EXAMINATION OF NUCLEUS ACCUMBENS MECHANISMS UNDERLYING THE MOTIVATION FOR PHYSICAL ACTIVITY ________________________________________________________________ A Dissertation presented to the Faculty of the Graduate School at the University of Missouri ________________________________________________________________ In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy ________________________________________________________________ by GREGORY NEAL RUEGSEGGER Dr. Frank W. Booth, PhD, Dissertation Supervisor May 2017 The undersigned, appointed by the Dean of the Graduate School, have examined the dissertation entitled: EXAMINATION OF NUCLEUS ACCUMBENS MECHANISMS UNDERLYING THE MOTIVATION FOR PHYSICAL ACTIVITY presented by Gregory Neal Ruegsegger, a candidate for the degree of Doctor of Philosophy and hereby certify that, in their opinion, it is worthy of acceptance. Frank W. Booth, Ph.D. M. Harold Laughlin, Ph.D. Douglas K. Bowles, Ph.D. Eileen M. Hasser, Ph.D. Matthew J. Will, Ph.D. To my family: my parents Bill and Linda, my sister Natalie, as well as my extended family. Your support and encouragement make even the most challenging endeavors attainable. ACKNOWLEDGEMENTS Knowledge is like money: to be of value it must circulate, and in circulating it can increase in quantity and, hopefully, in value. -Louis L’Amour Foremost, I would like thank my Heavenly Father for blessing me with the gift of curiosity that steered me to pursue a career in research, as well the countless other blessings I have in my life. I am grateful for all of the scientists whose hard work, passion, and intellectual curiosity provided the framework upon which this research constitutes. I am grateful and privileged to have trained with Dr. Frank Booth for my doctoral studies. Dr. Booth’s support has been undeviating, and I will always be indebted to Dr. Booth for his inspiring, nurturing, and caring environment. Most importantly, Dr. Booth’s passion for science has instilled in me a passion for my own pursuits in life, both at and away from the bench. Additionally, I consider it a great honor to have been guided by a wonderful dissertation committee during my doctoral studies: Dr. Harold Laughlin, Dr. Doug Bowles, Dr. Eileen Hasser, and Dr. Matt Will. Thank you all for your feedback, insight, and ‘open door’ attitudes toward my questions and concerns. It has been a pleasure to learn from each of you. I thank the members of Dr. Booth’s lab and the Dept. of Biomedical Sciences for their support and guidance. Specifically, I thank Tom Childs for his instrumental role in teaching me lab techniques and his continuous help with every aspect of my projects, as well as Ryan Toedebusch for his friendship, energy, contributions to my projects, and encouragement. I’ll always be thankful for the time we spent together. ii I am also very grateful to my close family: my grandparent, aunts, uncles, and cousins. Specifically, my aunt Sister Carol Wirtz for the sacrifices she continually makes for others, her commitment to our family in times of need, and for being a role model by which I strive to live my life. Finally, I want to thank my parents, Bill and Linda, for providing me with every resource and lesson I needed to achieve my goals. The knowledge and questions that came from your parenting were the catalyst that inspired my lifelong journey. While I didn’t at the time, I thank you for instilling our home with attitudes of hard work, dedication, and service that continue to inspire me to constantly improve myself and to use my talents and resources to the best of my ability. My sister, Natalie, has been equally supportive, and I hope that she has learned half as much from me as I have from her. I am so grateful to always have the three of you in my corner. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................ ii LIST OF TABLES ........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix LIST OF ABBREVIATIONS ............................................................................................ xi ABSTRACT ..................................................................................................................... xiv CHAPTER 1: Introduction ................................................................................................. 1 Physical inactivity is a major cause of chronic diseases ................................................ 1 The majority of individuals are not physically active despite believing exercise is good for health ........................................................................................................................ 2 Physical activity levels decline during juvenile and adolescent life .............................. 4 Is there a biological basis for physical inactivity? ......................................................... 6 Nucleus accumbens, dopamine, and physical activity motivation ................................. 9 Opioids influence nucleus accumbens dopamine signaling ......................................... 14 Opioids influence physical activity motivation............................................................ 15 Nucleus accumbens dopaminergic signaling diminishes during adolescence and adulthood ...................................................................................................................... 17 Maternal nutritional status influences offspring nucleus accumbens dopamine signaling as well as spontaneous activity ..................................................................... 19 A novel model of physical activity motivation: selective breeding for high or low voluntary running behavior .......................................................................................... 22 Aims of dissertation research ....................................................................................... 25 CHAPTER 2: Mu-opioid receptor inhibition decreases voluntary wheel running in a dopamine-dependent manner in rats bred for high voluntary running (published in Neuorscience. 2016, 339: 525-537.) ................................................................................. 29 ABSTRACT ................................................................................................................. 30 INTRODUCTION ....................................................................................................... 31 MATERIALS AND METHODS ................................................................................. 33 Experimental Animals ............................................................................................... 33 Experiment 1.............................................................................................................. 34 Experiment 2.............................................................................................................. 35 iv Experiment 3.............................................................................................................. 35 Experiment 4.............................................................................................................. 36 Tissue Extraction ....................................................................................................... 36 Western Blotting ........................................................................................................ 36 Tissue Dissociation and Electrophysiology ............................................................... 37 Thermal Stimulation Test .......................................................................................... 39 RNA isolation, cDNA synthesis, and qRT-PCR ....................................................... 39 Surgery and 6-OHDA administration ........................................................................ 40 Immunohistochemistry for 6-OHDA dopamine neuron lesion ................................. 41 Statistical Analysis .................................................................................................... 41 RESULTS .................................................................................................................... 42 Experiment 1.............................................................................................................. 42 Experiment 2.............................................................................................................. 44 Experiment 3.............................................................................................................. 46 Experiment 4.............................................................................................................. 48 DISCUSSION .............................................................................................................. 51 CHAPTER 3: Maternal Western diet promotes age-specific alterations in female offspring voluntary wheel running.................................................................................... 60 ABSTRACT ................................................................................................................. 61 INTRODUCTION ....................................................................................................... 62 MATERIALS AND METHODS ................................................................................