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Effects of access to voluntary wheel running on the development of stereotypy
Behavioural Processes 83 (2010) 242–246
Contents lists available at ScienceDirect
Behavioural Processes journal homepage: www.elsevier.com/locate/behavproc
Effects of access to voluntary wheel running on the development of stereotypy Artur Pawlowicz, Adam Demner, Mark H. Lewis ∗ Department of Psychiatry and McKnight Brain Institute, University of Florida, Gainesville, FL 32611, USA
a r t i c l e
i n f o
Article history: Received 26 August 2009 Received in revised form 12 November 2009 Accepted 15 November 2009 Keywords: Repetitive behavior Exercise Deer mice Environmental enrichment Peromyscus maniculatus
a b s t r a c t Stereotyped motor behaviors are a common consequence of environmental restriction in a wide variety of species. Although environmental enrichment has been shown to substantially reduce stereotypy levels, the various components of enrichment have not been evaluated independently to determine which is responsible for this effect. Exercise, particularly voluntary wheel running, is a promising candidate based on several lines of behavioral and neurobiological evidence. To test the hypothesis that access to wheel running will reduce stereotyped motor behavior, we reared deer mice from weaning with continuous access to either a functional running wheel or a locked wheel. We assessed running behavior throughout this time period and stereotypy levels in a test context at 30 and 45 days post-weaning. We found that exercise did not significantly affect stereotypy level nor was there an association between wheel running and stereotypy. Thus, exercise alone, unlike environmental enrichment, does not prevent the development of stereotypy. These results have important implications for animal welfare. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Abnormal repetitive behaviors are commonly displayed in animals housed in restricted (e.g., zoo, farm, laboratory) environments, as well as animals subjected to early social deprivation (Mason and Rushen, 2006). Indeed, repetitive behaviors are the most common category of abnormal behavior observed in confined animals (Wurbel, 2001). Some examples of aberrant repetitive behaviors observed in animals maintained in confinement include crib-biting and head-weaving in horses (Cooper et al., 2000), head-twirling in mink (Mason, 1993) and patterned locomotion in carnivores (Clubb and Vickery, 2006). Increasing the complexity of the environment has been a successfully employed strategy for reducing such stereotyped behavior in a number of species (e.g., Swaisgood and Sheperdson, 2006). For example, we have demonstrated repeatedly that increased environmental complexity (environmental enrichment) markedly attenuates the development of stereotyped behavior in deer mice (Hadley et al., 2006; Powell et al., 2000; Turner et al., 2002, 2003; Turner and Lewis, 2003). Typically, environmental enrichment involves larger, more complex housing, with increased novelty, and opportunities for exploration and exercise, areas for hiding and nesting and, sometimes, limited foraging as well (Hadley et al., 2006; Turner et al., 2003). This has made it difficult, however, to isolate the specific
∗ Corresponding author at: Department of Psychiatry, 100 S. Newell Drive, Mc Knight Brain Institute, Suite L4-100, Gainesville, FL 32611, USA. Tel.: +1 352 294 0415; fax: +1 352 294 0425. E-mail address: marklewis@ufl.edu (M.H. Lewis). 0376-6357/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2009.11.008
environmental features and mechanisms by which such experience induces its neurobiological and behavioral effects including attenuation of repetitive behaviors. A ubiquitous feature of more complex housing for laboratory rodents is a running wheel. Voluntary exercise in the form of wheel running has been advanced as being essential for the effects of environmental enrichment on certain behavioral and brain outcomes. For example, exercise has been shown to improve various measures of cognitive function in both intact rodents and mouse models of dementia and to result in increased neurogenesis and neurotrophin expression (see Cotman et al., 2007; van Praag, 2009 for recent reviews). These findings and the preeminent role posited for voluntary wheel running in the effects of environmental enrichment suggest its efficacy in attenuating the development of stereotypy. Indeed, two recent studies have concluded that wheel running significantly reduced the level of stereotypy in mice induced by environmental restriction (Howerton et al., 2008) or by a genetic mutation (Richter et al., 2008). Our own unpublished preliminary work, however, did not support such a conclusion. This study tested the effects in deer mice of either 30 or 60 days of access to voluntary wheel running starting at weaning (21–24 days of age). At both the 30 and 60 days post-weaning time points, we compared stereotypy scores from 21 mice which had access to running wheels with scores from 18 mice with no running wheels in their cages. At neither time point was there evidence for a difference between groups in stereotypy. This study must be considered preliminary, however, as we did not monitor or quantify wheel running nor did we employ a locked wheel as a control. Thus, the role of wheel running in enriched laboratory environments and its effects on stereotypic behavior remain unclear.
A. Pawlowicz et al. / Behavioural Processes 83 (2010) 242–246
To complicate the issue further, wheel running has been conceptualized as a stereotypic behavior in itself with the potential to become compulsive and self-reinforcing (Latham and Wurbel, 2006; Sherwin, 1998). Consistent with this perspective, wheel running upregulates Fos in striatum similar to the Fos expression pattern associated with drug-induced stereotypic behavior (Saka et al., 2004; Werme et al., 2002a,b). Moreover, running seems to activate many of the same pathways as drugs of abuse (Ferreira et al., 2006; Werme et al., 2000, 2002a,b). Accordingly, to investigate the effect of wheel running on stereotypy and reconcile the differing views of the role of exercise in enrichment, we directly examined the effect of voluntary access to a running wheel on the development of spontaneous stereotypy in deer mice. We hypothesized that frequent exercise on the running wheel would decrease stereotypy level, acting in a manner similar to environmental enrichment. Furthermore, we hypothesized that the amount of wheel running would correlate negatively with stereotypy level. 2. Methods 2.1. Subjects Deer mice (Peromyscus maniculatus) exhibit two major topographies of stereotypy (repetitive hindlimb jumping and backward somersaulting) as a consequence of being reared in standard laboratory caging. These behaviors do not require social isolation, specific cues or contexts, pharmacological agents, or specific CNS insult for induction. These behaviors occur at a high rate, persist across much of the life of the animal and appear relatively early in development, sometimes as early as weaning. As mentioned previously, we have shown repeatedly that environmental enrichment (either 30 or 60 days duration starting at weaning) markedly attenuates the development of stereotypy. For this study, 30 deer mice including 12 males and 18 females, were taken from the breeding colony maintained by our laboratory. Upon weaning at 21–24 days of age, groups of three same-sex deer mice were randomly assigned to either a standard cage with a functional running wheel (Wheel Running group) or a standard cage with a locked wheel (Control group). The groups were balanced for sex, with each having 6 males and 9 females. The experimental protocol was approved by the University of Florida Instititional Animal Care and Use Committee (IACUC) and complies with the NIH Guide for Care and Use of Laboratory Animals. 2.2. Housing and apparatus All deer mice were housed three per cage in standard polycarbonate rodent cages of dimensions 48 cm × 27 cm × 15 cm with pine bedding, nesting materials and access to rodent chow and water ad libitum. Deer mice housed in this way consistently exhibit high levels of repetitive behavior. Deer mice in the Wheel Running group were given unlimited access to a Bio-Serv Fast Trac dome-type running wheel placed on one side of their cage (BioServ, Frenchtown, NJ). The running wheels were checked daily to ensure proper and uninterrupted functioning. Subjects in the Control group were given unlimited access to the same type of wheel, but the wheel was immobilized with a wire attachment to prevent its use for running. The deer mice were housed in climate-controlled conditions maintained at 20–25 ◦ C and 50–70% humidity. The lighting conditions were controlled by an automatic timer, maintaining a 16 h light to 8 h dark cycle. This L:D cycle has been in effect for this colony for many years and our previous studies with deer mice have
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employed this cycle. Red lights during the dark cycle permitted video recording. Video recording during the dark cycles for both the wheel running and stereotypy assessments was conducted using infrared cameras. Stereotypy testing was conducted in separate, individual Plexiglass testing cages (22 cm × 25 cm × 28 cm). While in the testing cages, all mice had free access to the same food and water as in their home cages, but did not have access to running wheels. Stereotypy counts were calculated automatically in these cages using a photocell detection apparatus from Columbus Instruments (Columbus, OH). Photocells were placed such that vertical activity (jumping and somersaulting) resulted in beam breaks whereas rearing did not. Horizontal activity (e.g., locomotion) was not automatically recorded. 2.3. Assessment of wheel running behavior Each Wheel Running cage was inspected daily and recorded weekly throughout the course of the experiment during the dark cycle (8 h per week) using an infrared camera. Small areas of fur were clipped on two of the mice to aid in identification. Subsequently, the wheel running activity for each mouse was scored from the video using a partial interval scoring method. The occurrence or non-occurrence of wheel running for each 1-min interval was coded to determine the proportion of time that each mouse spent actively using the running wheel. If a mouse ran during a given minute, it received a score of 1; if it did not, it received a score of 0. The scores were added and divided by the total number of minutes to determine the proportion of time intervals spent wheel running. For data analysis purposes, wheel running intervals from two different dark cycles were averaged to generate a single score for each animal. 2.4. Assessment of spontaneous stereotypy Each mouse was tested at 30 and 45 days post-weaning in an individual Plexiglass cage during their 8-h dark cycle. These time points not only replicated the 30 days used in our preliminary study but also tested an intermediate point (45 days). In other studies from our lab we have determined that stereotypy in deer mice asymptotes to adult levels at approximately 45 days post-weaning. Mice were allowed to habituate to the testing environment after handling for at least 1 h prior to the onset of the dark cycle to allow recovery from the stress of handling and transition to a novel environment. During the entire testing period, the deer mice were individually caged and had access to food and water. Stereotypy counts, which equated to the number of vertical jumps or backwards somersaults a deer mouse performed during that 8 h, were automatically scored for each mouse using the photocell detection system. Photobeams were positioned to be interrupted by the vertical motion of jumping and somersaulting but not by rearing. All testing sessions were also recorded using infrared video cameras to insure accuracy of the automated recording. 2.5. Data analysis Following data verification, the stereotypy scores from each session (taken at 30 and 45 days post-weaning) for the Wheel Running and Control groups were analyzed for differences due to group and testing period, as well as their interaction using a 2 × 2 repeated measures analysis of variance (RMANOVA). For the Wheel Running group, the proportion of time spent running for each deer mouse (the average of two dark cycles) was also correlated with the mouse’s stereotypy scores using a Pearson product–moment correlation. An alpha value of less than 0.05 was used as the criterion for statistical significance in both analyses.
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A. Pawlowicz et al. / Behavioural Processes 83 (2010) 242–246
Fig. 1. The mean stereotypy scores for the Wheel Running and Control groups after 30 or 45 days of access to a running wheel or locked wheel following weaning. Error bars represent standard error of the mean.
Fig. 3. Association between stereotypy frequency at 30 days post-weaning and proportion of time spent running for the Wheel Running group.
3. Results
from mice which either had access to running wheels (n = 21) or no running wheels available (n = 18). No significant difference between groups were found at either time point.
3.1. Stereotypy assessment For the Wheel Running group (n = 15), total stereotypy counts for each 8-h period ranged from 972 to 14,114 at 30 days and from 1135 to 12,453 at 45 days. For the Control group (n = 15), scores ranged from 1241 to 16,117 at 30 days and from 1180 to 12,493 at 45 days. RMANOVA results indicated no main effect of group (F(1,27) = 1.87, p = 0.24), indicating that the stereotypy levels did not differ between the Wheel Running and Control groups irrespective of testing session. In addition, no main effect of testing session was found (F(1,27) = 0.35, p = 0.56), reflecting little change in stereotypy level between the day 30 and day 45 testing sessions. Fig. 1 shows the mean stereotypy counts for the Running and Control groups for both testing sessions. Finally, no significant interaction between group and testing session on stereotypy scores was found (F(1,27) = 0.37, p = 0.55). For comparison purposes, we have presented the results of our preliminary study in Fig. 2. This figure shows stereotypy scores, assessed as described in Section 2, at 30 and 60 days post-weaning
The deer mice assigned to the Wheel Running group spent from 9 to 71% of 1 min intervals of their active periods using the running wheel, averaging 43% (n = 15). Video records indicated that all 3 mice were able to use the running wheel at the same time and frequently did. For the deer mice in the Wheel Running group, the correlation between the proportion of 1 min intervals spent running and the 30 day stereotypy counts neared, but did not achieve, statistical significance (r = 0.50, p = 0.056, Fig. 3). However, when an outlying (greater than two standard deviations above the mean) stereotypy score was removed, the correlation was reduced considerably (r = 0.26, p = 0.37). The correlation between the proportion of time spent running and the 45-day stereotypy counts was also not significant (r = 0.11, p = 0.69, Fig. 4).
Fig. 2. The mean stereotypy scores for the Wheel Running and Control groups after 30 or 60 days of access to a running wheel or no running wheel available following weaning. Error bars represent standard error of the mean.
Fig. 4. Association between stereotypy frequency at 45 days post-weaning and proportion of time spent running for the Wheel Running group.
3.2. Wheel running assessment
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4. Discussion Initially, we hypothesized that wheel running would act similarly to environmental enrichment in reducing spontaneous motor stereotypy. We found that deer mice made ample use of the running wheels, spending, on average, almost half their time engaged in such activity. Nevertheless, we found no significant effect of voluntary access to a running wheel for either 30 or 45 days postweaning on stereotypy level. In addition, we found no evidence of an association between amount of wheel running and frequency of stereotypy assessed independently of the animal’s home cage. The lack of effect of exercise on the frequency of stereotyped behavior is consistent with the results of a previous study conducted in our lab (see Fig. 2). In addition to replication, a comparison of the two studies demonstrates that the no wheel condition does not differ from either the active or locked running wheel conditions. As mentioned previously, the preliminary study did not monitor or quantify wheel running. One important conclusion from these results is that wheel running alone does not appear to be responsible for the marked attenuation of stereotypy that we have consistently observed following environmental enrichment. This appears to be the case for all three durations of access to wheel running which equated to both the duration and developmental timing used in our enrichment studies. The present results are not consistent with findings from two recent studies that examined wheel running effects on stereotypy in mice, one using a model of environmental restriction (Howerton et al., 2008) and the other a transgenic mouse model of Alzheimer’s disease (Richter et al., 2008). Although both concluded that wheel running significantly reduced the level of stereotypy, the methodologies employed in each of these studies leave these conclusions open to interpretation. Specifically, both studies assessed stereotypy in the home cage during which the mice had access to running wheels. Given observations that mice spend a large proportion of their active periods using the running wheel, it may be difficult to conclude, unambiguously, that wheel running itself reduced stereotypy in these cases. Indeed, Richter et al. (2008) found that wheel running was inversely correlated with stereotypic behavior, suggesting that wheel running may have stereotypic qualities or certainly competed with the expression of stereotyped behavior. It would seem, then, that difference in outcome between our study and the two previously published studies is that we assessed stereotypy in the absence of access to the running wheel. Other explanations for the inconsistency, beyond choice of testing environments, may include genus differences as well as duration of access to voluntary wheel running, which in Howerton et al. (2008) was only 2 weeks. Deer mice in the Wheel Running group spent a large proportion of their active periods using the running wheel. Consequently, they reduced the time available for other active behaviors, including stereotyped jumping and backward somersaulting, when a wheel was present. Although we did observe noticeably reduced stereotypic behavior when the mice in the Wheel Running group had access to a running wheel (data not shown), our results show that this difference is not maintained once the deer mice no longer had access to a running wheel. Moreover, our results indicated no significant association between wheel running in the home cage and stereotypy exhibited in the testing cage. Moreover, since the three deer mice in any particular cage were able to use the wheel simultaneously and often did, it is unlikely that the amount of running was affected by competition or position in a dominance hierarchy. Physical exercise has been posited to be a critical component of the effects of environmental enrichment on brain and behavior. Voluntary wheel running has been shown to induce many of the same neurochemical and proliferative changes in the brain as complete environmental enrichment, in many cases to the same degree.
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Several studies have found that voluntary wheel running alone significantly increases hippocampal neurogenesis in rodents, reaching a level comparable to full environmental enrichment (Aberg et al., 2008; Bjornebekk et al., 2005; Brown et al., 2003; van Praag et al., 1999). Similar to enrichment, wheel running enhances learning ability and neurogenesis in both young and aged mice (van Praag et al., 2005). Wheel running also increases neuronal metabolism in the motor cortex and striatum (McCloskey et al., 2001). All of these effects have also been reported in environmental enrichment, suggesting that voluntary exercise may play a significant role in mediating these effects in enriched rodents. It may be, however, that wheel running may exert some of its effects in concert with other components of environmental enrichment. This was the conclusion of Pietropaolo et al. (2006) who found that whereas wheel running alone had few effects, it interacted significantly with other aspects of environmental enrichment when the two were combined. Indeed, some findings from this study suggest that the combined effects may be synergistic rather than simply additive. A review by Sherwin (1998) proposed that wheel running in rodents appears maladaptive in some circumstances, and might be an artifact of artificial laboratory conditions as well as being a stereotypic behavior in itself. Latham and Wurbel (2006) argue that while wheel running may occur in both stereotypic and nonstereotypic cases, there is sufficient evidence to claim that wheel running should be considered a stereotypy. Compulsive wheel running activates many of the same brain pathways as drugs of abuse (Ferreira et al., 2006). Cross-sensitization between wheel running and amphetamine has been demonstrated and wheel running increases preference for ethanol consumption, indicating a sensitization of reward pathways (Werme et al., 2002a,b). The present study was not designed to address the question of whether wheel running is itself a stereotyped behavior. We did not, however, find any significant correlation between time spent wheel running and stereotypy level. This lack of a correlation suggests that wheel running and repetitive jumping/somersaulting are likely not interchangeable stereotypies. From an animal welfare perspective, our results indicate that access to a running wheel is not a substitute for environmental enrichment, at least with respect to the development of repetitive behavior, but rather should be used in concert with other components of environmental enrichment. Conversely, we also found no evidence that providing access to a running wheel is in itself harmful, at least with respect to aberrant repetitive behavior. The components of environmental enrichment that affect repetitive behavior and its underlying neurobiology (i.e., cortico-basal ganglia circuitry) remain an important and unanswered question that have important implications for clinical disorders such as autism that have repetitive behavior as part of their presentation as well as for animal welfare.
References Aberg, E., Perlmann, T., Olson, L., Brene, S., 2008. Running increases neurogenesis without retinoic acid receptor activation in the adult mouse dentate gyrus. Hippocampus 18, 785–792. Bjornebekk, A., Mathe, A.A., Brene, S., 2005. The antidepressant effect of running is associated with increased hippocampal cell proliferation. Int. J. Neuropsychopharmacol. 8, 357–368. Brown, J., Cooper-Kuhn, C.M., Kempermann, G., Van Praag, H., Winkler, J., Gage, F.H., Kuhn, H.G., 2003. Enriched environment and physical activity stimulate hippocampal but not olfactory bulb neurogenesis. Eur. J. Neurosci. 17, 2042–2046. Clubb, R., Vickery, S., 2006. Locomotory stereotypies in carnivores: does pacing stem from hunting, ranging, or frustrated escape? In: Mason, G.J., Rushen, J. (Eds.), Stereotypic Animal Behaviour, 2nd ed. CABI, Oxford, pp. 58–85. Cooper, J., McDonald, L., Mills, D., 2000. The effect of increasing visual horizons on stereotypic weaving: implications for the social housing of stabled horses. Appl. Anim. Behav. Sci. 69, 67–83. Cotman, C., Berchtold, N., Christie, L.-A., 2007. Exercise builds brain health: key roles of growth factor cascades and inflamation. Trends Neurosci. 30, 464–472.
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Ferreira, A., Lamarque, S., Boyer, P., Perez-Diaz, F., Jouvent, R., Cohen-Salmon, C., 2006. Spontaneous appetence for wheel-running: a model of dependency on physical activity in rat. Eur. Psychiatr. 21, 580–588. Hadley, C., Hadley, B., Ephraim, S., Yang, M., Lewis, M.H., 2006. Spontaneous stereotypy and environmental enrichment in deer mice (Peromyscus maniculatus): reversibility of experience. Appl. Anim. Behav. Sci. 97, 312–322. Howerton, C.L., Garner, J.P., Mench, J.A., 2008. Effects of a running wheel-igloo enrichment on aggression, hierarchy linearity, and stereotypy in group-housed male CD-1 (ICR) mice. Appl. Anim. Behav. Sci. 115, 90–103. Latham, N., Wurbel, H., 2006. Wheel-running: a common rodent stereotypy? In: Mason, G.J., Rushen, J. (Eds.), Stereotypic Animal Behaviour, 2nd ed. CABI, Oxford, p. 91. Mason, G., Rushen, J. (Eds.), 2006. Stereotypic Animal Behaviour, 2nd ed. CABI, Oxford, 367 pp. Mason, G.J., 1993. Age and context affect the stereotypies of caged mink. Behaviour 127, 191–229. McCloskey, D.P., Adamo, D.S., Anderson, B.J., 2001. Exercise increases metabolic capacity in the motor cortex and striatum, but not in the hippocampus. Brain Res. 891, 168–175. Pietropaolo, S., Feldon, J., Alleva, E., Cirulli, F., Yee, B.K., 2006. The role of voluntary exercise in enriched rearing: a behavioral analysis. Behav. Neurosci. 120, 787–803. Powell, S.B., Newman, H.A., McDonald, T.A., Bugenhagen, P., Lewis, M.H., 2000. Development of spontaneous stereotyped behavior in deer mice: effects of early and late exposure to a more complex environment. Dev. Psychobiol. 37, 100–108. Richter, H., Ambree, O., Lewejohann, L., Herring, A., Keyvani, K., Paulus, W., Palme, R., Touma, C., Schabitz, W.R., Sachser, N., 2008. Wheel-running in a transgenic mouse model of alzheimer’s disease: protection or symptom? Behav. Brain Res. 190, 74–84. Saka, E., Goodrich, C., Harlan, P., Madras, B.K., Graybiel, A.M., 2004. Repetitive behaviors in monkeys are linked to specific striatal activation patterns. J. Neurosci. 24, 7557–7565.
Sherwin, C.M., 1998. Voluntary wheel running: a review and novel interpretation. Anim. Behav. 56, 11–27. Swaisgood, R., Sheperdson, D., 2006. Environmental enrichment for mitigating stereotypies in zoo animals. In: Mason, G.J., Rushen, J. (Eds.), Stereotypic Animal Behaviour, 2nd ed. CABI, Oxford, pp. 256–285. Turner, C.A., Lewis, M.H., 2003. Environmental enrichment: effects on stereotyped behavior and neurotrophin levels. Physiol. Behav. 80, 259–266. Turner, C.A., Lewis, M.H., King, M.A., 2003. Environmental enrichment: effects on stereotyped behavior and dendritic morphology. Dev. Psychobiol. 43, 20– 27. Turner, C.A., Yang, M.C., Lewis, M.H., 2002. Environmental enrichment: effects on stereotyped behavior and regional neuronal metabolic activity. Brain Res. 938, 15–21. van Praag, H., 2009. Exercise and the brain: something to chew on. Trends Neurosci. 32, 283–290. van Praag, H., Kempermann, G., Gage, F.H., 1999. Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat. Neurosci. 2, 266– 270. van Praag, H., Shubert, T., Zhao, C., Gage, F.H., 2005. Exercise enhances learning and hippocampal neurogenesis in aged mice. J. Neurosci. 25, 8680–8685. Werme, M., Messer, C., Olson, L., Gilden, L., Thoren, P., Nestler, E.J., Brene, S., 2002a. Delta FosB regulates wheel running. J. Neurosci. 22, 8133–8138. Werme, M., Thoren, P., Olson, L., Brene, S., 2000. Running and cocaine both upregulate dynorphin mRNA in medial caudate putamen. Eur. J. Neurosci. 12, 2967–2974. Werme, M., Lindholm, S., Thoren, P., Franck, J., Brene, S., 2002b. Running increases ethanol preference. Behav. Brain Res. 133, 301–308. Wurbel, H., 2001. Ideal homes? Housing effects on rodent brain and behaviour. Trends Neurosci. 24, 207–211.
Contents lists available at ScienceDirect
Behavioural Processes journal homepage: www.elsevier.com/locate/behavproc
Effects of access to voluntary wheel running on the development of stereotypy Artur Pawlowicz, Adam Demner, Mark H. Lewis ∗ Department of Psychiatry and McKnight Brain Institute, University of Florida, Gainesville, FL 32611, USA
a r t i c l e
i n f o
Article history: Received 26 August 2009 Received in revised form 12 November 2009 Accepted 15 November 2009 Keywords: Repetitive behavior Exercise Deer mice Environmental enrichment Peromyscus maniculatus
a b s t r a c t Stereotyped motor behaviors are a common consequence of environmental restriction in a wide variety of species. Although environmental enrichment has been shown to substantially reduce stereotypy levels, the various components of enrichment have not been evaluated independently to determine which is responsible for this effect. Exercise, particularly voluntary wheel running, is a promising candidate based on several lines of behavioral and neurobiological evidence. To test the hypothesis that access to wheel running will reduce stereotyped motor behavior, we reared deer mice from weaning with continuous access to either a functional running wheel or a locked wheel. We assessed running behavior throughout this time period and stereotypy levels in a test context at 30 and 45 days post-weaning. We found that exercise did not significantly affect stereotypy level nor was there an association between wheel running and stereotypy. Thus, exercise alone, unlike environmental enrichment, does not prevent the development of stereotypy. These results have important implications for animal welfare. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Abnormal repetitive behaviors are commonly displayed in animals housed in restricted (e.g., zoo, farm, laboratory) environments, as well as animals subjected to early social deprivation (Mason and Rushen, 2006). Indeed, repetitive behaviors are the most common category of abnormal behavior observed in confined animals (Wurbel, 2001). Some examples of aberrant repetitive behaviors observed in animals maintained in confinement include crib-biting and head-weaving in horses (Cooper et al., 2000), head-twirling in mink (Mason, 1993) and patterned locomotion in carnivores (Clubb and Vickery, 2006). Increasing the complexity of the environment has been a successfully employed strategy for reducing such stereotyped behavior in a number of species (e.g., Swaisgood and Sheperdson, 2006). For example, we have demonstrated repeatedly that increased environmental complexity (environmental enrichment) markedly attenuates the development of stereotyped behavior in deer mice (Hadley et al., 2006; Powell et al., 2000; Turner et al., 2002, 2003; Turner and Lewis, 2003). Typically, environmental enrichment involves larger, more complex housing, with increased novelty, and opportunities for exploration and exercise, areas for hiding and nesting and, sometimes, limited foraging as well (Hadley et al., 2006; Turner et al., 2003). This has made it difficult, however, to isolate the specific
∗ Corresponding author at: Department of Psychiatry, 100 S. Newell Drive, Mc Knight Brain Institute, Suite L4-100, Gainesville, FL 32611, USA. Tel.: +1 352 294 0415; fax: +1 352 294 0425. E-mail address: marklewis@ufl.edu (M.H. Lewis). 0376-6357/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2009.11.008
environmental features and mechanisms by which such experience induces its neurobiological and behavioral effects including attenuation of repetitive behaviors. A ubiquitous feature of more complex housing for laboratory rodents is a running wheel. Voluntary exercise in the form of wheel running has been advanced as being essential for the effects of environmental enrichment on certain behavioral and brain outcomes. For example, exercise has been shown to improve various measures of cognitive function in both intact rodents and mouse models of dementia and to result in increased neurogenesis and neurotrophin expression (see Cotman et al., 2007; van Praag, 2009 for recent reviews). These findings and the preeminent role posited for voluntary wheel running in the effects of environmental enrichment suggest its efficacy in attenuating the development of stereotypy. Indeed, two recent studies have concluded that wheel running significantly reduced the level of stereotypy in mice induced by environmental restriction (Howerton et al., 2008) or by a genetic mutation (Richter et al., 2008). Our own unpublished preliminary work, however, did not support such a conclusion. This study tested the effects in deer mice of either 30 or 60 days of access to voluntary wheel running starting at weaning (21–24 days of age). At both the 30 and 60 days post-weaning time points, we compared stereotypy scores from 21 mice which had access to running wheels with scores from 18 mice with no running wheels in their cages. At neither time point was there evidence for a difference between groups in stereotypy. This study must be considered preliminary, however, as we did not monitor or quantify wheel running nor did we employ a locked wheel as a control. Thus, the role of wheel running in enriched laboratory environments and its effects on stereotypic behavior remain unclear.
A. Pawlowicz et al. / Behavioural Processes 83 (2010) 242–246
To complicate the issue further, wheel running has been conceptualized as a stereotypic behavior in itself with the potential to become compulsive and self-reinforcing (Latham and Wurbel, 2006; Sherwin, 1998). Consistent with this perspective, wheel running upregulates Fos in striatum similar to the Fos expression pattern associated with drug-induced stereotypic behavior (Saka et al., 2004; Werme et al., 2002a,b). Moreover, running seems to activate many of the same pathways as drugs of abuse (Ferreira et al., 2006; Werme et al., 2000, 2002a,b). Accordingly, to investigate the effect of wheel running on stereotypy and reconcile the differing views of the role of exercise in enrichment, we directly examined the effect of voluntary access to a running wheel on the development of spontaneous stereotypy in deer mice. We hypothesized that frequent exercise on the running wheel would decrease stereotypy level, acting in a manner similar to environmental enrichment. Furthermore, we hypothesized that the amount of wheel running would correlate negatively with stereotypy level. 2. Methods 2.1. Subjects Deer mice (Peromyscus maniculatus) exhibit two major topographies of stereotypy (repetitive hindlimb jumping and backward somersaulting) as a consequence of being reared in standard laboratory caging. These behaviors do not require social isolation, specific cues or contexts, pharmacological agents, or specific CNS insult for induction. These behaviors occur at a high rate, persist across much of the life of the animal and appear relatively early in development, sometimes as early as weaning. As mentioned previously, we have shown repeatedly that environmental enrichment (either 30 or 60 days duration starting at weaning) markedly attenuates the development of stereotypy. For this study, 30 deer mice including 12 males and 18 females, were taken from the breeding colony maintained by our laboratory. Upon weaning at 21–24 days of age, groups of three same-sex deer mice were randomly assigned to either a standard cage with a functional running wheel (Wheel Running group) or a standard cage with a locked wheel (Control group). The groups were balanced for sex, with each having 6 males and 9 females. The experimental protocol was approved by the University of Florida Instititional Animal Care and Use Committee (IACUC) and complies with the NIH Guide for Care and Use of Laboratory Animals. 2.2. Housing and apparatus All deer mice were housed three per cage in standard polycarbonate rodent cages of dimensions 48 cm × 27 cm × 15 cm with pine bedding, nesting materials and access to rodent chow and water ad libitum. Deer mice housed in this way consistently exhibit high levels of repetitive behavior. Deer mice in the Wheel Running group were given unlimited access to a Bio-Serv Fast Trac dome-type running wheel placed on one side of their cage (BioServ, Frenchtown, NJ). The running wheels were checked daily to ensure proper and uninterrupted functioning. Subjects in the Control group were given unlimited access to the same type of wheel, but the wheel was immobilized with a wire attachment to prevent its use for running. The deer mice were housed in climate-controlled conditions maintained at 20–25 ◦ C and 50–70% humidity. The lighting conditions were controlled by an automatic timer, maintaining a 16 h light to 8 h dark cycle. This L:D cycle has been in effect for this colony for many years and our previous studies with deer mice have
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employed this cycle. Red lights during the dark cycle permitted video recording. Video recording during the dark cycles for both the wheel running and stereotypy assessments was conducted using infrared cameras. Stereotypy testing was conducted in separate, individual Plexiglass testing cages (22 cm × 25 cm × 28 cm). While in the testing cages, all mice had free access to the same food and water as in their home cages, but did not have access to running wheels. Stereotypy counts were calculated automatically in these cages using a photocell detection apparatus from Columbus Instruments (Columbus, OH). Photocells were placed such that vertical activity (jumping and somersaulting) resulted in beam breaks whereas rearing did not. Horizontal activity (e.g., locomotion) was not automatically recorded. 2.3. Assessment of wheel running behavior Each Wheel Running cage was inspected daily and recorded weekly throughout the course of the experiment during the dark cycle (8 h per week) using an infrared camera. Small areas of fur were clipped on two of the mice to aid in identification. Subsequently, the wheel running activity for each mouse was scored from the video using a partial interval scoring method. The occurrence or non-occurrence of wheel running for each 1-min interval was coded to determine the proportion of time that each mouse spent actively using the running wheel. If a mouse ran during a given minute, it received a score of 1; if it did not, it received a score of 0. The scores were added and divided by the total number of minutes to determine the proportion of time intervals spent wheel running. For data analysis purposes, wheel running intervals from two different dark cycles were averaged to generate a single score for each animal. 2.4. Assessment of spontaneous stereotypy Each mouse was tested at 30 and 45 days post-weaning in an individual Plexiglass cage during their 8-h dark cycle. These time points not only replicated the 30 days used in our preliminary study but also tested an intermediate point (45 days). In other studies from our lab we have determined that stereotypy in deer mice asymptotes to adult levels at approximately 45 days post-weaning. Mice were allowed to habituate to the testing environment after handling for at least 1 h prior to the onset of the dark cycle to allow recovery from the stress of handling and transition to a novel environment. During the entire testing period, the deer mice were individually caged and had access to food and water. Stereotypy counts, which equated to the number of vertical jumps or backwards somersaults a deer mouse performed during that 8 h, were automatically scored for each mouse using the photocell detection system. Photobeams were positioned to be interrupted by the vertical motion of jumping and somersaulting but not by rearing. All testing sessions were also recorded using infrared video cameras to insure accuracy of the automated recording. 2.5. Data analysis Following data verification, the stereotypy scores from each session (taken at 30 and 45 days post-weaning) for the Wheel Running and Control groups were analyzed for differences due to group and testing period, as well as their interaction using a 2 × 2 repeated measures analysis of variance (RMANOVA). For the Wheel Running group, the proportion of time spent running for each deer mouse (the average of two dark cycles) was also correlated with the mouse’s stereotypy scores using a Pearson product–moment correlation. An alpha value of less than 0.05 was used as the criterion for statistical significance in both analyses.
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Fig. 1. The mean stereotypy scores for the Wheel Running and Control groups after 30 or 45 days of access to a running wheel or locked wheel following weaning. Error bars represent standard error of the mean.
Fig. 3. Association between stereotypy frequency at 30 days post-weaning and proportion of time spent running for the Wheel Running group.
3. Results
from mice which either had access to running wheels (n = 21) or no running wheels available (n = 18). No significant difference between groups were found at either time point.
3.1. Stereotypy assessment For the Wheel Running group (n = 15), total stereotypy counts for each 8-h period ranged from 972 to 14,114 at 30 days and from 1135 to 12,453 at 45 days. For the Control group (n = 15), scores ranged from 1241 to 16,117 at 30 days and from 1180 to 12,493 at 45 days. RMANOVA results indicated no main effect of group (F(1,27) = 1.87, p = 0.24), indicating that the stereotypy levels did not differ between the Wheel Running and Control groups irrespective of testing session. In addition, no main effect of testing session was found (F(1,27) = 0.35, p = 0.56), reflecting little change in stereotypy level between the day 30 and day 45 testing sessions. Fig. 1 shows the mean stereotypy counts for the Running and Control groups for both testing sessions. Finally, no significant interaction between group and testing session on stereotypy scores was found (F(1,27) = 0.37, p = 0.55). For comparison purposes, we have presented the results of our preliminary study in Fig. 2. This figure shows stereotypy scores, assessed as described in Section 2, at 30 and 60 days post-weaning
The deer mice assigned to the Wheel Running group spent from 9 to 71% of 1 min intervals of their active periods using the running wheel, averaging 43% (n = 15). Video records indicated that all 3 mice were able to use the running wheel at the same time and frequently did. For the deer mice in the Wheel Running group, the correlation between the proportion of 1 min intervals spent running and the 30 day stereotypy counts neared, but did not achieve, statistical significance (r = 0.50, p = 0.056, Fig. 3). However, when an outlying (greater than two standard deviations above the mean) stereotypy score was removed, the correlation was reduced considerably (r = 0.26, p = 0.37). The correlation between the proportion of time spent running and the 45-day stereotypy counts was also not significant (r = 0.11, p = 0.69, Fig. 4).
Fig. 2. The mean stereotypy scores for the Wheel Running and Control groups after 30 or 60 days of access to a running wheel or no running wheel available following weaning. Error bars represent standard error of the mean.
Fig. 4. Association between stereotypy frequency at 45 days post-weaning and proportion of time spent running for the Wheel Running group.
3.2. Wheel running assessment
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4. Discussion Initially, we hypothesized that wheel running would act similarly to environmental enrichment in reducing spontaneous motor stereotypy. We found that deer mice made ample use of the running wheels, spending, on average, almost half their time engaged in such activity. Nevertheless, we found no significant effect of voluntary access to a running wheel for either 30 or 45 days postweaning on stereotypy level. In addition, we found no evidence of an association between amount of wheel running and frequency of stereotypy assessed independently of the animal’s home cage. The lack of effect of exercise on the frequency of stereotyped behavior is consistent with the results of a previous study conducted in our lab (see Fig. 2). In addition to replication, a comparison of the two studies demonstrates that the no wheel condition does not differ from either the active or locked running wheel conditions. As mentioned previously, the preliminary study did not monitor or quantify wheel running. One important conclusion from these results is that wheel running alone does not appear to be responsible for the marked attenuation of stereotypy that we have consistently observed following environmental enrichment. This appears to be the case for all three durations of access to wheel running which equated to both the duration and developmental timing used in our enrichment studies. The present results are not consistent with findings from two recent studies that examined wheel running effects on stereotypy in mice, one using a model of environmental restriction (Howerton et al., 2008) and the other a transgenic mouse model of Alzheimer’s disease (Richter et al., 2008). Although both concluded that wheel running significantly reduced the level of stereotypy, the methodologies employed in each of these studies leave these conclusions open to interpretation. Specifically, both studies assessed stereotypy in the home cage during which the mice had access to running wheels. Given observations that mice spend a large proportion of their active periods using the running wheel, it may be difficult to conclude, unambiguously, that wheel running itself reduced stereotypy in these cases. Indeed, Richter et al. (2008) found that wheel running was inversely correlated with stereotypic behavior, suggesting that wheel running may have stereotypic qualities or certainly competed with the expression of stereotyped behavior. It would seem, then, that difference in outcome between our study and the two previously published studies is that we assessed stereotypy in the absence of access to the running wheel. Other explanations for the inconsistency, beyond choice of testing environments, may include genus differences as well as duration of access to voluntary wheel running, which in Howerton et al. (2008) was only 2 weeks. Deer mice in the Wheel Running group spent a large proportion of their active periods using the running wheel. Consequently, they reduced the time available for other active behaviors, including stereotyped jumping and backward somersaulting, when a wheel was present. Although we did observe noticeably reduced stereotypic behavior when the mice in the Wheel Running group had access to a running wheel (data not shown), our results show that this difference is not maintained once the deer mice no longer had access to a running wheel. Moreover, our results indicated no significant association between wheel running in the home cage and stereotypy exhibited in the testing cage. Moreover, since the three deer mice in any particular cage were able to use the wheel simultaneously and often did, it is unlikely that the amount of running was affected by competition or position in a dominance hierarchy. Physical exercise has been posited to be a critical component of the effects of environmental enrichment on brain and behavior. Voluntary wheel running has been shown to induce many of the same neurochemical and proliferative changes in the brain as complete environmental enrichment, in many cases to the same degree.
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Several studies have found that voluntary wheel running alone significantly increases hippocampal neurogenesis in rodents, reaching a level comparable to full environmental enrichment (Aberg et al., 2008; Bjornebekk et al., 2005; Brown et al., 2003; van Praag et al., 1999). Similar to enrichment, wheel running enhances learning ability and neurogenesis in both young and aged mice (van Praag et al., 2005). Wheel running also increases neuronal metabolism in the motor cortex and striatum (McCloskey et al., 2001). All of these effects have also been reported in environmental enrichment, suggesting that voluntary exercise may play a significant role in mediating these effects in enriched rodents. It may be, however, that wheel running may exert some of its effects in concert with other components of environmental enrichment. This was the conclusion of Pietropaolo et al. (2006) who found that whereas wheel running alone had few effects, it interacted significantly with other aspects of environmental enrichment when the two were combined. Indeed, some findings from this study suggest that the combined effects may be synergistic rather than simply additive. A review by Sherwin (1998) proposed that wheel running in rodents appears maladaptive in some circumstances, and might be an artifact of artificial laboratory conditions as well as being a stereotypic behavior in itself. Latham and Wurbel (2006) argue that while wheel running may occur in both stereotypic and nonstereotypic cases, there is sufficient evidence to claim that wheel running should be considered a stereotypy. Compulsive wheel running activates many of the same brain pathways as drugs of abuse (Ferreira et al., 2006). Cross-sensitization between wheel running and amphetamine has been demonstrated and wheel running increases preference for ethanol consumption, indicating a sensitization of reward pathways (Werme et al., 2002a,b). The present study was not designed to address the question of whether wheel running is itself a stereotyped behavior. We did not, however, find any significant correlation between time spent wheel running and stereotypy level. This lack of a correlation suggests that wheel running and repetitive jumping/somersaulting are likely not interchangeable stereotypies. From an animal welfare perspective, our results indicate that access to a running wheel is not a substitute for environmental enrichment, at least with respect to the development of repetitive behavior, but rather should be used in concert with other components of environmental enrichment. Conversely, we also found no evidence that providing access to a running wheel is in itself harmful, at least with respect to aberrant repetitive behavior. The components of environmental enrichment that affect repetitive behavior and its underlying neurobiology (i.e., cortico-basal ganglia circuitry) remain an important and unanswered question that have important implications for clinical disorders such as autism that have repetitive behavior as part of their presentation as well as for animal welfare.
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