The effects of chronic treadmill and wheel running on behavior in rats

The effects of chronic treadmill and wheel running on behavior in rats

Brain Research 1019 (2004) 84 – 96 www.elsevier.com/locate/brainres Research report The effects of chronic treadmill and wheel running on behavior i...

346KB Sizes 0 Downloads 32 Views

Brain Research 1019 (2004) 84 – 96 www.elsevier.com/locate/brainres

Research report

The effects of chronic treadmill and wheel running on behavior in rats Paul R. Burghardt a,*, Laura J. Fulk b, Gregory A. Hand b, Marlene A. Wilson a a

Department of Pharmacology, Physiology and Neuroscience, University of South Carolina School of Medicine, Building 1 Room D26, Columbia, SC 29208, USA b Department of Exercise Science, University of South Carolina, Columbia, SC 29208, USA Accepted 23 May 2004 Available online 7 July 2004

Abstract In order to better understand the behavioral adaptations induced by physical activity, this set of experiments assessed the effects of two modes of running exercise on a battery of behavioral tests. The effects of 8 weeks of forced treadmill running and voluntary wheel running on behavior measures in the elevated plus maze, open field, social interaction and conditioned freezing paradigms were investigated. Eight weeks of treadmill running did not alter behavior in any test paradigm. Rats given unrestricted access to running wheels (WR) had a lower percent open arm time (6.0 F 2.3%) compared to locked wheel controls (LC) (20.7 F 5.7%) in the elevated plus maze. WR also showed decreased entries into center (0.2 F 0.2) and crossed fewer lines (61.0 F 14.9) in the open field compared to control groups. Both WR and LC groups showed increased social interaction; however, these differences are attributed to housing conditions. The effects of 4 weeks of wheel running on elevated plus maze and open field behavior were also investigated to address the possibility of a temporal effect of exercise on behavior. Four weeks of wheel running produced behavioral changes in the open field similar to those found at 8 weeks, but not in the elevated plus maze suggesting a temporal effect of wheel running on plus maze behavior. The behavioral adaptations found after 4 and 8 weeks of wheel running were not due solely to enriched environment and appear to be indicative of enhanced defensive behavior. D 2004 Elsevier B.V. All rights reserved. Theme: Neural basis of behavior Topic: Motivation and emotion Keywords: Exercise; Defensive behavior; Rat; Elevated plus maze; Open field; Social interaction; Conditioned freezing

1. Introduction Anecdotal reports and a variety of human studies support the concept that exercise can positively affect mood and modify neurochemical systems [5,15,17,18,38,41]. Numerous reports also indicate exercise-induced changes in behavior and neurochemical systems in rats (for reviews, see [10,15,35,49]). The interaction between neurochemical and behavioral changes induced by chronic physical activity, however, remains to be elucidated. Many of the changes seen in response to exercise have been reported in tests commonly used to assess anxiety-like or defensive responding behaviors in animals, including the open field, elevated plus maze, and social interaction tests [6,11,28]. For example, changes in anxiety-like behaviors * Corrresponding author. Tel.: +1-803-733-3196; fax: +1-803-7331523. E-mail address: [email protected] (P.R. Burghardt). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.05.086

have been seen after chronic treadmill training [23] and chronic wheel running [11], but these studies utilized distinct paradigms to assess behavioral outcomes. Fulk et al. utilized the elevated plus maze and entries into the center of the open field to assess anxiety-related behaviors, while the latter study assessed anxiety-related behaviors using experimentor-assessed ratings of behavior in the open field test. The use of these two models yielded divergent changes in anxiety related behaviors following treadmill training [11,23]. Chronic wheel running also prevented maladaptive behavioral changes (shuttle box escape deficit and exaggerated conditioned fear) seen in sedentary rats [24]. The use of a battery of behavioral paradigms that include ethological measurements allow for a more sensitive investigation of environmental manipulation, and have the added benefit of convergent validity when significant effects are found in several paradigms [1,45]. In order to better understand the behavioral adaptations induced by chronic physical activity and, ultimately, the underlying neurochem-

P.R. Burghardt et al. / Brain Research 1019 (2004) 84–96

istry, this set of experiments assessed the effects of two modes of running exercise (treadmill and running wheel) in the same battery of four behavioral tests that examined activity, anxiety-related and defensive forms of behavior. The effects of 8 weeks of forced treadmill running (experiment 1) and voluntary wheel running (experiment 2) on behavioral measures in the elevated plus maze, open field, social interaction, and conditioned freezing paradigms, were assessed. To address the possible role of a time course for the effects of exercise on behavior, 4 weeks of voluntary wheel running (experiment 3) was also assessed in elevated plus maze and open field. For experiment 1, both treadmill control animals and sedentary control animals were added as comparison groups to account for possible environmental enrichment/handling effects inherent in the treadmill running procedure. For experiments 2 and 3, a set of animals housed with a locked running wheel and a set of group housed environmentally enriched animals were added to distinguish the effects of exercise from environmental enrichment/housing effects inherent in the running wheel procedure. The results of experiment 1 suggest that 8 weeks of treadmill running does not alter anxiety-like behavior/defensive responding. Eight weeks of wheel running (experiment 2) produced changes in the elevated plus maze and open field, which suggests that wheel running enhanced defensive responding. Four weeks of wheel running (experiment 3) produced behavioral changes in the open field similar to those found at 8 weeks; however, behavior in the elevated plus maze was not altered suggesting a temporal effect of wheel running in the elevated plus maze.

85

cycle. During the first week of training animals were acclimated to the treadmill by gradually increasing running speed and time each day, as follows: Day 1—10 min at 10 m/min, Day 2—15 min at 12 m/min, Day 3—20 min at 15 m/min, Day 4—30 min at 18 m/min, Day 5—35 min at 20 m/min. By the end of week 2, animals were running for 55 min a day at 20 m/min, with the first 10 min consisting of a 12 m/min ‘‘warm-up’’. Neither electric shock nor severe physical prodding were used in this study. Treadmill controls were placed on a nonmoving treadmill for 45 min, 5 days/week. Sedentary controls remained in their home cages throughout the experiment and were handled in the vivarium on running days (5 days/week). Animals in all three groups were pair housed during this experiment. 2.2.2. Experiment 2: running wheel (8 week) Rats were randomly divided into three groups upon arrival: wheel runners (WR: n = 10), locked wheel controls (LC: n = 10), and group housed enriched environment (GHEE: n = 14). Wheel runners were singly housed and had 24-h access to a running wheel; locked wheel controls were singly housed and had 24-h access to a cage setting identical to the wheel runners, but with a locked running wheel; and group housed enriched environment rats were housed in groups of 4– 5 with an assortment of toys (tea cups, PVC tubing, etc.) in larger cages (75  54  24cm). Animals were handled twice per week by the investigators during cage changing, and were weighed on the first cage changing day of each week. GHEE animals were included in this experiment to assess if the ‘‘enriched’’ environment, rather than physical activity, contributed to changes in behavior.

2. Materials and methods 2.1. Animals For all experiments, Male Sprague – Dawley rats (Harlan), weighing approximately 175 g upon arrival, were housed in an environmentally controlled animal facility on a 12:12 light/dark cycle with food (Purina rat chow) and water available ad libitum. Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) approved animal facility, and all procedures were approved by the University of South Carolina Animal Care and Use Committee. 2.2. Training 2.2.1. Experiment 1: treadmill Rats were randomly divided into three groups: treadmill runners (TR: n = 10), treadmill controls (TC: n = 10), and sedentary controls (SC: n = 6). Treadmill runners ran on a motorized treadmill with individual Plexiglas lanes for 45 min, 5 days/week at 20 m/min, 5% grade [23,25]. Running took place during the early light portion of the light/dark

2.2.3. Experiment 3: running wheel (4 week) Rats were randomly divided into three groups upon arrival: wheel runners (WR: n = 10), locked wheel controls (LC: n = 10), and group housed enriched environment (GHEE: n = 10). Animals were housed and handled identically to the 8 week running wheel experiment. 2.2.4. Running wheel equipment WR and LC animals were housed in standard polypropylene cages (45  28  20 cm) with a Nalgene running wheel system (Nalgene) that was either free turning or locked, respectively. Running data was determined using the Mini-mitter data collection system (Mini Mitter Company, OR) connected to a Windows based PC. For each WR animal, the distance run over a 24-h period was calculated by summing the number of turns per bin (lights on to lights on) and multiplying the total number of turns by the circumference (108 cm) of the running wheel. The total distance for each 24-h period was then averaged for the ten WR animals. For the 8-week running wheel study, the number of turns was collected in 2-h bins. During the 4week running wheel study turns were collected in 20-min

86

P.R. Burghardt et al. / Brain Research 1019 (2004) 84–96

bins to more clearly illustrate the running patterns of the rats. 2.3. Behavioral testing All behavioral testing took place during the first third of the light portion of the light/dark cycle. Testing during this time period was employed to permit comparison with previous studies using both wheel [24,48] and treadmill running [23,65] protocols. In addition, animals performed the vast majority of wheel running during the middle third of their dark cycle. After 8 weeks of activity in the treadmill and first running wheel experiment, animals began a battery of four behavioral tests. Behavioral testing began with the elevated plus maze and open field test during the first week, followed by social interaction and conditioned freezing in the subsequent two weeks. For the treadmill experiment animals were tested on Monday, 72 h after the last Friday running session. For Experiment 1, animals resumed their treadmill training schedule immediately after behavioral testing, and were trained Monday through Friday during the weeks of behavioral testing. For wheel running experiments (2 and 3) animals were immediately returned to their home cages after behavioral testing, and resumed freeaccess to running wheels for the subsequent weeks between all behavioral testing. Animals ran for a total of 12 weeks in Experiments 1 and 2, but only 4 weeks in Experiment 3. In order to minimize carryover effect in the test battery, the test of conditioned freezing was administered last. McIlwain et al [34] have shown that conditioned freezing is not affected by testing order in murine behavioral test batteries. This sequence of behavioral tests might have affected the observation of changes in behavior due to exercise in the later testing paradigms. This design does not change the interpretation of the results in the current set of experiments, which demonstrate behavioral changes in the first tests (plus maze and open field) used in the sequence.

entries, percent unprotected head dipping (unprotected/ (unprotected + protected)), and closed arm entries. An arm entry required that all four of the animal’s paws entered into the arm. An increase in open arm time is suggestive of a decrease in a rat’s defensive responding [26,54], and anxiolytic compounds are known to increase the amount of time in the open arms [42]. 2.4.2. Open field The open field apparatus consisted of a square, black Plexiglas box (76  76  46 cm) with the floor divided into 25 squares (15  15 cm). Animals were placed into the center of the open field immediately following 5 min in the elevated plus maze, and allowed to explore freely for 5 min. Animals were video taped to allow scoring by observers’ blind to the treatment condition of the animal (average interrater reliability for all variables = 95%). The behaviors examined included: entries into the center of the field, number of lines crossed in the center of the field, time spent in the center of the field, and rearing (standing on hind legs). Line crossings were recorded when all four paws of the rat crossed a line. The total number of lines crossed was determined as a measure of activity. Exploration behavior in the open field has also been used as a measure of defensive behavior, where increased line crossings and rearing are suggestive of a decrease in defensive behaviors [47,54,60]. 2.4.3. Social interaction One week after elevated plus maze and open-field testing, each rat was placed with a novel male partner of the same age in a 60  60  30 cm chamber. The animals were allowed to freely interact for 5 min while their behavior was videotaped for subsequent scoring of time spent in active social interaction. Time spent in social interaction was defined as any interaction where the animals Table 1 Effect of 8 weeks of treadmill training on behavioral test battery measures Runners

2.4. Behavioral tests 2.4.1. Elevated plus maze This test was conducted as described in Stock et al [54], as modified from Pellow et al. [42], and can also be used to assess changes in defensive behavior [26]. The black Plexiglas elevated plus maze consisted of two opposing open and two opposing closed arms in the shape of a cross, connected by a central square. The maze was elevated 50 cm above the ground and had a 0.5-cm edge on the open arms. Animals were placed in the center square facing an open arm and allowed to explore the maze for five minutes while their behavior was videotaped and scored later by two trained observers blind to the treatment condition of the animal (average inter-rater reliability for all variables = 98%). The behaviors examined included: percent open arm time (open/(open + closed)), percent open arm

Treadmill control

Handled control

Elevated plus maze % Open arm time % Open arm entries Closed arm entries

11.3 F 2.8 18.3 F 4.6 11.7 F 1.0

16.1 F 4.1 21.6 F 5.1 9.7 F 1.0

14.8 F 5.3 23.0 F 8.0 8.0 F 2.0

Open field Total lines crossed Entries into center Time in center Center lines crossed Rears

113.2 F 12.4 1.3 F 0.8 7.1 F 1.7 1.2 F 0.9 9.9 F 5.1

81.7 F 13.8 1.2 F 0.6 6.8 F 1.7 1.9 F 1.3 13.1 F 7.8

94.2 F 19.7 0.5 F 0.3 6.2 F 1.1 0.8 F 0.7 10.7 F 4.9

50.5 F 8.6

41.9 F 3.5

35.5 F 3.5

506.1 F 28.6

524.5 F 24.4

448.3 F 44.6

Social interaction Total time Conditioned freezing Freezing

Values are reported as mean F SEM.

P.R. Burghardt et al. / Brain Research 1019 (2004) 84–96

were in contact or active pursuit. Decreased social interaction is indicative of increased anxiety/decreased defensive responding [7,20,45]. 2.4.4. Conditioned freezing Conditioned freezing was adapted from the method used by Primeaux and Holmes [44] and Petrovich et al. [43]. One week after the social interaction test, the animals were placed in a 46  24  22 cm shock box equipped with stainless steel floor rods (6.3 mm diameter) spaced 1.9 cm apart. The shock box was placed inside an isolation chamber equipped with ventilating fans to dampen external acoustic stimuli that might initiate freezing. Animals were placed in the box and allowed to explore for 4 min, and a scrambled shock was delivered to the floor grid by a solid state shock source (Coulborn Instruments, Allentown, PA). Animals were conditioned with three context-footshock pairings of 1 mA for a duration of 1 s per shock. The three shocks were delivered 20 s apart during the fifth minute of exposure to

87

the shock box. Rats were then removed from the context and placed back into their home cage. Twenty-four hours after the conditioning session, the animals were returned to the context and videotaped for 10 min in order to assess the amount of time spent freezing. Freezing behavior was defined as the lack of any movement except that related to respiration and was scored continuously with a stop-watch by reviewing videotapes. 2.5. Statistical analysis Behavioral measures, body weights, and running distance (Experiments 2 and 3) were compared using a oneway analysis of variance (ANOVA) with significance set at p < 0.05. A Newman – Keuls post-hoc test was used to determine differences between groups when a significant main effect was found. Experiment 1 compared TR, TC and SC groups, while experiments 2 and 3 compared WR, LC, and GHEE groups.

Fig. 1. Elevated plus maze. Each bar represents mean F S.E.M of 6 – 14 rats per group. Graphs represent the percent time spent in the open arms of the elevated plus maze for the treadmill experiment (A), 8-week running wheel experiment (B), and 4-week running wheel experiment (C). Graphs represent the number of closed arm entries executed during 5 min in the elevated plus maze for the treadmill experiment (D), 8 week running wheel experiment (E), and 4 week running wheel experiment (F). For the treadmill experiment groups consisted of treadmill runners (TR), treadmill controls (TC), and sedentary controls (SC). For wheel running experiments groups consisted of wheel runners (WR), locked wheel controls (LC), and group housed enriched environment (GHEE). Connecting line indicates significant difference from other group, p < 0.05.

88

P.R. Burghardt et al. / Brain Research 1019 (2004) 84–96

3. Results 3.1. Experiment 1: treadmill running In this set of experiments, behavior in several testing paradigms was compared among the groups of treadmill runners (TR: n = 10), treadmill controls (TC: n = 10), and sedentary controls (SC: n = 6). After 8 weeks of treadmill training, measures of exploratory/risk assessment, anxietylike behavior, and indicators of activity were determined in the elevated plus maze, open field, the test of social interaction, and conditioned freezing. Results are summarized in Table 1 and Figs. 1 –4. Chronic treadmill running failed to significantly affect anxiety-like/defensive responding behaviors in the four behavioral paradigms utilized in these experiments. Open arm behavior in the elevated plus maze was similar between groups. One-way ANOVA found no significant main effects

for percent open arm time ( F(2,23) = 0.16, Fig. 1A) or percent open arm entries ( F(2,23) = 0.18) in the elevated plus maze. No significant main effects were found in the open field for anxiety-like behaviors (see Table 1, Fig. 2A). Chronic treadmill training had no effect on the entries into the center of the open field ( F(2,23) = 0.31), time in the center of the open field ( F(2,23) = 0.10), or the number of center lines crossed ( F(2,23) = 0.25). There was also no difference found for rearing behavior between groups ( F(2,23) = 0.64). No significant main effects were found for the amount of time spent in social interaction ( F(2,23) = 1.26) or the amount of time spent freezing in the test of conditioned freezing ( F(2,23) = 1.43) (Table 1, Figs. 3A and 4A, respectively). Thus, chronic treadmill training failed to alter anxiety-like/ defensive responding behaviors in the four behavioral paradigms tested. Despite 8 weeks of moderate treadmill training, activityrelated parameters in these behavioral tests were comparable

Fig. 2. Open field. Each bar represents mean F S.E.M of 6 – 14 rats per group. Graphs represent the amount of time spent (in s) in the center of the open field for the treadmill experiment (A), 8-week running wheel experiment (B), and 4-week running wheel experiment (C). Graphs represent the number of lines crossed during five minutes in the open field for the treadmill experiment (D), 8-week running wheel experiment (E), and 4-week running wheel experiment (F). For the treadmill experiment groups consisted of treadmill runners (TR), treadmill controls (TC), and sedentary controls (SC). For wheel running experiments groups consisted of wheel runners (WR), locked wheel controls (LC), and group housed enriched environment (GHEE). * Indicates significant difference from other two groups, p < 0.05. Connecting line indicates significant difference from other group, p < 0.05.

P.R. Burghardt et al. / Brain Research 1019 (2004) 84–96

89

Fig. 3. Social interaction. Each bar represents mean ( F S.E.M. of 6 – 14 rats per group) time spent in social interaction during a 5-min social interaction test for animals in the treadmill experiment (A) and 8-week running wheel experiment (B). * Indicates significant difference from other two groups, p < 0.05.

in TR, TC and SC groups. No significant effect of training on the number of closed arm entries was found in the elevated plus maze ( F(2,23) = 1.98, Fig. 1D). There was no significant main effect of training on the total number of lines crossed in the open field ( F(2,23) = 1.36, Fig. 2D). Thus, no significant effects of chronic treadmill training were found on activity measures in either the elevated plus maze or the open field. No differences were found among groups for the final body weight over the training period ( F(2, 23) = 1.61, p = 0.22). Final body weights were similar in TR (389 F 19 g), TC (383 F 17 g) and SC (400 F 19 g) groups. 3.2. Experiment 2: running wheel (8 week) In this set of experiments animals were compared in the elevated plus maze, the open field, the test of social interaction, and conditioned freezing after eight weeks of unrestricted access to running wheels (WR, n = 10), having a locked wheel in their home cage (LC, n = 10), or being group housed in an enriched environment (GHEE, n = 14). Results are summarized in Table 2 and Figs. 1– 4. After 8 weeks animals with unrestricted access to running wheels had increased anxiety-like behaviors/defensive responding in the elevated plus maze and open field. A significant main effect was found for the percent open arm time in the elevated plus maze ( F(2,30) = 3.53, p < 0.05, Fig. 1B). Post hoc analysis revealed that wheel runners spent a smaller percentage of time in the open arms compared to animals with locked wheels ( p < 0.05). A trend existed ( p = 0.0578) for percent open arm entries, with wheel runners having less open arm entries compared to locked wheel animals. Significant main effects were also found for anxiety-like/ defensive responding behaviors in the open field. A significant main effect was found for the number of entries into the center of the field ( F(2,29) = 5.08, p < 0.05), with wheel running animals making fewer entries into the center of the field compared to locked and GHEE animals. A significant effect of training was found for the number of inner lines crossed in the open field ( F(2,29) = 4.68, p < 0.05), with fewer inner lines being crossed by wheel running animals compared to LC and GHEE groups. A significant main

effect was found for the amount of time spent in the center of the open field ( F(2,29) = 7.09, p < 0.05). Post hoc analysis determined that LC animals spent more time in the center of the field compared to wheel running and GHEE animals. A significant main effect of group was found for the number of rears in the open field ( F(2,29) = 3.97, p < 0.05), with wheel running animals performing fewer rears compared to locked animals. These groups failed to show any differences in time spent freezing ( F(2,31) = 1.60) in the conditioned freezing paradigm. A difference was also found for the amount of time spent in social interaction between groups. This effect, however, appeared to be a result of housing conditions rather than wheel running. A significant difference was found for the amount of time spent in social interaction ( F(2,29) = 15.23, p>0.05) between the three groups, with post hoc analysis indicating that GHEE animals spent less time in social interaction compared to WR and LC animals. Table 2 Effect of 8 weeks of wheel running on behavioral test battery measures Wheel runners Elevated plus maze Percent open arm time Percent open arm entries Percent unprotected head dips Closed arm entries

Locked control

Group housed enriched environment

6.0 F 2.3+ 9.6 F 2.7 20.7 F 7.1*

20.1 F 5.7+ 23.0 F 5.7 51.2 F 10.3

14.6 F 2.4 17.9 F 1.9 43.0 F 2.5

8.4 F 1.2+

10.1 F 0.8

12.2 F 0.6+

Open field Total lines crossed Entries into center Time in center Center lines crossed Rears

61.0 F 14.9* 0.2 F 0.2* 0.4 F 0.4 0.4 F 0.4* 5.3 F 1.6+

125.2 F + 9.7 2.0 F 0.5 6.6 F 1.8* 3.4 F 1.0 13.7 F 2.9+

Social interaction Total time

65.2 F 10.2

77.3 F 9.6

27.7 F 2.2*

476.7 F 30.5

424.9 F 16.2

467.6 F 15.9

Conditioned freezing Freezing

108.6 F 9.1 1.5 F 0.3 3.0 F 0.7 3.2 F 0.6 10.6 F 1.6

Values are reported as mean F SEM. * Indicates a difference from the two groups ( p < 0.05). +Indicates a difference between corresponding group with + ( p < 0.05), but no difference with the unlabeled group.

90

P.R. Burghardt et al. / Brain Research 1019 (2004) 84–96

Fig. 4. Conditioned freezing. Each bar represents mean ( F S.E.M. of 6 – 14 rats per group) time spent freezing during a ten minute conditioned freezing test for the treadmill experiment (A), and 8 week running wheel experiment (B).

In summary, 8 weeks of wheel running significantly increased anxiety-like behavior/defensive responding in the elevated plus maze and open field, while exposure to group housing and enriched environment significantly decreased the amount of time spent in social interaction. Freezing behavior was not affected by any treatment. Unlike 8 weeks of treadmill training, animals given access to running wheels for 8 weeks had lower levels of activity in the elevated plus maze and open field. A significant main effect was found for the number of closed arm entries in the elevated plus maze ( F(2,30) = 4.99, p < 0.05), and post hoc analysis revealed that wheel running animals made fewer closed arm entries compared to GHEE animals. A significant main effect was also found for the total number of lines crossed in the open field ( F(2,29) =7.97, p < 0.05). Post hoc analysis revealed that WR animals crossed fewer total lines compared to both LW animals and GHEE. Eight weeks of chronic wheel running inhibited weight gain, as indicated by lower final body weight ( F(2, 31) = 8.19, p < 0.002) in WR animals (353 F 27.6 g)

compared to both GHEE and LC (379 F 21.9 and 401 F 30.0 g, respectively). 3.3. Experiment 3: running wheel (4 weeks) An additional set of WR (n = 10), LC (n = 10), and GHEE (n = 10) animals were compared in the elevated plus maze and the open field at a four week time point to determine if the behavioral changes seen in experiment 2 required extended (8 weeks) wheel running. These two behavioral paradigms were chosen since effects of running were seen in these tests after 8 weeks. The 4-week time point was selected since it was the earliest time point at which animals were running distances similar to the 8-week time point, and was half of the amount of training time used in experiment 2 (see Fig. 5). Behavioral results are summarized in Table 3 and Figs. 1– 2. Unrestricted access to running wheels for four weeks failed to affect anxiety-like/defensive responding behaviors in the elevated plus maze (Table 3). No significant main effects were found for the percent open arm time in the

Fig. 5. Running wheel distance from experiment 2, eight weeks of running wheel access (diamond marker, solid line), and experiment 3, four weeks of running wheel access (open square marker, dashed line). Average distance run in the freely turning running wheels in a 24-h period per week. Error bars represent SEM.

P.R. Burghardt et al. / Brain Research 1019 (2004) 84–96 Table 3 Effect of 4 weeks of wheel running on elevated plus maze and open field behavior Wheel runners Elevated plus maze % Open arm time % Open arm entries % Unprotected head dips Closed arm entries Open field Total lines crossed Entries into center Time in center Center lines crossed Rears

Locked control

Group house enriched environment

9.6 F 2.0 8.7 F 2.8 32.2 F 5.5

14.6 F 3.3 21.7 F 2.3 48.3 F 5.0

21.1 F 5.7 24.4 F 5.5 37.0 F 8.4

9.8 F 1.0

9.9 F 1.4

11.9 F 0.6

66.7 F 9.9* 0.2 F 0.1* 1.6 F 1.3* 0.4 F 0.3 5.9 F 0.9*

124.6 F 12.0 2.2 F 0.6 8.3 F 2.8 4.7 F 1.3 13.2 F 2.4

111.7 F 9.2 3.2 F 0.8 9.8 F 4.0 9.1 F 2.2 11.0 F 1.5

Values are reported as mean F SEM. * Indicates a difference from the other two groups ( p < 0.05). +Indicates a difference between corresponding group with + ( p < 0.05), but no difference with the unlabeled group.

elevated plus maze ( F(2,27) = 1.53, Fig. 1C) or percent open arm entries ( F(2,27) = 0.14) after 4 weeks of access to running wheels. Unrestricted access to running wheels for four weeks produced changes in anxiety-like behaviors/defensive responding in the open field similar to those seen in the eight-week running wheel experiment. A significant main effect was found for the number of entries into the center of the field ( F(2,26) = 6.84, p < 0.0001), with WR animals making fewer entries into the center of the field compared to LC and GHEE animals. A significant effect of training was found for the number of inner lines crossed in the open field ( F(2,26) = 7.54, p < 0.003), with fewer inner lines crossed by WR animals compared to LC and GHEE. The amount of time spent in the center of the open field ( F(2,26) = 5.11, p < 0.0001, Fig. 2C) differed among groups. Post hoc analysis determined that LC animals spent more time in the center of the field compared to WR and GHEE animals. A main effect was also found for the number of rears in the open field ( F(2,26) = 8.21, p < 0.002), with WR animals performing fewer rears compared to LC animals. These results suggest that 4 and 8 week access to running wheels differentially influences anxiety-like behaviors/defensive responding in the elevated plus maze, whereas four weeks of unrestricted access to the wheels is sufficient to produce changes in open area behavior in the open field. Four weeks of wheel running resulted in a lower final body weight ( F(2,27) = 10.13, p < 0.0005) in WR animals (287 F 17.4 g) compared to both GHEE and LC (304 F 8.20 and 312 F 9.86 g, respectively). Unlike 8 weeks of wheel running, four weeks of wheel running failed to decrease activity in the elevated plus maze as indicated by the number of closed arm entries ( F(2,27) = 1.55, Fig. 1F). A significant main effect was found for the total number of lines crossed in the open field

91

( F(2,26) = 15.19, p < 0.0001, Fig. 2F). Post hoc analysis revealed that WR and GHEE animals crossed fewer total lines compared to LC animals. As with anxiety-like behaviors in the elevated plus maze, 4 weeks of wheel running failed to alter activity measures in the elevated plus maze, but did decrease total activity in the open field. 3.4. Running distance and work Since the distance run by the rats in the treadmill experiment (1.02 km/day from week 2 on) was significantly less than that covered by the rats allowed free access to running wheels in experiments 2 and 3 (see Fig. 5) the total work performed by rats in each mode of exercise in a 24-h period was determined for the week preceding the start of behavioral testing. Rats running on the treadmill in experiment 1 ran for 55 min a day (10 min at 12 m/min and the remaining 45 min at 20 m/min) at a 5% incline. Work was calculated as follows for the treadmill experiment [Work = Force (N)  distance (m)] where force is the weight of the animal times the distance [Distance = velocity (m/min)  time (min)  0.05]. Total distance was calculated as the sum of the first 10 min at 12 m/min plus the remaining 45 min at 20 m/min. For experiment 1, the average amount of work performed by TR rats during each running session the week prior to testing was 172 F 2.6 J (Joule). For experiment 2 (wheel running), animals ran an average of 5.25 F 1.1 km/24 h/week (mean F SEM) during the 8-week running wheel period. A peak distance of 8.02 F 0.96 km/24 h/week occurred during the sixth week of running (Fig. 5). A good deal of individual variability existed for the distance run between rats at a given time point (3.47 – 14.0 km/24 h period during week 6). In Experiment 3, rats given access to running wheels for four weeks exhibited a running pattern similar to that of the 8week WR rats (Fig. 5); running an average distance of 3.28 F 0.65 km/24 h/week. The 4-week WR rats also showed similar levels of individual variability compared to the animals in experiment 2 (1.14 –12.8 km/24 h period during the fourth week). No difference was found ( F(2,27) = 0.14) between the weekly distance run per 24 h for animals in experiment 3 during week 4 (5.81 F 1.25 km/24 h/week), and animals in experiment 2 during week 4 (6.67 F 1.23 km/24 h/week) or week 8 (6.36 F 1.04 km/24 h/week). This indicates that animals in experiments 2 and 3 were running similar distances at weeks 8 and 4 when they began behavioral testing. The running wheels in experiments 2 and 3 did not have any external resistance placed on them. A mass of 6 g was required to overcome the inertia of the wheel, and therefore work was calculated [Work = force (N)  distance (m)] as previously described [29]. With distance determined by multiplying the number of revolutions in a 24-h period by the circumference of the wheel, and force determined by the 6 g mass required to overcome the inertia of the wheel. For experiments 2 and 3 the average amount of work performed

92

P.R. Burghardt et al. / Brain Research 1019 (2004) 84–96

by WR rats the week prior to testing was 375 F 61 and 342 F 74 J, respectively. Therefore in a 24-h period (mainly during the middle third of the dark phase of the light/dark cycle) rats with free access to running in experiments 2 and 3 performed approximately twice as much work as the rats running on the treadmill in experiment 1. It is notable that the vast majority of work performed by WR animals in experiments 2 and 3 was completed over a significantly longer period of time (3 –4 h), compared to the running duration of TR animals in experiment 1, and it is possible that the disparity between TR and WR are related to differences in relative intensity or duration of the training paradigms.

4. Discussion It is generally believed that exercise has mood-enhancing effects [5,15,17,18,38,41], as well as being advantageous for other CNS responses including cognition [9,58]. These effects have been suggested to occur in humans, and in some cases in animals [9,12,48,58], although conflicting effects of exercise are seen in many studies. These discrepancies may be due to several factors, including the use of forced or voluntary exercise protocols, the duration and severity of physical activity employed and the behavioral paradigms used to assess responses. Several behavioral paradigms are available to test anxiety like/defensive behaviors [1,33], and the use of behavioral test batteries that include several paradigms comprised of more ethological measures are gaining favor in terms of mimicking human behavioral pathologies [1,45]. The present study addressed several questions, including assessing if the effects of chronic voluntary and chronic forced physical activity were similar and if distinct changes are seen in different animal models of anxiety-like behaviors/defensive responding. The results of experiment 1 suggest that 8 weeks of treadmill running does not alter anxiety-like behavior or defensive responding in the elevated plus maze, open field, social interaction or conditioned freezing paradigms. Eight weeks of wheel running (experiment 2) produced changes in the elevated plus maze and open field behaviors. Four weeks of wheel running (experiment 3) produced behavioral changes in the open field similar to those found at 8 weeks, however, behavior in the elevated plus maze was not altered. This suggests that prolonged access to wheel running is necessary to alter behavior in the elevated plus maze. In contrast to the predicted effects, however, chronic wheel running induced decreased open arm time in the plus maze and center time in the open field. Although these changes are consistent with increased anxiety-like behaviors, this interpretation is structured around the analysis of pharmacological effects in these behavioral paradigms [45]. An alternative interpretation is that shifts in defensive responding and risk assessment behaviors occur in response to chronic access to running wheels [47,54,60]. This is sup-

ported by indications that runners show changes in several measures of defensive responding, including unprotected head dipping in the elevated plus maze (Table 2) and entries into the center of the field (Tables 2 and 3). Animals with access to freely turning wheels were also more aggressive during handling (biting and struggling) compared to LC and GHEE groups. 4.1. Treadmill running Since training protocols have varied in past studies of treadmill exercise, and it was not known if behavioral adaptations to exercise occurred in a time dependent manner, an 8-week protocol was employed since longer periods of running are known to evoke neurochemical (for reviews, see Refs. [15,35,49]) and physiological adaptations [37]. Eight weeks of treadmill running failed to change the behavior of animals in any of the behavioral paradigms employed in this set of experiments. The findings of this study are in agreement with other studies that have investigated the effects of 4 days of treadmill running on behavior for the elevated plus maze and social interaction [6]. Fulk et al. [23] have shown that eight weeks of treadmill running increases the amount of time spent in the open areas of the elevated plus maze. Differences in protocol make comparisons between this and the previous study difficult. In the previous Fulk et al. [23] study, animals were tested under more aversive testing conditions, plus animals were chronically stressed by recurring cage-mate switches during the training period. The stress related effects of differences in protocol are evidenced by the overall lower baseline open arm values in the previous Fulk et al. study compared with those in this report. Under such testing conditions, control subjects were at a basal level of open arm time that would have precluded observing increases in anxiety-like behaviors. Chronic treadmill training failed to modify open field behavior, which is inconsistent with previous studies showing changes in locomotion after treadmill training [11,52,55]. Although Tharp and Carson [55] interpreted the increased locomotion in the running animals as evidence of decreased anxiety, no differences were found for the number of central squares crossed in the open field or emergence latency from the tunnel test. Therefore the interpretation of decreased anxiety lacks the corroboration of other open field measures or the support of other tests of anxiety-like behavior, and may actually reflect changes in defensive behaviors [47,60]. Further, Dishman et al. (1996) demonstrated that treadmill training decreased open field locomotion in the center area, a measure that can be indicative of increased defensive or anxiety-like behavior. This study, however, used repeated testing in the open field, with a habituation period prior to assessment of open field behavior. As indicated in this previous study, the differences in the influences of treadmill training on open field behavior differ depending on the use of habituation trials or single post-test only design features. This could suggest that

P.R. Burghardt et al. / Brain Research 1019 (2004) 84–96

chronic treadmill training alters habituation to novel environments. In contrast to the findings in this study, Skalicky et al. [52] reported that chronic treadmill training prevented a decline in spontaneous open field activity that occurs with aging. A possible cause for this discrepancy may be the different age of the animals used in the studies, since Skalicky et al. [52] started the training regime when their rats were 5 months old, whereas the rats in the present study had completed their training regime by that age. The training protocol employed by Skalicky et al. [52] was also considerably different than the protocol used for these studies, in that the animals were trained for 20 minutes, twice a day, for 18 months. The results of the current study indicate that chronic (8 week) treadmill training with the current protocol employed does not elicit changes in anxiety-related behaviors/defensive responding in the four behavioral models tested. One of the concerns of treadmill training in rodents is that the nature of the training may impart a significant amount of ‘‘stress’’, and therefore any favorable behavioral adaptations due to exercise would be counteracted by the chronic stress inherent in forced running (for references see Refs. [37,63]). The nature of the stress that may be associated with treadmill training has been difficult to determine due to the nature of the treadmill training protocols that are typically employed. For example the use of intermittent running schedules [23,52], and high levels of intensity [37], make it difficult to determine if the interruption of the training schedule yields some psychological stress, or high-intensity exercise imparts psychological stress secondary only to physiologic stress induced by high-intensity exercise. The increased extracellular release of corticotropin releasing factor (CRF) in the amygdala and hypothalamus during the first and final third of an exhaustive run [25], however implicate forced treadmill running as a stressful exercise paradigm since CRF is a neuropeptide known to be released in response to various stressors (for review, see Ref. [32]). Previous studies [25,37,55] seem to suggest that there is a stressful component to forced treadmill training that is not due to physical exhaustion, even though there is a fair deal of heterogeneity between training protocols. Another potential problem of the training protocol employed in this study is the interrupted running schedule used during treadmill training, and the fact that animals were tested after their two-day hiatus from treadmill training. This potentially retarded, or eliminated, the effects of running especially if the effects on behavior are due to an accumulation of acute effects (e.g., several acute bouts) analogous to insulin’s response to exercise [27]. It is also possible that a 5 days per week running schedule may require a longer total duration for subtle behavioral effects to manifest similar to those shown by Skalicky et al. [52]. The break in scheduled training may have also been stressful due to a change in expected routines, and it has been shown that continuous running protocols are more effective in eliciting changes in the open field compared to

93

intermittent protocols [51]. Treadmill training has been shown to enhance responses to stress [14,63,64], and the behavioral adaptations to chronic treadmill training may require more noxious stimuli than that which is inherent in the plus maze or open field tests in order to become apparent. Chronic treadmill training, however, also failed to modify behaviors in the forced swim test or induce an antidepressant-like effect in the neonatal clomipramine model [65], so it is unclear what role the stress severity plays in the expression of the behavioral adaptation to chronic exercise. Additionally, although animals were retrained on the treadmill between the various behavioral tests, it is possible that testing of the same animals sequentially in several different paradigms influenced their performance in the later tests, however this is unlikely for conditioned freezing [34]. Since a variety of neurochemical [15,25,35,49] and physiological [37] adaptations have been reported after chronic treadmill running, it is possible that this type of activity influences anxiety-related/defensive behaviors, however, the stress-related variables associated with the treadmill running protocol may have confounded subtle changes in behavior. It is also possible that behavioral changes are related to the intensity and/or duration of the daily training protocol, which may be responsible for the divergent behavioral changes observed after 8 weeks of treadmill training and eight weeks of wheel running. 4.2. Wheel running Wheel running has been shown to elicit behavioral, neurochemical and physiological [10,13,16,28,40,46, 48,53,58,62,66] adaptations, and may be a better method for studying the effect of long term exercise on behavior due to the voluntary nature of wheel running. Since no behavioral effects were seen after 8 weeks of treadmill running, the effect of 8 weeks of voluntary wheel running on behavior was investigated to attempt to circumvent several of the caveats associated with forced treadmill training. In this study control groups consisted of animals exposed to a locked wheel (LC) and animals group housed in a slightly enriched environment (GHEE). The latter control group was included to assess the potential role of enriched environment, as opposed to physical exertion, induced by chronic unrestricted access to a running wheel. Several changes in behavior were seen in the elevated plus maze and open field in response to wheel running, while social interaction and conditioned freezing behaviors were unaltered by chronic wheel running. Eight weeks of voluntary wheel running decreased open area behavior in both the elevated plus maze and open field, which might be suggestive of increased defensive behaviors in these paradigms. It is known that animals housed in ethological settings tend to have enhanced defensive behaviors, analogous to those of their feral counterparts [2]. Animals given access to running wheels for eight weeks spent less time in the open areas of the elevated plus maze and open field

94

P.R. Burghardt et al. / Brain Research 1019 (2004) 84–96

compared to LC animals indicating that the testing conditions were more stressful to WR rats resulting in a suppression of exploration while intensifying risk-assessment behaviors [1,26,47,60]. This type of open arm avoidance is also exhibited by the dominant animal when mice are group housed and is attributed to enhanced risk assessment [19]. Increases in defensive behavior are also supported by the lower frequency of unprotected head dipping performed by the WR rats. The consistent differences in elevated plus maze and open field measures between LC and WR animals are suggestive of an enhancement of defensive behavior in WR animals due to running. In the present study, animals with access to freely turning wheels were also more aggressive during handling (biting and struggling) compared to LC and GHEE. In some measures the GHEE animals fall between LC and WR groups, which suggests that although some of the behavioral adaptations in WR animals are likely due to exercise, that exercise itself may serve as a means of environmental enrichment [59]. GHEE animals were also found to spend less time in social interaction compared to LC and WR animals. This effect, however, is attributed to housing conditions and has been reported by other groups [7,39]. Thus, measures in the elevated plus maze (unprotected head dipping, Table 2) and open field (entries into the center of the field, Tables 2 and 3) along with decreased activity in both novel environments indicate that WR animals showed enhanced defensive responding compared to GHEE and LC animals. It is likely that housing and wheel running elicit complex behavioral and neurochemical changes that may be paradigm specific [48,59], analogous to those seen in visible burrow system animals [3,4]. Wheel running animals showed decreased activity as indicated by the total lines crossed in the open field, and closed arm entries in the plus maze. Wheel runners differed from both locked wheel animals and GHEE rats in the open field, but only showed decreased activity compared to GHEE animals in the elevated plus maze. The decrease in open field activity induced by wheel running is in agreement with other studies [28], although there are conflicting reports [10,11]. These divergent changes in the open field may be related to utilizing a single novel exposure to the open field, versus analysis of open field behavior following habituation to the testing apparatus [11]. Moreover, in this familiar environment animals showed a decrease in experimenter assessed anxiety-like behaviors after 8 weeks of wheel running [11]. This might suggest that chronic access to running wheels produces divergent behaviors in a novel environment when compared to a familiar environment, and suggests that the adaptations observed are critically dependent upon the testing conditions. The changes seen in a single open field test in a novel environment would be more comparable to tests of anxiety-like behaviors in the elevated plus maze, that similarly rely on novelty of the testing situation. In fact, repeated exposure to the elevated plus maze results in distinct states of ‘‘anxiety’’, accompanied by

different neurochemical and pharmacological responses (see Ref. [21]), and it is likely that similar habituation events occur in the open field test. The differences may also be related to the fact that the animals were tested in the final open field test after a 2-day hiatus from access to the wheels in the previous study [11]. Several lines of evidence indicate this decreased activity is not due to fatigue, but to a more specific change associated with these tests that may reflect increases in defensive behavior. Analysis of the peak running period during the rat’s dark cycle indicated that the vast majority of running occurred during the middle third of the dark cycle, several hours prior to testing. Further, as reported by other groups [28] animals immediately began running in wheels upon return to home cage after behavioral testing, suggesting behavioral changes were not indicative of fatigue. Moreover, animals that had access to running wheels for 4 and 8 weeks were running the same distance in a 24-h period at the beginning of behavioral testing, but behavioral changes in the plus maze were only seen following 8 weeks of running. In addition, there was no correlation between distance run 24 h prior to behavioral testing, and measures of activity in the elevated plus maze and open field. In order to discern if the effects on behavior required prolonged physical activity another set of animals was given access to running wheels for four weeks. The 4-week time point was chosen since running distance was similar at 4 and 8 weeks in experiment 2. The animals were only tested in the open field and elevated plus maze, since effects were found in these tests after 8 weeks of running. Four weeks of wheel running failed to shift open arm behavior or activity (closed arm) measures in the plus maze, such as that seen after 8 weeks of wheel running. This suggests that the reduction in open arm behavior in the elevated plus maze induced by unrestricted access to running wheels is timedependent and requires longer than 4 weeks to manifest a significant behavioral change. In contrast, 4 weeks of wheel running produced similar effects in the open field tests as those seen after 8 weeks. Wheel running animals crossed fewer lines in the open field compared to locked wheel and GHEE rats. Taken together these findings suggest a temporal effect of running on elevated plus maze behavior, and a dissociation between the effects of chronic voluntary physical activity between these two behavioral models. This might suggest that defensive behaviors assessed in the open field are more sensitive to alterations induced by wheel running. It also suggests that temporally or anatomically distinct neurochemical alterations might underlie the behavioral changes observed in these two behavioral models. For example, septal and amygdalar lesions have divergent effects on open field locomotion and elevated plus maze behavior [8,56]. In this set of experiments, it was not feasible to directly address whether these effects were due to carryover from an acute bout of running (e.g., running on the previous night), or an accumulation of acute bouts of running (analogous to

P.R. Burghardt et al. / Brain Research 1019 (2004) 84–96

insulin responses) [27,36] on behavior. It seems that the lack of effect in the elevated plus maze after four weeks of running would argue against carryover from an acute bout of running, since it would be expected that running the night before the behavioral test would be sufficient to alter behavior. The results of these experiments suggest that some of the behavioral effects of exercise are due to chronic adaptations requiring a long period of physical activity, or an accumulation of acute bouts of running after chronic adaptations have occurred. Therefore it is reasonable to suggest that at the very least these behavioral adaptations require chronic wheel running and are likely not due to an acute bout of exercise. A variety of neurochemical changes are likely to mediate the behavioral changes seen after wheel running, and distinct neurochemical changes are likely to mediate the behavioral changes seen in the elevated plus maze and open field. Changes in opioidergic [30,49,50,61], serotonergic [24], GABAergic [11], and catacholaminergic [10,35] systems have also been observed after wheel running. Six weeks of wheel running increased basal expression of serotonin 5-HT1A inhibitory autoreceptors in the dorsal raphe nucleus, an area involved in anxiety and depression [24]. Wheel running also appears to change BDNF [48,57] which may affect antidepressant efficacy [48]. Many of the neurochemical systems affected by wheel running are also known to be involved in elevated plus maze and open field behavior [22,31]. These neurochemical and behavioral changes suggest that exercise may be useful as an intervention for anxiety and depression, and possibly as an adjunctive therapy in preventing drug relapse. However interpretation of behavioral measures may not be as straightforward when pharmacological agents are absent from the experimental design, which would otherwise lend predictive validity to the test paradigms. In conclusion, it is apparent that chronic voluntary running produces behavioral changes in the elevated plus maze and open field. These behavioral changes are dependent upon the number of weeks of running, and specific to the behavioral test paradigm employed. In particular, behavioral changes in response to wheel running appear to be enhanced defensive behaviors that are likely related to adaptations in a variety of neurochemical systems. These behavioral adaptations are not due solely to enriched environment due to the discrepancy between WR and GHEE animals on several behavioral measures. Chronic treadmill running failed to produce behavioral changes with the running protocol and testing conditions used in this set of experiments.

Acknowledgements This work was supported by School of Medicine/School of Public Health, Collaborative Research Incentive from the University of South Carolina; and RO1 MH63344 to MAW.

95

References [1] C. Belzung, G. Griebel, Measuring normal and pathological anxietylike behaviour in mice: a review, Behav. Brain Res. 125 (2001) 141 – 149. [2] R.J. Blanchard, K.J. Flannelly, D.C. Blanchard, Defensive behavior of laboratory and wild Rattus norvegicus, J. Comp. Psychol. 100 (1986) 101 – 107. [3] R.J. Blanchard, M.A. Hebert, P. Ferrari, P. Palanza, R. Figueira, D.C. Blanchard, S. Parmigiani, Defensive behaviors in wild and laboratory (Swiss) mice: the mouse defense test battery, Physiol. Behav. 65 (1998) 201 – 209. [4] D.C. Blanchard, G. Griebel, R.J. Blanchard, Mouse defensive behaviors: pharmacological and behavioral assays for anxiety and panic, Neurosci. Biobehav. Rev. 25 (2001) 205 – 218. [5] A. Broocks, T. Meyer, M. Opitz, U. Bartmann, U. Hillmer-Vogel, A. George, G. Pekrun, D. Wedekind, E. Ruther, B. Bandelow, 5-HT1A responsivity in patients with panic disorder before and after treatment with aerobic exercise, clomipramine or placebo, Eur. Neuropsychopharmacol. 13 (2003) 153 – 164. [6] F. Chaouloff, Influence of physical exercise on 5-HT1A receptor- and anxiety-related behaviours, Neurosci. Lett. 176 (1994) 226 – 230. [7] S. Cheeta, E. Irvine, S.E. File, Social isolation modifies nicotine’s effects in animal tests of anxiety, Br. J. Pharmacol. 132 (2001) 1389 – 1395. [8] C.D. Corman, P.M. Meyer, D.R. Meyer, Open-field activity and exploration in rats with septal and amygdaloid lesions, Brain Res. 5 (1967) 469 – 476. [9] C.W. Cotman, N.C. Berchtold, Exercise: a behavioral intervention to enhance brain health and plasticity, Trends Neurosci. 25 (2002) 295 – 301. [10] R.K. Dishman, Brain monoamines, exercise, and behavioral stress: animal models, Med. Sci. Sports Exerc. 29 (1997) 63 – 74. [11] R.K. Dishman, A.L. Dunn, S.D. Youngstedt, J.M. Davis, M.L. Burgess, S.P. Wilson, M.A. Wilson, Increased open field locomotion and decreased striatal GABAA binding after activity wheel running, Physiol. Behav. 60 (1996) 699 – 705. [12] R.K. Dishman, K.J. Renner, S.D. Youngstedt, T.G. Reigle, B.N. Bunnell, K.A. Burke, H.S. Yoo, E.H. Mougey, J.L. Meyerhoff, Activity wheel running reduces escape latency and alters brain monoamine levels after footshock, Brain Res. Bull. 42 (1997) 399 – 406. [13] R.K. Dishman, B.N. Bunnell, S.D. Youngstedt, H.S. Yoo, E.H. Mougey, J.L. Meyerhoff, Activity wheel running blunts increased plasma adrenocorticotrophin (ACTH) after footshock and cage-switch stress, Physiol. Behav. 63 (1998) 911 – 917. [14] R.K. Dishman, K.J. Renner, J.E. White-Welkley, K.A. Burke, B.N. Bunnell, Treadmill exercise training augments brain norepinephrine response to familiar and novel stress, Brain Res. Bull. 52 (2000) 337 – 342. [15] A.L. Dunn, R.K. Dishman, Exercise and the neurobiology of depression, Exerc. Sport Sci. Rev. 19 (1991) 41 – 98. [16] A.L. Dunn, T.G. Reigle, S.D. Youngstedt, R.B. Armstrong, R.K. Dishman, Brain norepinephrine and metabolites after treadmill training and wheel running in rats, Med. Sci. Sports Exerc. 28 (1996) 204 – 209. [17] A.L. Dunn, M.H. Trivedi, H.A. O’Neal, Physical activity dose – response effects on outcomes of depression and anxiety, Med. Sci. Sports Exerc. 33 (2001) S587 – S597. [18] P.A. Farrell, A.B. Gustafson, W.P. Morgan, C.B. Pert, Enkephalins, catecholamines, and psychological mood alterations: effects of prolonged exercise, Med. Sci. Sports Exerc. 19 (1987) 347 – 353. [19] P.F. Ferrari, P. Palanza, S. Parmigiani, R.J. Rodgers, Interindividual variability in Swiss male mice: relationship between social factors, aggression, and anxiety, Physiol. Behav. 63 (1998) 821 – 827. [20] S.E. File, J.R. Hyde, Can social interaction be used to measure anxiety? Br. J. Pharmacol. 62 (1978) 19 – 24.

96

P.R. Burghardt et al. / Brain Research 1019 (2004) 84–96

[21] S.E. File, H. Zangrossi Jr., M. Viana, F.G. Graeff, Trial 2 in the elevated plus-maze: a different form of fear? Psychopharmacology (Berl.) 111 (1993) 491 – 494. [22] S.E. File, L.E. Gonzalez, N. Andrews, Comparative study of pre- and postsynaptic 5-HT1A receptor modulation of anxiety in two ethological animal tests, J. Neurosci. 16 (1996) 4810 – 4815. [23] L.J. Fulk, H.S. Stock, J.D. Marshall, A.J. Lynn, M.A. Wilson, G.A. Hand, Chronic treadmill training reduces acute anxiety-related behaviors in rats, Int. J. Sports Med. 24 (2003) 1 – 5. [24] B.N. Greenwood, T.E. Foley, H.E. Day, J. Campisi, S.H. Hammack, S. Campeau, S.F. Maier, M. Fleshner, Freewheel running prevents learned helplessness/behavioral depression: role of dorsal raphe serotonergic neurons, J. Neurosci. 23 (2003) 2889 – 2898. [25] G.A. Hand, C.B. Hewitt, L.J. Fulk, H.S. Stock, J.A. Carson, J.M. Davis, M.A. Wilson, Differential release of corticotropin-releasing hormone (CRH) in the amygdala during different types of stressors, Brain Res. 949 (2002) 122 – 130. [26] S.L. Handley, J.W. McBlane, An assessment of the elevated X-maze for studying anxiety and anxiety-modulating drugs, J. Pharmacol. Toxicol. Methods 29 (1993) 129 – 138. [27] J. Henriksson, Influence of exercise on insulin sensitivity, J. Cardiovasc. Risk 2 (1995) 303 – 309. [28] P. Hoffmann, P. Thoren, D. Ely, Effect of voluntary exercise on openfield behavior and on aggression in the spontaneously hypertensive rat (SHR), Behav. Neural Biol. 47 (1987) 346 – 355. [29] A. Ishihara, R.R. Roy, Y. Ohira, Y. Ibata, V.R. Edgerton, Hypertrophy of rat plantaris muscle fibers after voluntary running with increasing loads, J. Appl. Physiol. 84 (1998) 2183 – 2189. [30] R.B. Kanarek, A.V. Gerstein, R.P. Wildman, W.F. Mathes, K.E. D’Anci, Chronic running-wheel activity decreases sensitivity to morphine-induced analgesia in male and female rats, Pharmacol. Biochem. Behav. 61 (1998) 19 – 27. [31] W. Kang, S.P. Wilson, M.A. Wilson, Overexpression of proenkephalin in the amygdala potentiates the anxiolytic effects of benzodiazepines, Neuropsychopharmacology 22 (2000) 77 – 88. [32] G.F. Koob, Corticotropin-releasing factor, norepinephrine, and stress, Biol. Psychiatry 46 (1999) 1167 – 1180. [33] R.G. Lister, Ethologically-based animal models of anxiety disorders, Pharmacol. Ther. 46 (1990) 321 – 340. [34] K.L. McIlwain, M.Y. Merriweather, L.A. Yuva-Paylor, R. Paylor, The use of behavioral test batteries: effects of training history, Physiol. Behav. 73 (2001) 705 – 717. [35] R. Meeusen, K. De Meirleir, Exercise and brain neurotransmission, Sports Med. 20 (1995) 160 – 188. [36] K.J. Mikines, B. Sonne, B. Tronier, H. Galbo, Effects of training and detraining on dose – response relationship between glucose and insulin secretion, Am. J. Physiol. 256 (1989) E588 – E596. [37] A. Moraska, T. Deak, R.L. Spencer, D. Roth, M. Fleshner, Treadmill running produces both positive and negative physiological adaptations in Sprague – Dawley rats, Am. J. Physiol., Regul. Integr. Comp. Physiol. 279 (2000) R1321 – R1329. [38] W.P. Morgan, Affective beneficence of vigorous physical activity, Med. Sci. Sports Exerc. 17 (1985) 94 – 100. [39] R.J. Niesink, J.M. Van Ree, Short-term isolation increases social interactions of male rats: a parametric analysis, Physiol. Behav. 29 (1982) 819 – 825. [40] H.A. O’Neal, J.D. Van Hoomissen, P.V. Holmes, R.K. Dishman, Prepro-galanin messenger RNA levels are increased in rat locus coeruleus after treadmill exercise training, Neurosci. Lett. 299 (2001) 69 – 72. [41] S.A. Paluska, T.L. Schwenk, Physical activity and mental health: current concepts, Sports Med. 29 (2000) 167 – 180. [42] S. Pellow, P. Chopin, S.E. File, M. Briley, Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat, J. Neurosci. Methods 14 (1985) 149 – 167. [43] G.D. Petrovich, A.P. Scicli, R.F. Thompson, L.W. Swanson, Associative fear conditioning of enkephalin mRNA levels in central amygdalar neurons, Behav. Neurosci. 114 (2000) 681 – 686.

[44] S.D. Primeaux, P.V. Holmes, Role of aversively motivated behavior in the olfactory bulbectomy syndrome, Physiol. Behav. 67 (1999) 41 – 47. [45] R.J. Rodgers, Animal models of ‘anxiety’: where next? Behav. Pharmacol. 8 (1997) 477 – 496. [46] K.J. Rodnick, G.M. Reaven, W.L. Haskell, C.R. Sims, C.E. Mondon, Variations in running activity and enzymatic adaptations in voluntary running rats, J. Appl. Physiol. 66 (1989) 1250 – 1257. [47] J.R. Royce, On the construct validity of open-field measures, Psychol. Bull. 84 (1977) 1098 – 1106. [48] A. Russo-Neustadt, T. Ha, R. Ramirez, J.P. Kesslak, Physical activityantidepressant treatment combination: impact on brain-derived neurotrophic factor and behavior in an animal model, Behav. Brain Res. 120 (2001) 87 – 95. [49] G.A. Sforzo, Opioids and exercise. An update, Sports Med. 7 (1989) 109 – 124. [50] H.M. Sisti, M.J. Lewis, Naloxone suppression and morphine enhancement of voluntary wheel-running activity in rats, Pharmacol. Biochem. Behav. 70 (2001) 359 – 365. [51] M. Skalicky, A. Viidik, Comparison between continuous and intermittent physical exercise on aging rats: changes in patterns of spontaneous activity and connective tissue stability, Aging (Milano) 11 (1999) 227 – 234. [52] M. Skalicky, H. Bubna-Littitz, A. Viidik, Influence of physical exercise on aging rats: I. Life-long exercise preserves patterns of spontaneous activity, Mech. Ageing Dev. 87 (1996) 127 – 139. [53] J. Soares, P.V. Holmes, K.J. Renner, G.L. Edwards, B.N. Bunnell, R.K. Dishman, Brain noradrenergic responses to footshock after chronic activity-wheel running, Behav. Neurosci. 113 (1999) 558 – 566. [54] H.S. Stock, G.A. Hand, K. Ford, M.A. Wilson, Changes in defensive behaviors following olfactory bulbectomy in male and female rats, Brain Res. 903 (2001) 242 – 246. [55] G.D. Tharp, W.H. Carson, Emotionality changes in rats following chronic exercise, Med. Sci. Sports 7 (1975) 123 – 126. [56] D. Treit, J. Menard, Dissociations among the anxiolytic effects of septal, hippocampal, and amygdaloid lesions, Behav. Neurosci. 111 (1997) 653 – 658. [57] J.D. Van Hoomissen, H.O. Chambliss, P.V. Holmes, R.K. Dishman, Effects of chronic exercise and imipramine on mRNA for BDNF after olfactory bulbectomy in rat, Brain Res. 974 (2003) 228 – 235. [58] H. van Praag, B.R. Christie, T.J. Sejnowski, F.H. Gage, Running enhances neurogenesis, learning, and long-term potentiation in mice, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 13427 – 13431. [59] H. van Praag, G. Kempermann, F.H. Gage, Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus, Nat. Neurosci. 2 (1999) 266 – 270. [60] R.N. Walsh, R.A. Cummins, The open-field test: a critical review, Psychol. Bull. 83 (1976) 482 – 504. [61] M. Werme, P. Thoren, L. Olson, S. Brene, Running and cocaine both upregulate dynorphin mRNA in medial caudate putamen, Eur. J. Neurosci. 12 (2000) 2967 – 2974. [62] M. Werme, S. Lindholm, P. Thoren, J. Franck, S. Brene, Running increases ethanol preference, Behav. Brain Res. 133 (2002) 301 – 308. [63] J.E. White-Welkley, B.N. Bunnell, E.H. Mougey, J.L. Meyerhoff, R.K. Dishman, Treadmill exercise training and estradiol differentially modulate hypothalamic – pituitary – adrenal cortical responses to acute running and immobilization, Physiol. Behav. 57 (1995) 533 – 540. [64] J.E. White-Welkley, G.L. Warren, B.N. Bunnell, E.H. Mougey, J.L. Meyerhoff, R.K. Dishman, Treadmill exercise training and estradiol increase plasma ACTH and prolactin after novel footshock, J. Appl. Physiol. 80 (1996) 931 – 939. [65] H.S. Yoo, B.N. Bunnell, J.B. Crabbe, L.R. Kalish, R.K. Dishman, Failure of neonatal clomipramine treatment to alter forced swim immobility: chronic treadmill or activity-wheel running and imipramine, Physiol. Behav. 70 (2000) 407 – 411. [66] H.S. Yoo, J.B. Tackett, J.B. Crabbe, B.N. Bunnell, R.K. Dishman, Antidepressant-like effects of physical activity versus imipramine: neonatal clomipramine model, Psychobiology 28 (2000) 540 – 549.