6IBG mice, we did not observe a significant effect on alcohol consumption in the presence of a locked wheel (although the mean amount consumed by the females with a locked wheel was slightly less). Interestingly, male mice drank less alcohol (g/kg) when the wheel was locked, but this was confounded by the fact that they drank less total liquid under that condition. For the alcohol PR, we might hypothesized that there was a decrease in how often the males who could run were going to the alcohol bottle compared with those with a locked wheel. Furthermore, it is important to note that the cages used for alcohol preference alone (i.e., no wheels) were slightly smaller than the cages with wheels. Most current protocols for environmental enrichment (EE) include housing rodents in larger cages with other animals and providing complex, varied objects, which often includes running wheels (Nithianantharajah and Hannan, 2006). Future studies examining different EE conditions need to be carried out to help elucidate the relative effectiveness of enrichment and exercise in reducing voluntary alcohol consumption. For example, it is not clear whether the effects of EE and wheel running might be additive. When wheel running has been examined separately
451
from other enrichments, it was found that running increased neuronal cell proliferation, cell survival, and net neurogenesis, whereas EE only increased cell survival (van Praag et al., 1999b), suggesting that the ability to exercise may be a key component in behavioral outcome. In summary, the unlimited access two-bottle choice mouse model presented here should serve as a useful tool for research aimed at understanding the possible neuronal mechanisms common to these behaviors. Future work should focus on examining which genes and proteins may be differentially expressed under different environmental conditions, and whether different responses are observed in other inbred strains of mice that do not show naturally high levels of alcohol preference and wheel running. Such work may provide valuable insight into possible alternative forms of prevention and treatment of human alcohol abuse and dependence.
Acknowledgments The authors would like to thank Dr. Jeanne Wehner, Hilary Clegg, Jessica Godfrey, Stacy Romero, Isabel Schlaepfer, Priyanka Thummalapally, and Joshua Wilcox for technical assistance and helpful discussion of this work.
References Bachmanov, A. A., Tordoff, M. G., and Beauchamp, G. K. (1996). Ethanol consumption and taste preferences in C57BL/6ByJ and 129/J mice. Alcohol. Clin. Exp. Res. 20, 201–206. Bamber, D. J., Cockerill, I. M., Rodgers, S., and Carroll, D. (2003). Diagnostic criteria for exercise dependence in women. Br. J. Sports Med. 37, 393–400. Belknap, J. K., Crabbe, J. C., and Young, E. R. (1993). Voluntary consumption of ethanol in 15 inbred mouse strains. Psychopharmacology (Berl) 112, 503–510. Boehm, S. L. 2nd, Moore, E. M., Walsh, C. D., Gross, C. D., Cavelli, A. M., Gigante, E., et al. (2008). Using drinking in the dark to model prenatal binge-like exposure to ethanol in C57BL/6J mice. Dev. Psychobiol. 50, 566–578. Carelli, R. M., Ijames, S. G., and Crumling, A. J. (2000). Evidence that separate neural circuits in the nucleus accumbens encode cocaine versus ‘‘natural’’ (water and food) reward. J. Neurosci. 20, 4255–4266. Chan, C. S., and Grossman, H. Y. (1988). Psychological effects of running loss on consistent runners. Percept. Mot. Skills 66, 875–883. Chapman, C. L., and De Castro, J. M. (1990). Running addiction: measurement and associated psychological characteristics. J. Sports Med. Phys. Fitness 30, 283–290. Chester, J. A., de Paula Barrenha, G., DeMaria, A., and Finegan, A. (2006). Different effects of stress on alcohol drinking behaviour in male and female mice selectively bred for high alcohol preference. Alcohol Alcohol. 41, 44–53. Crews, F. T., Nixon, K., and Wilkie, M. E. (2004). Exercise reverses ethanol inhibition of neural stem cell proliferation. Alcohol 33, 63–71. Dishman, R. K., Berthoud, H. R., Booth, F. W., Cotman, C. W., Edgerton, V. R., Fleshner, M. R., et al. (2006). Neurobiology of exercise. Obesity (Silver Spring) 14, 345–356. Ferreira, A., Lamarque, S., Boyer, P., Perez-Diaz, F., Jouvent, R., and Cohen-Salmon, C. (2006). Spontaneous appetence for wheel-running:
452
M.A. Ehringer et al. / Alcohol 43 (2009) 443e452
a model of dependency on physical activity in rat. Eur. Psychiatry 21, 580–588. Freed, C. R., and Yamamoto, B. K. (1985). Regional brain dopamine metabolism: a marker for the speed, direction, and posture of moving animals. Science 229, 62–65. Fuller, J. L. (1964). Measurement of alcohol preference in genetic experiments. J. Comp. Physiol. Psychol. 57, 85–88. Furst, D. M., and Germone, K. (1993). Negative addiction in male and female runners and exercisers. Percept. Mot. Skills 77, 192–194. Goldman, D., Oroszi, G., and Ducci, F. (2005). The genetics of addictions: uncovering the genes. Nat. Rev. Genet. 6, 521–532. Greenwood, B. N., and Fleshner, M. (2008). Exercise, learned helplessness, and the stress-resistant brain. Neuromolecular Med. 10, 81–98. Greenwood, B. N., Foley, T. E., Burhans, D., Maier, S. F., and Fleshner, M. (2005). The consequences of uncontrollable stress are sensitive to duration of prior wheel running. Brain Res. 1033, 164–178. Greenwood, B. N., Foley, T. E., Day, H. E., Campisi, J., Hammack, S. H., Campeau, S., et al. (2003). Freewheel running prevents learned helplessness/behavioral depression: role of dorsal raphe serotonergic neurons. J. Neurosci. 23, 2889–2898. Gutgesell, M. E., Timmerman, M., and Keller, A. (1996). Reported alcohol use and behavior in long-distance runners. Med. Sci. Sports Exerc. 28, 1063–1070. Kamdar, N. K., Miller, S. A., Syed, Y. M., Bhayana, R., Gupta, T., and Rhodes, J. S. (2007). Acute effects of Naltrexone and GBR 12909 on ethanol drinking-in-the-dark in C57BL/6J mice. Psychopharmacology (Berl) 192, 207–217. Kanarek, R. B., Marks-Kaufman, R., D’Anci, K. E., and Przypek, J. (1995). Exercise attenuates oral intake of amphetamine in rats. Pharmacol. Biochem. Behav. 51, 725–729. Larson, E. B., and Carroll, M. E. (2005). Wheel running as a predictor of cocaine self-administration and reinstatement in female rats. Pharmacol. Biochem. Behav. 82, 590–600. Lerman, I., Harrison, B. C., Freeman, K., Hewett, T. E., Allen, D. L., Robbins, J., et al. (2002). Genetic variability in forced and voluntary endurance exercise performance in seven inbred mouse strains. J. Appl. Physiol. 92, 2245–2255. Lett, B. T., Grant, V. L., Byrne, M. J., and Koh, M. T. (2000). Pairings of a distinctive chamber with the aftereffect of wheel running produce conditioned place preference. Appetite 34, 87–94. Lightfoot, J. T., Turner, M. J., Daves, M., Vordermark, A., and Kleeberger, S. R. (2004). Genetic influence on daily wheel running activity level. Physiol. Genomics 19, 270–276. Mathes, W. F., and Kanarek, R. B. (2001). Wheel running attenuates the antinociceptive properties of morphine and its metabolite, morphine6-glucuronide, in rats. Physiol. Behav. 74, 245–251. McMillan, D. E., McClure, G. Y., and Hardwick, W. C. (1995). Effects of access to a running wheel on food, water and ethanol intake in rats bred to accept ethanol. Drug Alcohol Depend. 40, 1–7. Meliska, C. J., Bartke, A., McGlacken, G., and Jensen, R. A. (1995). Ethanol, nicotine, amphetamine, and aspartame consumption and preferences in C57BL/6 and DBA/2 mice. Pharmacol. Biochem. Behav. 50, 619–626. Middaugh, L. D., Kelley, B. M., Bandy, A. L., and McGroarty, K. K. (1999). Ethanol consumption by C57BL/6 mice: influence of gender and procedural variables. Alcohol 17, 175–183. Molteni, R., Zheng, J. Q., Ying, Z., Gomez-Pinilla, F., and Twiss, J. L. (2004). Voluntary exercise increases axonal regeneration from sensory neurons. Proc. Natl. Acad. Sci. U.S.A. 101, 8473–8478. Nestler, E. J. (2005). Is there a common molecular pathway for addiction? Nat. Neurosci. 8, 1445–1449.
Nithianantharajah, J., and Hannan, A. J. (2006). Enriched environments, experience-dependent plasticity and disorders of the nervous system. Nat. Rev. Neurosci. 7, 697–709. Nixon, K. (2006). Alcohol and adult neurogenesis: roles in neurodegeneration and recovery in chronic alcoholism. Hippocampus 16, 287–295. Ozburn, A. R., Harris, R. A., and Blednov, Y. A. (2008). Wheel running, voluntary ethanol consumption, and hedonic substitution. Alcohol 42, 417–424. Pierce, E. F., Rohaly, K. A., and Fritchley, B. (1997). Sex differences on exercise dependence for men and women in a marathon road race. Percept. Mot. Skills 84, 991–994. van Praag, H., Christie, B. R., Sejnowski, T. J., and Gage, F. H. (1999a). Running enhances neurogenesis, learning, and long-term potentiation in mice. Proc. Natl. Acad. Sci. U.S.A. 96, 13427–13431. van Praag, H., Kempermann, G., and Gage, F. H. (1999b). Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat. Neurosci. 2, 266–270. Rhodes, J. S., Ford, M. M., Yu, C. H., Brown, L. L., Finn, D. A., Garland, T. Jr., et al. (2007). Mouse inbred strain differences in ethanol drinking to intoxication. Genes Brain Behav. 6, 1–18. Rhodes, J. S., and Garland, T. (2003). Differential sensitivity to acute administration of Ritalin, apomorphine, SHC 23390, but not racolpride in mice selectively bred for hyperactive wheel-running behavior. Psychopharmacology 167, 242–250. Rhodes, J. S., Garland, T. Jr., and Gammie, S. C. (2003). Patterns of brain activity associated with variation in voluntary wheel-running behavior. Behav. Neurosci. 117, 1243–1256. Sacchetti, P., Carpentier, R., Segard, P., Olive-Cren, C., and Lefebvre, P. (2006). Multiple signaling pathways regulate the transcriptional activity of the orphan nuclear receptor NURR1. Nucleic Acids Res. 34, 5515–5527. Smith, M. A., Schmidt, K. T., Iordanou, J. C., and Mustroph, M. L. (2008). Aerobic exercise decreases the positive-reinforcing effects of cocaine. Drug Alcohol Depend. 98, 129–135. Smolen, A., Marks, M. J., Smolen, T. N., and Collins, A. C. (1986). Dose and route of administration alter the relative elimination of ethanol by long-sleep and short-sleep mice. Alcohol. Clin. Exp. Res. 10, 198–204. Ussher, M., Sampuran, A. K., Doshi, R., West, R., and Drummond, D. C. (2004). Acute effect of a brief bout of exercise on alcohol urges. Addiction 99, 1542–1547. Vaynman, S., and Gomez-Pinilla, F. (2006). Revenge of the ‘‘sit’’: how lifestyle impacts neuronal and cognitive health through molecular systems that interface energy metabolism with neuronal plasticity. J. Neurosci. Res. 84, 699–715. Vogel, R. A. (2002). Alcohol, heart disease, and mortality: a review. Rev. Cardiovasc. Med. 3, 7–13. Volkow, N. D., and Wise, R. A. (2005). How can drug addiction help us understand obesity? Nat. Neurosci. 8, 555–560. Werme, M., Hermanson, E., Carmine, A., Buervenich, S., Zetterstrom, R. H., Thoren, P., et al. (2003). Decreased ethanol preference and wheel running in Nurr1-deficient mice. Eur. J. Neurosci. 17, 2418–2424. Werme, M., Lindholm, S., Thoren, P., Franck, J., and Brene, S. (2002). Running increases ethanol preference. Behav. Brain Res. 133, 301– 308. Yoneyama, N., Crabbe, J. C., Ford, M. M., Murillo, A., and Finn, D. A. (2008). Voluntary ethanol consumption in 22 inbred mouse strains. Alcohol. 42, 149e160 Zito, K., and Svoboda, K. (2002). Activity-dependent synaptogenesis in the adult Mammalian cortex. Neuron 35, 1015–1017.
Reduced alcohol consumption in mice with access to a running wheel Marissa A. Ehringera,b,*, Nicole R. Hofta, Matthias Zunhammerc a
University of Colorado, Institute for Behavioral Genetics, 1480 30th Street, 447 UCB, Boulder, CO 80309-0447, USA b Department of Integrative Physiology, 354 UCB, Boulder, CO 80309-0354, USA c University of Regensburg, Department of Psychiatry, Psychosomatics, and Psychotherapy, 93053 Regensburg, Germany Received 11 August 2008; received in revised form 8 May 2009; accepted 20 June 2009
Abstract Studies of the behavioral effects of alcohol in humans and rodent models have implicated a number of neurological pathways and genes. Separate studies have shown that certain regions of the brain are involved in behavioral responses to exercise. The aim of this study was to determine whether mice which normally voluntarily consume high amounts of alcohol (C57BL/6 strain) would exhibit reduced alcohol consumption when given access to a running wheel under two different models of voluntary consumption: unlimited access two-bottle choice and limited access drinking in the dark (DID). Under the two-bottle choice model, the animals voluntarily consumed less alcohol when a wheel was present in their cage. However, sex-specific differences emerged because female mice voluntarily consumed less alcohol when they have the opportunity to exercise on a running wheel, whereas male mice consumed less alcohol even if the running wheel was locked. There were no significant differences observed in alcohol metabolism or food consumption. Under the DID protocol, no differences in alcohol consumption were observed in the presence of a running wheel. These results suggest that exercise may be a useful approach to consider for treatment for some types of chronic human alcohol problem behaviors, but may be less applicable to human binge drinking. Ó Published by Elsevier Inc. Keywords: Alcohol preference; Exercise; Wheel running; Mouse model
Introduction In recent years, it has become apparent that common neuronal pathways may be involved in a wide range of addictive behaviors, including drugs of abuse, overeating, gambling, and sexual addictions, and that these behaviors are often comorbid with other psychiatric disorders such as depression, stress, and anxiety (Goldman et al., 2005; Nestler, 2005; Volkow and Wise, 2005). Although other neurobiological pathways are likely to be involved, the mesolimbic dopaminergic system has become the focus of study for understanding reward responses. Dopaminergic cell bodies in the ventral tegmental area project to forebrain nuclei, including the nucleus accumbens, and it has been shown that these neurons can be activated by reward stimuli (Carelli et al., 2000).
This study was supported by University of Colorado Council on Research and Creative Work, Junior Faculty Development Award to M.A.E. * Corresponding author. University of Colorado, Institute for Behavioral Genetics, 1480 30th Street, 447 UCB, Boulder, CO 80309-0447, USA. Tel.: þ1-303-492-1463; fax: þ1-303-492-8063. E-mail address: [email protected] (M.A. Ehringer). 0741-8329/09/$ e see front matter Ó Published by Elsevier Inc. doi: 10.1016/j.alcohol.2009.06.003
A number of studies have shown that exercise has addictive properties among people who intensely exercise on a regular basis (Chan and Grossman, 1988; Chapman and De Castro, 1990; Furst and Germone, 1993; Pierce et al., 1997). These studies have shown that exercise can become a chronic activity, that the need to continue to exercise persists despite physical and psychological symptoms, and that ‘‘dependency’’ is associated with quantitative factors, including frequency, duration, and intensity. Recently, Bamber et al. have begun developing specific diagnostic criteria for exercise dependence that includes two main components: impaired function and withdrawal (Bamber et al., 2003). Gutgesell et al. (1996) have found that although runners typically report more occasions of drinking than nonrunners, among those who have a history of problem drinking, runners report drinking less alcohol than nonrunners. Similarly, intense exercise has been shown to lead to a significant decrease in alcohol cravings in recovering alcoholics (Ussher et al., 2004). These effects suggest that running and alcohol may be activating shared neural pathways. Wheel running in rodents has been used in connection with models of hyperactivity (Rhodes et al., 2003), stress response (Greenwood et al., 2003, 2005), response to
444
M.A. Ehringer et al. / Alcohol 43 (2009) 443e452
novelty (Ferreira et al., 2006), food deprivation, and drugs of abuse (Larson and Carroll, 2005; Mathes and Kanarek, 2001). A landmark study by Freed and Yamamoto (1985) demonstrated that dopamine signaling could be selectively activated by voluntary motor behavior, but not by passive movement of the head. Similarly, studies of mice selectively bred for hyperactive wheel running will decrease their running when administered dopamine reuptake blockers (Rhodes and Garland, 2003). Wheel running has been used as a model of physical activity dependence (Ferreira et al., 2006) and shown to confer conditioned place preference after only 2 h of access (Lett et al., 2000). The role of exercise in modulating neuronal plasticity and regeneration has become a topic of great interest (Dishman et al., 2006; Vaynman and Gomez-Pinilla, 2006), and is in contrast to the neurodegenerative effects of alcohol (Nixon, 2006). A recent report by Crews et al. (Crews et al., 2004) demonstrated that access to a running wheel could reverse alcohol’s inhibitory effect on neural stem-cell proliferation. The authors suggest ‘‘the opposing effects of ethanol consumption and exercise on neurogenesis could contribute to the CNS pathology and health benefits, respectively, of these two behaviors.’’ It has been shown that wheel running can prime adult sensory neurons for enhanced axonal regeneration (Molteni et al., 2004) and that activity-dependent plasticity is retained into adulthood (Zito and Svoboda, 2002). At least some of the positive effects observed with running, including increased neurogenesis, learning, and long-term potentiation appear to be mediated via hippocampal mechanisms (van Praag et al., 1999a). In animal research, several studies have been conducted to examine possible effects of exercise on voluntary alcohol consumption. Rats bred to accept ethanol (P rats) show decreased ethanol intake and increased water intake when given access to a running wheel (McMillan et al., 1995), with no effect on food consumption. Werme et al. (2002) showed that Lewis rats, which ran during a withdrawal period following alcohol exposure showed increased alcohol consumption compared with those that did not have access to wheels during the withdrawal period, concluding that running increases ethanol preference following withdrawal. The same group also showed that Nurr1 deficient mice (heterozygous for the Nurr1 gene deletion) displayed lower alcohol consumption and less running behavior than their littermate wild-type controls (Werme et al., 2003). The Nurr1 protein is a member of the NGFI-B family of transcription factors and plays an important role in signaling pathways involved in differentiation and phenotypic expression of dopaminergic neurons (Sacchetti et al., 2006). However, Crews et al. (2004) reported no difference in voluntary alcohol consumption in male C57BL/6 mice with access to a running wheel. This is in agreement with Ozburn et al. (Ozburn et al., 2008), who found that voluntary ethanol consumption of female C57BL/6J mice remained stable under a protocol involving alternating periods of access to running wheels.
In addition to alcohol, a few studies have examined selfadministration of other drugs, including amphetamine and cocaine. Kanarek et al. (Kanarek et al., 1995) reported that male Sprague-Dawley rats consumed significantly less amphetamine when the animals had access to running wheels compared with no access to a wheel. Likewise, Smith et al. (Smith et al., 2008) found that wheel running was associated with decreased positive-reinforcing effects of cocaine in female Long-Evans rats, as measured using a progressive ratio schedule. This collection of studies supports the possibility that hedonic substitution of alcohol by exercise might be a useful approach for preventing or treating alcohol and drug dependence. However, variation in study designs makes it difficult to draw overarching conclusions regarding the interaction of these two behaviors. Most of the studies have used rats, but four different strains have been used and in most cases only one sex was tested. Among the three mouse studies using different behavioral paradigms, only females were used in two studies (Ozburn et al., 2008; Werme et al., 2003), whereas only males were tested in the third study (Crews et al., 2004). The purpose of the present study was to examine whether providing C57BL/6 mice access to a running wheel would lead to decreased alcohol intake using two common measures of voluntary consumption. The C57BL/6 strain was selected for these pilot studies because among commonly used inbred mouse strains they show high levels of alcohol consumption and high levels of voluntary wheel running (Belknap et al., 1993; Lerman et al., 2002; Lightfoot et al., 2004). An unlimited access two-bottle choice (water or ethanol) protocol was selected because it has been well characterized as a model of alcohol behavior for over 30 years (Belknap et al., 1993; Fuller, 1964; Yoneyama et al., 2008). In addition, we also used the relatively new drinking-in-the-dark (DID) protocol, believed to be a model of binge drinking in which the animals have limited access to alcohol during the dark cycle (Boehm et al., 2008; Kamdar et al., 2007; Rhodes et al., 2007). Experimental procedures Animals and housing The C57BL/6IBG mice were bred at the Institute for Behavioral Genetics at the University of Colorado, Boulder. On collection, mice were in group housing on a 12-h light:12-h dark cycle (lights on at 0700 h) and provided with ad libitum access to food and water. When they were 60e90 days old, the animals were placed in individual cages, with or without running wheels (as noted in the following) and allowed to acclimate for 3e7 days. The cages without wheel were standard 30 13 17-cm polycarbonate cages, whereas the cages with wheel were slightly larger to accommodate the wheels (30.3 20.6 26 cm with wheels 242 mm in diameter), available from Respironics Mini Mitter (Bend, OR, USA). The cages were
M.A. Ehringer et al. / Alcohol 43 (2009) 443e452
checked for food and water daily, and animals were weighed every 4 days. The local Institutional Animal Care and Use Committee approved all procedures. Ethanol preference testing The same animals were used for Ethanol preference testing and Combined ethanol preference and voluntary wheel running. The mice were given a choice between tap water and different concentrations of ethanol diluted in tap water in two 15-mL tubes with a metal sipper tube. Three percent ethanol solution was provided in the ethanol tube on days 1 and 2, 7% solution was provided on days 3 and 4, and then the solution remained at 10% for days 5 through 13. A diagram of the behavioral testing procedure is shown in Fig. 1A. The placement of the tubes was reversed daily during weighing and refilling to prevent the mice from developing placement preference. To account for evaporation, calibration tubes containing water and ethanol were placed in empty cages in the testing room, measured daily, and these amounts were subtracted from all data for individual mice. All together, 37 animals (19 males and 18 females) were tested. A Student’s two-tailed t-test was used to compare drinking levels (g alcohol/kg body weight) in males and females (SPSS 16.0, Chicago, IL). Voluntary wheel running Mice were placed in cages containing free wheels for 13 days with free access to food and water. The distance run by the mice was recorded daily using a magnet system attached to the wheel for which number of revolutions was collected by a counter, next to the cage. All together, 31 animals (16 males and 15 females) were used. A Student’s two-tailed t-test was used to compare running distances in males and females. Combined ethanol preference and voluntary wheel running The ethanol paradigm described as previously mentioned, introducing 3%, 7%, and 10% ethanol, was conducted with mice in cages that contained movable running wheels and locked running wheels (Fig. 1A). Daily ethanol consumption and distance run data were collected. SPSS 16.0 was used to conduct a one-way analysis of variance (ANOVA) within each sex to compare mean alcohol consumption using two measures: grams of alcohol per kilogram of body weight and ethanol preference ratio (PR), the ratio of volume of alcohol consumed divided by the total volume of liquid consumed. Tukey’s post hoc tests were used to determine significant differences between the three cage condition groups (sedentary, locked wheel, or movable wheel). One-way ANOVAs were also used to examine whether the total volume of liquid consumed differed by cage condition within each sex. Finally, a Student’s two-tailed t-test was used to compare running distances in the presence or absence of alcohol within each
445
sex. All together, 108 animals (59 males and 49 females) were used, including the 37 baseline-drinking animals used for the sex comparison of baseline ethanol drinking (as previously mentioned). A similar two-bottle choice procedure was repeated with a different set of animals whereby the alcohol concentration was increased gradually to 20% ethanol (Fig. 1B). Animals had access to water and ethanol each day, where the ethanol bottle concentrations were 3% on days 1e2, 7% on days 3e4, 10% on days 5e6, 13% on days 7e8, 17% on days 9e10, and 20% on days 11e14. Data from days 11e14 were analyzed using a Student’s t-test to compare ethanol consumption between sedentary and running animals.
Alcohol metabolism Mice were individually housed in cages with or without running wheels for 2 weeks to determine if animals that experienced a ‘‘pretreatment’’ of running showed faster rates of alcohol metabolism. At the end of the 2-week period, animals were injected with ethanol into the intraperitoneal cavity (3 g/kg). Retro-orbital sinus blood was collected nine times following the injection to determine blood ethanol concentrations (BECs) (10, 20, 30, 45, 60, 90, 120, 180, and 240 min). The BEC was measured using a modified enzymatic assay, which couples the conversion of ethanol to acetaldehyde and the conversion of nicotinamide adenine dinucleotide (NAD) to NAD with hydrogen (NADH) by the addition of alcohol dehydrogenase (ADH from yeast). The amount of NADH produced is measured at 340 nm using a spectrophotometer, and ethanol concentrations can be calculated in reference to a standard curve (Smolen et al., 1986). All together, 16 animals (8 males and 8 females) were used. Linear regression curves were fit to estimate a slope of alcohol decay for each animal. A Student’s two-tailed t-test was used to test for differences in these slopes by sex and cage condition (sedentary or running).
Food consumption The standard 13-day 10% alcohol protocol was administered to an additional set of mice with and without access to running wheels. This experiment was conducted twice. In the first batch, food pellets were glued into small petri dishes (60 15 mm) and attached to the side of the cage using Velcro. The petri dishes were removed, weighed, and replenished daily (10 males, 5 sedentary and 5 running; 20 females, 10 sedentary and 10 running). In the second experiment, food pellets were placed in the hopper next to the liquid bottles. These pellets were removed, weighed, and replenished daily. In this second batch, there were 15 males (8 sedentary, 7 running) and 15 females (8 sedentary, 7 running).
446
M.A. Ehringer et al. / Alcohol 43 (2009) 443e452
A 3-7 days single housing
Days 1-2 3% EtOH
Days 3-4 7%EtOH
Days 5-13 10%EtOH
15 days with free access to wheel or locked wheel
B 3-7 days single housing
Days 1-2 3% EtOH
Days 3-4 7%EtOH
Days 5-6 10% EtOH
Days 7-8 13%EtOH
Days 11-14 20%EtOH
Days 9-10 17%EtOH
14 days free access to wheel
C Days 1-7 group housing Reverse light cycle
Days 8-14 single housing Reverse light cycle
Days 15-18 2 hour limited access to 20% alcohol
11 days with free access to wheel
Fig. 1. Schematic of behavioral testing protocols. (A) Two-bottle choice unlimited access to 10% ethanol. Animals were allowed to acclimate in their individual cages with or without running wheels for 48 h prior to introduction of alcohol. (B) Two-bottle choice unlimited access to 20% ethanol. (C) Drinking in the dark.
DID and voluntary wheel running Animal breeding, housing, and cage conditions were as previously mentioned, except animals for this experiment were acclimated to a reverse light cycle (lights OFF at 0700 h, lights ON at 1900 h) for 7 days in group housing (5 mice per cage), following by 7 additional acclimation days in their individual cages. Mice had unlimited access to water and food at all times up, until the 4-day DID protocol began. Three hours after the lights were off (1000 h), water bottles were replaced with bottles containing 20% (vol/vol) ethanol for 2 h, at which time the amount of ethanol consumed was recorded and the water bottle replaced. A diagram of the behavioral testing protocol is shown in Fig. 1C. For those animals in cages with moveable wheels, distance run was recorded daily. A Student’s t-test was used to compare amount of alcohol consumed during the limited access period within each sex by cage condition (sedentary or running). Results All reported values are mean 6 standard error for the 10% ethanol testing days, unless otherwise noted. Baseline ethanol consumption and wheel running As has been shown previously (Middaugh et al., 1999), females consumed significantly more ethanol than males (P 5 .01, t 5 2.725). The females drank 20.77 6 1.48 g/kg (n 5 18) ethanol daily, whereas the males drank 15.75 6 1.12 g/kg (n 5 19) ethanol (Table 1). These consumption levels are slightly higher than reports ranging
from approximately 9 g/kg daily in males to 13 g/kg in females (Bachmanov et al., 1996; Belknap et al., 1993; Meliska et al., 1995; Middaugh et al., 1999), but they have remained consistent over approximately 3 years of testing. Likewise, female C57BL/6IBG mice ran significantly farther than males (P 5 .017, t 5 2.522). Females ran 5722.37 6 806.37 m/day (n 5 16), whereas males ran only 3527.91 6 373.54 m/day (n 5 16). All subsequent analyses were performed in males and females separately. Similar results were found for 3% and 7% ethanol testing days as shown in Fig. 2. Ethanol consumption in presence of running wheel One-way ANOVA revealed a significant difference in total liquid consumption among females that was dependent on cage condition (F 5 4.68, P 5 .014), where Tukey’s post hoc test indicated the mice with a movable wheel drank more total liquid (6.28 6 0.33 g) than either of the other two groups (5.34 6 0.22 g and 5.36 6 0.77 g; P ! .05). This increase in overall drinking was associated with a decrease in ethanol intake because a significant difference in alcohol consumption between animals for the three cage conditions was also present (F 5 4.64, P 5 .015 for g/kg and F 5 4.75, P 5 .013 for PR). Tukey’s post hoc test revealed that female mice with access to a running wheel consumed significantly less alcohol (13.7 6 2.31 g/kg or 0.51 6 0.07 PR) than sedentary mice (20.7 6 1.5 g/kg or 0.78 6 0.05 PR) (P ! .05), or mice with a locked wheel (20.3 6 1.6 g/kg or 0.73 6 0.05 PR) (P ! .05) (Table 1). There was no difference in the mean distance (m) run per day in mice with or without access
M.A. Ehringer et al. / Alcohol 43 (2009) 443e452 Table 1 Summary of all experiments Experiment and variable
Males
N
Two-bottle choice ethanol and wheel running Mean g/kg alcohol at 10% Sedentary 15.75 6 1.12 19 Locked wheel 11.84 6 1.44 16 Free wheel 9.11 6 1.02 24
Females
N
20.77 6 1.48 20.32 6 1.64 13.74 6 2.13
18 18 13
Mean preference ratio (PR) at 10% Sedentary 0.66 6 0.05 Locked wheel 0.62 6 0.07 Free wheel 0.40 6 0.05
19 16 24
0.78 6 0.05 0.73 6 0.05 0.51 6 0.07
18 18 13
Total liquid consumption at 10% (g) Sedentary 5.63 6 0.21 Locked wheel 4.85 6 0.27 Free wheel 6.06 6 0.18
19 16 24
5.34 6 0.22 5.36 6 0.18 6.28 6 0.33
18 18 13
Mean g/kg alcohol at 20% Sedentary 10.16 6 2.67 Free wheel 7.74 6 1.92
17 12
19.15 6 3.22 8.35 6 2.37
13 11
Mean PR at 20% Sedentary Free wheel
0.25 6 0.06 0.18 6 0.05
17 12
0.40 6 0.07 0.15 6 0.04
13 11
Total liquid consumption at 20% (g) Sedentary 5.02 6 0.17 Free wheel 6.18 6 0.37
17 12
5.33 6 0.77 6.54 6 0.32
13 11
Alcohol metabolism Slope of regression line for alcohol clearance Sedentary 0.63 6 0.09 8 8 Free wheel 0.86 6 0.09
0.69 6 0.09 0.86 6 0.09
8 8
Food consumption Mean g food consumed daily (10% alcohol) Sedentary 3.67 6 0.12 13 Free wheel 4.27 6 0.20 12
3.39 6 0.14 3.86 6 0.11
18 17
Mean g food consumed daily (7% alcohol) Sedentary 4.10 6 0.23 13 Free wheel 3.60 6 0.36 12
3.44 6 0.24 3.67 6 0.18
18 17
Mean g food consumed daily (3% alcohol) Sedentary 3.60 6 0.11 13 Free wheel 4.22 6 0.15 12
3.30 6 0.11 4.08 6 0.16
18 17
5.68 6 0.48 5.38 6 0.32
12 10
Drinking in the dark Mean g/kg alcohol (2-h limited access) Sedentary 5.39 6 0.26 Free wheel 5.47 6 0.37
10 10
to alcohol (data not shown). Fig. 2A, B shows daily alcohol consumption means by cage condition group for the 3%, 7%, and 10% ethanol days. One-way ANOVA showed a significant difference in alcohol consumption among males between animals for the three cage conditions (F 5 8.723, P 5 .001 for g/kg and F 5 7.37, P 5 .001 for PR) (Fig. 2C, D). Results differed slightly for the g/kg consumption measure compared with the PR measure. Tukey’s post hoc test revealed a significant difference between the sedentary group and the movable wheel group (P ! .001), a trend between sedentary mice and the locked wheel group (P 5 .07), and no difference
447
between the locked wheel and movable wheel (P 5 .241). The mean alcohol consumption values were 9.1 6 1.0 g/kg (free wheel), 11.8 6 1.4 g/kg (locked wheel), and 15.7 6 1.1 g/kg (sedentary) (Table 1). However, there was a statistically significant difference in the total amount of liquid consumed dependent on cage type (F 5 7.89, P 5 .001). Tukey’s post hoc analysis revealed that the mice with a locked wheel drank less total liquid than either of the other two groups. Those with a locked wheel drank 4.85 6 0.27 g compared with 5.6 6 0.21 g for the sedentary animals and 6.06 6 0.18 g for the running mice (P 5 .047 and P 5 .001, respectively). Using PR as the alcohol consumption measure revealed significant differences between the sedentary group (0.66 6 0.05 PR) and the free running group (0.40 6 0.05 PR) where P 5 .03, and between the locked wheel group (0.62 6 0.07 PR) and the free running group (P 5 .014). There was no difference in the mean distance (m) run per day in mice with and without access to alcohol (data not shown). Fig. 2C, D shows daily alcohol consumption means by cage condition group for the 3%, 7%, and 10% ethanol days. Repeated-measure ANOVA showed that for days 5e13 (when the concentration of ethanol is 10%), the variables alcohol g/kg, PR, and distance run do not differ significantly within subject (all P O .05), suggesting that these behaviors are fairly consistent across this time period for an individual animal. In addition, there were no significant differences in body weight changes over the course of the experiment (F 5 0.864, P 5 .427). Results for the two-bottle choice at 20% ethanol were similar to those for the 10% concentration. Fig. 3 shows the daily alcohol consumption means for females (A and B) and males (C and D) at all concentrations of ethanol (3%, 7%, 10%, 13%, 17%, and 20%) in the sedentary and running mice. There was a significant difference in g/kg of ethanol consumed (t 5 2.62, P 5 .016) and PR (t 5 3.08, P 5 .005), but no difference in total amount of liquid consumed (t 5 1.36, P 5 .19) among the females. Sedentary females consumed 19.15 6 3.22 g/kg of 20% ethanol daily, whereas running mice consumed 8.35 6 2.37 g/kg. Their respective PRs for ethanol were 0.40 6 0.07 and 0.15 6 0.04 (Table 1). In the males, there were no significant differences in g/kg alcohol consumed or in PR (both P O .40). However, as can be seen from the mean values in Table 1 and Fig. 3, the trend toward decreased alcohol consumption with access to a running wheel could be seen. In addition, there was a significant difference in total amount of liquid consumed (t 5 2.84, P 5 .012). The sedentary males drank a total volume of 5.02 6 0.17 g, whereas the running mice drank 6.18 6 0.37 g. Alcohol metabolism Two groups of mice were individually housed with water and food, half with access to a running wheel and half in a standard cage, which were subjected to eight retro-orbital
448
M.A. Ehringer et al. / Alcohol 43 (2009) 443e452
B
Females
Female 1.0
no wheel wheel free wheel w/ brake
no wheel wheel free wheel w/ brake
20
0.8
Mean volume ethanol / total volume consumed
Mean g/kg Ethanol Consumed
A 25
15
10
5
0.6
0.4
0.2
0.0
0 2
4
6
8
10
2
4
8
D
Males
10
Males
1.0
no wheel wheel free wheel w/ brake
20
no wheel wheel free wheel w/ brake
0.8
Mean volume ethanol / total volume consumed
Mean g/kg Ethanol Consumed
C 25
6
% ethanol (v/v)
% ethanol (v/v)
15
10
5
0
0.6
0.4
0.2
0.0 2
4
6
8
10
% ethanol (v/v)
2
4
6
8
10
% ethanol (v/v)
Fig. 2. (A) Mean daily g/kg of 3%, 7%, and 10% ethanol consumed in female C57BL/6 mice in a standard cage (black circle), with access to a locked wheel (black triangle) or a running wheel (open circle). (B) Mean daily ethanol preference ratio (PR) (vol/vol) consumed in female C57BL/6 mice in a standard cage (black circle), with access to a locked wheel (black triangle) or a running wheel (open circle). (C) Mean daily g/kg of 3%, 7%, and 10% ethanol consumed in male C57BL/6 mice in a standard cage (black circle), with access to a locked wheel (black triangle) or a running wheel (open circle). (D) Mean daily ethanol PR (vol/vol) consumed in male C57BL/6 mice in a standard cage (black circle), with access to a locked wheel (black triangle) or a running wheel (open circle). Error bars show 61 standard error.
sinus blood collections at the end of the 2 weeks of ‘‘treatment.’’ This experiment was repeated twice, first with eight mice (four male and four female) and again 2 years later with eight additional mice. There was no significant difference in the metabolism of ethanol by sex (F 5 2.29, P 5 .16) nor by treatment (F 5 0.20, P 5 .67). Fig. 4 shows the mean BEC levels for each time point for each group, with female runners and nonrunners in panel A and males in panel B. Food consumption To determine if food consumption played a role in the drinking behavior between mice with or without a running wheel, food consumption (g) was measured daily (Table 1). As described in the Experimental procedures, this experiment was conducted twice. In the first batch, there was no significant difference in the amount of alcohol consumed between sedentary and running mice. We hypothesized that perhaps the process of opening the cage twice daily to remove and replace the petri dish with food might have led to additional stress that affected their drinking. Therefore, we repeated the experiment but placed the food in the
hopper next to their liquid bottles. However, we noted that the removal and replacement of food from the hopper did create some ‘‘loud’’ metallic noises, which often awakened and visibly agitated the mice. Results from both batches were comparable, so the data were combined. There was no significant difference in mean g/kg of 10% alcohol consumed, and no difference in alcohol PR (all P O .50). Among the females, the runners (17) consumed 16.8 6 2.3 g/kg or a ratio of 0.57 6 0.09 ethanol, whereas the sedentary mice (18) consumed 18.6 6 2.1 g/kg or a ratio of 0.61 6 0.08. Although not significant, there is a directional trend for the runners to drink less alcohol than the sedentary mice. However, among the males, the runners (12) consumed 12.5 6 2.0 g/kg or 0.52 6 0.10 PR of alcohol, and the sedentary animals (13) drank only 11.7 6 2.1 g/kg or 0.51 6 0.10 PR. Examining the food data, there were no significant differences in the amount of food eaten between runners and sedentary mice, although there was a trend for the runners to eat more at all alcohol concentrations except in the males at 7% alcohol. These data are presented in Table 1.
M.A. Ehringer et al. / Alcohol 43 (2009) 443e452
B
Females
Female 1.0
no wheel wheel free
no wheel wheel free
20
Mean volume ethanol / total volume consumed
Mean g/kg Ethanol Consumed
A 25
449
15
10
5
0.8
0.6
0.4
0.2
0 0.0
5
10
15
5
20
D
Males no wheel wheel free
15
20
Males 1.0
Mean volume ethanol / total volume consumed
Mean g/kg Ethanol Consumed
C 25
10
% ethanol (v/v)
% ethanol (v/v)
20
15
10
no wheel wheel free 0.8
0.6
0.4
0.2
5
0.0
0
5
10
15
20
% ethanol (v/v)
5
10
15
20
% ethanol (v/v)
Fig. 3. (A) Mean daily g/kg of 3%, 7%, 10%, 13%, 17%, and 20% ethanol consumed in female C57BL/6 mice in a standard cage (black circle) or with a running wheel (open circle). (B) Mean daily ethanol preference ratio (PR) (vol/vol) consumed in female C57BL/6 mice in a standard cage (black circle) or with a running wheel (open circle). (C) Mean daily g/kg of 3%, 7%, 10%, 13%, 17%, and 20% ethanol consumed in male C57BL/6 mice in a standard cage (black circle) or with a running wheel (open circle). (D) Mean daily ethanol PR (vol/vol) consumed in male C57BL/6 mice in a standard cage (black circle) or with a running wheel (open circle). Error bars show 61 standard error.
DID and voluntary wheel running Under standard cage conditions (no wheel), there was no difference between males and females in the amount of alcohol consumed during the 2-h limited access period (females, 5.68 6 0.48 g/kg; males, 5.39 6 0.26 g/kg; t 5 0.51, P 5 .62), so both sexes were analyzed together. There were no differences in the daily running distances between animals with and without access to alcohol (data not shown). Sedentary mice consumed 5.55 6 0.28 g/kg of alcohol, whereas the mice with access to a running wheel drank 5.43 6 0.24 g/kg (t 5 0.34, P 5 .74). Data for each sex separately are presented in Table 1. A total of 20 mice (10 males and 10 females) were tested with the running wheels and 22 sedentary mice (10 males and 12 females), for a total of 42 mice.
Discussion Here, we have shown that C57BL/6IBG mice alter their alcohol consumption patterns when given unlimited access to a running wheel over several days, and that these behavioral responses differ in males and females. C57BL/6IBG
females drank significantly less alcohol when a running wheel was provided and movable, but not when the wheel was locked, suggesting that the decreased consumption is likely to be (at least in part) due to exercise and not simply a novel environment. In C57BL/6IBG males, the mice drank significantly less alcohol (g/kg of body weight) in the presence of a wheel, including showing a trend to drink less when it was locked, suggesting that a novel environment may be a key factor. However, it was noted that the males also drank less total liquid when the brake was engaged on the wheel. This led to the idea that perhaps the males were using the locked wheel as a ‘‘jungle gym’’ and perhaps increased activity simply limited the time they spent drinking in general. However, when the data were analyzed on the basis of comparing ethanol PR, males showed a similar pattern as the females. They drank a lower ratio of alcohol when they could run, but not when the wheel was locked, providing support to the notion that exercise is a key component. To further characterize these behaviors, we examined other aspects of the behavioral testing procedure, including increasing the alcohol concentration to 20%, and possible differences in alcohol metabolism and food consumption.
450
M.A. Ehringer et al. / Alcohol 43 (2009) 443e452
A 400
Females no wheel wheel free
mg % ethanol
300
200
100
0 0
20
40
60
80
100
120
140
160
180
200
Minutes post injection
B
Males
400
no wheel wheel free
mg % ethanol
300
200
100
0 0
20
40
60
80
100
120
140
160
180
200
Minutes post injection Fig. 4. Pharmacokinetic profile of blood ethanol concentrations in female (A) and male (B) mice with or without access to a running wheel prior to an intraperitoneal injection of alcohol.
The female mice behaved similarly when presented with 20% ethanol, showing significant decreases in alcohol consumption when they had access to a running wheel. Results were not significant for the male mice drinking 20% ethanol, but such as the 10% ethanol experiments, the data were confounded by the fact that the running mice drank significantly more total liquid than did the sedentary males. Our data suggest that these differences in alcohol consumption are not likely to reflect differences in alcohol metabolism between sedentary and running mice. Animals given 2 weeks of ‘‘pretreatment’’ of voluntary running prior to an intraperitoneal injection of alcohol did not differ in their rate of metabolism compared with ‘‘untreated’’ sedentary animals in a cage with no wheel. Furthermore, we did not find a significant difference between sedentary and running mice in the amount of food consumed. However, the two experiments examining food intake revealed an unexpected result because the process of weighing the food each day appeared to affect the drinking results, whereby there was no significant difference in alcohol consumption between sedentary and running mice. In our ongoing work using this behavioral model, we continue to observe a significant decrease in alcohol consumption among females in the presence of a running
wheel, using a protocol where the food is weighed every 4 days, corresponding to the same days the mice are weighed. Furthermore, the experiments testing a range of concentrations up to 20% ethanol (Fig. 3) consistently show decreased ethanol intake in the presence of a wheel. This finding highlights the challenges and complexities of studying animal behavior, but it is an interesting observation because it is in agreement with work showing connections between alcohol and stress (Chester et al., 2006), and exercise and stress (Greenwood and Fleshner, 2008). In our standard experiment, we provide an excess of food pellets in the bottom of the cage, so it is only necessary to replenish them every few days. One explanation for differing results for the food experiments might be related to a stress response induced by daily contact with their food, leading to high-alcohol consumption even in the presence of a running wheel. These observations support the hypothesis that common neuronal pathways may be activated by alcohol, exercise, and stress, and that access to a running wheel may provide an alternative neurological reward to those rewards normally stimulated by alcohol consumption. Results from testing using the DID protocol, believed to be a model of binge drinking, revealed an interesting difference. Using this limited access model, neither male nor female C57BL/6IBG mice showed any differences in alcohol consumption. This finding is important because it reflects potential important differences in the underlying neurobiology related to chronic alcohol intake over long periods of time compared with binge drinking. Our findings suggest that although regular exercise may have the potential to be a useful treatment component for some alcohol problem behaviors, it may be less effective for others, such as binge drinking (Vogel, 2002). These results are in agreement with several of the previous studies supporting the idea that the rewarding properties of exercise may substitute in part for the rewarding properties of alcohol and other substances. Wheel running has been shown to lead to decreased intake of amphetamine (Kanarek et al., 1995) and decreased positive-reinforcing effects of cocaine (Smith et al., 2008) in rats. The C57BL/6IBG mice are similar to the selectively bred alcohol-preferring P rats in that they voluntary drink high levels of alcohol, and they both show decreased intake of alcohol in the presence of a running wheel (McMillan et al., 1995). As access to a running wheel had no effect on alcohol intake in the nonpreferring rats, it would be interesting to test other strains of mice with different baseline voluntary drinking and wheel-running behaviors to determine whether individual genetic differences may contribute to this response. There is support for this idea from the work conducted in the Nurr1 deficient mice, which show lower alcohol intake and less running than their wild-type counterparts (Werme et al., 2003). In contrast to our work, Crews et al. (Crews et al., 2004) did not observe any difference in alcohol intake in male C57BL/6 mice, but this analysis was not the main focus of their study and very few animals were tested. As the effect
M.A. Ehringer et al. / Alcohol 43 (2009) 443e452
in our study is less pronounced in males, this discrepancy may be due in part to differences in testing procedure and perhaps modest genetic differences between the two different sources of the C57BL/6 mice. Likewise, it has been shown that within an individual animal, alternating access periods to running wheels does not lead to differences in alcohol intake (Ozburn et al., 2008). In a small number of animals, we examined this phenomenon also by locking the wheels for 5 days at the end of our standard 13-day 10% two-bottle choice experiment and did not see any evidence for increased drinking (unpublished data). However, it is possible that there may be important temporal considerations for these types of study designs, because we cannot predict whether 5 or 7 days (as used by Ozburn et al., 2008) would constitute sufficient length of exposure to a different environment to lead to meaningful differences in alcohol consumption. It is difficult to make a direct comparison to the findings of Werme et al. (Werme et al., 2002) because they were examining ethanol preference in rats following an ethanol withdrawal period. The experimental design used here differs dramatically because there was no attempt to induce alcohol dependency in the mice, given the availability of water at all times. Although one explanation for the connection between voluntary alcohol consumption and wheel running might be common neuronal reward pathway activation by both behaviors, alternative reasons for this decreased alcohol consumption exist. For example, ethanol is a diuretic, and wheel running may result in additional loss of water. Alcohol consumption may have decreased to maintain water balance. Future studies should attempt to address these possibilities. A major limitation of this work is the relative inability to disentangle the effect of exercise from a possible effect of environmental enrichment, particularly for the male animals. In the female C57BL/6IBG mice, we did not observe a significant effect on alcohol consumption in the presence of a locked wheel (although the mean amount consumed by the females with a locked wheel was slightly less). Interestingly, male mice drank less alcohol (g/kg) when the wheel was locked, but this was confounded by the fact that they drank less total liquid under that condition. For the alcohol PR, we might hypothesized that there was a decrease in how often the males who could run were going to the alcohol bottle compared with those with a locked wheel. Furthermore, it is important to note that the cages used for alcohol preference alone (i.e., no wheels) were slightly smaller than the cages with wheels. Most current protocols for environmental enrichment (EE) include housing rodents in larger cages with other animals and providing complex, varied objects, which often includes running wheels (Nithianantharajah and Hannan, 2006). Future studies examining different EE conditions need to be carried out to help elucidate the relative effectiveness of enrichment and exercise in reducing voluntary alcohol consumption. For example, it is not clear whether the effects of EE and wheel running might be additive. When wheel running has been examined separately
451
from other enrichments, it was found that running increased neuronal cell proliferation, cell survival, and net neurogenesis, whereas EE only increased cell survival (van Praag et al., 1999b), suggesting that the ability to exercise may be a key component in behavioral outcome. In summary, the unlimited access two-bottle choice mouse model presented here should serve as a useful tool for research aimed at understanding the possible neuronal mechanisms common to these behaviors. Future work should focus on examining which genes and proteins may be differentially expressed under different environmental conditions, and whether different responses are observed in other inbred strains of mice that do not show naturally high levels of alcohol preference and wheel running. Such work may provide valuable insight into possible alternative forms of prevention and treatment of human alcohol abuse and dependence.
Acknowledgments The authors would like to thank Dr. Jeanne Wehner, Hilary Clegg, Jessica Godfrey, Stacy Romero, Isabel Schlaepfer, Priyanka Thummalapally, and Joshua Wilcox for technical assistance and helpful discussion of this work.
References Bachmanov, A. A., Tordoff, M. G., and Beauchamp, G. K. (1996). Ethanol consumption and taste preferences in C57BL/6ByJ and 129/J mice. Alcohol. Clin. Exp. Res. 20, 201–206. Bamber, D. J., Cockerill, I. M., Rodgers, S., and Carroll, D. (2003). Diagnostic criteria for exercise dependence in women. Br. J. Sports Med. 37, 393–400. Belknap, J. K., Crabbe, J. C., and Young, E. R. (1993). Voluntary consumption of ethanol in 15 inbred mouse strains. Psychopharmacology (Berl) 112, 503–510. Boehm, S. L. 2nd, Moore, E. M., Walsh, C. D., Gross, C. D., Cavelli, A. M., Gigante, E., et al. (2008). Using drinking in the dark to model prenatal binge-like exposure to ethanol in C57BL/6J mice. Dev. Psychobiol. 50, 566–578. Carelli, R. M., Ijames, S. G., and Crumling, A. J. (2000). Evidence that separate neural circuits in the nucleus accumbens encode cocaine versus ‘‘natural’’ (water and food) reward. J. Neurosci. 20, 4255–4266. Chan, C. S., and Grossman, H. Y. (1988). Psychological effects of running loss on consistent runners. Percept. Mot. Skills 66, 875–883. Chapman, C. L., and De Castro, J. M. (1990). Running addiction: measurement and associated psychological characteristics. J. Sports Med. Phys. Fitness 30, 283–290. Chester, J. A., de Paula Barrenha, G., DeMaria, A., and Finegan, A. (2006). Different effects of stress on alcohol drinking behaviour in male and female mice selectively bred for high alcohol preference. Alcohol Alcohol. 41, 44–53. Crews, F. T., Nixon, K., and Wilkie, M. E. (2004). Exercise reverses ethanol inhibition of neural stem cell proliferation. Alcohol 33, 63–71. Dishman, R. K., Berthoud, H. R., Booth, F. W., Cotman, C. W., Edgerton, V. R., Fleshner, M. R., et al. (2006). Neurobiology of exercise. Obesity (Silver Spring) 14, 345–356. Ferreira, A., Lamarque, S., Boyer, P., Perez-Diaz, F., Jouvent, R., and Cohen-Salmon, C. (2006). Spontaneous appetence for wheel-running:
452
M.A. Ehringer et al. / Alcohol 43 (2009) 443e452
a model of dependency on physical activity in rat. Eur. Psychiatry 21, 580–588. Freed, C. R., and Yamamoto, B. K. (1985). Regional brain dopamine metabolism: a marker for the speed, direction, and posture of moving animals. Science 229, 62–65. Fuller, J. L. (1964). Measurement of alcohol preference in genetic experiments. J. Comp. Physiol. Psychol. 57, 85–88. Furst, D. M., and Germone, K. (1993). Negative addiction in male and female runners and exercisers. Percept. Mot. Skills 77, 192–194. Goldman, D., Oroszi, G., and Ducci, F. (2005). The genetics of addictions: uncovering the genes. Nat. Rev. Genet. 6, 521–532. Greenwood, B. N., and Fleshner, M. (2008). Exercise, learned helplessness, and the stress-resistant brain. Neuromolecular Med. 10, 81–98. Greenwood, B. N., Foley, T. E., Burhans, D., Maier, S. F., and Fleshner, M. (2005). The consequences of uncontrollable stress are sensitive to duration of prior wheel running. Brain Res. 1033, 164–178. Greenwood, B. N., Foley, T. E., Day, H. E., Campisi, J., Hammack, S. H., Campeau, S., et al. (2003). Freewheel running prevents learned helplessness/behavioral depression: role of dorsal raphe serotonergic neurons. J. Neurosci. 23, 2889–2898. Gutgesell, M. E., Timmerman, M., and Keller, A. (1996). Reported alcohol use and behavior in long-distance runners. Med. Sci. Sports Exerc. 28, 1063–1070. Kamdar, N. K., Miller, S. A., Syed, Y. M., Bhayana, R., Gupta, T., and Rhodes, J. S. (2007). Acute effects of Naltrexone and GBR 12909 on ethanol drinking-in-the-dark in C57BL/6J mice. Psychopharmacology (Berl) 192, 207–217. Kanarek, R. B., Marks-Kaufman, R., D’Anci, K. E., and Przypek, J. (1995). Exercise attenuates oral intake of amphetamine in rats. Pharmacol. Biochem. Behav. 51, 725–729. Larson, E. B., and Carroll, M. E. (2005). Wheel running as a predictor of cocaine self-administration and reinstatement in female rats. Pharmacol. Biochem. Behav. 82, 590–600. Lerman, I., Harrison, B. C., Freeman, K., Hewett, T. E., Allen, D. L., Robbins, J., et al. (2002). Genetic variability in forced and voluntary endurance exercise performance in seven inbred mouse strains. J. Appl. Physiol. 92, 2245–2255. Lett, B. T., Grant, V. L., Byrne, M. J., and Koh, M. T. (2000). Pairings of a distinctive chamber with the aftereffect of wheel running produce conditioned place preference. Appetite 34, 87–94. Lightfoot, J. T., Turner, M. J., Daves, M., Vordermark, A., and Kleeberger, S. R. (2004). Genetic influence on daily wheel running activity level. Physiol. Genomics 19, 270–276. Mathes, W. F., and Kanarek, R. B. (2001). Wheel running attenuates the antinociceptive properties of morphine and its metabolite, morphine6-glucuronide, in rats. Physiol. Behav. 74, 245–251. McMillan, D. E., McClure, G. Y., and Hardwick, W. C. (1995). Effects of access to a running wheel on food, water and ethanol intake in rats bred to accept ethanol. Drug Alcohol Depend. 40, 1–7. Meliska, C. J., Bartke, A., McGlacken, G., and Jensen, R. A. (1995). Ethanol, nicotine, amphetamine, and aspartame consumption and preferences in C57BL/6 and DBA/2 mice. Pharmacol. Biochem. Behav. 50, 619–626. Middaugh, L. D., Kelley, B. M., Bandy, A. L., and McGroarty, K. K. (1999). Ethanol consumption by C57BL/6 mice: influence of gender and procedural variables. Alcohol 17, 175–183. Molteni, R., Zheng, J. Q., Ying, Z., Gomez-Pinilla, F., and Twiss, J. L. (2004). Voluntary exercise increases axonal regeneration from sensory neurons. Proc. Natl. Acad. Sci. U.S.A. 101, 8473–8478. Nestler, E. J. (2005). Is there a common molecular pathway for addiction? Nat. Neurosci. 8, 1445–1449.
Nithianantharajah, J., and Hannan, A. J. (2006). Enriched environments, experience-dependent plasticity and disorders of the nervous system. Nat. Rev. Neurosci. 7, 697–709. Nixon, K. (2006). Alcohol and adult neurogenesis: roles in neurodegeneration and recovery in chronic alcoholism. Hippocampus 16, 287–295. Ozburn, A. R., Harris, R. A., and Blednov, Y. A. (2008). Wheel running, voluntary ethanol consumption, and hedonic substitution. Alcohol 42, 417–424. Pierce, E. F., Rohaly, K. A., and Fritchley, B. (1997). Sex differences on exercise dependence for men and women in a marathon road race. Percept. Mot. Skills 84, 991–994. van Praag, H., Christie, B. R., Sejnowski, T. J., and Gage, F. H. (1999a). Running enhances neurogenesis, learning, and long-term potentiation in mice. Proc. Natl. Acad. Sci. U.S.A. 96, 13427–13431. van Praag, H., Kempermann, G., and Gage, F. H. (1999b). Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat. Neurosci. 2, 266–270. Rhodes, J. S., Ford, M. M., Yu, C. H., Brown, L. L., Finn, D. A., Garland, T. Jr., et al. (2007). Mouse inbred strain differences in ethanol drinking to intoxication. Genes Brain Behav. 6, 1–18. Rhodes, J. S., and Garland, T. (2003). Differential sensitivity to acute administration of Ritalin, apomorphine, SHC 23390, but not racolpride in mice selectively bred for hyperactive wheel-running behavior. Psychopharmacology 167, 242–250. Rhodes, J. S., Garland, T. Jr., and Gammie, S. C. (2003). Patterns of brain activity associated with variation in voluntary wheel-running behavior. Behav. Neurosci. 117, 1243–1256. Sacchetti, P., Carpentier, R., Segard, P., Olive-Cren, C., and Lefebvre, P. (2006). Multiple signaling pathways regulate the transcriptional activity of the orphan nuclear receptor NURR1. Nucleic Acids Res. 34, 5515–5527. Smith, M. A., Schmidt, K. T., Iordanou, J. C., and Mustroph, M. L. (2008). Aerobic exercise decreases the positive-reinforcing effects of cocaine. Drug Alcohol Depend. 98, 129–135. Smolen, A., Marks, M. J., Smolen, T. N., and Collins, A. C. (1986). Dose and route of administration alter the relative elimination of ethanol by long-sleep and short-sleep mice. Alcohol. Clin. Exp. Res. 10, 198–204. Ussher, M., Sampuran, A. K., Doshi, R., West, R., and Drummond, D. C. (2004). Acute effect of a brief bout of exercise on alcohol urges. Addiction 99, 1542–1547. Vaynman, S., and Gomez-Pinilla, F. (2006). Revenge of the ‘‘sit’’: how lifestyle impacts neuronal and cognitive health through molecular systems that interface energy metabolism with neuronal plasticity. J. Neurosci. Res. 84, 699–715. Vogel, R. A. (2002). Alcohol, heart disease, and mortality: a review. Rev. Cardiovasc. Med. 3, 7–13. Volkow, N. D., and Wise, R. A. (2005). How can drug addiction help us understand obesity? Nat. Neurosci. 8, 555–560. Werme, M., Hermanson, E., Carmine, A., Buervenich, S., Zetterstrom, R. H., Thoren, P., et al. (2003). Decreased ethanol preference and wheel running in Nurr1-deficient mice. Eur. J. Neurosci. 17, 2418–2424. Werme, M., Lindholm, S., Thoren, P., Franck, J., and Brene, S. (2002). Running increases ethanol preference. Behav. Brain Res. 133, 301– 308. Yoneyama, N., Crabbe, J. C., Ford, M. M., Murillo, A., and Finn, D. A. (2008). Voluntary ethanol consumption in 22 inbred mouse strains. Alcohol. 42, 149e160 Zito, K., and Svoboda, K. (2002). Activity-dependent synaptogenesis in the adult Mammalian cortex. Neuron 35, 1015–1017.