Robert S. Bowen

Altered dopaminergic profiles: implications for the regulation of voluntary physical activity

The biological regulating factors of physical activity in animals are not well understood. This study investigated differences in the central mRNA expression of seven dopamine genes (Drd1, Drd2, Drd3, Drd4, Drd5, TH, and DAT) between high active C57/LJ (n=17) male mice and low active C3H/HeJ (n=20) male mice, and between mice with access to a running wheel and without running wheel access within strain. Mice were housed with running wheels interfaced with a computer for 21 days with distance and duration recorded every 24 h. On day 21, the striatum and nucleus accumbens were removed during the active period (approximately 9 pm) for dopaminergic analysis. On average, the C57L/J mice with wheels ran significantly farther (10.25+/-1.37 km/day vs. 0.01+/-0.09 km/day, p<0.001), longer (329.73+/-30.52 min/day vs. 7.81+/-6.32 min/day, p<0.001), and faster (31.27+/-3.13 m/min vs. 11.81+/-1.08 m/min, p<0.001) than the C3H/HeJ mice with wheels over the 21 day period. No differences in gene expression were found between mice in either strain with wheels and those without wheels suggesting that access to running wheels did not alter dopaminergic expression. In contrast, relative expression for two dopamine genes was significantly lower in the C57L/J mice compared to the C3H/HeJ mice. These results indicate that decreased dopaminergic functioning is correlated with increased activity levels in C57L/J mice and suggests that D1-like receptors as well as tyrosine hydroxylase (an indicator of dopamine production), but not D2-like receptors may be associated with the regulation of physical activity in inbred mice.

Strain screen and haplotype association mapping of wheel running in inbred mouse strains.

Previous genetic association studies of physical activity, in both animal and human models, have been limited in number of subjects and genetically homozygous strains used as well as number of genomic markers available for analysis. Expansion of the available mouse physical activity strain screens and the recently published dense single-nucleotide polymorphism (SNP) map of the mouse genome (approximately 8.3 million SNPs) and associated statistical methods allowed us to construct a more generalizable map of the quantitative trait loci (QTL) associated with physical activity. Specifically, we measured wheel running activity in male and female mice (average age 9 wk) in 41 inbred strains and used activity data from 38 of these strains in a haplotype association mapping analysis to determine QTL associated with activity. As seen previously, there was a large range of activity patterns among the strains, with the highest and lowest strains differing significantly in daily distance run (27.4-fold), duration of activity (23.6-fold), and speed (2.9-fold). On a daily basis, female mice ran further (24%), longer (13%), and faster (11%). Twelve QTL were identified, with three (on Chr. 12, 18, and 19) in both male and female mice, five specific to males, and four specific to females. Eight of the 12 QTL, including the 3 general QTL found for both sexes, fell into intergenic areas. The results of this study further support the findings of a moderate to high heritability of physical activity and add general genomic areas applicable to a large number of mouse strains that can be further mined for candidate genes associated with regulation of physical activity. Additionally, results suggest that potential genetic mechanisms arising from traditional noncoding regions of the genome may be involved in regulation of physical activity.

Repeatability of exercise behaviors in mice

PURPOSE Measurements of exercise behaviors in rodents such as maximal treadmill endurance and physical activity are often used in the literature; however, minimal data are available regarding the repeatability of measurements used for these exercise behaviors. This study assessed the repeatability of a commonly used maximal exercise endurance treadmill test as well as voluntary physical activity measured by wheel running in mice. METHODS Repeatability of treadmill tests were analyzed for both inbred and outbred mice in addition to a 10 week repeatability analysis using Balb/cJ mice (n=20). Voluntary daily physical activity was assessed by distance, duration, and speed of wheel running (WR). Physical activity measurements on days 5 and 6 of WR in a large cohort (n=739) of both inbred and outbred mice were compared. RESULTS No significant differences (p>0.05) in exercise endurance were found between different cohorts of Balb/cJ and DBA/2J mice indicating strains overall generally test the same; however, significant differences between tests were seen within BaD2F(2) animals (p<0.001). Bland-Altman analysis revealed a lack of agreement between weekly endurance tests within mouse, and correlation analysis showed lack of consistent correlations between weekly endurance tests within mouse. No significant differences were found for WR measurements within mouse between days (p=0.99). High correlations between days within mouse for WR were found (r=0.74-0.85). CONCLUSIONS High intra-mouse variability between repeated endurance tests suggests that treadmill testing in an enclosed chamber with shock grid for motivation to run in mice is not repeatable. Conversely, high correlation and agreement between days of wheel-running measurements suggest that voluntary activity (WR) is repeatable and stable within individual mice.

Sex Hormone Effects on Physical Activity Levels: Why Doesn't Jane Run as Much as Dick?

The relationship between physical activity levels and disease rates has become an important health-related concern in the developed world. Heart disease, certain cancers and obesity persist at epidemic rates in the US and Western Europe. Increased physical activity levels have been shown to reduce the occurrence of many chronic diseases leading to reductions in the burden on the healthcare system. Activity levels in humans are affected by many cultural and environmental factors; nevertheless, current research points to a strong biological input with potential genetic, neurological and endocrinological origins. Of unique interest, the sex hormones appear to have a very strong influence on activity levels. The current animal literature suggests that females tend to be more active than males due to biological pathways of estrogenic origin. The majority of human epidemiological and anthropological data, on the contrary, suggest women are less active than men in spite of this inherent activity-increasing mechanism.The purpose of this study is to review the current literature regarding the control of physical activity levels by the sex hormones in humans. Using the natural transitional phases of the aging endocrine system, natural periodicity of the menstrual cycle and pharmacological/hormone replacement therapy as variable experimental stages, some authors have been able to provide some information regarding the existence of an inherent activity-increasing mechanism in humans. In brief, activity levels during life stages prior to and after menopause do not significantly differ, despite the vast changes in sex hormone levels and function. Sex hormone differences throughout a regular menstrual cycle do not appear to influence activity levels in humans either; an effect that is pronounced in the female rodent. The use of hormone replacement therapies provides researchers with more systematic controls over hormone modulation in human subjects; however, this benefit comes with additional confounding variables, mostly due to disease or other states of malfunction. Despite the addition of these confounding factors, minor changes to the activity pattern have been observed in women, especially during the initial administration of the therapy. Observations are yet to be made in male subjects during replacement therapy.In general, some evidence exists suggesting that a biological mechanism extending from the sex hormones influences activity in humans. Unfortunately, despite a small number of investigative reports, the paucity of human research investigating how the sex hormones affect activity levels in humans prevents conclusive delineation of the mechanisms involved. Future research in this unique sub-field of endocrinology and exercise science utilizing more appropriate research protocols and effective techniques will provide definitive evidence of such mechanisms.

Effects of Supraphysiological Doses of Sex Steroids on Wheel Running Activity in Mice.

The regulatory mechanisms of physical activity are postulated to include environmental and biological/genetic factors. In particular, the sex steroids appear to have profound effects on wheel running in rodents. The purpose of this project was to investigate the effects of 17β-estradiol and testosterone on wheel running distance, duration, and speed in male and female C57BL/6J mice. The mice (N=46) were provided free access to running wheels interfaced with computers to track daily running distance, duration, and speed. Activity was assessed at baseline in intact mice, after surgical gonadectomy, and after replacement with either 17β-estradiol or testosterone. Upon removal of the gonads, physical activity levels were significantly reduced in both males and females. Distance (10–30% of baseline) and duration (20–47% of baseline) measures were most affected by the loss of endogenous steroids, while running speed (60–77% of baseline) though significantly reduced-decreased by a much lower magnitude. Testosterone replacement fully recovered running distance, duration, and speed to pre-surgical levels in both sexes (100% of baseline). Distance (30–42% of baseline) and duration (43–47% of baseline) were partially recovered by 17β-estradiol, but not to baseline levels. Speed (100% of baseline) was fully recovered by 17β-estradiol replacement in males and females. This study suggests that physical activity in mice is affected by endogenous steroids and can be altered by exogenous steroid replacement. The differences in the recovery abilities of 17β-estradiol and testosterone suggest that both estrogenic and androgenic pathways may be involved to variable degrees in activity regulation.

Effects of Aromatase Inhibition on the Physical Activity Levels of Male Mice.

Increasing activity levels in an inactive population can lead to associative increases in health and well-being. Both biologic and genetic factors have been identified that alter physical activity levels in humans and rodents with an extensive early literature regarding sex steroid effects on physical activity. Currently, it is suggested that the androgens require conversion to estrogens prior to eliciting any effects on activity patterns. Recent data contradicts this assertion; thus, the purpose of this study was to evaluate the necessity of the aromatase complex in activity regulation. Wheel running was assessed in male C57BL/6J mice under various sex steroid-disrupted and aromatase-inhibited conditions. Inhibition of the aromatase complex was achieved through administration of two different aromatase inhibiting substances-letrozole and exemestane. Wheel running was unaffected by aromatase inhibition in reproductively intact and sex steroid supplemented mice. Orchidectomy significantly reduced wheel running activity. Steroid replacement recovered wheel running to pre-surgical levels; however, aromatase inhibition did not further affect wheel running levels. The recovery of wheel running in mice with androgen supplementation and the further persistence of wheel running in mice with compromised aromatase function suggests that the androgens-testosterone in particular-may directly affect wheel running patterns in male mice.

Stabilization of the wheel running phenotype in mice.

PURPOSE Increased physical activity is well known to improve health and wellness by modifying the risks for many chronic diseases. Rodent wheel running behavior is a beneficial surrogate model to evaluate the biology of daily physical activity in humans. Upon initial exposure to a running wheel, individual mice differentially respond to the experience, which confounds the normal activity patterns exhibited in this otherwise repeatable phenotype. To promote phenotypic stability, a minimum seven-day (or greater) acclimation period is utilized. Although phenotypic stabilization is achieved during this 7-day period, data to support acclimation periods of this length are not currently available in the literature. The purpose of this project is to evaluate the wheel running response in C57BL/6j mice immediately following exposure to a running wheel. METHODS Twenty-eight male and thirty female C57BL/6j mice (Jackson Laboratory, Bar Harbor, ME) were acquired at eight weeks of age and were housed individually with free access to running wheels. Wheel running distance (km), duration (min), and speed (m∙min(-1)) were measured daily for fourteen days following initial housing. One-way ANOVAs were used to evaluate day-to-day differences in each wheel running character. Limits of agreement and mean difference statistics were calculated between days 1-13 (acclimating) and day 14 (acclimated) to assess day-to-day agreement between each parameter. RESULTS Wheel running distance (males: F=5.653, p=2.14 × 10(-9); females: F=8.217, p=1.20 × 10(-14)), duration (males: F=2.613, p=0.001; females: F=4.529, p=3.28 × 10(-7)), and speed (males: F=7.803, p=1.22 × 10(-13); females: F=13.140, p=2.00 × 10(-16)) exhibited day-to-day differences. Tukeys HSD post-hoc testing indicated differences between early (males: days 1-3; females: days 1-6) and later (males: days >3; females: days >6) wheel running periods in distance and speed. Duration only exhibited an anomalous difference between wheel running on day 13 and wheel running on days 1 through 4 in males. In females, duration exhibited anomalous differences due to abnormally depressed wheel running on day 6 and abnormally elevated wheel running on day 14. Limits of agreement and mean difference statistics indicated stable phenotypic variability with an up-trending daily mean for distance and speed that stabilized within the first three days in males and within eight days in females. Duration exhibited stable variability after nine days in males and after seven days in females. CONCLUSION Although it is common practice to allow a prolonged (≥ seven day) acclimation period prior to recording wheel running data, the current study suggests that phenotypic stabilization of all three indices is achieved at different times with distance and speed exhibiting stability by day three in males and day eight in females. Duration exhibits stability by day nine in males and day seven in females.

Effects of varying doses of estrogen and caudal pressure on wheel running in orchidectomized male mice

Physical inactivity is a leading cause of hypokinetic diseases – obesity, heart disease, diabetes, and certain types of cancers. Increased city walkability, better access to fitness facilities, and remediation of socioeconomic barriers prove successful for limited populations within the confines of stringently controlled environments; however, these strategies fail to reverse the ever‐increasing physical inactivity epidemic on a global scale indicating the existence of other unidentified factors. These purported biological factors remain critical targets to understand the regulation of this complex phenotype. An estrogenic mechanism that incompletely or slowly adjusts physical activity levels following reintroduction of estrogenic compounds to surgically gonadectomized mice has been postulated to exist. Currently, this mechanism remains scrutinized due to concerns that elevated estrogen levels induce urinary bladder distension. The distension of the urinary bladder may mechanically disrupt physical activity, masking any physiological effects estrogen has on physical activity. The purpose of this study was to evaluate the effects of estrogen on physical activity levels while employing dose‐related strategies to alleviate distension in mice. Wheel running data were collected under normal physiological conditions, following removal of endogenous sex steroids via orchidectomy, and during estrogen replacement at various doses (0%, 10%, 50% or 100% estrogen‐containing implants) to induce varying degrees of urinary bladder distension. Wheel running distance (P = 0.005) and duration (P = 0.006) decreased after orchidectomy, but slowly increased following estrogen replacement. During the study, wheel running did not return to the levels observed in physiologically intact mice. Significant distension was not observed between estrogen treatment groups indicating that a slow‐responding estrogen effect exists in male mice that prevents wheel running from returning to normal levels immediately following steroid reintroduction. The limited increase in wheel running during estrogen treatment following orchidectomy is not an artifact of induced urinary bladder distension.