Jessica L. Malisch

Circadian pattern of total and free corticosterone concentrations, corticosteroid-binding globulin, and physical activity in mice selectively bred for high voluntary wheel-running behavior

In vertebrates, baseline glucocorticoid concentrations vary predictably on a diel basis, usually peaking shortly before the onset of activity. Presumably, circadian patterns in glucocorticoid secretion have evolved to match predictable rises in energetic need. In mice from lines selectively bred for high voluntary wheel-running, previous studies have reported that baseline plasma corticosterone concentrations at two different times during the photophase are elevated twofold above those of non-selected control lines. Here, we tested the hypothesis that the elevated daytime corticosterone levels could be explained by a shift in the circadian pattern of corticosterone levels. We measured baseline total plasma corticosterone levels, corticosteroid-binding globulin (CBG) capacity, and calculated free corticosterone levels (corticosterone not bound to corticosteroid-binding globulin and potentially biologically active) at six points during the 24-hour cycle in males on a 12:12 photoperiod. We also examined the daily pattern of both wheel-running and home-cage activity. Based on combined analysis of all six points, the circadian pattern of total corticosterone, corticosteroid-binding globulin, and free corticosterone levels did not significantly differ between high-runner and control mice (linetype * time interaction P=0.56, 0.45, and 0.55, respectively); however, all varied with time (all P<0.0001) and mice from the selected lines had significantly elevated total (P=0.0125) and free (P=0.0140) corticosterone, with no difference in CBG binding capacity (P=0.77). All mice were active primarily during the dark phase, and the factorial increase in activity of selected relative to controls lines was 2.33 for total daily wheel revolutions and 2.76 for total daily home-cage activity. The onset of the active period for both measures of locomotor activity coincided with peak total and free corticosterone levels in both selected and control lines. These findings lend support to our hypothesis that elevated circulating corticosterone levels have evolved as an adaptation to support increased locomotor activity in the selected lines.

Baseline and stress-induced plasma corticosterone concentrations of mice selectively bred for high voluntary wheel running.

The hypothalamic‐pituitary‐adrenal (HPA) axis is important in regulating energy metabolism and in mediating responses to stressors, including increasing energy availability during physical exercise. In addition, glucocorticoids act directly on the central nervous system and influence behavior, including locomotor activity. To explore potential changes in the HPA axis as animals evolve higher voluntary activity levels, we characterized plasma corticosterone (CORT) concentrations and adrenal mass in four replicate lines of house mice that had been selectively bred for high voluntary wheel running (HR lines) for 34 generations and in four nonselected control (C) lines. We determined CORT concentrations under baseline conditions and immediately after exposure to a novel stressor (40 min of physical restraint) in mice that were housed without access to wheels. Resting daytime CORT concentrations were approximately twice as high in HR as in C mice for both sexes. Physical restraint increased CORT to similar concentrations in HR and C mice; consequently, the proportional response to restraint was smaller in HR than in C animals. Adrenal mass did not significantly differ between HR and C mice. Females had significantly higher baseline and postrestraint CORT concentrations and significantly larger adrenal glands than males in both HR and C lines. Replicate lines showed significant variation in body mass, length, baseline CORT concentrations, and postrestraint CORT concentrations in one or both sexes. Among lines, both body mass and length were significantly negatively correlated with baseline CORT concentrations, suggesting that CORT suppresses growth. Our results suggest that selection for increased locomotor activity has caused correlated changes in the HPA axis, resulting in higher baseline CORT concentrations and, possibly, reduced stress responsiveness and a lower growth rate.

How to run far: multiple solutions and sex-specific responses to selective breeding for high voluntary activity levels

The response to uniform selection may occur in alternate ways that result in similar performance. We tested for multiple adaptive solutions during artificial selection for high voluntary wheel running in laboratory mice. At generation 43, the four replicate high runner (HR) lines averaged 2.85-fold more revolutions per day as compared with four non-selected control (C) lines, and females ran 1.11-fold more than males, with no sex-by-linetype interaction. Analysis of variance indicated significant differences among C lines but not among HR for revolutions per day. By contrast, average speed varied significantly among HR lines, but not among C, and showed a sex-by-linetype interaction, with the HR/C ratio being 2.02 for males and 2.45 for females. Time spent running varied among both HR and C lines, and showed a sex-by-linetype interaction, with the HR/C ratio being 1.52 for males but only 1.17 for females. Thus, females (speed) and males (speed, but also time) evolved differently, as did the replicate selected lines. Speed and time showed a trade-off among HR but not among C lines. These results demonstrate that uniform selection on a complex trait can cause consistent responses in the trait under direct selection while promoting divergence in the lower-level components of that trait.

Maximal metabolic rates during voluntary exercise, forced exercise, and cold exposure in house mice selectively bred for high wheel-running

SUMMARY Selective breeding for high wheel-running activity has generated four lines of laboratory house mice (S lines) that run about 170% more than their control counterparts (C lines) on a daily basis, mostly because they run faster. We tested whether maximum aerobic metabolic rates (V̇O2max) have evolved in concert with wheel-running, using 48 females from generation 35. Voluntary activity and metabolic rates were measured on days 5+6 of wheel access (mimicking conditions during selection), using wheels enclosed in metabolic chambers. Following this, V̇O2max was measured twice on a motorized treadmill and twice during cold-exposure in a heliox atmosphere (HeO2). Almost all measurements, except heliox V̇O2max, were significantly repeatable. After accounting for differences in body mass (S<C) and variation in age at testing, S and C did not differ in V̇O2max during forced exercise or in heliox, nor in maximal running speeds on the treadmill. However, running speeds and V̇O2max during voluntary exercise were significantly higher in S lines. Nevertheless, S mice never voluntarily achieved the V̇O2max elicited during their forced treadmill trials, suggesting that aerobic capacity per se is not limiting the evolution of even higher wheel-running speeds in these lines. Our results support the hypothesis that S mice have genetically higher motivation for wheel-running and they demonstrate that behavior can sometimes evolve independently of performance capacities. We also discuss the possible importance of domestication as a confounding factor to extrapolate results from this animal model to natural populations.

Effects of reproductive status on behavioral and endocrine responses to acute stress in a biparental rodent, the California mouse (Peromyscus californicus)

In several mammalian species, lactating females show blunted neural, hormonal, and behavioral responses to stressors. It is not known whether new fathers also show stress hyporesponsiveness in species in which males provide infant care. To test this possibility, we determined the effects of male and female reproductive status on stress responsiveness in the biparental, monogamous California mouse (Peromyscus californicus). Breeding (N=8 females, 8 males), nonbreeding (N=10 females, 10 males) and virgin mice (N=12 females, 9 males) were exposed to a 5-min predator-urine stressor at two time points, corresponding to the early postpartum (5-7 days postpartum) and mid/late postpartum (19-21 days postpartum) phases, and blood samples were collected immediately afterwards. Baseline blood samples were obtained 2 days prior to each stress test. Baseline plasma corticosterone (CORT) concentrations did not differ among male or female groups. CORT responses to the stressor did not differ among female reproductive groups, and all three groups showed distinct behavioral responses to predator urine. Virgin males tended to increase their CORT response from the first to the second stress test, while breeding and nonbreeding males did not. Moreover, virgin and nonbreeding males showed significant behavioral changes in response to predator urine, whereas breeding males did not. These results suggest that adrenocortical responses to a repeated stressor in male California mice may be modulated by cohabitation with a female, whereas behavioral responses to stress may be blunted by parental status.

Selection for increased voluntary wheel-running affects behavior and brain monoamines in mice.

Selective-breeding of house mice for increased voluntary wheel-running has resulted in multiple physiological and behavioral changes. Characterizing these differences may lead to experimental models that can elucidate factors involved in human diseases and disorders associated with physical inactivity, or potentially treated by physical activity, such as diabetes, obesity, and depression. Herein, we present ethological data for adult males from a line of mice that has been selectively bred for high levels of voluntary wheel-running and from a non-selected control line, housed with or without wheels. Additionally, we present concentrations of central monoamines in limbic, striatal, and midbrain regions. We monitored wheel-running for 8 weeks, and observed home-cage behavior during the last 5 weeks of the study. Mice from the selected line accumulated more revolutions per day than controls due to increased speed and duration of running. Selected mice exhibited more active behaviors than controls, regardless of wheel access, and exhibited less inactivity and grooming than controls. Selective-breeding also influenced the longitudinal patterns of behavior. We found statistically significant differences in monoamine concentrations and associated metabolites in brain regions that influence exercise and motivational state. These results suggest underlying neurochemical differences between selected and control lines that may influence the observed differences in behavior. Our results bolster the argument that selected mice can provide a useful model of human psychological and physiological diseases and disorders.

Selective Breeding for a Behavioral Trait Changes Digit Ratio

The ratio of the length of the second digit (index finger) divided by the fourth digit (ring finger) tends to be lower in men than in women. This 2D∶4D digit ratio is often used as a proxy for prenatal androgen exposure in studies of human health and behavior. For example, 2D∶4D ratio is lower (i.e. more “masculinized”) in both men and women of greater physical fitness and/or sporting ability. Lab mice have also shown variation in 2D∶4D as a function of uterine environment, and mouse digit ratios seem also to correlate with behavioral traits, including daily activity levels. Selective breeding for increased rates of voluntary exercise (wheel running) in four lines of mice has caused correlated increases in aerobic exercise capacity, circulating corticosterone level, and predatory aggression. Here, we show that this selection regime has also increased 2D∶4D. This apparent “feminization” in mice is opposite to the relationship seen between 2D∶4D and physical fitness in human beings. The present results are difficult to reconcile with the notion that 2D∶4D is an effective proxy for prenatal androgen exposure; instead, it may more accurately reflect effects of glucocorticoids, or other factors that regulate any of many genes.

Exercise training effects on hypoxic and hypercapnic ventilatory responses in mice selected for increased voluntary wheel running

What is the central question of this study? We used experimental evolution to determine how selective breeding for high voluntary wheel running and exercise training (7–11 weeks) affect ventilatory chemoreflexes of laboratory mice at rest. What is the main finding and its importance? Selective breeding, although significantly affecting some traits, did not systematically alter ventilation across gas concentrations. As with most human studies, our findings support the idea that endurance training attenuates resting ventilation. However, little evidence was found for a correlation between ventilatory chemoreflexes and the amount of individual voluntary wheel running. We conclude that exercise ‘training’ alters respiratory behaviours, but these changes may not be necessary to achieve high levels of wheel running.

Acute Restraint Stress Alters Wheel-Running Behavior Immediately Following Stress and up to 20 Hours Later in House Mice

In vertebrates, acute stressors—although short in duration—can influence physiology and behavior over a longer time course, which might have important ramifications under natural conditions. In laboratory rats, for example, acute stress has been shown to increase anxiogenic behaviors for days after a stressor. In this study, we quantified voluntary wheel-running behavior for 22 h following a restraint stress and glucocorticoid levels 24 h postrestraint. We utilized mice from four replicate lines that have been selectively bred for high voluntary wheel-running activity (HR mice) for 60 generations and their nonselected control (C) lines to examine potential interactions between exercise propensity and sensitivity to stress. Following 6 d of wheel access on a 12L∶12D photo cycle (0700–1900 hours, as during the routine selective breeding protocol), 80 mice were physically restrained for 40 min, beginning at 1400 hours, while another 80 were left undisturbed. Relative to unrestrained mice, wheel running increased for both HR and C mice during the first hour postrestraint (P < 0.0001) but did not differ 2 or 3 h postrestraint. Wheel running was also examined at four distinct phases of the photoperiod. Running in the period of 1600–1840 hours was unaffected by restraint stress and did not differ statistically between HR and C mice. During the period of peak wheel running (1920–0140 hours), restrained mice tended to run fewer revolutions (−11%; two-tailed P = 0.0733), while HR mice ran 473% more than C (P = 0.0008), with no restraint × line type interaction. Wheel running declined for all mice in the latter part of the scotophase (0140–0600 hours), restraint had no statistical effect on wheel running, but HR again ran more than C (+467%; P = 0.0122). Finally, during the start of the photophase (0720–1200 hours), restraint increased running by an average of 53% (P = 0.0443) in both line types, but HR and C mice did not differ statistically. Mice from HR lines had statistically higher plasma corticosterone concentrations than C mice, with no statistical effect of restraint and no interaction between line type and restraint. Overall, these results indicate that acute stress can affect locomotor activity (or activity patterns) for many hours, with the most prominent effect being an increase in activity during a period of typical inactivity at the start of the photophase, 15–20 h poststressor.

Within-lifetime trade-offs but evolutionary freedom for hormonal and immunological traits: evidence from mice bred for high voluntary exercise.

SUMMARY Chronic 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 ‘stressors’) that alter baseline CORT during an individual’s lifetime. In particular, evolved increases in circulating levels of ‘stress hormones’ 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.