High Runner Mice Research Articles

A meta-analysis of whole-body and heart mass effect sizes from a long-term artificial selection experiment for high voluntary exercise.

Selection experiments play an increasingly important role in comparative and evolutionary physiology. However, selection experiments can be limited by relatively low statistical power, in part because replicate line is the experimental unit for analyses of direct or correlated responses (rather than number of individuals measured). One way to increase the ability to detect correlated responses is through a meta-analysis of studies for a given trait across multiple generations. To demonstrate this, we applied meta-analytic techniques to two traits (body mass and heart ventricle mass, with body mass as a covariate) from a long-term artificial selection experiment for high voluntary wheel-running behavior. In this experiment, all four replicate High Runner (HR) lines reached apparent selection limits around generations 17-27, running approximately 2.5- to 3-fold more revolutions per day than the four non-selected Control (C) lines. Although both traits would also be expected to change in HR lines (relative heart size expected to increase, expected direction for body mass is less clear), their statistical significance has varied, despite repeated measurements. We compiled information from 33 unique studies and calculated a measure of effect size (Pearson's R). Our results indicate that, despite a lack of statistical significance in most generations, HR mice have evolved larger hearts and smaller bodies relative to controls. Moreover, plateaus in effect sizes for both traits coincide with the generational range during which the selection limit for wheel-running behavior was reached. Finally, since reaching the selection limit, absolute effect sizes for body mass and heart ventricle mass have become smaller (i.e. closer to 0).

Selective breeding for high levels of voluntary exercise in mice does not reduce lifetime reproductive success or lifespan

The ways locomotor abilities may interact with life history traits are complicated and may involve shifts in resource allocation. For example, individuals that have large litters may have reduced energy available to support the organ systems required for high locomotor performance abilities. Alternatively, energy spent on excess physical activity might subtract from that available for somatic maintenance, and thus be negatively related to lifespan or to other fitness components that contribute to lifetime reproductive success (LRS). We studied lifetime reproductive successes, life span, and health span in mice from a long-term (>100 generations) artificial selection experiment that includes 4 replicate High Runner (HR) lines bred for voluntary locomotor activity on wheels and 4 lines that serve as non-selected Controls (C). Since reaching selection limits at around generation 17-27 (depending on line and sex), the HR mice have been running ~3-fold farther on a daily basis as compared with C mice, for both sexes, which leads to substantially increased energy expenditure and food consumption. Importantly, when housed without wheels, HR mice are not only more active, but also consume more food than C mice. In the present study, we tested for differences in LRS between the HR and C lines. We are also comparing lifespan (median, maximum) and healthspan (e.g., by measurement of maximal oxygen consumption [VO2max] during forced exercise and maximal sprint speed. Twelve male-female pairs per line from generation 97 (born Oct. 2021) are being maintained until their end of life. In addition to total number of pups weaned per pair, we record components of LRS, such as age at first and last litter for the pairs, number of litters, and inter-birth intervals. Here, we present a subset of results through the first 2+ years of the experiment. Only two of 100 pairs never gave birth to any litters, and 89 of 100 pairs weaned at least one litter. The total number of litters born to pairs ranged from 0 to 12, and the number of litters from which at least one pup was weaned ranged from 0 to 11, with a mean of 3.96+0.73 (SE) for C lines and 3.43+0.65 for HR lines (P=0.61). The total number of pups weaned (our measure of LRS) ranged from 0 to 80, with a mean of 29.1+8.1 (SE) for C lines and 25.8+4.4 for HR lines (P=0.73). Thus, we found no evidence that Darwinian fitness has been reduced by artificial selection for high voluntary exercise. As of 1 Nov. 2023, approximately 17% of the breeders are still alive, and preliminary analyses do not indicate significant differences between the C and HR lines in median or maximum lifespan. Supported by U.S. National Science Foundation grant IOS-2038528. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A Meta Analysis of Whole-Body and Heart Mass Effect Sizes From a Long-Term Artificial Selection Experiment for High Voluntary Exercise

Effect sizes are a quantitative measure of the magnitude and direction of a phenomenon. These estimates can be compiled across multiple studies to summarize the weight of evidence for a particular alternative hypothesis, which is generally termed a meta-analysis. A meta-analytic approach allows one to summarize information from a wide array of similar studies and reach conclusions that may not be apparent on a case-by-case basis (e.g., to determine the overall effectiveness of a drug). Meta-analyses may be particularly useful in selection experiments, where the magnitude of a correlated response is necessarily expected to increase across generations, perhaps eventually reaching a plateau. Here, we examined the effect of artificial selection for high voluntary wheel running on heart mass in mice, which was expected to increase with selection for aerobic exercise behavior. The experiment spans nearly 30 years and 100 generations. All 4 of the replicate High Runner (HR) lines reached selection limits around generations 17-27, running approximately 2.5-3-fold more revolutions per day than the 4 non-selected Control (C) lines. These selection limits cannot be explained simply by a complete loss of heritability. Functional studies have tried to elucidate subordinate traits that affect motivational and/or ability for endurance exercise and have shown that HR mice differ from C in several other behavioral, physiological, and morphological traits. Several studies of heart ventricle mass have reported larger values in HR mice (with body mass as a covariate), but the differences often do not reach statistical significance (p<0.05). We compiled Least-Square Means, standard errors, F-statistics, degrees of freedom, and p-values from multiple studies. We also recorded pertinent information, such as: covariates used, age, generation, and whether other main effects were present (e.g., sex, wheel access). We calculated a common measure of effect size (Pearson’s R) and associated confidence intervals. This work is part of a larger meta-analysis to quantify effects of selection on various traits across multiple levels of biological organization. Preliminary results indicate that, despite a lack of statistical significance in many generations, HR mice have evolved larger hearts and smaller bodies relative to Controls. Moreover, a plateau in effect sizes for both of these traits coincide with the generational range during which the selection limit for wheel-running behavior was reached. NSF IOS-2038528. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of long-term voluntary wheel running and selective breeding for wheel running on femoral nutrient canals.

The nutrient artery provides ~50%-70% of the total blood volume to long bones in mammals. Studying the functional characteristics of this artery invivo can be difficult and expensive, so most researchers have measured the nutrient foramen, an opening on the outer surface of the bone that served as the entry point for the nutrient artery during development and bone ossification. Others have measured the nutrient canal (i.e., the passage which the nutrient artery once occupied), given that the external dimensions of the foramen do not necessarily remain uniform from the periosteal surface to the medullary cavity. The nutrient canal, as an indicator of blood flow to long bones, has been proposed to provide a link to studying organismal activity (e.g., locomotor behavior) from skeletal morphology. However, although external loading from movement and activity causes skeletal remodeling, it is unclear whether it affects the size or configuration of nutrient canals. To investigate whether nutrient canals can exhibit phenotypic plasticity in response to physical activity, we studied a mouse model in which four replicate high runner (HR) lines have been selectively bred for high voluntary wheel-running behavior. The selection criterion is the average number of wheel revolutions on days 5 and 6 of a 6-day period of wheel access as young adults (~6-8 weeks old). An additional four lines are bred without selection to serve as controls (C). For this study, 100 female mice (half HR, half C) from generation 57 were split into an active group housed with wheels and a sedentary group housed without wheels for 12 weeks starting at ~24 days of age. Femurs were collected, soft tissues were removed, and femora were micro-computed tomography scanned at a resolution of 12 μm. We then imported these scans into AMIRA and created 3D models of femoral nutrient canals. We tested for evolved differences in various nutrient canal traits between HR and C mice, plastic changes resulting from chronic exercise, and the selection history-by-exercise interaction. We found few differences between the nutrient canals of HR versus C mice, or between the active and sedentary groups. We did find an interaction between selection history and voluntary exercise for the total number of nutrient canals per femur, in which wheel access increased the number of canals in C mice but decreased it in HR mice. Our results do not match those from an earlier study, conducted at generation 11, which was prior to the HR lines reaching selection limits for wheel running. The previous study found that mice from the HR lines had significantly larger total canal cross-sectional areas compared to those from C lines. However, this discrepancy is consistent with studies of other skeletal traits, which have found differences between HR and C mice to be somewhat inconsistent across generations, including the loss of some apparent adaptations with continued selective breeding after reaching a selection limit for wheel-running behavior.

Selective breeding for high voluntary exercise in mice increases maximal (VO2max), but not basal metabolic rate.

In general, sustained high rates of physical activity require a high maximal aerobic capacity (VO2max), which may also necessitate a high basal aerobic metabolism (BMR), given that the two metabolic states are linked via shared organ systems, cellular properties, and metabolic pathways. We tested the hypotheses that (a) selective breeding for high voluntary exercise in mice would elevate both VO2max and BMR, and (b) these increases are accompanied by increases in the sizes of some internal organs (ventricle, triceps surae muscle, liver, kidney, spleen, lung, brain). We measured 72 females from generations 88 and 96 of an ongoing artificial selection experiment comprising 4 replicate High Runner (HR) lines bred for voluntary daily wheel-running distance and 4 non-selected Control (C) lines. With body mass as a covariate, HR lines as a group had significantly higher VO2max (+13.6%, P<0.0001), consistent with previous studies, but BMR did not significantly differ between HR and C lines (+6.5%, P=0.181). Additionally, HR mice did not statistically differ from C mice for whole-body lean or fat mass, or for the mass of any organ collected (with body mass as a covariate). Finally, mass-independent VO2max and BMR were uncorrelated (r=0.073, P=0.552) and the only statistically significant correlation with an organ mass was for VO2max and ventricle mass (r=0.285, P=0.015). Overall, our results indicate that selection for a behavioral trait can yield large changes in behavior without proportional modifications to underlying morphological or physiological traits.

Interplay between Learning and Voluntary Wheel Running in Male C57BL/6NCrl Mice.

Exercise is shown to improve cognitive function in various human and animal studies. Laboratory mice are often used as a model to study the effects of physical activity and running wheels provide a voluntary and non-stressful form of exercise. The aim of the study was to analyze whether the cognitive state of a mouse is related to its wheel-running behavior. Twenty-two male C57BL/6NCrl mice (9.5 weeks old) were used in the study. The cognitive function of group-housed mice (n = 5-6/group) was first analyzed in the IntelliCage system followed by individual phenotyping with the PhenoMaster with access to a voluntary running wheel. The mice were divided into three groups according to their running wheel activity: low, average, and high runners. The learning trials in the IntelliCage showed that the high-runner mice exhibited a higher error rate at the beginning of learning trials but improved their outcome and learning performance more compared to the other groups. The high-runner mice ate more compared to the other groups in the PhenoMaster analyses. There were no differences in the corticosterone levels between the groups, indicating similar stress responses. Our results demonstrate that high-runner mice exhibit enhanced learning capabilities prior to access to voluntary running wheels. In addition, our results also show that individual mice react differently when introduced to running wheels, which should be taken into consideration when choosing animals for voluntary endurance exercise studies.

Hippocampal, Whole Midbrain, Red Nucleus, and Ventral Tegmental Area Volumes Are Increased by Selective Breeding for High Voluntary Wheel-Running Behavior.

Uncovering relationships between neuroanatomy, behavior, and evolution are important for understanding the factors that control brain function. Voluntary exercise is one key behavior that both affects, and may be affected by, neuroanatomical variation. Moreover, recent studies suggest an important role for physical activity in brain evolution. We used a unique and ongoing artificial selection model in which mice are bred for high voluntary wheel-running behavior, yielding four replicate lines of high runner (HR) mice that run ∼3-fold more revolutions per day than four replicate nonselected control (C) lines. Previous studies reported that, with body mass as a covariate, HR mice had heavier whole brains, non-cerebellar brains, and larger midbrains than C mice. We sampled mice from generation 66 and used high-resolution microscopy to test the hypothesis that HR mice have greater volumes and/or cell densities in nine key regions from either the midbrain or limbic system. In addition, half of the mice were given 10 weeks of wheel access from weaning, and we predicted that chronic exercise would increase the volumes of the examined brain regions via phenotypic plasticity. We replicated findings that both selective breeding and wheel access increased total brain mass, with no significant interaction between the two factors. In HR compared to C mice, adjusting for body mass, both the red nucleus (RN) of the midbrain and the hippocampus (HPC) were significantly larger, and the whole midbrain tended to be larger, with no effect of wheel access nor any interactions. Linetype and wheel access had an interactive effect on the volume of the periaqueductal gray (PAG), such that wheel access increased PAG volume in C mice but decreased volume in HR mice. Neither linetype nor wheel access affected volumes of the substantia nigra, ventral tegmental area, nucleus accumbens, ventral pallidum (VP), or basolateral amygdala. We found no main effect of either linetype or wheel access on neuronal densities (numbers of cells per unit area) for any of the regions examined. Taken together, our results suggest that the increased exercise phenotype of HR mice is related to increased RN and hippocampal volumes, but that chronic exercise alone does not produce such phenotypes.

Mice from lines selectively bred for voluntary exercise are not more resistant to muscle injury caused by either contusion or wheel running.

Muscle injury can be caused by strenuous exercise, repetitive tasks or external forces. Populations that have experienced selection for high locomotor activity may have evolutionary adaptations that resist exercise-induced injury and/or enhance the ability to cope with injury. We tested this hypothesis with an experiment in which mice are bred for high voluntary wheel running. Mice from four high runner lines run ~three times more daily distance than those from four non-selected control lines. To test recovery from injury by external forces, mice experienced contusion via weight drop on the calf. After injury, running distance and speed were reduced in high runner but not control lines, suggesting that the ability of control mice to run exceeds their motivation. To test effects of injury from exercise, mice were housed with/without wheels for six days, then trunk blood was collected and muscles evaluated for injury and regeneration. Both high runner and control mice with wheels had increased histological indicators of injury in the soleus, and increased indicators of regeneration in the plantaris. High runner mice had relatively more central nuclei (regeneration indicator) than control in the soleus, regardless of wheel access. The subset of high runner mice with the mini-muscle phenotype (characterized by greatly reduced muscle mass and type IIb fibers) had lower plasma creatine kinase (indicator of muscle injury), more markers of injury in the deep gastrocnemius, and more markers of regeneration in the deep and superficial gastrocnemius than normal-muscled individuals. Contrary to our expectations, high runner mice were not more resistant to either type of injury.

DNA Methylation Analysis of Imprinted Genes in the Cortex and Hippocampus of Cross-Fostered Mice Selectively Bred for Increased Voluntary Wheel-Running

We have previously shown that high runner (HR) mice (from a line genetically selected for increased wheel-running behavior) have distinct, genetically based, neurobiological phenotypes as compared with non-selected control (C) mice. However, developmental programming effects during early life, including maternal care and parent-of-origin-dependent expression of imprinted genes, can also contribute to variation in physical activity. Here, we used cross-fostering to address two questions. First, do HR mice have altered DNA methylation profiles of imprinted genes in the brain compared to C mice? Second, does maternal upbringing further modify the DNA methylation status of these imprinted genes? To address these questions, we cross-fostered all offspring at birth to create four experimental groups: C pups to other C dams, HR pups to other HR dams, C pups to HR dams, and HR pups to C dams. Bisulfite sequencing of 16 imprinted genes in the cortex and hippocampus revealed that the HR line had altered DNA methylation patterns of the paternally imprinted genes, Rasgrf1 and Zdbf2, as compared with the C line. Both fostering between the HR and C lines and sex modified the DNA methylation profiles for the paternally expressed genes Mest, Peg3, Igf2, Snrpn, and Impact. Ig-DMR, a gene with multiple paternal and maternal imprinted clusters, was also affected by maternal upbringing and sex. Our results suggest that differential methylation patterns of imprinted genes in the brain could contribute to evolutionary increases in wheel-running behavior and are also dependent on maternal upbringing and sex.

Oral antibiotics reduce voluntary exercise behavior in athletic mice

The gut microbiome can affect various aspects of both behavior and physiology, including exercise ability, but effects on voluntary exercise have rarely been studied. We studied females from a selection experiment in which 4 replicate High Runner (HR) lines of mice are bred for voluntary exercise and compared with 4 non-selected control (C) lines. HR and C mice differ in several traits that likely interact with the gut microbiome, including higher daily running distance, body temperatures when running, spontaneous physical activity when housed without wheels, and food consumption. After two weeks of wheel access to reach a stable plateau in daily running, mice were administered broad-spectrum antibiotics for 10 days. Antibiotic treatment caused a significant reduction in daily wheel-running distance in the HR mice (−21%) but not in the C mice. Antibiotics did not affect body mass or food consumption in either HR or C mice, and we did not observe sickness behavior. Wheel running by HR mice did not recover during the 12 days following cessation of antibiotics. The decreased wheel-running in HR but not C mice, with no apparent negative side effects of antibiotics, suggests that the HR microbiome is an important component of their high-running phenotype.

Effects of Selective Breeding, Voluntary Exercise, and Sex on Endocannabinoid Levels in the Mouse Small-Intestinal Epithelium

The endocannabinoid (eCB) system in the gut communicates with the body and brain as part of the homeostatic mechanisms that affect energy balance. Although perhaps best known for its effects on energy intake, the eCB system also regulates voluntary locomotor behavior. Here, we examined gut eCB concentrations in relation to voluntary exercise, specifically in mice selectively bred for high voluntary wheel running behavior. We measured gut eCBs in four replicate non-selected Control (C) lines and four replicate lines of High Runner (HR) mice that had been selectively bred for 74 generations based on the average number of wheel revolutions on days 5 and 6 of a 6-day period of wheel access when young adults. On average, mice from HR lines run voluntarily on wheels ∼3-fold more than C mice on a daily basis. A recent study showed that circulating levels of primary endocannabinoids 2-arachidonoyl-sn-glycerol (2-AG) and anandamide (AEA) are altered by six days of wheel access, by acute wheel running, and differ between HR and C mice in sex-specific ways [1]. We hypothesized that eCBs in the upper small-intestinal epithelium (i.e., proximal jejunum), a region firmly implicated in eCB signaling, would differ between HR and C mice (linetype), between the sexes, between mice housed with vs. without wheels for six days, and would covary with amounts of acute running and/or home-cage activity (during the previous 30 minutes). We used the same 192 mice as in [1] , half males and half females, half HR and half C (all 8 lines), and half either given or not given access to wheels for six days. We assessed the eCBs, 2-AG and AEA, and their analogs docosahexaenoylglycerol (DHG), docosahexaenoylethanolamide (DHEA), and oleoylethanolamide (OEA). Both 2-AG and DHG showed a significant 3-way interaction of linetype, wheel access, and sex. In addition, HR mice had lower concentrations of 2-AG in the small-intestinal epithelium when compared to C mice, which may be functionally related to differences in locomotor activity or to differences in body composition and/or food consumption. Moreover, the amount of home-cage activity during the prior 30 min was a negative predictor of 2-AG and AEA concentrations in jejunum mucosa, particularly in the mice with no wheel access. Lastly, 2-AG, but not AEA, was significantly correlated with 2-AG in plasma in the same mice.

Effects of selective breeding for voluntary exercise, chronic exercise, and their interaction on muscle attachment site morphology in house mice.

Skeletal muscles attach to bone at their origins and insertions, and the interface where tendon meets bone is termed the attachment site or enthesis. Mechanical stresses at the muscle/tendon-bone interface are proportional to the surface area of the bony attachment sites, such that a larger attachment site will distribute loads over a wider area. Muscles that are frequently active and/or are of larger size should cause attachment sites to hypertrophy (training effect); however, experimental studies of animals subjected to exercise have provided mixed results. To enhance our ability to detect training effects (a type of phenotypic plasticity), we studied a mouse model in which 4 replicate lines of High Runner (HR) mice have been selectively bred for 57generations. Selection is based on the average number of wheel revolutions on days 5 & 6 of a 6-day period of wheel access as young adults (6-8weeks old). Four additional lines are bred without regard to running and serve as non-selected controls (C). On average, mice from HR lines voluntarily run ~3 times more than C mice on a daily basis. For this study, we housed 50 females (half HR, half C) with wheels (Active group) and 50 (half HR, half C) without wheels (Sedentary group) for 12weeks starting at weaning (~3weeks old). We tested for evolved differences in muscle attachment site surface area between HR and C mice, plastic changes resulting from chronic exercise, and their interaction. We used a precise, highly repeatable method for quantifying the three-dimensional (3D) surface area of four muscle attachment sites: the humerus deltoid tuberosity (the insertion point for the spinodeltoideus, superficial pectoralis, and acromiodeltoideus), the femoral third trochanter (the insertion point for the quadratus femoris), the femoral lesser trochanter (the insertion point for the iliacus muscle), and the femoral greater trochanter (insertion point for the middle gluteal muscles). In univariate analyses, with body mass as a covariate, mice in the Active group had significantly larger humerus deltoid tuberosities than Sedentary mice, with no significant difference between HR and C mice and no interaction between exercise treatment and linetype. These differences between Active and Sedentary mice were also apparent in the multivariate analyses. Surface areas of the femoral third trochanter, femoral lesser trochanter, and femoral greater trochanter were unaffected by either chronic wheel access or selective breeding. Our results, which used robust measurement protocols and relatively large sample sizes, demonstrate that muscle attachment site morphology can be (but is not always) affected by chronic exercise experienced during ontogeny. However, contrary to previous results for other aspects of long bone morphology, we did not find evidence for evolutionary coadaptation of muscle attachments with voluntary exercise behavior in the HR mice.

Effects of early-life exposure to Western diet and voluntary exercise on adult activity levels, exercise physiology, and associated traits in selectively bred High Runner mice

Exercise behavior is under partial genetic control, but it is also affected by numerous environmental factors, potentially including early-life experiences whose effects persist into adulthood. We studied genetic and early-life environmental effects on wheel-running behavior in a mouse model that includes four replicate high runner (HR) lines selectively bred for increased voluntary wheel running as young adults and four non-selected control (C) lines. In a full factorial design, mice from each line were granted wheel access or not and administered either standard or Western diet (WD) from weaning (3 weeks old) to 6 weeks of age (sexual maturity). In addition to acute effects, after a washout period of 8 weeks (∼6 human years) in which all mice had standard diet and no wheel access, we found both beneficial and detrimental effects of these early-life exposures. During the first week of treatments, WD increased distance run by 29% in C mice and 48% in HR mice (significant Diet × Linetype interaction), but diet effects disappeared by the third week. Across the three weeks of juvenile treatment, WD significantly increased fat mass (with lean mass as a covariate). Tested as adults, early-life exercise increased wheel running of C mice but not HR mice in the first week. Early-life exercise also reduced adult anxiety-like behavior and increased adult fasted blood glucose levels, triceps surae mass, subdermal fat pad mass, and brain mass, but decreased heart ventricle mass. Using fat mass as a covariate, early-life exercise treatment increased adult leptin concentration. In contrast, early-life WD increased adult wheel running of HR mice but not C mice. Early-life WD also increased adult lean mass and adult preference for Western diet in all groups. Surprisingly, early-life treatment had no significant effect on adult body fat or maximal aerobic capacity (VO2max). No previous study has tested for combined or interactive effects of early-life WD and exercise. Our results demonstrate that both factors can have long-lasting effects on adult voluntary exercise and related phenotypes, and that these effects are modulated by genetic background. Overall, the long-lasting effects of early-life exercise were more pervasive than those of WD, suggesting critical opportunities for health intervention in childhood habits, as well as possible threats from modern challenges. These results may be relevant for understanding potential effects of activity reductions and dietary changes associated with the obesity epidemic and COVID-19 pandemic.

Rapid and longer-term effects of selective breeding for voluntary exercise behavior on skeletal morphology in house mice.

Selection experiments can elucidate the varying course of adaptive changes across generations. We examined the appendicular skeleton of house mice from four replicate High Runner (HR) lines bred for physical activity on wheels and four non-selected Control (C) lines. HR mice reached apparent selection limits between generations 17 and 27, running ~3-fold more than C. Studies at generations 11, 16, and 21 found that HR mice had evolved thicker hindlimb bones, heavier feet, and larger articular surface areas of the knee and hip joint. Based on biomechanical theory, any or all of these evolved differences may be beneficial for endurance running. Here, we studied mice from generation 68, plus a limited sample from generation 58, to test whether the skeleton continued to evolve after selection limits were reached. Contrary to our expectations, we found few differences between HR and C mice for these later generations, and some of the differences in bone dimensions identified in earlier generations were no longer statistically significant. We hypothesize that the loss of apparently coadapted lower-level traits reflects (1) deterioration related to a gradual increase in inbreeding and/or (2) additional adaptive changes that replace the functional benefits of some skeletal changes.

Effects of Selective Breeding, Exercise, and Sex on Endocannabinoid Levels in the Mouse Small Intestine

Only half of adults in the United States get the physical activity they need to help prevent certain chronic diseases. Such diseases as cardiovascular disease and diabetes are metabolism‐related and closely tied to insufficient physical activity. The endocannabinoid (EC) system serves many physiological roles, including in the regulation of voluntary locomotion, energy balance, and food reward. Signaling at the cannabinoid type 1 receptor (CB1) has been specifically implicated in acute and long‐term rodent voluntary exercise on wheels. Plasma levels of endocannabinoids in mice are known to change in association with wheel running, but EC concentrations in the gut have not been studied in the context of exercise. We studied four replicate lines of high runner (HR) mice that had been selectively bred for 74 generations based on the average number of wheel revolutions on days 5 and 6 of a 6‐day period of wheel access. Four additional replicate lines have been bred without regard to wheel running and serve as controls (C). On average, mice from HR lines voluntarily run on wheels ~3 times more than C mice on a daily basis. HR mice have higher home‐cage activity when housed without wheels, reduced body mass and fat, eat more, and their wheel running responds differently to a CB1 agonist &amp; antagonist compared to C mice. A recent study showed that circulating levels (during peak running) of primary endocannabinoids 2‐arachidonoylglycerol (2‐AG) and anandamide (AEA) are altered by wheel running, differ between HR and C mice, and have sex‐specific interactions (Thompson et al. 2017. Physiology &amp; Behavior 170:141–150). The EC system in the gut is of particular interest because consuming dietary fats leads to increased EC content in rodent small‐intestinal epithelium, which in turn, drives overeating. A focus on HR gut endocannabinoids may present novel insights concerning exercise metabolism of fats and gut‐brain reward signaling. We hypothesized that small intestine [EC] would differ between HR and C mice, between the sexes, between mice housed with vs. without wheels for 6 days, and would covary with acute running amounts (during the previous 30 minutes). We used 196 total mice, half males and half females, half HR and half C, and half either given or not given access to wheels for 6 days. Jejunum gut mucosa was collected for lipid extractions and subsequent EC analysis via ultra‐performance liquid chromatography coupled to tandem mass spectrometry (UPLC/MS/MS). 2‐AG, AEA, as well as their analogs docosahexaenoylglycerol (DHG), oleoylethanolamide (OEA) and docosahexaenoylethanolamide (DHEA), were measured. DHG showed a significant 3‐way interaction of linetype, wheel access, and sex, with no consistent effects of any of these factors. A trend also showed HR mice having lower 2‐AG, which suggests that HR mice may have lower rates of production of intestinal ECs and/or faster rates of degradation by monoacylglycerol lipase (MGL). Functionally, lower 2‐AG in HR jejunum might be related to differences from C mice with respect to locomotor activity, body composition, and/or food consumption.

Effects of Selective Breeding for Voluntary Exercise, Chronic Exercise, and Their Interaction on Muscle Attachment Site Morphology in House Mice

Skeletal muscles attach to bone at their origins and insertions, and the interface where tendon meets bone is termed the muscle attachment site (or enthesis). Muscle attachment sites function to anchor muscles/tendons to bone and to dissipate stress (force per unit area) resulting from force transmission. Mechanical stresses at the muscle/tendon‐bone interface are proportional to surface area of the bony attachment sites, so a larger muscle attachment site will distribute loads over a wider surface area. Muscles that are frequently active and/or are of larger size should cause muscle attachment sites to hypertrophy (training effect), so paleontological studies have often used characteristics of attachment sites as indicators of locomotor behavior.In studies of humans, muscle attachment sites have been used to infer physical activity of fossil specimens, with larger attachment sites assumed to indicate larger muscles and/or habitual use. For example, muscle attachment site morphology was examined in prehistoric Andean fishing and agricultural populations. From this data, anthropologists concluded that the fishing population had more muscular upper bodies while the agricultural population had more muscular lower bodies. Such differences in muscle attachment site morphology have been attributed to differences in physical activity levels and loading history of limb bones experienced by individuals during their lifetime. However, bone performance during loading is governed by both genetic factors and loading history, but also by age, sex, and epigenetic factors. Thus, controversy arises when paleontologists attempt to infer the past loading history (i.e., activity levels) of individuals from bone morphology.We studied 4 replicate lines of high runner (HR) mice that had been selectively bred for 57 generations based on the average number of wheel revolutions on days 5 &amp; 6 of a 6‐day period of wheel access. Four additional lines have been bred without regard to wheel running and serve as controls (C). On average, mice from HR lines voluntarily run ~3 times more than C mice on a daily basis. We housed 100 mice with or without wheels for 12 weeks starting at weaning. We quantified the evolved differences in muscle attachment site morphology between HR and C mice as well as plastic changes resulting from chronic exposure to exercise by measuring the cortical cross‐sectional areas, linear dimensions, and total surface area for muscles that attach on the femur and humerus. Sites chosen for subsequent analysis included the femoral third trochanter, an insertion point for the quadratus femoris, and the humerus deltoid crest, an insertion point for the spinodeltoideus, superficial pectoralis, and acromiodeltoideus.With body mass as a covariate, HR mice had significantly larger humerus deltoid tuberosities than C, with no significant training effect. Morphology of the femoral third trochanter did not differ between HR and C mice and was unaffected by chronic wheel access. Our results suggest that muscle attachment site morphology is largely determined by genetic background rather than exercise during ontogeny.3D Model of the Femoral Third Trochanter of a MouseFigure 1

Effects of Maternal Exercise During Pregnancy on Offspring Body Composition and Voluntary Exercise

The Developmental Origins of Health and Disease hypothesis posits that various factors experienced early in life can predispose individuals to being healthy or unhealthy as adults. For example, children with high birth weight, either by being born to obese mothers or mothers who experience high gestational weight gain during pregnancy, are more likely to be obese as adults. Although maternal diet has been shown to affect offspring body weight in both humans and rodents, the role of maternal exercise remains unclear.We studied a novel animal model, mice from 4 replicate High Runner (HR) lines selectively bred for ~85 generations for voluntary wheel running and 4 non‐selected Control (C) lines. HR mice run ~3‐fold longer distances than C on a daily basis. In the present study, 80 female mice (prospective dams) were given wheel access starting at 12 weeks of age, paired with males after 2 weeks for a period of 18 days, and allowed continued wheel access until 10 days after giving birth. An additional 80 dams were not given wheels. Offspring body composition was measured at weaning (3 weeks of age), after sexual maturity (7 weeks of age), and after breeding (20 weeks of age for males, 24 weeks of age for females). Offspring wheel running was measured at 7 weeks of age for 6 days, as is used in the selection experiment.During the 14 days prospective dams were housed individually, and consistent with many previous studies, HR dams ran ~3‐fold more than C (HR mean = 16,215 rev/day, range = 10,898 to 21,653 revs/day; C mean = 5,478 rev/day, range = 3,338 to 8,422 revs/day, p &lt; 0.0001). During the first 5 days after giving birth, dams ran very little (HR mean = 1,758 rev/day, range = 145 to 2,816 revs/day; C mean = 1,445 rev/day, range = 707 to 2,574 revs/day; p = 0.1519) and during days 6–10 after birth they ran even less (HR mean = 1,377 rev/day, range = 92 to 3,851 revs/day; C mean = 1,109 rev/day, range = 210 to 2,408 revs/day; p = 0.5229).Maternal wheel access did not significantly affect either time from pairing to giving birth or litter size. Maternal wheel access did not significantly affect offspring wheel‐running distance, but HR offspring ran ~2.75‐fold more than C for males (HR mean = 8,495 rev/day, range = 3,051 to 16,733 revs/day; C mean = 3,080 rev/day, range = 376 to 6,002 revs/day; p = 0.0012) and ~3‐fold more for females (HR mean = 11,448 rev/day, range = 4,316 to 21,128 revs/day; C mean = 3,703 rev/day, range = 448 to 7,894 revs/day; p = 0.0002), as expected. At 7 weeks of age (after sexual maturity), maternal wheel access had an interactive effect on female offspring (p = 0.0117), increasing lean mass for C mice but decreasing it for HR mice.Overall, our results show that although maternal wheel access during pregnancy did not alter litter size or offspring wheel‐running behavior, it did have effects on offspring body composition at sexual maturity, but the effects varied between the sexes and in relation to genetic background (linetype).

Exercise-induced loading increases ilium cortical area in a selectively bred mouse model.

Little is known about how ilium cortical bone responds to loading. Using a mouse model, this study presents data testing the hypothesis that iliac cross-sectional properties are altered in response to increased activity. The sample derives from lines of High Runner (HR) mice bred for increased wheel-running activity. Four treatment groups of female mice were tested: non-selected control lines housed without (N = 19) and with wheels (N = 20), and HR mice housed without (N = 17) and with wheels (N = 18) for 13 weeks beginning at weaning. Each pelvis was μCT-scanned, cross-sectional properties (cortical area-Ct.Ar, total area-Tt.Ar, polar moment of area, and polar section modulus) were determined from the ilium midshaft, and robusticity indices (ratio of the square root of Ct.Ar or Tt.Ar to caudal ilium length) were calculated. Mixed models were implemented with linetype, wheel access, and presence of the mini-muscle phenotype as fixed effects, replicate line nested within linetype as a random effect, and body mass as a covariate. Results demonstrate that the mouse ilium morphologically resembles a long bone in cross section. Body mass and the mini-muscle phenotype were significant predictors of iliac cross-sectional properties. Wheel access only had a statistically significant effect on Ct.Ar and its robusticity index, with greater values in mice with wheel access. These results suggest that voluntary exercise increases cortical area, but does not otherwise strengthen the ilium in these mice, corroborating previous studies on the effect of increased wheel-running activity on femoral and humeral cross-sectional properties in these mice.

Influence of corticosterone on growth, home-cage activity, wheel running, and aerobic capacity in house mice selectively bred for high voluntary wheel-running behavior

Glucocorticoids, a class of metabolic hormones, impact a wide range of traits (e.g., behavior, skeletal growth, muscle maintenance, glucose metabolism), and variation in concentrations of circulating glucocorticoids (such as corticosterone), at the level of natural individual variation, in relation to endocrine disorders, or from exogenous supplementation, have manifold effects. Changes in circulating corticosterone concentrations can also impact multiple aspects of locomotor behavior, including both motivation and physical ability for exercise. To examine further the role of corticosterone in locomotor behavior and associated traits, we utilized laboratory house mice from a long-term experiment that selectively breeds for high levels of voluntary exercise. As compared with four non-selected control (C) lines, mice from the four replicate High Runner (HR) lines have ~2-fold higher baseline circulating corticosterone concentrations as well as ~3-fold higher voluntary wheel running on a daily basis, higher home-cage activity when deprived of wheels, higher maximal aerobic capacity, and smaller body size; potentially, all of these differences could be modulated by circulating corticosterone. We administered 50 μg/mL corticosterone-21-hemisuccinate in the drinking water of both HR and C male mice from weaning through ~8 weeks of age. As compared with mice from C lines, HR mice had higher endogenous corticosterone levels; higher daily wheel-running distance, duration, and speed; higher maximal oxygen consumption during forced exercise (VO2max); spent more time in the closed arms of an elevated plus maze; and had larger reproductive fat pads. For both HR and C mice, corticosterone treatment strongly suppressed endogenous circulating corticosterone levels, decreased growth rate and adult body mass, increased food and water consumption (both adjusted for body mass), increased entries into closed arms of an elevated plus maze, decreased home-cage activity (total and average intensity), decreased wheel-running distance and maximum speed, and decreased VO2max. At the suborganismal level, corticosterone treatment decreased relative adrenal, liver, and triceps surae muscle mass, as well as tail length, but increased both subdermal and reproductive fat pad masses, as well as hematocrit. Overall, the responses of both HR and C mice to corticosterone supplementation were “negative” from a health perspective. These results have significant implications for understanding both the evolution of baseline corticosterone levels and stress-related effects on activity levels. They also suggest that patients experiencing extended periods of glucocorticoid treatment might benefit from attempts to increase their physical activity as an adjuvant.

Effects of selective breeding for high voluntary wheel-running behavior on femoral nutrient canal size and abundance in house mice.

Bone modeling and remodeling are aerobic processes that entail relatively high oxygen demands. Long bones receive oxygenated blood from nutrient arteries, epiphyseal-metaphyseal arteries, and periosteal arteries, with the nutrient artery supplying the bulk of total blood volume in mammals (~50-70%). Estimates of blood flow into these bones can be made from the dimensions of the nutrient canal, through which nutrient arteries pass. Unfortunately, measuring these canal dimensions non-invasively (i.e. without physical sectioning) is difficult, and thus researchers have relied on more readily visible skeletal proxies. Specifically, the size of the nutrient artery has been estimated from dimensions (e.g. minimum diameters) of the periosteal (external) opening of the nutrient canal. This approach has also been utilized by some comparative morphologists and paleontologists, as the opening of a nutrient canal is present long after the vascular soft tissue has degenerated. The literature on nutrient arteries and canals is sparse, with most studies consisting of anatomical descriptions from surgical proceedings, and only a few investigating the links between nutrient canal morphology and physiology or behavior. The primary objective of this study was to evaluate femur nutrient canal morphology in mice with known physiological and behavioral differences; specifically, mice from an artificial selection experiment for high voluntary wheel-running behavior. Mice from four replicate high runner (HR) lines are known to differ from four non-selected control (C) lines in both locomotor and metabolic activity, with HR mice having increased voluntary wheel-running behavior and maximal aerobic capacity (VO2 max) during forced treadmill exercise. Femora from adult mice (average age 7.5months) of the 11th generation of this selection experiment were μCT-scanned and three-dimensional virtual reconstructions of nutrient canals were measured for minimum cross-sectional area as a skeletal proxy of blood flow. Gross observations revealed that nutrient canals varied far more in number and shape than prior descriptions would indicate, regardless of sex or genetic background (i.e. HR vs. C lines). Canals adopted non-linear shapes and paths as they traversed from the periosteal to endosteal borders through the cortex, occasionally even branching within the cortical bone. Additionally, mice from both HR and C lines averaged more than four nutrient canals per femur, in contrast to the one to two nutrient canals described for femora from rats, pigs, and humans in prior literature. Mice from HR lines had significantly larger total nutrient canal area than C lines, which was the result not of an increase in the number of nutrient canals, but rather an increase in their average cross-section size. This study demonstrates that mice with an evolutionary history of increased locomotor activity and maximal aerobic metabolic rate have a concomitant increase in the size of their femoral nutrient canals. Although the primary determinant of nutrient canal size is currently not well understood, the present results bolster use of nutrient canal size as a skeletal indicator of aerobically supported levels of physical activity in comparative studies.