1. 1 Born to Run: Experimental Evolution of High Voluntar y Exercise Mice Professor Theodore Garland, Jr., Ph.D. Depar tment of Biology

2. 2 Collaborators in Our Lab or at U.C., Riverside: Collaborators Elsewhere (partial list): Wendy Acosta Doug Altshuler Anne M. Bronikowski Vincent Careau Patrick A. Carter Mark A. Chappell Gerald C. Claghorn Mark A. Coleman Elizabeth Dlugosz Isabelle Girard Fernando R. Gomes Robert M. Hannon Brooke K. Keeney Scott A. Kelly Erik M. Kolb Terry Belke Abel Bult-Ito Hannah Carey Greg Cartee Lynn Copes Serge Daan Brigitte Demes Gary Diffee Cynthia Downs Joey C. Eisenmann Patricia A. Freeman Fred H. (Rusty) Gage Stephen C. Gammie Helga Guderley Russ Hepple Peter Hurd Aaron M. Janowsky Denis Joanisse Isabella Jonas Grant McClelland Kevin M. Middleton Gordon S. Mitchell Andy Obenaus Mark Olfert Steve F. Perry Daniel Pomp Thomas A. Prolla Gary Sieck Sharon Swartz Doug Syme Nicole Templeman Lobke M. Vaanholt Gertjan van Dijk Henriette van Praag G. Henk Visser Ben B. Yaspelkis III This selection experiment began in 1993, > 100 publications. Pawel Koteja Guo Li Chris Lytle Jessica L. Malisch Morris & Gina Maduro Thomas H. Meek Kevin Middleton Tricia Radojcic Enrico L. Rezende Justin S. Rhodes Derek Roff Heidi Schutz John Shyy John G. Swallow Shizhong Xu Many Undergrads

3. 3 Background&Introduction

4. 4 Evolutionary Physiology How do organisms work? How can we use "model organisms" to understand how organisms work? Where is the line between "normal" individual variation and pathology? How do we promote health and cure disease? How do populations change over time? How can we use "model organisms" to understand how evolution and genetics work? How important is natural selection in shaping biological diversity? How does the way organisms work influence the way they evolve? Copyright by Theodore Garland, Jr. May 2007

5. We are studying: Animal Locomotion from the perspective of Evolutionary Physiology 5

6. 6 An Observation about Behavior: Animals exhibit tremendous diversity in locomotor behavior (e.g., home range size).

7. 7 An Observation about Behavior: Animals are diverse in locomotor behavior (e.g., home range size). "Motivation" vs. per formance ability? Evolutionary causes: Why has this diversity evolved? Natural selection: what is the benefit? Physiological causes: How do animals work? Body structure and physiology: what is the limit?

8. 8 log 10 Body Mass (kg) log 10 Home Range Area (km 2 ) Diversity in Locomotor Behavior 49 Species of Mammals But, even for different animals that are the same size, home ranges are very diverse and can be 100-times different. Large animals tend to have large home ranges. Note the logarithmic axes.

9. 9 log 10 Body Mass (kg) log 10 Home Range Area (km 2 ) Diversity in Locomotor Behavior 49 Species of Mammals Carnivora ungulates Carnivores tend to have larger home ranges than herbivores.

10. 10 An Observation about Behavior: Animals are diverse in locomotor behavior (e.g., home range size). "Motivation" vs. per formance ability? Evolutionary causes: Why has this diversity evolved? Natural selection: what is the benefit? Physiological causes: How do animals work? Body structure and physiology: what is the limit?

11. 11 log 10 Body Mass (kg) log 10 Home Range Area (km 2 ) Diversity in Locomotor Behavior 49 Species of Mammals Carnivora Eat Herbivores Eat Plants ungulates Might the kind of diet be a reason for the diversity? Garland, T., Jr., A. W. Dickerman, C. M. Janis, and J. A. Jones. 1993. Phylogenetic analysis of covariance by computer simulation. Systematic Biology 42:265-292.

12. 12 log 10 Body Mass (kg) log 10 Home Range Area (km 2 ) Diversity in Locomotor Behavior 49 Species of Mammals Carnivora Eat Herbivores Eat Plants ungulates Possibly just a direct environmental effect?

13. 13 An Observation about Behavior: Animals are diverse in locomotor behavior (e.g., home range size). "Motivation" vs. per formance ability? Evolutionary causes: Why has this diversity evolved? Natural selection: what is the benefit? Physiological causes: How do animals work? Body structure and physiology: what is the limit?

14. We can attempt to answer these questions with experimentation.

15. 15 Lions, and tigers, and bears (oh my!) are great, but not the most convenient organisms to study …

16. 16 "…research in which populations are studied across multiple generations under defined and reproducible conditions, whether in the laboratory or in nature."

17. 17 Charles Darwin did not recognize this as a way to study evolution because he thought the process was too slow.

18. Humans can also show diversity in locomotor behavior.

19. 19 ● Individual differences in voluntary activity levels are large in both human beings and other animals (both in the lab and in the wild). Locomotion & Activity Levels

20. 20 An Observation about Behavior: Animals are diverse in locomotor behavior (e.g., home range size). "Motivation" vs. per formance ability? Evolutionary causes: Why has this diversity evolved? Natural selection: what is the benefit? Physiological causes: How do animals work? Body structure and physiology: what is the limit?

21. 21 Locomotion & Activity Levels

22. 22 ● Both motivation & ability must be affected by 1) multiple alleles (genes), 2) numerous environmental factors, 3) interactions of genes and environment. You can lead a horse to water, but you can't make him drink! Locomotion & Activity Levels

23. Genetic Factors in Locomotion and Activity Levels

24. 24 Locomotion & Activity Levels

25. 25 Locomotion & Activity Levels

26. Experimental Evolution Evolution: –Can we cause activity levels to change across generations by selective breeding? –Are genes important in determining activity levels? Physiology: –How is body structure affected? Bones, muscles, circulatory system? –What is the role of motivation? Brain structure? –What is the role of metabolism?

27. 27 Methods

28. 28 Why Select on Wheel-Running? Why Select on Wheel-Running? http://school.discovery.com/clipart/clip/ani-mouse.html 1.potentially physiologically taxing - did you ever have a pet hamster? 2.individual dif ferences are highly repeatable (consistent) from day-to-day 3.genetically inherited to some extent 4.easy to automate measurement 5.an important regulator of body mass and composition 6.analogous to human voluntary activity? (e.g., Eikelboom, 1999)

29. 29 Star ting (Base) Population: 112 male & 112 female outbred mice Design: 8 lines: 4 Selected for High Running (HR lines), 4 non-selected Control 10 mating pairs in each (lit ter size ~10) Within-family selection Selection Criterion: Wheel revolutions on days 5 + 6 This is a completely "voluntary" behavior. Experimental Design

30. 30 Impor tant point! ● We did not select for low voluntary running because it might not be the same trait as high wheel running. ● For example, selection for low running might simply increase fear of entering the wheels. ● For humans, active behaviors may not be opposites of sedentary behaviors. ● Jogging is not the opposite of playing a video game. ● Also, selection for low per formance might be accomplished by increasing frequency of “harmful” alleles (deleterious recessives), thus leading to sickly mice from a variety of causes.

31. 31 Wheels are Attached to Standard Housing Cages

32. 32 200 total wheels, 100 in each of two rooms Food and water are always available.

33. 33 Results

34. 34 Revolutions/Day on days 5 + 6 Selected Control 14FRUN56.DSF 0 5 10 15 20 25 30 35 40 45 50 Generation Wheel Circum- ference = 1.12 m Selected run almost 3X more than control Our lab moved from U. Wisconsin to U. Calif.

35. 35 Revolutions/Day on days 5 + 6 Selected Control 0 5 10 15 20 25 30 35 40 45 50 Generation 14MRUN56.DSF Wheel Circum- ference = 1.12 m Males always tend to run less than females, but the fold difference between selected and control lines is the same as in females.

36. 36 How do Mice from the Selected Lines Run More?

37. Data are collected about the wheel revolutions logged by the mice. Selected mice run more revolutions/day than controls Selected mice do not run for longer time period than controls Selected mice do run faster than controls

38. 38 Ratio of Selected/Control Lines 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 Generation Total Revolutions Females Remember, for these ratios: 1 means that selected & control are the same <1 means control is higher than selected >1 means selected is higher than control

39. 39 Ratio of Selected/Control Lines 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 Generation Min/Day Total Revolutions Females

40. 40 Ratio of Selected/Control Lines 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 Generation Min/Day R.P.M. Total Revolutions Females

41. 41 Show movie that accompanies: Girard, I., M. W. McAleer, J. S. Rhodes, and T. Garland, Jr. 2001. Selection for high voluntary wheel running increases intermittency in house mice (Mus domesticus). Journal of Experimental Biology 204:4311-4320. http://www.biology.ucr.edu/people/faculty/Garland/Girard01.mov Mice from the selected lines mainly run faster than those from control lines.

42. 42 HCA_sensors_and_computer_2_shrunk.jpg What about activity in ordinary cages? What about activity in ordinary cages? Force plates detect any movement in the cage.

43. 43 g44_circadian_hca.xls S & C Lines Also Differ in Regular Cage Activity Activity (normalized units) Lights Off Time (hours) Malisch et al., 2008, 2009 Cage activity is much higher in mice from the Selected lines when they do not have wheels.

44. 44 How Else do the Selected and Control Lines Differ?

45. 45 Something to consider What makes an individual a good distance runner?

46. 46 Mice from the Selected lines: are smaller in body size have less body fat, even when housed without wheels eat more for their body weight, even when housed without wheels show no statistically significant difference from the Control lines in basal or resting metabolic rate Body Size and Composition

47. 47 Exercise Adaptations? Exercise Adaptations?

48. 48 Using Oxygen = Maximal Aerobic Capacity http://nyvelocity.com/files/old/pictures/image/cadence/collete3.jpg http://farm4.staticflickr.com/3108/2325682863_f41a385d5c.jpg Single best indicator of cardiopulmonary function Single best indicator of cardiopulmonary function Sets upper limit to exercise intensity you can sustain for a long time Sets upper limit to exercise intensity you can sustain for a long time

49. 49 Maximal Aerobic Capacity or or VO 2 max Mice generally won’t cooperate to wear a mask ….

50. 50 VO 2 max of Males at Generation 32 Body-mass adjusted Mean and Standard Error (average) (a measure of uncertainty) ml O 2 min x gram Control Selected. = 33% higher Rezende, E. L., S. A. Kelly, F. R. Gomes, M. A. Chappell, and T. Garland, Jr. 2006. Effects of size, sex, and voluntary running speeds on costs of locomotion in lines of laboratory mice selectively bred for high wheel-running activity. Physiological and Biochemical Zoology 79:83-99.

51. 51 Treadmill Endurance Capacity Meek, T. H., B. P. Lonquich, R. M. Hannon, and T. Garland, Jr. 2009. Endurance capacity of mice selectively bred for high voluntary wheel running. Journal of Experimental Biology 212: 2908-2917.

52. 52 Treadmill Endurance (gen. 49) Means Endurance Time (minutes) FemaleMale S/C Selected35.032.6 Control28.126.3 1.24 Mice from the Selected lines have higher endurance capacity.

53. 53 Increased Insulin-Stimulated Glucose Uptake of Isolated Extensor Digitorum Longus Muscles (extends toes 2-5 and dorsiflexes ankle) Gen. 17 Males Wheels & No wheels for 2 months Dumke et al. 2001. Journal of Applied Physiology 91:1289-1297. Exercise Adaptations Mice from the Selected lines are better able to utilize some of the energy stores in their muscles.

54. 54 Exercise Adaptations Larger Femoral Heads Also true for humans! Garland, T., Jr., and P. A. Freeman. 2005. Selective breeding for high endurance running increases hindlimb symmetry. Evolution 59:1851-1854. Kelly, S. A., P. P. Czech, J. T. Wight, K. M. Blank, and T. Garland, Jr. 2006. Experimental evolution and phenotypic plasticity of hindlimb bones in high-activity house mice. Journal of Morphology 267:360-374.

55. 55 Exercise Adaptations Thicker Femurs and Tibiafibulas (but not longer) Gen. 21B Males Wheels & No Wheels for 2 months Kelly, S. A., P. P. Czech, J. T. Wight, K. M. Blank, and T. Garland, Jr. 2006. Experimental evolution and phenotypic plasticity of hindlimb bones in high-activity house mice. Journal of Morphology 267:360-374. Maybe you expected the Selected lines to have evolved longer legs?

56. 56 Exercise Adaptations More Symmetrical Hindlimb Bone Lengths Also true for English thoroughbred horses! Garland, T., Jr., and P. A. Freeman. 2005. Selective breeding for high endurance running increases hindlimb symmetry. Evolution 59:1851-1854.

57. 57 Increased Hear t Mass (adjusted for differences in body mass) Gen. 35 Females 5 days of Wheel Access Gomes et al. 2004. SICB Talk. Gen. 37 Females Wheels & No Wheels for 3 months Kelly et al. HVR In preparation. Gen. 49 Females Wheels & No Wheels for 2 months Kelly et al. Cotman In preparation. Exercise Adaptations

58. 58 Exercise Adaptations Reduced Hindlimb Muscle Mass This difference is similar to what we see for human marathon runners. Garland, T., Jr., M. T. Morgan, J. G. Swallow, J. S. Rhodes, I. Girard, J. G. Belter, and P. A. Carter. 2002. Evolution of a small-muscle polymorphism in lines of house mice selected for high activity levels. Evolution 56:1267-1275.

59. 59 The Brain & Motivation blue: hippocampus red: caudate-putamen purple: ventricles blue: hippocampus yellow: midbrain orange: cerebellum Mice from the Selected lines have larger brains, but not the cerebellum. MRI scan done at Loma Linda University

60. 60 What neurotransmitters (brain chemicals) are involved in motivation? Dopamine is a logical candidate. Dopamine is a logical candidate. Initial Experimental Test: compare response to drugs that target dopaminergic function. compare response to drugs that target dopaminergic function.

61. 61 Dopamine Transporter Blockers Ritalin, GBR 12909, Cocaine (also serotonin) Receptor Agonist Apomorphine (non-selective) Receptor Antagonists Raclopride (D-2), SCH23390 (D-1) Serotonin Transporter Blocker Prozac Endogenous Opioids Receptor Antagonists Naloxone Naltrexone Rhodes, J. S., S. C. Gammie, and T. Garland, Jr. 2005. Neurobiology of mice selected for high voluntary wheel-running activity. Integrative and Comparative Biology 45:438-455.

62. 62 Results: Pharmacology Several dopamine drugs had dif ferential ef fects on C and S lines.

63. 63 Results: Pharmacology Several dopamine drugs had dif ferential ef fects on C and S lines. For example, Ritalin increased running in C lines, but decreased it in S lines, almost down to level of controls.

64. 64 Rit alin Inject ion Time (min) Selected Lines Control Lines Wheel Running Distance (m) open = Saline closed = Ritalin

65. 65 Rit alin Inject ion Time (min) Selected Lines Control Lines Wheel Running Distance (m) open = Saline closed = Ritalin Therefore, the Selected lines are a potentially useful model for studying human ADHD.

66. 66 Results: Pharmacology Several dopamine drugs had dif ferential ef fects on C and S lines. For example, Ritalin increased running in C lines, but decreased it in S lines, down to level of controls. No drug increased wheel running in the selected lines. Therefore, motivation and/or ability to run may be at a physiological limit. Therefore, motivation and/or ability to run may be at a physiological limit.

67. 67 Conclusions& Future Directions

68. 68 ● Experimental evolution can be a rapid and effective way to alter the characteristics of organisms and increase our understanding of both physiology and evolution. ● Evolutionary increases in voluntary activity levels entail increases in both motivation and ability to per form physical activity. ● Some traits that have changed seem to represent clear adaptations to support high locomotor activity (e.g., higher VO 2 max). ● Others might be maladaptive byproducts (not discussed today). Conclusions.

69. 69 Future Directions ● Figure out what is limiting further increases in wheel running. ● Figure out what is limiting further increases in wheel running. Exercise ability vs. motivation? ● Unravel the neural and psychological mechanisms of motivation for exercise. ● Unravel the neural and psychological mechanisms of motivation for exercise. Is this like an addiction? ● Find the genes that cause higher running. ● Find the genes that cause higher running. What are they doing?

70. 70 ● Consider translation to human beings. Might we develop drugs that make it more pleasurable for people to exercise? Or less pleasurable to be sedentary? And Maybe Even …

71. 71 Resources

72. 72 Resources ● The scientific papers on Dr. Garland's mice can be found as PDF files at his website: ● The scientific papers on Dr. Garland's mice can be found as PDF files at his website: http://www.biology.ucr.edu/people/faculty/Garland.html ● 1-hour lecture on the mouse experiment: http://cnas.ucr.edu/sciencelectures/garlandlecture.html ● Wikipedia has various relevant sites: ● Wikipedia has various relevant sites: http://en.wikipedia.org/wiki/Experimental_evolution http://en.wikipedia.org/wiki/Evolutionary_physiology http://en.wikipedia.org/wiki/Exercise_physiology http://en.wikipedia.org/wiki/Evolution

73. 73 Extra Slides Follow

74. 74 Ratio of Selected/Control Lines 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 Generation Total Revolutions Females Wild Females The Real "Wild Type" This is the average wheel running for selected females divided by the average Wheel running in control females per generation

75. 75 Body Mass (grams) Selected Control 10 20 30 40 50 15000 10000 5000 0 Revolutions/Day S & C Lines at Gen. 24 vs. 13 Other Species This is a substantial evolutionary difference. But the Selected lines are not "abnormal"

76. 76 Daily Pat tern Does Not Dif fer Much... Daily Pat tern Does Not Dif fer Much... Given that we keep selecting, why don’t they also run for more minutes per night? Selected Control

77. 77 Why a Selection Limit? Functional Answers: Motivation or Reward at a Maximum Ability to Run at a Maximum Energetic Cost is too High Not mutually exclusive, May elucidate mechanisms of evolutionary “constraints”

78. 78 Generation 49 Endurance Day 1 (minutes) Endurance Day 2 (minutes) N = 74 r = 0.785 P = 1 -16 paired t = 4.57 P = 0.00002 Treadmill Endurance Capacity The selected lines have greater endurance than the control lines

79. 79 Maximal Sprint Speed on a 6-meter Photocell- timed Racetrack Generation 48, Meek et al., unpublished results

80. 80 Maximal Sprint Speed Sprint Speed Day 1 (m/s) Sprint Speed Day 2 (m/s) N = 118 r = 0.640 P = 6 -15 paired t = 6.79 P = 5 -10 But there is no difference in sprinting ability Generation 48, Meek et al., unpublished results

81. 81 Increased GLUT- 4 Glucose Transporter Concentration in Gastrocnemius Muscles (af ter wheel access) (extends ankle) Gen. 35 Females 5 days of Wheel Access Sampled in Afternoon Gomes et al. 2009 Exercise Adaptations Gastroc- nemius

82. 82 Tim Noakes Nature 2006 444:1000-1001

83. 83 Hypothesis from the business world: Exceptional per formance is 50% attitude & 50% aptitude. "What separates high per formers from lesser competitors isn’t just talent. It's the way they fuse their capability and mindset."

84. 84 Const ant Dark Const ant Light

85. 85 It occurs when the wheel is harder to turn. If wheels are locked to prevent rotation, selected mice climb more. Based on these behavioral data, we hypothesize that selected mice possess higher motivation to run.

86. 86 The Increased Wheel Running Lasts for Much of the Lifespan and is Quite General: and is Quite General:

87. 87 Does the brain show evidence of increased motivation for running? Mice from both Control and Selected lines were given wheel access for 6 days. for 6 days. On day 7, two hours before lights off, wheels were blocked for 1/2 of the mice. 14 of 23 brain regions showed higher activity in mice with blocked motivation wheels, which may indicate motivation, stress, anticipation, frustration, anxiety or depression. The data also showed an interaction between wheel type (blocked and free) and line type (control and selected), suggesting greater motivation in selected mice greater motivation in selected mice.

88. 88 c-fos Immunoreactivity in Striatum (CPu) Interaction P = 0.03 Fos Cell Count Control Selected Blocked Free Selected with Blocked Wheel Selected with Free Wheel

89. 89 The Effects of Diet

90. 90 Effects of a "Western" Diet Males from generation 52 Weaned at 21 days of age, housed individually At 25 days of age, divided into: SedentaryWheel Access Standard Diet 5050 Western Diet 5050 42% kcal from fat plus added sucrose 42% kcal from fat plus added sucrose Analyses of blood and tissue samples are currently in progress …

91. 91 Control Selected Wheel No Wheel S W S W Retroperitoneal Fat Pad Mass (g) Least Squares Means ± SE Retroperitoneal Fat: 82 days old Diet

92. 92 Control Selected Std. Western Std. Western Days 17-30 of wheel access Revolutions/Day 52% Increase Adjusted Means + Standard Errors +36% Time +18% Speed These mice are unique! Effects of a "Western" Diet

93. 93 Introduction, v ersion 1.0 (more evolutionary)

94. 94 From either perspective, as Darwin wrote: "The whole organism is so tied together that when slight variations in one par t occur, and are accumulated through natural selection, other par ts become modified." Charles Dar win (1859) The Origin of Species "This is a very important subject, most imper fectly understood."

95. 95 Future Directions 1 ● Figure out what "Western" diet is doing to the selected lines. Are they protected from its adverse effects (development of metabolic syndrome, etc.)? ● Unravel the neural and psychological mechanisms of motivation for exercise. Is this like an addiction? ● Find the genes that cause higher running. What is their mechanism of action?

96. 96 Future Directions 2 ● Consider translation to human beings. Might we develop drugs that make it more pleasurable for people to exercise?