Experimental evolution and phenotypic plasticity of hindlimb bones in high-activity house mice

Studies of rodents have shown that both forced and voluntary chronic exercise cause increased hindlimb bone diameter, mass, and strength. Among species of mammals, "cursoriality" is generally associated with longer limbs as well as relative lengthening of distal limb segments, resulting in an increased metatarsal/femur (MT/F) ratio. Indeed, we show that phylogenetic analyses of previously published data indicate a positive correlation between body mass-corrected home range area and both hindlimb length and MT/F in a sample of 19 species of Carnivora, although only the former is statistically significant in a multiple regression. Therefore, we used an experimental evolution approach to test for possible adaptive changes (in response to selective breeding and/or chronic exercise) in hindlimb bones of four replicate lines of house mice bred for high voluntary wheel running (S lines) for 21 generations and in four nonselected control (C) lines. We examined femur, tibiafibula, and longest metatarsal of males housed either with or without wheel access for 2 months beginning at 25-28 days of age. As expected from previous studies, mice from S lines ran more than C (primarily because the former ran faster) and were smaller in body size (both mass and length). Wheel access reduced body mass (but not length) of both S and C mice. Analysis of covariance (ANCOVA) revealed that body mass was a statistically significant predictor of all bone measures except MT/F ratio; therefore, all results reported are from ANCOVAs. Bone lengths were not significantly affected by either linetype (S vs. C) or wheel access. However, with body mass as a covariate, S mice had significantly thicker femora and tibiafibulae, and wheel access also significantly increased diameters. Mice from S lines also had heavier feet than C, and wheel access increased both foot and tibiafibula mass. Thus, the directions of evolutionary and phenotypic adaptation are generally consistent. Additionally, S-line individuals with the mini-muscle phenotype (homozygous for a Mendelian recessive allele that halves hindlimb muscle mass [Garland et al., 2002, Evolution 56:1,267-1,275]) exhibited significantly longer and thinner femora and tibiafibulae, with no difference in bone masses. Two results were considered surprising. First, no differences were found in the MT/F ratio (the classic indicator of cursoriality). Second, we did not find a significant interaction between linetype and wheel access for any trait, despite the higher running rate of S mice.     

Initiation of limb regeneration: the critical steps for regenerative capacity

While urodele amphibians (newts and salamanders) can regenerate limbs as adults, other tetrapods (reptiles, birds and mammals) cannot and just undergo wound healing. In adult mammals such as mice and humans, the wound heals and a scar is formed after injury, while wound healing is completed without scarring in an embryonic mouse. Completion of regeneration and wound healing takes a long time in regenerative and non-regenerative limbs, respectively. However, it is the early steps that are critical for determining the extent of regenerative response after limb amputation, ranging from wound healing with scar formation, scar-free wound healing, hypomorphic limb regeneration to complete limb regeneration. In addition to the accumulation of information on gene expression during limb regeneration, functional analysis of signaling molecules has recently shown important roles of fibroblast growth factor (FGF), Wnt/beta-catenin and bone morphogenic protein (BMP)/Msx signaling. Here, the routine steps of wound healing/limb regeneration and signaling molecules specifically involved in limb regeneration are summarized. Regeneration of embryonic mouse digit tips and anuran amphibian (Xenopus) limbs shows intermediate regenerative responses between the two extremes, those of adult mammals (least regenerative) and urodele amphibians (more regenerative), providing a range of models to study the various abilities of limbs to regenerate.     

Selective breeding for high endurance running increases hindlimb symmetry

Comparative studies provide correlational evidence of morphological adaptations for high locomotor performance, such as the classical indicators of cursoriality in mammals, long limbs and high metatarsal/femur ratios. More recently, enlarged femoral condyles have been suggested as an adaptation for high endurance running in the genus Homo. Asymmetry of locomotor appendages should adversely affect locomotor abilities, but this has not been studied in a rigorous evolutionary context. We used experimental evolution to test for morphological adaptations associated with high voluntary wheel running in selectively bred lines of mice. Surprisingly, the classical indicators of cursoriality had not evolved in concert with high activity levels. Instead, high runners had larger femoral condyles and reduced directional asymmetry of hindlimb bones. We hypothesize that greater limb symmetry and larger femoral heads are general adaptations associated with sustained, high-speed locomotion.     

Bone modeling response to voluntary exercise in the hindlimb of mice

The functional adaptation of juvenile mammalian limb bone to mechanical loading is necessary to maintain bone strength. Diaphyseal size and shape are modified during growth through the process of bone modeling. Although bone modeling is a well-documented response to increased mechanical stress on growing diaphyseal bone, the effect of proximodistal location on bone modeling remains unclear. Distal limb elements in cursorial mammals are longer and thinner, most likely to conserve energy during locomotion because they require less energy to move. Therefore, distal elements are hypothesized to experience greater mechanical loading during locomotion and may be expected to exhibit a greater modeling response to exercise. In this study, histomorphometric comparisons are made between femora and tibiae of mice treated with voluntary exercise and a control group (N = 20). We find that femora of exercised mice exhibit both greater bone growth rates and growth areas than do controls (P < 0.05). The femora of exercised mice also have significantly greater cortical area, bending rigidity, and torsional rigidity (P < 0.05), although bending and torsional rigidity are comparable when standardized by bone length. Histomorphometric and cross-section geometric properties of the tibial midshaft of exercised and control mice did not differ significantly, although tibial length was significantly greater in exercised mice (P < 0.05). Femora of exercised mice were able to adapt to increased mechanical loading through increases in compressive, bending, and torsional rigidity. No such adaptations were found in the tibia. It is unclear if this is a biomechanical adaptation to greater stress in proximal elements or if distal elements are ontogenetically constrained in a tradeoff of bone strength of distal elements for bioenergetic efficiency during locomotion.     

Reliability of the method of levels for determining cutaneous temperature sensitivity

Determination of the thermal thresholds is used clinically for evaluation of peripheral nervous system function. The aim of this study was to evaluate reliability of the method of levels performed with a new, low cost device for determining cutaneous temperature sensitivity. Nineteen male subjects were included in the study. Thermal thresholds were tested on the right side at the volar surface of mid-forearm, lateral surface of mid-upper arm and front area of mid-thigh. Thermal testing was carried out by the method of levels with an initial temperature step of 2°C. Variability of thermal thresholds was expressed by means of the ratio between the second and the first testing, coefficient of variation (CV), coefficient of repeatability (CR), intraclass correlation coefficient (ICC), mean difference between sessions (S1-S2diff), standard error of measurement (SEM) and minimally detectable change (MDC). There were no statistically significant changes between sessions for warm or cold thresholds, or between warm and cold thresholds. Within-subject CVs were acceptable. The CR estimates for warm thresholds ranged from 0.74°C to 1.06°C and from 0.67°C to 1.07°C for cold thresholds. The ICC values for intra-rater reliability ranged from 0.41 to 0.72 for warm thresholds and from 0.67 to 0.84 for cold thresholds. S1-S2diff ranged from -0.15°C to 0.07°C for warm thresholds, and from -0.08°C to 0.07°C for cold thresholds. SEM ranged from 0.26°C to 0.38°C for warm thresholds, and from 0.23°C to 0.38°C for cold thresholds. Estimated MDC values were between 0.60°C and 0.88°C for warm thresholds, and 0.53°C and 0.88°C for cold thresholds. The method of levels for determining cutaneous temperature sensitivity has acceptable reliability.     

Wheel running in the wild

The importance of exercise for health and neurogenesis is becoming increasingly clear. Wheel running is often used in the laboratory for triggering enhanced activity levels, despite the common objection that this behaviour is an artefact of captivity and merely signifies neurosis or stereotypy. If wheel running is indeed caused by captive housing, wild mice are not expected to use a running wheel in nature. This however, to our knowledge, has never been tested. Here, we show that when running wheels are placed in nature, they are frequently used by wild mice, also when no extrinsic reward is provided. Bout lengths of running wheel behaviour in the wild match those for captive mice. This finding falsifies one criterion for stereotypic behaviour, and suggests that running wheel activity is an elective behaviour. In a time when lifestyle in general and lack of exercise in particular are a major cause of disease in the modern world, research into physical activity is of utmost importance. Our findings may help alleviate the main concern regarding the use of running wheels in research on exercise.     

Regeneration potency of mouse limbs

Mammalians have a low potency for limb regeneration compared to that of amphibians. One explanation for the low potency is the deficiency of cells for regenerating amputated limbs in mammals. Amphibians can form a blastema with dedifferentiated cells, but mammals have few such cells. In this paper, we report limb formation, especially bone/cartilage formation in amputated limbs, because bone/cartilage formation is a basic step in limb pattern regeneration. After the amputation of limbs of a neonatal mouse, hypertrophy of the stump bone was observed at the amputation site, which was preceded by cell proliferation and cartilage formation. However, no new elements of bone/cartilage were formed. Thus, we grafted limb buds of mouse embryo into amputated limbs of neonatal mice. When the intact limb bud of a transgenic green fluorescent protein (GFP) mouse was grafted to the limb stump after amputation at the digit joint level, the grafted limb bud grew and differentiated into bone, cartilage and soft tissues, and it formed a segmented pattern that was constituted by bone and cartilage. The skeletal pattern was more complicated when limb buds at advanced stages were used. To examine if the grafted limb bud autonomously develops a limb or interacts with stump tissue to form a limb, the limb bud was dissociated into single cells and reaggregated before grafting. The reaggregated limb bud cells formed similar digit-like bone/cartilage structures. The reaggregated grafts also formed segmented cartilage. When the reaggregates of bone marrow mesenchymal cells were grafted into the stump, these cells formed cartilage, as do limb bud cells. Finally, to examine the potency of new bone formation in the stump tissue without exogenously supplied cells, we grafted gelatin gel containing BMP-7. BMP induced formation of several new bone elements, which was preceded by cartilage formation. The results suggest that the environmental tissues of the stump allow the formation of cartilage and bone at least partially, and that limb formation will be possible by supplying competent cells endogenously or exogenously in the future.     

Plasma corticosterone response to acute and chronic voluntary exercise in female house mice.

Plasma levels of corticosterone (B) respond acutely to exercise in all mammals that have been studied, but the literature contains conflicting reports regarding how chronic activity alters this response. We measured acute and chronic effects of voluntary activity on B in a novel animal model, mice selectively bred for high voluntary wheel running. Female mice were housed with or without wheels for 8 wk beginning at 26 days of age. Wheel-access selection mice had significantly higher B at night 8, day 15, and night 29, compared with wheel-access controls. Elevation of B was an acute effect of voluntary exercise. When adjusted for running in the previous 20 min, no difference between wheel-access selection and control animals remained. No training effect on B response was observed. These results are among the strongest evidence that, in some animals, the acute B response is unaffected by chronic voluntary exercise. In mice without wheels, selection mice had significantly higher B than controls at day 15, night 29, and night 50, suggesting that selection resulted in a modulation of the hypothalamic-pituitary-adrenal axis. Growth over the first 4 wk of treatment was significantly and inversely related to average night B levels within each of the four treatment groups.     

Epigenetic Effects on Integration of Limb Lengths in a Mouse Model: Selective Breeding for High Voluntary Locomotor Activity

Mammals exhibit a similar pattern of integration among homologous limb elements, the strength of which is believed to vary in response to selection for functional coordination or similarity. Although integration is hypothesized to primarily reflect the effect of genes intrinsic to limbs, extrinsic genetic or epigenetic factors may also affect the strength of integration through their impact on the magnitude and direction of skeletal variance or covariance. Such factors as neuromuscular coordination or bone-muscle interactions may therefore play a role in both canalization and the structure or magnitude of limb integration. If this were the case, then increased levels of locomotor activity would be predicted to increase canalization and the magnitude of covariation between limbs. To investigate whether postnatal activity levels can have a significant effect on variance within or covariance among homologous limb elements, we compared four groups of male mice from a long-term selective breeding experiment: (1) mice from lines bred for increased voluntary activity on running wheels and allowed free access to a wheel for 8 weeks beginning at weaning (“active”), (2) selected mice that did not have wheel access (“sedentary”), (3) active mice from non-selected control lines, and (4) sedentary control mice. Mice from selected lines that had wheel access ran significantly more than control-line mice. However, when controlled for activity, linetype, and body mass, results indicate few significant differences in means, variance, or covariation structure, and no significant differences in integration between limbs, suggesting that postnatal activity levels do not significantly affect canalization or integration of limb lengths. A possible explanation for this result is that whereas baseline levels of postnatal activity may help to maintain patterns of variance and integration, increased levels of activity do not further increase these measures. Investigations into disrupted epigenetic processes (e.g., via models in which neuromuscular coordination is impaired) are required to further test hypotheses about how canalization or integration of limb variation is affected by epigenetic factors.     

Loss of desmin leads to impaired voluntary wheel running and treadmill exercise performance.

We examined voluntary wheel running and forced treadmill running exercise performance of wild-type mice and mice null for the desmin gene. When given access to a cage wheel, desmin null mice spent less time running and ran less far than wild-type mice. Wild-type mice showed a significant training effect with prolonged voluntary wheel running, as evidenced by an increase in mean running speed across the 3-wk exercise period, whereas desmin null mice did not. Desmin null mice also performed less well in acute treadmill stress and endurance tests compared with wild-type mice. We also evaluated serum creatine kinase (CK) activity in wild-type and desmin null mice in response to running. Voluntary running did not result in elevated CK activity in either wild-type or desmin null mice, whereas downhill treadmill running caused significant increases in serum CK activity in both wild-type and desmin null mice. However, the increase in serum CK was significantly less in desmin null mice than in wild-type mice. These results suggest that the lack of desmin adversely affects the ability of mice to engage in both chronic and acute bouts of endurance running exercise but that this decrement in performance is not associated with an increase in serum CK activity.     

Behavioral assessment of intermittent wheel running and individual housing in mice in the laboratory

Physical cage enrichment—exercise devices for rodents in the laboratory—often includes running wheels. This study compared responses of mice in enriched physical and social conditions and in standard social conditions to wheel running, individual housing, and open-field test. The study divided into 6 groups, 48 female BALB/c mice group housed in enriched and standard conditions. On alternate days, the study exposed 2 groups to individual running wheel cages. It intermittently separated from their cage mates and housed individually 2 groups with no running wheels; 2 control groups remained in enriched or standard condition cages. There were no significant differences between enriched and standard group housed mice in alternate days' wheel running. Over time, enriched, group housed mice ran less. Both groups responded similarly to individual housing. In open-field test, mice exposed to individual housing without running wheel moved more and faster than wheel running and home cage control mice. They have lower body weights than group housed and wheel running mice. Intermittent withdrawal of individual housing affects the animals more than other commodities. Wheel running normalizes some effects of intermittent separation from the enriched, social home cage.     

Genetic variations and physical activity as determinants of limb bone morphology: an experimental approach using a mouse model

To gain insight into past human physical activity, anthropologists often infer functional loading history from the morphology of limb bone remains. It is assumed that, during life, loading had a positive, dose-dependent effect on bone structure that can be identified despite other effects. Here, we investigate the effects of genetic background and functional loading on limb bones using mice from an artificial selection experiment for high levels of voluntary wheel running. Growing males from four replicate high runner (HR) lines and four replicate nonselected control (C) lines were either allowed or denied wheel access for 2 months. Using μCT, femoral morphology was assessed at two cortical sites (mid-diaphysis, distal metaphysis) and one trabecular site (distal metaphysis). We found that genetic differences between the linetypes (HR vs. C), between the replicate lines within linetype, and between individuals with and without the so-called "mini-muscle" phenotype (caused by a Mendelian recessive gene that halves limb muscle mass) gave rise to significant variation in nearly all morphological indices examined. Wheel access also influenced femoral morphology, although the functional response did not generally result in enhanced structure. Exercise caused moderate periosteal enlargement, but relatively greater endocortical expansion, resulting in significantly thinner cortices and reduced bone area in the metaphysis. The magnitude of the response was independent of distance run. Mid-diaphyseal bone area and area moments, and trabecular morphology, were unaffected by exercise. These results underscore the strong influence of genetics on bone structure and the complexity by which mechanical stimuli may cause alterations in it.     

Muscle-bone properties after prolonged voluntary wheel running in a mouse model of dominant severe osteogenesis imperfecta

Objective:The objective of the current study is to assess the effect of a seven-week voluntary wheel running intervention on muscles and bones properties in a mouse model mimicking dominant severe osteogenesis imperfecta (OI).Methods:Female wild-type (WT) and OI (Col1a1^Jrt/+) mice either performed voluntarily wheel-running exercise for 7-weeks or remained sedentary. Running distance and speed, forelimb grip strength, isolated muscle force and fatigability as well as bone morphology and mechanical properties were assessed.Results:We demonstrate that female WT and OI mice voluntarily performed exercise, although OI mice exercised less than WT littermates. The exercise regimen increased soleus muscle masses in WT and OI but increased relative grip strength in WT mice only. Specific muscle force and fatigability were similar between WT and OI mice and did not improve with exercise. Furthermore, the exercise regimen did not improve the femoral architectural and biomechanical properties in OI mice.Conclusion:Our study suggests that voluntary wheel running is not appropriate to assess the effects of exercise in a mouse model of OI. Findings from exercising OI mice model studies may not necessarily be transferable to humans.     

Running-induced anxiety is dependent on increases in hippocampal neurogenesis

Exercise, specifically voluntary wheel running, is a potent stimulator of hippocampal neurogenesis in adult mice. In addition, exercise induces behavioral changes in numerous measures of anxiety in rodents. However, the physiological underpinnings of these changes are poorly understood. To investigate the role of neurogenesis in exercise-mediated anxiety, we examined the cellular and behavioral effects of voluntary wheel running in mice with a reduction in hippocampal neurogenesis, achieved through conditional deletion of ataxia telangiectasia-mutated and rad-3-related protein (ATR), a cell cycle checkpoint kinase necessary for normal levels of neurogenesis. Following hippocampal microinjection of an adeno-associated virus expressing Cre recombinase to delete ATR, mice were exposed to 4 weeks of voluntary wheel running and subsequently evaluated for anxiety-like behavior. Wheel running resulted in increased cell proliferation and neurogenesis, as measured by bromodeoxyuridine and doublecortin, respectively. Wheel running also resulted in heightened anxiety in the novelty-induced hypophagia, open field and light-dark box tests. However, both the neurogenic and anxiogenic effects of wheel running were attenuated following hippocampal ATR deletion, suggesting that increased neurogenesis is an important mediator of exercise-induced anxiety.     

Use of helium-oxygen to examine the effect of cold acclimation on the summit metabolism of a marsupial, Dasyuroides byrnei

To determine whether marsupial mammals increase their metabolic capabilities during cold acclimation, the metabolism of both warm and cold acclimated Dasyuroides byrnei was examined by exposure to cold in a helium-oxygen atmosphere. Mean values of heat production and conductance were significantly higher in a helium-oxygen atmosphere than in air. Body temperature did not change until metabolic capacity was exhausted. Both cold and warm acclimated groups could maintain a metabolic scope of 10-11 times the basal or standard level for this species. Such a metabolic scope is much higher than levels recorded for placental mammals. At very low ambient temperatures cold acclimated D. byrnei could sustain a high level of heat production longer than could warm acclimated animals. While there are some similarities between marsupial mammals and placental mammals in their responses to cold acclimation, an increase in maximum metabolism, as reported for placentals, does not seem to occur in marsupials.     

Evolution of posture in amniotes–Diving into the trabecular architecture of the femoral head

Extant amniotes show remarkable postural diversity. Broadly speaking, limbs with erect (strongly adducted, more vertically oriented) posture are found in mammals that are particularly heavy (graviportal) or show good running skills (cursorial), while crouched (highly flexed) limbs are found in taxa with more generalized locomotion. In Reptilia, crocodylians have a “semi-erect” (somewhat adducted) posture, birds have more crouched limbs and lepidosaurs have sprawling (well-abducted) limbs. Both synapsids and reptiles underwent a postural transition from sprawling to more erect limbs during the Mesozoic Era. In Reptilia, this postural change is prominent among archosauriforms in the Triassic Period. However, limb posture in many key Triassic taxa remains poorly known. In Synapsida, the chronology of this transition is less clear, and competing hypotheses exist. On land, the limb bones are subject to various stresses related to body support that partly shape their external and internal morphology. Indeed, bone trabeculae (lattice-like bony struts that form the spongy bone tissue) tend to orient themselves along lines of force. Here, we study the link between femoral posture and the femoral trabecular architecture using phylogenetic generalized least squares. We show that microanatomical parameters measured on bone cubes extracted from the femoral head of a sample of amniote femora depend strongly on body mass, but not on femoral posture or lifestyle. We reconstruct ancestral states of femoral posture and various microanatomical parameters to study the “sprawling-to-erect” transition in reptiles and synapsids, and obtain conflicting results. We tentatively infer femoral posture in several hypothetical ancestors using phylogenetic flexible discriminant analysis from maximum likelihood estimates of the microanatomical parameters. In general, the trabecular network of the femoral head is not a good indicator of femoral posture. However, ancestral state reconstruction methods hold great promise for advancing our understanding of the evolution of posture in amniotes.     

Selective breeding as a tool to probe skeletal response to high voluntary locomotor activity in mice

We present a novel mouse-model for the study of skeletal structureand evolution, based on selective breeding for high levels ofvoluntary wheel running. Whereas traditional models (originallyinbred strains, more recently knockouts and transgenics) relyon the study of mutant or laboratory-manipulated phenotypes,we have studied changes in skeletal morphometrics resultingfrom many generations of artificial selection for high activityin the form of wheel running, in which mice engage voluntarily.Mice from the four replicate High Runner (HR) lines run nearlythree times as many revolutions during days 5 and 6 of a 6-dayexposure to wheels (1.12 m circumference). We have found significantchanges in skeletal dimensions of the hind limbs, includingdecreased directional asymmetry, larger femoral heads, and widerdistal femora. The latter two have been hypothesized as evolutionaryadaptations for long-distance locomotion in hominids. Exercise-trainingstudies involving experimental groups with and without accessto wheels have shown increased diameters of both femora andtibiafibulae, and suggest genetic effects on trainability (genotype-by-environmentinteractions). Reanalysis of previously published data on bonemasses of hind limbs revealed novel patterns of change in bonemass associated with access to wheels for 2 months. Withoutaccess to wheels, HR mice have significantly heavier tibiafibulaeand foot bones, whereas with chronic access to wheels, a significantincrease in foot bone mass that was linearly related to increasesin daily wheel running was observed. Mice exhibiting a recentlydiscovered small-muscle phenotype ("mini-muscle," [MM] causedby a Mendelian recessive gene), in which the mass of the tricepssurae muscle complex is 50% lower than in normal individuals,have significantly longer and thinner bones in the hind limb.We present new data for the ontogenetic development of musclemass in Control, HR, and MM phenotypes in mice of 1–7weeks postnatal age. Statistical comparisons reveal highly significantdifferences both in triceps surae mass and mass-corrected tricepssurae mass between normal and MM mice at all but the postnatalage of 1 week. Based on previously observed differences in distributionsof myosin isoforms in adult MM mice, we hypothesize that a reductionof myosin heavy-chain type-IIb isoforms with accounts for ourobserved ontogenetic changes in muscle mass.     

Solute transport in growth plate cartilage: in vitro and in vivo

Bone elongation originates from cartilaginous discs (growth plates) at both ends of a growing bone. Here chondrocytes proliferate and subsequently enlarge (hypertrophy), laying down a matrix that serves as the scaffolding for subsequent bone matrix deposition. Because cartilage is generally avascular, all nutrients, oxygen, signaling molecules, and waste must be transported relatively long distances through the tissue for it to survive and function. Here we examine the transport properties of growth plate cartilage. Ex vivo, fluorescence photobleaching recovery methods are used in tissue explants. In vivo, multiphoton microscopy is used to image through an intact perichondrium and into the cartilage of anesthetized mice. Systemically introduced fluorescent tracers are monitored directly as they move from the vasculature into the cartilage. We demonstrate the existence of a relatively permissive region at the midplane of the growth plate, where chondrocytes transition from late proliferative to early hypertrophic stages and where paracrine communication is known to occur between chondrocytes and cells in the surrounding perichondrium. Transport in the living mouse is also significantly affected by fluid flow from the two chondro-osseus junctions, presumably resulting from a pressure difference between the bone vasculature and the cartilage.     

Training Nonhuman Primates Using Positive Reinforcement Techniques

Physical cage enrichment—exercise devices for rodents in the laboratory—often includes running wheels. This study compared responses of mice in enriched physical and social conditions and in standard social conditions to wheel running, individual housing, and open-field test. The study divided into 6 groups, 48 female BALB/c mice group housed in enriched and standard conditions. On alternate days, the study exposed 2 groups to individual running wheel cages. It intermittently separated from their cage mates and housed individually 2 groups with no running wheels; 2 control groups remained in enriched or standard condition cages. There were no significant differences between enriched and standard group housed mice in alternate days' wheel running. Over time, enriched, group housed mice ran less. Both groups responded similarly to individual housing. In open-field test, mice exposed to individual housing without running wheel moved more and faster than wheel running and home cage control mice. They have lower body weights than group housed and wheel running mice. Intermittent withdrawal of individual housing affects the animals more than other commodities. Wheel running normalizes some effects of intermittent separation from the enriched, social home cage.