We evaluated the effect of voluntary exercise on spontaneous physical activity (SPA) and food consumption in mice from 4 replicate lines bred for 57 generations for high voluntary wheel running (HR) and from 4 non-selected control (C) lines. Beginning at ~24 days of age, mice were housed in standard cages or in cages with attached wheels. Wheel activity and SPA were monitored in 1-min intervals. Data from the 8th week of the experiment were analyzed because mice were sexually mature and had plateaued in body mass, weekly wheel running distance, SPA, and food consumption. Body mass, length, and masses of the retroperitoneal fat pad, liver, and heart were recorded after the 13th week. SPA of both HR and C mice decreased with wheel access, due to reductions in both duration and average intensity of SPA. However, total activity duration (SPA+wheel running; min/day) was ~1/3 greater when mice were housed with wheels, and food consumption was significantly increased. Overall, food consumption in both HR and C mice was more strongly affected by wheel running than by SPA. Duration of wheel running had a stronger effect than average speed, but the opposite was true for SPA. With body mass as a covariate, chronic wheel access significantly reduced fat pad mass and increased heart mass in both HR and C mice. Given that both HR and C mice housed with wheels had increased food consumption, the energetic cost of wheel running was not fully compensated by concomitant reductions in SPA. The experiment demonstrates that both duration and intensity of both wheel running and SPA were significant predictors of food consumption. This sort of detailed analysis of the effects of different aspects of physical activity on food consumption has not previously been reported for a non-human animal, and it sets the stage for longitudinal examination of energy balance and its components in rodent models.

SUMMARY We compiled published values of mammalian maximum oxygen consumption during exercise (V ·O2Supplementary material available online at http://jeb.biologists.org/lookup/suppl

Key messageA switchgrass protoplast system was developed, achieving a cost reduction of ~1000-fold, a threefold increase in transformation efficiency, and a fourfold reduction in required DNA quantity compared to previous methods.AbstractIn recent years, there has been a resurgence in the use of protoplast systems for rapid screening of gene silencing and genome-editing targets for siRNA, miRNA, and CRISPR technologies. In the case of switchgrass (Panicum virgatum L.), to achieve economic feasibility for biofuel production, it is necessary to develop plants with decreased cell wall recalcitrance to reduce processing costs. To achieve this goal, transgenic plants have been generated with altered cell wall chemistry; however, with limited success owing to the complexity of cell walls. Because of the considerable cost, time, and effort required to screen transgenic plants, a protoplast system that can provide data at an early stage has potential to eliminate low performing candidate genes/targets prior to the creation of transgenic plants. Despite the advantages of protoplast systems, protoplast isolation in switchgrass has proven costly, requiring expensive lab-grade enzymes and high DNA quantities. In this paper, we describe a low-cost protoplast isolation system using a mesophyll culture approach and a cell suspension culture. Results from this work show a cost reduction of ~1000-fold compared to previous methods of protoplast isolation in switchgrass, with a cost of $0.003 (USD) per reaction for mesophyll protoplasts and $0.018 for axenic cell culture-derived protoplasts. Further, the efficiency of protoplast transformation was optimized threefold over previous methods, despite a fourfold reduction in DNA quantity. The methods developed in this work remove the cost barrier previously limiting high-throughput screening of genome-editing and gene silencing targets in switchgrass, paving the way for more efficient development of transgenic plants.

SUMMARYWe investigated sprint performance and running economy of a unique 'mini-muscle' phenotype that evolved in response to selection for high voluntary wheel running in laboratory mice (Mus domesticus). Mice from four replicate selected (S) lines run nearly three times as far per day as four control lines. The mini-muscle phenotype, resulting from an initially rare autosomal recessive allele, has been favoured by the selection protocol, becoming fixed in one of the two S lines in which it occurred. In homozygotes, hindlimb muscle mass is halved, mass-specific muscle oxidative capacity is doubled, and the medial gastrocnemius exhibits about half the mass-specific isotonic power, less than half the mass-specific cyclic work and power, but doubled fatigue resistance. We hypothesized that mini-muscle mice would have a lower whole-animal energy cost of transport (COT), resulting from lower costs of cycling their lighter limbs, and reduced sprint speed, from reduced maximal force production. We measured sprint speed on a racetrack and slopes (incremental COT, or iCOT) and intercepts of the metabolic rate versus speed relationship during voluntary wheel running in 10 mini-muscle and 20 normal S-line females. Mini-muscle mice ran faster and farther on wheels, but for less time per day. Mini-muscle mice had significantly lower sprint speeds, indicating a functional trade-off. However, contrary to predictions, mini-muscle mice had higher COT, mainly because of higher zero-speed intercepts and postural costs (intercept-resting metabolic rate). Thus, mice with altered limb morphology after intense selection for running long distances do not necessarily run more economically.

The glucocorticoid hormones corticosterone (CORT) and cortisol influence numerous physiological, morphological, and behavioral functions. However, few studies have addressed possible relationships between individual differences in glucocorticoid concentrations and whole-animal performance or metabolism. Because CORT is important in glucose regulation and energy metabolism and can influence activity levels, we hypothesized that individual variation in baseline circulating CORT levels would correlate with individual differences in energy expenditure (routine and maximal), aerobic physiology, voluntary exercise on wheels, and organ masses. We tested this hypothesis in the California mouse (Peromyscus californicus). We collected data from 54 adult, colony-bred mice on baseline CORT levels (measured near both the circadian peak and the circadian trough), voluntary wheel running and its energetic costs, maximal oxygen consumption during forced treadmill exercise ([Formula: see text]), basal metabolic rate, and relative organ masses. We found surprisingly few statistically significant relationships among CORT, energy metabolism, behavior, and organ masses, and these relationships appeared to differ between males and females. These findings suggest that individual differences in baseline CORT levels are not an important determinant of voluntary activity levels or aerobic performance in California mice.

The purpose of this study was to evaluate the effects of early-life exercise on adult physical activity (wheel running, home-cage activity), body mass, food consumption, and circulating leptin levels in males from four replicate lines of mice selectively bred for high voluntary wheel running (High Runner or HR) and their four non-selected control (C) lines. Half of the mice were given wheel access shortly after weaning for three consecutive weeks. Wheel access was then removed for 52 days, followed by two weeks of adult wheel access for all mice. A blood sample taken prior to adult wheel testing was analyzed for circulating leptin concentration. Early-life wheel access significantly increased adult voluntary exercise on wheels during the first week of the second period of wheel access, for both HR and C mice, and HR ran more than C mice. During this same time period, activity in the home cages was not affected by early-age wheel access, and did not differ statistically between HR and C mice. Throughout the study, all mice with early wheel access had lower body masses than their sedentary counterparts, and HR mice had lower body masses than C mice. With wheel access, HR mice also ate significantly more than C mice. Early-life wheel access increased plasma leptin levels (adjusted statistically for fat-pad mass as a covariate) in C mice, but decreased them in HR mice. At sacrifice, early-life exercise had no statistically significant effects on visceral fat pad, heart (ventricle), liver or spleen masses (all adjusted statistically for variation in body mass). Results support the hypothesis that early-age exercise in mice can have at least transitory positive effects on adult levels of voluntary exercise, in addition to reducing body mass, and may be relevant for the public policy debates concerning the importance of physical education for children.

SUMMARYChronic increases in circulating corticosterone (CORT) generally suppress immune function, but it is not known whether evolved increases necessarily have similar adverse effects. Moreover, the evolution of immune function might be constrained by the sharing of signaling molecules, such as CORT, across numerous physiological systems. Laboratory house mice (Mus domesticus Linnaeus) from four replicate lines selectively bred for high voluntary wheel running (HR lines) generally had baseline circulating CORT approximately twofold higher than in four non-selected control (C) lines. To test whether elevated baseline CORT suppresses the inflammatory response in HR mice, we injected females with lipopolysaccharide (LPS). All mice injected with LPS exhibited classic signs of an inflammatory response, including sickness behavior, loss of body mass, reduced locomotor activity (i.e. voluntary wheel running), enlarged spleens and livers, elevated hematocrit and elevated inflammatory cytokines. However, as compared with C mice, the inflammatory response was not suppressed in HR mice. Our results, and those of a previous study, suggest that selective breeding for high voluntary exercise has not altered immune function. They also suggest that the effects of evolved differences in baseline CORT levels may differ greatly from effects of environmental factors (often viewed as ʻstressorsʼ) that alter baseline CORT during an individualʼs lifetime. In particular, evolved increases in circulating levels of ʻstress hormonesʼ are not necessarily associated with detrimental suppression of the inflammatory response, presumably as a result of correlated evolution of other physiological systems (counter-measures). Our results have important implications for the interpretation of elevated stress hormones and of immune indicators in natural populations. Supplementary material available online at

SUMMARYMice from lines selectively bred for high levels of voluntary wheel running express a high incidence of a small muscle phenotype ('mini-muscles') that may confer an adaptive advantage with respect to endurance-running capacity. Plantar flexors in the minimuscle phenotype exhibit a high capacity for aerobic activity, including altered enzyme activities, loss of expression of type II b myosin heavy chain (MHC), increased expression of type I, II x and II a MHC, and mechanical performance consistent with slower, more fatigue-resistant muscles. We hypothesized that these changes may accompany enhanced efficiency of contraction, perhaps in support of the enhanced capacity for endurance running. To assess efficiency, we measured work and associated oxygen consumption from isolated soleus and medial gastrocnemius muscles from mice with mini-muscle and normal phenotypes. We also measured the MHC expression of the plantar flexor muscles to better understand the physiological basis of any differences in efficiency. The proportion of the various MHC isoforms in the soleus was shifted toward a slightly faster phenotype in the mini-muscle mice, whereas in the gastrocnemius and plantaris it was shifted toward a markedly slower phenotype, with large reductions in type II b MHC and large increases in type I, II a , and II x MHC. Soleus muscles from normal and mini-muscle mice showed no statistical differences in efficiency, but medial gastrocnemius from mini-muscle mice were significantly less efficient than those from normal mice, despite the distinctly slower MHC phenotype in mini-muscle mice. Thus, based on measures of efficiency from isolated muscles under conditions near optimal for power output, the shift toward a slower phenotype in 'mini' gastrocnemius muscles does not appear to confer advantages directly through increased efficiency. Rather, the slower phenotype may reduce energy used by the muscles and be permissive to enhanced running ability, perhaps by reducing reliance on anaerobic metabolism.