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The Antidepressant-like Effect of Physical Activity on a Voluntary Running Wheel

CUNHA, MAURICIO P.1; OLIVEIRA, ÁGATHA1; PAZINI, FRANCIS L.1; MACHADO, DANIELE G.1; BETTIO, LUIS E. B.1; BUDNI, JOSIANE1; AGUIAR, ADERBAL S. JR1; MARTINS, DANIEL F.2; SANTOS, ADAIR R. S.2; RODRIGUES, ANA LÚCIA S.1

Medicine & Science in Sports & Exercise: May 2013 - Volume 45 - Issue 5 - p 851–859
doi: 10.1249/MSS.0b013e31827b23e6
BASIC SCIENCES: Special Report

Purpose Physical activity is currently being considered an effective alternative in the treatment of depression. At the preclinical level, the voluntary running wheel is a useful method of increasing physical activity in rodents and induces an antidepressant-like effect in some behavioral paradigms.

Methods This study investigated the effect of physical activity on a voluntary running wheel in mice submitted to the forced swimming test (FST) and tail suspension test, two predictive tests of antidepressant properties. Moreover, the influence of the inhibition of serotonin and noradrenaline synthesis as well as the inhibition of protein kinase A (PKA) and calcium/calmodulin-dependent protein kinase II (CAMK-II) activity by pharmacological agents in the antidepressant-like action of physical activity was investigated.

Results Physical activity on a voluntary running wheel by 21 d produced a reduction in the immobility time in the FST and tail suspension test, without producing alteration on locomotor activity in the open-field test. The antidepressant-like effect in the FST elicited by physical activity lasted for 7 d after removal of the running wheel. The anti-immobility effect of physical activity was prevented by the pretreatment of mice with p-chlorophenylalanine methyl ester (100 mg·kg−1, i.p., once a day, for four consecutive days, inhibitor of serotonin synthesis), α-methyl-p-tyrosine (100 mg·kg−1, i.p., an inhibitor of noradrenaline and dopamine synthesis), H-89 (1 μg per site, i.c.v., a PKA inhibitor), and KN-62 (1 μg per site, i.c.v., a CAMK-II inhibitor).

Conclusions Taken together, these results first suggest that the effect of physical activity on the FST is dependent on either the increase in the bioavailability of monoamines in the synaptic cleft or an activation of intracellular signaling pathways mediated by PKA and CAMK-II.

1Department of Biochemistry, Center of Biological Sciences, Universidade Federal de Santa Catarina, Florianópolis, BRAZIL; and 2Department of Physiological Sciences, Center of Biological Sciences, Universidade Federal de Santa Catarina, Florianópolis, BRAZIL

Address for correspondence: Ana Lúcia S. Rodrigues, Ph.D., Department of Biochemistry, Center of Biological Sciences, Universidade Federal de Santa Catarina, 88040-900, Florianópolis, SC, Brazil; E-mail: alsrodri@gmail.com.

Submitted for publication May 2012.

Accepted for publication October 2012.

Depression is a disorder characterized by a broad range of symptoms, including altered mood and cognitive functions and recurrent thoughts of death or suicide. In contrast with the normal experiences of sadness, clinical depression is a chronic disease that can interfere significantly in the individual’s life quality. The World Health Organization ranks unipolar depression as the fourth most important cause of mortality and disability (23).

Many studies reveal important roles for monoaminergic systems in the pathophysiology and treatment of depression. Specifically, deficiencies in serotonin (5-HT) and noradrenaline (NA) metabolism have often been associated with clinical depression, supporting the role of these neurotransmitters in the pathogenesis of this disease (7).

Furthermore, the activation of cAMP signaling pathways may occur through 5-HT or NA receptors, including the β-adrenergic or the 5-HT7 receptors, which couple to stimulatory G protein (Gs). Antidepressants up-regulate Gs coupling to adenylate cyclase, which increases the intracellular cAMP levels, causing the activation of cAMP-dependent protein kinase (PKA) (3). Furthermore, other protein kinases are being associated with antidepressant properties, like calcium/calmodulin-dependent protein kinase II (CAMK-II) (32).

Although classical antidepressant therapies are beneficial, a sizeable number of patients remain resistant to their therapeutic effects (31). Thus, there is an urgent need to develop strategies for antidepressant treatment with fewer side effects. On the basis of an improved understanding of the neurobiology of depression, several novel nonpharmacological interventions are being proposed. Physical activity is defined as bodily movement produced by skeletal muscles that generate energy expenditures. Thus, the term encompasses physical activity–related activities, as daily activities correlated with self-care and home care and sports activities, among others (5), and is currently being proposed as an effective alternative in the treatment of depression. In humans, a reduced risk of depression was reported in individuals engaged in high activity (>2500 kcal·wk−1, 28% reduced risk) and moderate activity (1000–2499 kcal·wk−1, 17% reduced risk) as compared with those at low activity (25). At the preclinical level, the voluntary running wheel is a useful method of increasing physical activity in rodents. It induces an antidepressant-like effect in some behavioral paradigms, such as forced swimming test (FST), tail suspension test (TST), and learned helplessness (13), and is useful for investigating the mechanisms underlying the antidepressant effects of physical activity.

It has been shown that physical activity on the running wheel reduces levels of messenger ribonucleic acid coding for 5-HT1B autoreceptors and 5-HT transporter (16) and increases brain NA levels (12) in rats. Furthermore, free access to a running wheel up-regulates genes correlated with the signal transduction pathways, normally associated with antidepressant treatment, including CAMK-II and PKA genes in rats (22). Nevertheless, it is unclear how all these changes could contribute to the antidepressant effect elicited by physical activity on the running wheel.

Taking into account that few studies have been conducted to clarify the mechanisms underlying the antidepressant-like action elicited by physical activity in animal models, the present work aimed at extending the literature by investigating the involvement of the noradrenergic and serotonergic systems and coupled intracellular targets (PKA and CAMK-II pathways) in the effect of physical activity on the voluntary running wheel in the FST in mice using pharmacological inhibitors of the synthesis of serotonin and noradrenaline (p-chlorophenylalanine methyl ester [PCPA] and α-methyl-p-tyrosine [AMPT], respectively) as well as PKA and CAMK-II inhibitors (H-89 and KN-62, respectively).

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METHODS

Animals

Male Swiss mice (30–40 g) were used in the present study. Animals were obtained from the Central Biothery of Universidade Federal de Santa Catarina. They were housed in groups of three animals per plastic cage under controlled conditions of light (from 07:00 to 19:00 h) and temperature (21°C ± 1°C). Mice were allowed free access to standard laboratory food and tap water and to adapt to the laboratory environment for at least 1 wk before the behavioral studies. Animals were randomly distributed into specified experimental groups. All manipulations were carried out between 14:00 and 17:00 h, with each animal used only once. All procedures in this study were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the American College of Sports Medicine animal care and approved by the ethics committee of the institution. All efforts were made to minimize the animals’ suffering and to reduce the number of animals used in the experiments.

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Drugs

The following drugs were used: AMPT, PCPA, N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide (H-89), and 4-[2-[(5-isoquinolinyl-sulfonyl) methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl) propyl] phenyl ester (KN-62) (Sigma Chemical Co., St. Louis, MO). All compounds were dissolved in saline solution (NaCl 0.9%). AMPT and PCPA were administered by intraperitoneal route (10 mL·kg−1 body weight), whereas H-89 and KN-62 were administered by intracerebroventricular route.

Intracerebroventricular administration was performed under ether anesthesia as previously described (2). Briefly, a 0.4-mm external diameter hypodermic needle attached to a cannula, which was linked to a 25-μL Hamilton syringe, was inserted perpendicularly through the skull and no more than 2 mm into the brain of the mouse. A volume of 5 μL was then administered in the left lateral ventricle. The injection was given for 30 s, and the needle remained in place for another 30 s to avoid the reflux of the substances injected. The injection site was 1 mm to the right or left from the midpoint on a line drawn through to the anterior base of the ears. After completion of the experiments, all animals were decapitated, and their brains were examined freshly. Results from mice presenting incorrect injection or any sign of cerebral hemorrhage were excluded from the statistical analysis (overall less than 5% of the total animals used).

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Voluntary Running Wheel Paradigm

Mice in the physical activity group had free access to a running wheel (12.6 cm in diameter), which was attached to the side of the home cage and accessible through an opening in the wall of the cage, as adapted from Duman et al. (13). The control group (sedentary) had no access to a running wheels in the cages.

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Experimental Design

Effect of physical activity on the voluntary running wheel (14 or 21 d) in the FST, TST, or open-field test.

To investigate a possible antidepressant-like effect elicited by physical activity on the voluntary running wheel, mice were given free continual wheel access for 2 or 3 wk (independent groups). A further 24 h (without running wheel) was allowed to elapse before mice were tested in the FST, TST, or open-field test. The control group (sedentary) had no access to running wheels in the cages. The number of animals in this set of experiment was seven to nine animals per group.

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Effect of the running wheel removal on the effect of physical activity in the FST.

To investigate the effect of the running wheel removal on the antidepressant-like effect induced by physical activity, mice were given continual wheel access for 3 wk up until the time of behavioral testing. After 1 or 7 d of running wheel removal (independent groups), mice were tested in the FST or open-field test. The control group (sedentary) had no access to running wheels in the cages. In this research paradigm, seven to nine animals per group were used.

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Effect of PCPA on the physical activity–induced reduction in the immobility time in the FST.

To investigate a possible contribution of 5-HT system in the anti-immobility effect of physical activity on the voluntary running wheel in the FST, animals (sedentary or physical activity) were treated with PCPA (100 mg·kg−1, i.p., an inhibitor of 5-HT synthesis) or vehicle, once a day, for four consecutive days. Twenty-four hours after the last PCPA or saline injection, mice were tested in the FST or open-field test. For this protocol, animals were divided into four experimental groups as follows: (i) mice that did not have access to a running wheel and that were administered with vehicle (saline), (ii) mice that did not have access to a running wheel and that were administered with PCPA, (iii) mice that had free access to a running wheel (physically active) and that were administered with vehicle, and (iv) mice that had free access to a running wheel and that were administered with PCPA. In this experiment, seven to 10 animals per experimental group were used. The doses of the PCPA and the protocol used were based on the literature and previous studies from our laboratory (19,29).

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Effect of AMPT on the physical activity–induced reduction in the immobility time in the FST.

To test the hypothesis that the antidepressant-like effect of physical activity on the voluntary running wheel is mediated through an interaction with the noradrenergic system, animals (sedentary or physical activity) were treated with AMPT (100 mg·kg−1, i.p., an inhibitor of the enzyme tyrosine hydroxylase) or vehicle 4 h before the FST or open-field test. Behavioral tests were performed 24 h after running wheel removal. For this protocol, mice were divided into four groups: (i) mice that did not have access to a running wheel (sedentary) and that were administered with vehicle (saline), (ii) mice that did not have access to a running wheel and that were administered with AMPT, (iii) animals that had free access to a running wheel (physically active) and that were administered with vehicle (saline), and (iv) mice that had free access to a running wheel and that were administered with AMPT. The doses of AMPT and the protocol used were selected on the basis of the literature and previous results from our laboratory (19,20). In this experiment, eight to 11 animals per experimental group were used.

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Effect of H-89 or KN-62 on the physical activity–induced reduction in the immobility time in the FST.

In the experiments designed to verify the involvement of the PKA and CAMK-II in the mechanisms underlying the antidepressant-like effect of physical activity on the voluntary running wheel in the FST, mice were treated with H-89 (1 μg per site, i.c.v., a PKA inhibitor), KN-62 (1 μg per site, i.c.v., a CAMK-II inhibitor), or vehicle. Fifteen minutes later, the FST or open-field test was carried out. Moreover, 24 h before the behavioral tests, the running wheel was removed. The doses of the kinase inhibitors and the protocols used were selected on the basis of previous studies from our group (2). The experimental groups to develop these experiments are as follows: (i) mice did not have access to a running wheel and that were administered with vehicle (saline), (ii) mice that did not have access to a running wheel and that were administered with kinase inhibitors (KN-62 or H-89), (iii) mice that had free access to a running wheel (physically active) and that were administered with vehicle, and (iv) mice that had free access to a running wheel and which were administered with kinase inhibitors (KN-62 or H-89). In these experiments, eight to nine animals per experimental group were used.

In all experimental protocols, the FST, TST, or open-field test were performed in independent groups of animals.

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Behavioral Tests

Forced swimming test.

FST has been used as a model predictive of antidepressant-like properties (28). The test procedure was previously standardized and validated in our laboratory (2,19). Mice were individually forced to swim in an open cylindrical container (diameter, 10 cm; height, 25 cm), containing 19 cm of water (depth) at 25°C ± 1°C; the total duration of immobility was recorded during a 6-min period. Each mouse was judged to be immobile when it ceased struggling and remained floating motionless in the water, making only those movements necessary to keep its head above water in a slightly hunched but upright position, its nose above the water surface. A decrease in the duration of immobility is indicative of an antidepressant-like effect (28). After the initial 2–3 min of vigorous activity, the animals showed a period of immobility by floating with minimum movements. The mouse FST is sensitive to all classes of antidepressants and to nonpharmacological therapies, such as electroconvulsive shock and physical activity (13,28).

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Tail suspension test.

The TST has become one of the most widely used models for assessing antidepressant-like activity in mice. The test is based on the fact that animals subjected to the short-term inescapable stress of being suspended by their tail will develop an immobile posture. The total duration of immobility induced by tail suspension was measured according to the method described by Steru et al. (33). Briefly, mice both acoustically and visually isolated were suspended 50 cm above the floor by adhesive tape placed approximately 1 cm from the tip of the tail. Immobility time was recorded during a 6-min period. Mice were considered immobile only when they hung passively and completely motionless, and the immobility was recorded by observers blind to the drug treatment. Antidepressant treatments reduce the immobility time in the TST (19,33).

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Open-field test.

To assess the possible effects of physical activity on the voluntary running wheel on locomotor activity, mice were evaluated in the open-field paradigm as previously described (19). Mice were individually placed in a wooden box (40 × 60 × 50 cm) with the floor divided into 12 equal rectangles. The number of rectangles crossed by the animal with its four paws (crossing), rising of the front paws (rearing), and grooming were registered during a period of 6 min. The number of crossings was considered as indicative of locomotor activity, and the number of rearings was indicative of exploratory behavior. The behavior was recorded by an observer blind to the drug treatment. The floor of the open-field apparatus was cleaned between tests.

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Statistical Analysis.

Comparisons between experimental and control groups were performed by one-way or two-way ANOVA followed by the Tukey HSD test when appropriate. A value of P < 0.05 was considered to be significant.

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RESULTS

Effect of physical activity on the voluntary running wheel (14–21 d) on immobility time in the FST, TST, and locomotor activity in the open-field test.

Figure 1A shows that mice submitted to physical activity for 14 d on the voluntary running wheel did not exhibit a reduction in the immobility time in the FST, but an antidepressant-like behavior in the FST was observed in an independent group of mice that was submitted for 21 d to the voluntary running wheel. No significant differences were observed in the control groups (sedentary mice). A two-way ANOVA revealed a main effect of physical activity (F 1,28 = 30.68, P < 0.001), period (F 1,28 = 5.16, P = 0.03) and physical activity–period interaction (F 1,28 = 4.47, P = 0.04).

FIGURE 1

FIGURE 1

Figure 1B shows that immobility time in the TST was reduced in mice, which had free access to a running wheel for 21 d as compared with the immobility time in the sedentary group. The one-way ANOVA revealed significant differences of physical activity intervention (F 1,13 = 5.48, P = 0.04). Moreover, free access to a running wheel for 21 or 14 d did not affect the number of crossings in the open-field test (Fig. 1C). A two-way ANOVA revealed no main effect of physical activity (F 1,32 = 3.98, P = 0.055), period (F 1,32 = 0.00055, P > 0.10), and physical activity–period interaction (F 1,32 = 0.00055, P > 0.10).

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Effects of running wheel removal on the effect of physical activity (21 d) in the FST and locomotor activity in the open-field test.

Figure 2A shows that mice submitted to physical activity for 21 d on the voluntary running wheel followed by 1 or 7 d running wheel removal exhibit an antidepressant-like behavior in the FST. A two-way ANOVA revealed a main effect of physical activity (F 1,31 = 37.15, P < 0.001), removal time (F 1,31 = 74.95, P < 0.001), and physical activity–removal time interaction (F 1,31 = 6.24, P = 0.01). The running wheel removal for 1 or 7 d did not affect the number of crossings in the open-field test (Fig. 2B). A two-way ANOVA revealed no main effect of time of wheel removal (F 1,27 = 8.96, P = 0.06), physical activity treatment (F 1,27 = 0.52, P > 0.10), and physical activity–time of wheel removal interaction (F 1,27 = 0.40, P > 0.10).

FIGURE 2

FIGURE 2

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Effect of treatment of mice with PCPA or AMPT on physical activity–induced anti-immobility effect in the FST and locomotor activity in the open-field test.

Figure 3A shows that the treatment of mice with PCPA (100 mg·kg, i.p., once a day, for four consecutive days) was able to reverse the antidepressant-like effect in the FST produced by 21 d of physical activity. A two-way ANOVA revealed a significant main effect of physical activity (F 1,35 = 5.06, P = 0.02) and physical activity–PCPA treatment interaction (F 1,35 = 13.86, P < 0.001), but not of PCPA treatment (F 1,35 = 13.86, P = 0.07). The administration of PCPA in sedentary mice or in mice that had free access to a running wheel did not affect the number of crossings in the open-field test (Fig. 3C). A two-way ANOVA revealed no significant differences of physical activity (F 1,34 = 0.42, P > 0.10), PCPA treatment (F 1,34 = 0.11, P > 0.10), and physical activity–PCPA treatment interaction (F 1,34 = 0.64, P > 0.10).

FIGURE 3

FIGURE 3

Figure 3B shows that the treatment of mice with AMPT (100 mg·kg−1, i.p., 4 h before of the behavior analysis) was able to abolish the antidepressant-like effect in the FST elicited by 21 d of physical activity on the running wheel. A two-way ANOVA revealed significant differences of physical activity (F 1,27 = 21.74, P < 0.001) and physical activity–AMPT treatment interaction (F 1,27 = 11.60, P = 0.002), but not of AMPT treatment (F 1,27 = 1.38, P > 0.10). The administration of AMPT did not affect the number of crossings in the open-field test (Fig. 3D) in sedentary mice or in mice that had free access to a running wheel. A two-way ANOVA revealed no significant differences of physical activity (F 1,30 = 0.42, P > 0.10), AMPT treatment (F 1,30 = 1.08, P > 0.10), and physical activity–AMPT treatment interaction (F 1,30 = 0.83, P > 0.10).

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Effect of treatment of mice with H-89 or KN-62 on physical activity–induced reduction in the immobility time in the FST and locomotor activity in the open-field test.

Figure 4A shows that the treatment of mice with H-89 (1 μg per site, i.c.v., 15 min before the behavioral analysis) was able to abolish the antidepressant-like effect in the FST elicited by 21 d of physical activity. A two-way ANOVA revealed significant differences of physical activity (F 1,28 = 41.34, P < 0.001) and H-89 treatment (F 1,27 = 5.08, P = 0.03) in addition to a significant physical activity–H-89 treatment interaction (F 1,28 = 6.73, P = 0.01). The administration of H-89 did not affect the number of crossings in the open-field test (Fig. 4C) in sedentary mice or in mice that had free access to a running wheel. A two-way ANOVA revealed no significant differences of physical activity (F 1,26 = 1.71, P > 0.10), H-89 treatment (F 1,26 = 0.10, P > 0.10), and physical activity–H-89 treatment interaction (F 1,26 = 1.71, P > 0.10).

FIGURE 4

FIGURE 4

Figure 4B shows that the treatment of mice with KN-62 (1μg per site, i.c.v., 15 min before the behavioral analysis) was able to abolish the antidepressant-like action in the FST produced by 21 d of physical activity. A two-way ANOVA revealed significant main effects of physical activity (F 1,27 = 7.10, P = 0.01) and physical activity–KN-62 treatment interaction (F 1,27 = 5.02, P = 0.03), but not KN-62 treatment (F 1,27 = 3.80, P = 0.06). The administration of KN-62 did not affect the number of crossings in the open-field test (Fig. 4D) in sedentary mice or in mice that had free access to a running wheel. A two-way ANOVA revealed no significant differences of physical activity (F 1,30 = 2.72, P > 0.10), KN-62 treatment (F 1,30 = 1.27, P > 0.10), and physical activity–KN-62 treatment interaction (F 1,30 = 0.08, P > 0.10).

It is worth noting that neither the physical activity in the voluntary running wheel nor its interaction with pharmacological agents altered the number of rearings and groomings in the open-field test (data not shown).

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DISCUSSION

The behavioral effects elicited by physical activity in rodents have been widely investigated using two experimental paradigms: (i) a forced approach (using a treadmill or even forced swimming) (ii) and a voluntary intervention (free access to a voluntary running wheel). The physiological, morphological, and behavioral effects of voluntary physical activity do not seem to correlate with the effects of forced physical activity (18). The forced exercise on a treadmill, or even forced swimming, is associated with psychophysiological changes correlated with chronic stress, as adrenal hypertrophy, elevated basal corticosterone, and immunosuppression, whereas voluntary physical activity does not seem to produce changes in the levels of corticotrophin releasing hormone, adrenocorticotropic hormone, and plasma corticosterone (18). This study was aimed at investigating the antidepressant-like action elicited by the experimental paradigm of voluntary physical activity in the running wheel because this intervention cause a smaller change in the hypothalamic–pituitary–adrenal axis.

The present study clearly shows that voluntary physical activity in the running wheel produced a reduction in the immobility time in the TST and FST, an effect consistent with an antidepressant profile. These results are in line with several studies that have indicated that physical activity produces antidepressant-like effects in mice in animal models, including FST and TST (13). Furthermore, physical activity may enhance mood in healthy (30) and depressive individuals (10).

The behavioral antidepressant profile in learned helplessness, but not in the FST, elicited by different periods of free access to the voluntary running wheel has been demonstrated. A study by Greenwood et al. (15) showed that 6 wk, but not 3 wk, of free access to a running wheel prevented the deficit in shuttle box escape. In addition, the same group demonstrated that 6 wk, but not 2 wk, of access to a running wheel reversed a modified version of learned helplessness (17). In the present study, we show for the first time that physical activity on the voluntary running wheel by 21 d, but not 14 d, is effective in reducing the immobility time in the FST and TST, an indicative of an antidepressant-like effect. FST and TST are widely used to assess the antidepressant properties of new antidepressant interventions, as they are sensitive to all major classes of antidepressant drugs, including tricyclics, selective 5-HT reuptake inhibitors, monoamine oxidase inhibitors, and atypicals (28,33). Also, some works have demonstrated that nonpharmacological strategies, including physical activity and exercise, are detectable in these predictive tests (13,35). However, FST and TST have some limitations. One of these is the short duration of drug treatment required to produce an antidepressant effect in these tests. In mice, a single dose of antidepressant treatment is sufficient to reduce immobility time in FST and TST, whereas in patients, it may take several weeks before the effects of antidepressants become apparent (14). Moreover, pharmacological or nonpharmacological interventions that enhance locomotor activity may give a false-positive effect in these tests. In our study, the reduction in the immobility time elicited by the voluntary running wheel cannot be attributable to a psychostimulant action of this intervention. This conclusion derives from the fact that physical activity in the voluntary running wheel did not alter the locomotor activity of mice in the open-field test. Despite the limitations mentioned earlier, the FST and the TST are currently the most widely used preclinical tests of antidepressant efficacy (28,33).

The antidepressant-like effect elicited by physical activity in the voluntary running wheel is well established. Herein, we show that this effect remains after 7 d delay after the physical activity period.

The neurotransmitter NA has long been implicated both in the pathogenesis of depression and in the mechanism of action of antidepressant medications (7). The NA depletion paradigm has been successfully used to investigate the mechanism of action of antidepressants in preclinical and clinical studies (7,19,21). Studies reported the ability of the selective inhibitor of tyrosine hydroxylase (the rate-limiting enzyme in the synthesis of NA and dopamine) AMPT to cause a relapse of depression in depressed patients currently treated with a noradrenaline reuptake inhibitor, but not with patients treated with selective 5-HT reuptake inhibitors (21). Other studies show that AMPT had very little effect on mood in normal subjects but, when given to depressed patients in remission, produced a return of depressive symptoms (7). Of note is a study by Mayorga et al. (20), which showed that AMPT, at the same administration protocol used in our study, reduces dopamine and NA levels (57% and 53%, respectively) in mice, without affecting 5-HT levels. In addition, the acute administration of AMPT temporarily reversed the antidepressant response to desipramine, mazindol, and mirtazapine in patients (21). Moreover, our group has reported that the administration of AMPT abolished the antidepressant-like action of antidepressant compounds in the TST in mice (19). In the present work, the pretreatment of mice with AMPT was able to prevent the anti-immobility effect of physical activity in the voluntary running wheel in the FST, suggesting that its effect in the FST may be dependent on the bioavailability of NA and/or DA in the synaptic cleft. In line with this, physical activity in the voluntary running wheel confers resistance to learned helplessness, another paradigm for antidepressant activity screening, associated with the increase of NA concentrations in locus caeruleus and dorsal raphe nuclei (12).

5-HT depletion is a widely used paradigm to study 5-HT system-related mechanisms in the pathophysiology and treatment of depression in animal models. In the present study, the involvement of the 5-HT system in the antidepressant-like effect of physical activity in the FST was investigated by PCPA pretreatment. PCPA, a tryptophan hydroxylase inhibitor, at same dose used in the present study produced partial but highly significant reductions on brain 5-HT levels (∼60%), without affecting NA and DA levels in mice (29). PCPA was also reported to reduce serotonin levels (93.1%) in rats (26). Therefore, the reversal of the anti-immobility effect of physical activity by the treatment of mice with PCPA suggests that its effect in the FST may be dependent on the availability of 5-HT in the synaptic cleft, somewhat in agreement with a study that indicates that physical activity increases 5-HT release and metabolism (6). Moreover, other studies have shown that PCPA on its own did not alter the immobility time of control animals but significantly blocked the anti-immobility effect of fluoxetine, a 5-HT reuptake inhibitor, leaving the antidepressant action caused by imipramine unaffected (26). Noteworthy, PCPA treatment did not produce reduction in running behavior during the 4 d of injections (data not shown). Reinforcing the notion presented herein that the increase of 5-HT levels in the synaptic cleft could be an important target in the antidepressant-like effect of physical activity in mice that had free access to a running wheel, a study showed that voluntary running wheel decreased 5-HT transporter and 5-HT1B autoreceptors messenger ribonucleic acid in the dorsal and median raphe nuclei (16).

In line with the notion that 5-HT and NA availability is essential for the antidepressant-like effect elicited by physical activity, it was reported that 5-HT and NA lesions suppressed the enhancing effect of physical activity in the voluntary running wheel in another behavioral paradigm. Physical activity in the voluntary running wheel during pregnancy was able to enhance learning and memory in rat pups, an effect blocked by 5-HT and NA lesions, suggesting that NA and 5-HT systems in offspring brain have a crucial role in mediating the effects of maternal physical activity in the cognitive function of rat pups (1).

Levels and activity of PKA are reported to be influenced by antidepressant treatment. Chronic but not acute treatment with antidepressants leads to the increase on cyclic adenosine monophosphate response element-binding (CREB) expression (24) and enhanced brain PKA activity (32). Furthermore, PKA activator elicited an antidepressant-like effect in the FST (4). The major mechanism of PKA-mediated function is through the phosphorylation of specific substrates, which includes CREB. The results presented herein show that the reduction of immobility time in the FST elicited by physical activity was partially blocked by the pretreatment of mice with H-89, an isoquinolinesulfonamide that acts as a competitive inhibitor against ATP for binding to the catalytic subunit of PKA (8), administered at a dose that per se produced no effect in the FST. This result suggests that the PKA signaling pathway is activated by 21 d of physical activity on the voluntary running wheel. Our results are in line with a study which reported that physical activity up-regulated 1.67-fold the PKA type I-alpha regulatory chain gene (22).

CAMK-II, the most abundant protein kinase in the brain (27), has been shown to be activated by long-term treatment with some antidepressant drugs, suggesting that the modulation of CAMK-II may also have important beneficial effects in the treatment of depressive disorders (32). The present study provides novel evidence that CAMK-II serves a refined role in mediating the antidepressant-like effect of physical activity on the voluntary running wheel because KN-62, an inhibitor that interacts with the regulatory domain of CAMK-II blocking the active form of this enzyme (34), completely abolished the antidepressant-like effect of physical activity in the FST. Our results are in agreement with the fact that physical activity on the voluntary running wheel up-regulated 2.5- to 3.0-fold the CAMK-II gene measured by microarray analysis (22). Also, physical activity on the voluntary running wheel was reported to increase the expression of p-CAMK-II (11). Moreover, KN-62 suppresses the enhancing of learning and memory induced by physical activity on the voluntary running wheel, suggesting that CAMK-II may have a crucial specific role in mediating the effects of physical activity in the cognitive function (36).

A study by Cinquanta et al. (9) reported that KN-62 significantly inhibited the [3H]5-HT release induced by D-fenfluramine in the hippocampal synaptosomes of rats, suggesting that the exocytotic-like 5-HT release induced by D-fenfluramine is dependent on Ca2+-influx with the subsequent activation of CAMK-II. Thus, we cannot rule out the possibility that the reversal of the anti-immobility effect of physical activity in the FST by the treatment of mice with KN-62 is dependent on a reduced 5-HT release, probably through the blockage of either Ca2+ influx or activation of CAMK-II. Also, we cannot rule out the possibility that intracellular signaling pathways mediated by other kinases as well as a cross talking between them may play a role in the mechanism underlying the antidepressant-like effect elicited by physical activity. Indeed, a role for PKA and CAMK-II signaling pathways in the antidepressant-like effect of the NMDA receptor antagonist memantine in the FST was reported. Interestingly, its anti-immobility effect in the FST was prevented by the pretreatment of mice with the same doses of H-89 and KN-62 used in the present study (2).

In conclusion, the present study provides evidence that physical activity on the voluntary running wheel for 21 d produces significant effects in the FST and TST, compatible with an antidepressant profile. In addition, we clearly show the involvement of the monoaminergic systems as well as intracellular signaling pathways mediated by PKA and CAMK-II in the effect of physical activity in the FST (Fig. 5). Thus, the present results suggest that physical activity is an effective approach to produce antidepressant-like effects whose mechanisms of action are similar to the ones produced by conventional antidepressants but devoid of the side effects produced by the classical therapy with these drugs.

FIGURE 5

FIGURE 5

M.P. Cunha, D.F. Martins, A.R.S. Santos, and A.L.S. Rodrigues designed the study, wrote the protocol, and wrote the manuscript. M.P. Cunha, D.F. Martins, and A.S. Aguiar Jr. built the cages containing the voluntary running wheel. J. Budni performed the intracerebroventricular administrations. M.P. Cunha, F.L. Pazini, A. Oliveira, J. Budni, L.E.B. Bettio, D.G. Machado, A.S. Aguiar Jr., and D.F. Martins performed the behavioral experiments. M.P. Cunha, F.L. Pazini, D.G. Machado, A. Oliveira, and L.E.B. Bettio undertook the statistical analysis. A.L.S. Rodrigues, J. Budni, A.S. Aguiar Jr., and A.R.S. Santos managed the literature searches and performed data and interpretation analysis. All authors contributed to and have approved the manuscript.

The present study was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Apoio à Pesquisa Científica Tecnológica do Estado de Santa Catarina (FAPESC), Rede Instituto Brasileiro de Neurociência (IBN-Net/CNPq), and Núcleo de Excelência em Neurociências Aplicadas de Santa Catarina (NENASC) Project/PRONEX Program CNPq/FAPESC, Brazil. M.P. Cunha, J. Budni, A.S. Aguiar Jr., L.E.B. Bettio, D.G. Machado, and D.F. Martins are Ph.D. students. A. Oliveira and F.L. Pazini are M.Sc. students. ALS Rodrigues and A.R.S. Santos are recipients of CNPq fellowship.

The results of the present study do not constitute endorsement by the American College of Sports Medicine.

The authors declare no conflict of interest.

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Keywords:

CAMK-II; FORCED SWIMMING TEST; NORADRENALINE; PKA; SEROTONIN

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