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. Author manuscript; available in PMC: 2013 Nov 1.
Published in final edited form as: Pharmacol Biochem Behav. 2012 Nov;103(1):18–25. doi: 10.1016/j.pbb.2012.07.012

Voluntary wheel running attenuates ethanol withdrawal-induced increases in seizure susceptibility in male and female rats

Leslie L Devaud *, Shawn A Walls, Walter D McCulley III, Alan M Rosenwasser
PMCID: PMC3537856  NIHMSID: NIHMS402863  PMID: 22871538

Abstract

We recently found that voluntary wheel running attenuated ethanol withdrawal-induced increased susceptibility to chemoconvulsant-induced seizures in male rats. Since female rats recover from ethanol withdrawal (EW) more quickly than male rats across several behavioral measures, this study was designed to determine whether the effects of exercise on EW seizures also exhibited sex differences. Animals were maintained under No-Wheel, Locked-Wheel or Free-Wheel conditions and ethanol was administered by liquid diet for 14 days with control animals pair-fed an isocaloric diet, after which seizure thresholds were determined at 1 day or 3 days of EW. Consistent with previous reports, females ran significantly more than males, regardless of diet condition. Introduction of the ethanol-containing liquid diet dramatically increased running for females during the day (rest) phase, with little impact on night phase activity. Consistent with previous reports, EW increased seizure susceptibility at 1 day in non-exercising males and females and at 3 days in males. These effects were attenuated by access to running wheels in both sexes. We also assessed the effects of sex, ethanol diet and exercise on ethanol clearance following an acute ethanol administration at 1 day EW in a separate set of animals. Blood ethanol concentrations at 30 min post-injection were lower in males, ethanol-exposed animals, and runners, but no interactions among these factors were detected. Interestingly, females displayed more rapid ethanol clearance than males and there were no effects of either diet or wheel access on clearance rates. Taken together, these data suggest that voluntary wheel running during ethanol administration provides protective effects against EW seizures in both males and females. This effect may be mediated, in part, in male, but not female rat, by effects of exercise on early pharmacokinetic contributions. This supports the idea that encouraging alcoholics to exercise may benefit their recovery.

Keywords: ethanol withdrawal, wheel running, ethanol liquid diet, sex differences

1. Introduction

Researchers have long understood the value of exercise and fitness for human health. In 1990, Pollock and Wilmore published a literature review supporting the utility of exercise in prevention and rehabilitation for numerous major health disorders (Pollock and Wilmore, 1990). More recently, exercise has been shown to antagonize the effects of drugs of abuse in both animal and human studies. For example, chronic running wheel exercise reduced cocaine seeking both during initial extinction and subsequent reinstatement tests (Cosgrove et al, 2002; Lynch et al, 2010; Smith et al, 2008). Similarly, both morphine (Hosseini et al, 2009; Lett et al, 2002) and ethanol (Ehringer et al, 2009; Ozburn et al, 2008) self-administration were reduced with exercise. In a study of human smokers, cigarette cravings and brain responses by smokers were also reduced with exercise (Janse Van Rensburg et al, 2008). These, and similar findings support the suggestion that exercise may be beneficial in the treatment of addictions, including alcoholism.

We recently reported that unlimited access to running wheels during the course of ethanol administration protected against increases in EW seizure susceptibility in male rats, despite the fact that ethanol consumption itself reduced wheel running (McCulley et al 2012). In studies with human alcoholics, Palmer and coworkers reported the potential utility of physical exercise as a treatment intervention, especially for reducing the anxiogenic effects of ethanol withdrawal (Palmer et al, 1988). In contrast, when exercise was only encouraged for heavy drinkers, there was no effect on treatment outcomes (Kendzor et al, 2008). Another report saw improved abstinence in recovering alcoholics when more structured exercise was incorporated as part of the overall intervention (Brown et al, 2009). These clinical studies suggest that exercise is likely to be of therapeutic benefit to recovering alcoholics.

One unresolved question is how exercise improves outcomes for ethanol dependence and withdrawal. Exercise appeared to reduce the intoxicating effects of acute ethanol (Mollenauer et al, 1991; 1992), possibly due to enhanced ethanol metabolism (Ardies, 1989). In contrast, a recent report by Leasure and Nixon (2010) found a neuroprotective effect of exercise in a binge model of ethanol exposure in the absence of exercise-induced changes in ethanol clearance rates.

We have investigated sex differences in the effects of ethanol dependence and EW in an animal model for a number of years. We, and others, have found important sex differences in EW across several measures (Alele et al, 2009; Devaud et al, 1996; Devaud and Chadda, 2001; Koirala et al, 2008; Reilly et al, 2009; Veatch et al, 2007). In general, females tend to show reduced severity and more rapid recovery from EW compared to their male counterparts. This correlates with the observation that alcoholic women present with reduced EW symptomology compared to alcoholic men, even when levels of consumption are equivalent (Deshmukh et al, 2003). Evidence suggests that these sex differences may result, at least in part, from the differing hormonal milieu. Progesterone-derived neurosteroids are present at higher levels in females than males and share some overlap with ethanol in their mechanisms of action (see Finn et al, 2010 for review). However, possible sex differences in the effects of exercise on EW have not been studied previously. Therefore, the current study was initiated to determine if exercise would be of benefit to EW females as well as males. We examined the effect of free access to running wheels on ethanol consumption by liquid diet, the effects of ethanol consumption on running levels and the consequence of this extended exercise on EW seizure susceptibility in female and male rats. We also assessed the effects of wheel running on basal seizure thresholds and blood ethanol concentrations, to determine if there was evidence for direct effects of exercise on seizure risk and/or changes in ethanol clearance.

2. Methods

2.1 Animals

Male and female CR rats (Charles River Labs, Wilmington, MA) were approximately 42 days old at the start of experimental procedures. A total of 156 animals (81 males and 75 females) were used for the PTZ seizure threshold experiments and an additional 16 males and 14 females were used for the ethanol clearance experiments. We controlled for age, rather than weight. Estrus cyclicity was not directly monitored as previous studies found that cycles for females synchronize over the course of the experimental procedures when housed in the same room as males. Additionally, individual housing and liquid diet procedure tended to prolong estrus in the female rats (Alele et al, 2007).

2.2 Materials

Pentylenetetrazol (PTZ) from Sigma-Aldrich (St. Louis, MO) was dissolved in normal saline at a concentration of 5.0 mg/ml. Ethanol was dissolved in normal saline at 20% v/v for blood ethanol concentration determinations.

2.3 Activity Wheel Procedure

Animals were individually housed and randomly assigned to one of three running wheel conditions: (1) standard rat cages without wheels (No-Wheel), (2) standard rat cages with mechanically locked running wheels (Locked-Wheel) or (3) standard rat cages with functioning running wheels (Free-Wheel). The Locked-Wheel condition was included to separate the possible effects of environmental complexity from those of exercise. Running wheels were available continuously 24 hr per day throughout the course of the experiment in the Free-Wheel condition. Running wheel activity was recorded by use of an external electronic LCD counter that was attached to the side of each Free-Wheel cage. Activity was recorded twice daily at 7:00 a.m. (start of rest phase) and at 5:00 p.m. (near start of active phase). We chose these two times as lights on and approaching lights off. Counters were manually reset after obtaining counts. All animals were housed under their respective conditions for 10 days prior to introduction of the liquid diets to allow for acclimation and adaptation to running wheels in Free-Wheel animals and this is presented as baseline wheel turns.

2.4 Liquid diet procedure

Animals were made ethanol-dependent by administration of 6% ethanol, v/v, in a nutritionally complete liquid diet, which was slightly modified from the Frye liquid diet (Devaud et al, 1994). Diet components were purchased individually with diet made at least twice per week (MP Biomedical, Costa Mesa, CA). Fresh diet was provided daily and administered for 14 days as previously described (Alele and Devaud, 2007; Devaud et al, 1994; Koirala et al, 2008; McCulley et al, 2011; Walls et al, 2011). Control animals were pair-fed the same liquid diet but with dextrose substituted isocalorically for the ethanol to ensure equivalent caloric intake and comparable nutritional status. The amount of liquid diet consumed was recorded daily.

After 14 days, liquid diet administration was terminated and regular lab chow provided ad libitum to all animals to maintain equivalent diet conditions. Seizure threshold testing was scheduled at 1 day (1d) or 3 days (3d) EW. All procedures were conducted in accordance with approved University of Maine Animal Welfare Protocols and NIH guidelines for the humane care and use of animals in an AAALAC-accredited facility.

2.5 Pentylenetetrazol (PTZ) Seizure threshold procedure

Constant tail vein infusion of the chemoconvulsant, PTZ, was used for the induction of seizures. A 25 g butterfly needle was inserted into a lateral tail vein while the animal was gently restrained and needle taped into place. The animal was then allowed to move freely while the observer gently held the tip of its tail. PTZ was infused at 1.6 ml/min and the time to the first myoclonic twitch of the face and/or neck indicated the endpoint of infusion (Alele and Devaud, 2007; McCulley et al, 2011). Seizure thresholds were calculated by multiplying the latency (minutes) to the first sign of a seizure by the PTZ infusion rate and concentration (5 mg/ml × 1.6 ml/min) and are presented as mg PTZ/kg.

2.6 Blood ethanol determinations (BEC)

In a separate experiment, ethanol (as a 20% solution in saline) was given as a bolus intraperitoneal injection at 2.0 g/kg to No-Wheel or Free-Wheel animals at 1d EW. No Locked-Wheel animals were studied in this experiment. Tail blood was then collected at 30 min, 60 min, 2 hr and 4 hr post-injection to assess changes in BEC over time. BECs were determined using an Analox AM1 analyzer (Analox Instruments Co, Lunenberg, MA). Blood ethanol concentrations were not collected as part of the seizure susceptibility experiments because of the disruptive nature of the procedure and, more importantly, when animals are fed a liquid diet, they drink in unpredictable bouts during the active (night) phase with minimal drinking during the day (rest) phase. Therefore, blood ethanol concentrations taken during the course of the liquid diet administration are highly variable and will not provide an accurate assessment of intake nor of clearance. Daily consumption provides a better reflection of ethanol intake, with animals needing to consume at least 8 g/kg/day to achieve and maintain dependence (Devaud et al, 1995; Devaud and Chadda, 2001; Janis et al, 1998).

2.7 Data analysis

Data for wheel running and PTZ seizure thresholds were analyzed using standard factorial ANOVA, and post-hoc pairwise comparisons were performed using the least-significant difference (LSD) procedure to control type-1 error rate as described below for each dependent measure (SPSS, Chicago IL, USA). The BEC data were analyzed by factorial ANOVA to determine the effects of sex, diet and exercise on initial BECs, and then by repeated measures ANOVA to explore ethanol clearance rates. Finally, regression analysis was used to express clearance rates in mg/dl/hour.

3. Results

3.1 Body weights and ethanol consumption

Animals in all groups displayed substantial weight gain over the course of the experiment (Table 1). A 3-factor ANOVA was conducted to examine the effects of sex, housing conditions (No-Wheel, Locked-Wheel, Free-Wheel), and liquid diet type (ethanol-containing vs. non-ethanol containing) on weight gain. This analysis revealed a significant main effect of sex [F(1,170) = 385.249, p < 0.001], indicating that males gained proportionately more weight (over 100%) than females (approximately 60%). Neither wheel running or ethanol administration significantly impacted weight gain.

TABLE 1.

Average weights during the course of the experiment (as g).

MALES
Control Initial Weight Final weight % Increase
No-Wheel 165.0 ± 2.3 358.6 ± 3.2 117%
Locked-Wheel 166.0 ± 2.1 351.5 ± 2.9 112%
Free-Wheel 157.5 ± 3.6 323.8 ± 2.8 106%
Ethanol Fed Initial Weight Final Weight % Increase
No-Wheel 164.0 ± 0.9 349.8 ± 2.6 113%
Locked-Wheel 164.8 ± 1.1 345.8 ± 3.4 110%
Free-Wheel 159.9 ± 2.6 341.3 ± 2.0 113%
FEMALES
Control Initial Weight Final weight % Increase
No-Wheel 146.3 ± 1.8 238.3 ± 3.8 63%
Locked-Wheel 146.9 ± 4.6 234.8 ± 6.7 60%
Free-Wheel 140.0 ± 1.8 225.5 ± 5.6 61%
Ethanol Fed Initial Weight Final Weight % Increase
No-Wheel 145.5 ± 1.9 241.6 ± 3.5 66%
Locked-Wheel 141.8 ± 1.4 237.6 ± 4.6 68%
Free-Wheel 141.5 ± 1.9 224.7 ± 5.0 59%
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Two-factor ANOVA was used to examine the effects of sex and housing conditions on ethanol intake (g/kg/day) in animals exposed to the ethanol-containing liquid diet (Figure 1). This analysis showed significant main effects for sex [F(1,119) = 135.137, p < 0.001] and housing conditions [F(2,141) = 10.415, p < 0.001], but no sex-X-housing interaction. Thus, females consumed significantly more ethanol than males, consistent with previous reports. Post-hoc pairwise tests showed that Free-Wheel animal of both sexes consumed significantly more ethanol than either of their Locked-Wheel or No-Wheel counterparts, while Locked-Wheel and No-Wheel animals did not differ.

Figure 1.

Figure 1

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Mean ± SEM daily ethanol consumption by sex and housing conditions averaged for the final 7 days of liquid diet administration. Both males and females in the Free-Wheel condition drank significantly more diet than their No-Wheel or Locked-Wheel counterparts. N= 17–18 per wheel condition for males and 14–17 per wheel condition for females. *: P < 0.05 compared to No-Wheel or Locked-Wheel conditions within sex.

3.2 Running-wheel activity

Figure 2 shows the mean number of wheel turns for the night (A) and day (B) phases of the daily cycle. Running wheel activity in Free-Wheel animals was analyzed using a 3-factor mixed ANOVA with feeding condition (chow-fed baseline vs. liquid diet) as a repeated-measures factor and with sex and diet type as between-subjects factors.

Figure 2.

Figure 2

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Mean ± SEM night phase (A) and day phase (B) wheel turns for comparisons by sex and diet conditions. Females ran significantly more than males for each diet condition. N= 12–20 per wheel condition for males and 12–19 per wheel condition for females. *: P < 0.05 compared to baseline running; #: P < 0.05 and ##: P < 0.01 compared to control diet running within sex condition.

Analysis of night-phase activity revealed a significant main effect of sex [F(1,53) = 30.276, p < 0.001], and a significant feeding condition X diet type interaction [F(1,53) = 6.916, p < 0.011] indicating that night-phase activity increased under liquid diet conditions in animals exposed to the control (non-ethanol containing) liquid diet. In sharp contrast, night-phase activity was unchanged for animals exposed to the ethanol-containing diet.

Analysis of day-phase activity revealed complex interactions among the various experimental treatments. Significant main effects were seen for sex [F(1,53) = 9.447, p = 0.003], feeding condition [F(1,53) = 14.583, p < 0.001], and diet type [F(1,53) = 6.256, p < 0.02], while significant 2-way interactions were observed between feeding condition and diet type [F(1,53) = 5.373, p = 0.02] and feeding condition and sex [F(1,53) = 7.182, p = 0.01]. Finally, a marginally significant interaction was also seen between diet type and sex [F(1,53) = 3.927, p = 0.053]. To summarize these effects, females displayed more activity than males across all treatment conditions, consistent with previous reports (Eikelboom and Mills, 1988: Pietropaolo et al, 2008). Additionally, day-phase activity (normally the rest period) was increased under liquid diet, and this effect was pronounced in ethanol-exposed animals and especially in females. These analyses indicate that exposure to ethanol-containing liquid diet selectively increased day-phase activity, suggesting a blunting of the normal day-night variation in activity.

3.3 PTZ Seizure thresholds

As shown in Figure 3, EW increased seizure susceptibility (reduced seizure thresholds) by more than 20% for males at 1d and 3d in both No-Wheel and Locked-Wheel conditions. For females, there was also an approximate 20% decrease in seizure threshold at 1d EW with an 8% decrease at 3d EW for both the No-Wheel and Locked-Wheel conditions. These responses are similar to what we have previously reported for females housed without wheels and confirm that females recover more quickly than males from EW (Devaud and Chadda, 2001). In contrast, both males and females in the Free-Wheel condition showed attenuated EW responses relative to those seen in non-exercising groups. Thus, male runners displayed a 14% reduction in seizure thresholds and a 9% change at 1d EW and 3d EW, respectively. For EW females, seizure thresholds were reduced by just 3% at 1d EW and 2% at 3d EW compared to ethanol naïve control levels.

Figure 3.

Figure 3

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PTZ seizure thresholds (mean ± SEM) for ethanol-fed and pair-fed control animals at 1 or 3 d of EW. For males: control values, N= 10 No Wheel, 9 Locked Wheel and 9 Free Wheel; 1d EW: N= 9 for No Wheel, 8 for Locked Wheel and 9 for Free Wheel; 3d EW: N= 9 for No Wheel, 9 for Locked Wheel and 9 for Free Wheel. For females: control values, N= 11 No Wheel, 9 Locked Wheel and 8 Free Wheel; for 1d EW: N= 9 for No Wheel, 8 for Locked Wheel and 7 for Free Wheel; for 3d EW: N= 8 for No Wheel, 8 for Locked Wheel and 7 for Free Wheel. *: P < 0.05 compared to control (basal) thresholds within sex and wheel condition.

A 3-factor AVOVA was conducted to determine the effects of sex, ethanol treatment (control, 1d EW, 3d EW) and housing conditions (No-Wheel, Locked-Wheel, Free-Wheel) on seizure thresholds. This analysis showed significant main effects of all three factors, indicating that males had lower thresholds than females [F(1,141) = 72.903, p < 0.001], EW animals had lower thresholds than ethanol-naive control animals [F(2,141) = 25.012, p < 0.001], and Free-Wheel animals had higher thresholds than Locked-Wheel or No-Wheel animals [F(2,141) = 12.987, p < 0.001]. In addition, this analysis revealed a significant sex X treatment interaction [F(2,141) = 5.799, p = 0.004]. On the other hand, there were no sex X housing or sex X housing X treatment effects, indicating that access to running wheels had similar effects for both males and females.

In order to compare the sex-by-treatment interaction on seizure thresholds with earlier findings, we conducted separate post-hoc pairwise comparisons between ethanol conditions for females and males in non-exercising animals. These comparisons showed that 1d EW females had lower thresholds than either control or 3d EW females (p < 0.05), while control and 3d EW females did not differ. In contrast, both 1d and 3d EW males exhibited lower seizure thresholds than controls (p < 0.05), while 1d and 3d EW males did not differ. Thus, in agreement with previous reports, seizure thresholds returned to control levels by day 3 EW in females, but remained significantly lowered at that time-point in males.

Since post-hoc tests showed that Free-Wheel animals had higher seizure thresholds than either Locked-Wheel or No-Wheel animals, while the latter two groups did not differ, we conducted an additional 3-factor ANOVA in which housing conditions were collapsed into two levels: runners (Free-Wheel animals) and non-runners (Locked-Wheel and No-Wheel animals). The results of this analysis were virtually identical to the original ANOVA with the exception that the housing-by-treatment interaction was now robustly significant [F(2,147) = 4.206, p = 0.02]. This interaction indicates that wheel running modulated seizure thresholds primarily in EW animals, rather than in controls.

3.4 Blood ethanol concentrations and ethanol clearance

One postulated explanation for the ability of running wheel access to attenuate EW signs is that exercise may alter the metabolic processing of ethanol. As there are contradictory findings as to whether exercise enhances ethanol clearance, we ran a separate experiment following identical dietary treatments as described above, utilizing No-Wheel and Free-Wheel housing conditions. All animals were administered a single, bolus administration of 2.0 g/kg ethanol at 1d EW and tail blood was then collected at several time points to measure changes in BECs over time. At the initial time point, 30 min after administration, 3-factor ANOVA (sex, diet, exercise) showed that BECs were significantly lower in males than females [F(1,18) = 4.877, P = 0.04], lower in ethanol diet than control diet animals [F(1,18) = 17.134, P = 0.001], and lower in runners than non-runners [F(1,18) = 8.461, P = 0.009], but no interactions among these factors were detected (Figure 4). Thus, while exercise reduced initial BECs, this effect was not specific for EW animals. BECs were next analyzed with a 4-factor ANOVA (time, sex, diet, exercise), and linear regression was used to estimate clearance rates for each group (Table 2). ANOVA revealed a significant main effect of time, reflecting the expected overall decrease in BEC values over time for all groups [F(3,15) = 104.296, P < 0.001], and a time X sex interaction [F(3,15) = 13.243, P < 0.001] indicating that BECs declined more rapidly in females than males (Figure 5, Table 2). Importantly, there was no time X wheel or time X diet interactions, indicating that neither ethanol liquid diet nor running wheel access significantly altered metabolic clearance rates. Significant main effect of diet [F(1,5) = 9.859, P = 0.03] and wheel access reflected the fact that the effects of these factors observed at 30 minutes post-injection persisted over the course of assessment. Finally, the ANOVA analysis revealed a significant sex X wheel interaction [F(1,32) = 11.396, P = 0.02] showing that running wheel activity lowered BECs for control males, but not for females.

Figure 4.

Figure 4

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Blood ethanol concentrations (mean ± SEM) during EW at the initial (30 min) time point following the bolus ethanol injection. N= 4 per treatment condition for males (con No Wheel, con Free Wheel, EtOH fed No Wheel and EtOH fed Free Wheel) and 3–4 per treatment condition for females (con No Wheel, con Free Wheel, EtOH fed No Wheel and EtOH fed Free Wheel). *: P < 0.05 compared to no wheel control levels within sex.

TABLE 2.

Ethanol clearance rates (mg/dl/hr)

MALES
No-Wheel Con 0.231 ± 0.066
No-wheel EtOH 0.149 ±0.039
Free-wheel con 0.230 ± 0.044
Free-wheel EtOH 0.238 ± 0.048
FEMALES
No-Wheel Con 0.332 ± 0.096
No-wheel EtOH 0.320 ± 0.064
Free-wheel con 0.413± 0.068
Free-wheel EtOH 0.239 ± 0.058
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Figure 5.

Figure 5

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Blood ethanol concentrations (mean ± SEM) during EW across 4 time points following a bolus ethanol injection.

3.5 Correlation between wheel turns and basal seizure thresholds

Correlation analyses were conducted to determine if individual levels of wheel running in animals were related to seizure thresholds. The analysis was conducted using data pooled across all running wheel and EW experiments. This produced a significant correlation (p = 0.01, n=17) between amount of wheel activity and PTZ-induced seizure thresholds for control males (Figure 6). A nearly significant correlation was also observed for 3d EW males (p = 0.057) but not for 1d EW males, nor for control or EW females (data not shown). This suggests that activity alone may have some influence on basal PTZ-induced seizures even in the absence of ethanol withdrawal.

Figure 6.

Figure 6

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Correlation between number of wheel turns per day and PTZ seizure threshold in control (non-ethanol exposed) and EW male rats. Data was summarized from a total of six experiments (three with males only and three with males and females). P = 0.01 for control males but was not significant for either 1d or 3d EW males. There were no significant correlations for females (data not shown).

4. Discussion

We recently found that voluntary wheel running attenuated the EW-induced increases in seizure susceptibility in male rats (McCulley et al, 2012). A number of previous investigations have shown that female rats recover more quickly from EW than do male rats across several behavioral measures (Devaud and Chadda, 2001; Reilly et al, 2009; Walls et al, 2011). Therefore, the present study investigated the effects of voluntary wheel running on seizure susceptibility in EW female as well as male rats. Results from the present set of experiments confirm and build upon previous findings showing that seizure risk in non-running EW female rats returned to basal levels by 3d EW whereas EW male rats continued to show reduced seizure thresholds at this time. Nevertheless, voluntary wheel running increased seizure thresholds (attenuated the EW-induced increases in seizure susceptibility) for both sexes and at both EW time points, without effecting seizure thresholds in non-ethanol exposed control animals. This suggests that exercise could prove to be especially beneficial for both men and women as they undergo detoxification and recover from alcoholism. These findings add further support to the possibility that exercise may be generally beneficial against addiction and its consequences, as suggested by several clinical studies (Brown et al, 2009; Palmer et al, 1988). The finding that voluntary exercise protected against the increased seizure susceptibility of EW is especially important as withdrawal-induced seizures pose a serious risk for alcoholics.

Several potential mechanisms may contribute to the protective effect of exercise in EW. The effects of exercise on EW may arise from pharmacokinetic factors, such as reduced absorption and/or increased metabolism and clearance in exercising animals (Ardies et al, 1989). While we did observe reduced BECs in exercising animals at the initial 30-minute time point, there was no significant effect of exercise on subsequent clearance rates. Further, the effects of exercise on initial BEC was not specific for EW, and indeed, was more prominent in male controls than any other group. Thus, we conclude that while early pharmacokinetic factors such as absorption may have contributed to mediating the effects of exercise on withdrawal seizures, it seems unlikely that alterations in metabolic clearance were involved. One limitation of the present studies was that BECs were not measured during the induction of dependence, so the potential contribution of lower BECs during the initial days of ethanol administration on withdrawal severity could not be taken into account. There was also evidence for reduced BECs in the ethanol-fed animals, even at 1d EW and this was much greater for males than females. These data support the suggestion that additional neurobiological factors contribute to the protective effect of wheel running as BECs were only slightly reduced further in EW and running animals compared to their non-running counterparts. The sex differences could arise from higher metabolism or reduced gastric absorption for males compared to females but, again, does not completely explain the attenuation of the enhanced PTZ seizure susceptibility seen during ethanol withdrawal.

Research has shown that exercise is beneficial for overall brain health, with compelling evidence supporting increased neural plasticity and adaptability, especially in response to stress (Novak et al, 2012; Pietropaolo et al, 2008). For example, extended exercise promoted habituation of the hypothalamic-pituitary-adrenal axis response to stress (Greenwood et al, 2007; Nyhius et al, 2010; Sasse et al, 2008) and protected against hippocampal cell death elicited by a toxic insult following repeated stress (Kim et al, 2011). Additional reports found that exercise may be even more beneficial under pathological situations. Exercise had a significant anti-depressant action in several animal models of depression (Bjornebekk et al, 2006; Duman et al, 2008; Sartori et al, 2011). It reduced the severity of kainic acid-induced seizures (Reiss et al, 2009), supporting current findings of a significant protective effect of voluntary wheel running on seizure susceptibility. The correlation analysis for control males suggested that running wheel activity appeared to reduce susceptibility to PTZ-induced seizures in non-ethanol exposed animals. At the present time, this effect was selective for male, but not female rats. This may be due to the smaller sample size available for females, but may also involve innate sex differences.

At the cellular level, these effects may involve increased neurogenesis (van Praag et al, 1999) and increased dendritic spine density (Stranahan et al, 2007), both of which may involve increases in the release of neurotrophins, such as brain-derived neurotrophic factor (Cotman and Berchtold, 2002; Duman et al, 2008; Neeper et al, 1995). These actions appear to be especially pronounced in the hippocampus (Bednarczyk et al, 2009; Farmer et al, 2004), which is a brain area involved in seizure expression and ethanol withdrawal. Voluntary wheel running altered expression of cognitive behaviors in an Alzheimer’s disease mouse model (Pietropaolo et al, 2008), suggested to result from running-induced increases in neuroplasticity. Additionally, extended exercise was found to promote mitochondrial health (Steiner et al, 2011), which also would provide general protection against challenges such as oxidative stress. Therefore, the beneficial effects of exercise on neuronal activity likely contribute to its observed benefits during chronic ethanol consumption and withdrawal.

Prior research has revealed important sex differences in responses to ethanol, likely involving both organizational and activational influences (see Devaud et al, 2003; Finn et al 2010 for review). Hypothalamic areas that are important for endocrine control of circadian rhythms are known to be sexually dimorphic. Additionally, it has been established that ovarian hormones also influence circadian rhythms (see Mong et al, 2011 for review). Thus, innate sex differences in regulation of circadian rhythms may play a role in the differential effects of the ethanol diet on changes in voluntary running during the rest phase.

Future studies are needed to determine the amount and timing of exercise required for effects observed in the present study. It is particularly intriguing to note that voluntary exercise altered acute ethanol clearance in control males but not females, and it will be interesting to further explore this finding. Furthermore, it will be important to test whether this beneficial action generalizes to other EW symptoms. Research has shown that exercise could have additional benefit to help reduce relapse, an important risk of EW, because of its innate rewarding action (Greenwood et al, 2011). In summary, these new findings show that voluntary exercise is protective for both female and male rats against the increased seizure susceptibility of EW. This adds further support to the suggested value of incorporating exercise in treatment of alcoholism and other addictions.

HIGHLIGHTS.

  1. Wheel running protected against ethanol withdrawal seizure sensitivity in both male and female rats.

  2. The ethanol diet altered wheel-running activity during both active and rest phases.

  3. There were sex differences in wheel running and ethanol withdrawal responses.

Acknowledgements

The excellent technical assistance of Jacob Wilson is gratefully acknowledged.

Footnotes

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