Nervous System Compensation Following Tail Loss and Regeneration in the Leopard Gecko (Eublepharis macularius) By Stefanie Simone Bradley A Thesis presented to the University of Guelph In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Science Guelph, Ontario, Canada © Stefanie Simone Bradley, January, 2019 ABSTRACT NERVOUS SYSTEM COMPENSATION FOLLOWING TAIL LOSS AND REGENERATION IN THE LEOPARD GECKO (EUBLEPHARIS MACULARIUS) Stefanie Bradley Advisor: University of Guelph, 2018 Dr. M.K. Vickaryous Mass change is a physical phenomenon with important implications for biomechanics and locomotion. Here, we used the leopard gecko (Eublepharis macularius) to investigate the effect of a drastic change in mass following tail loss (autotomy), and subsequent regeneration of the tail. We assessed two components of the nervous system: tactile sensitivity, and Purkinje cell neuromorphology. Using Semmes-Weinstein monofilaments, we found regional differences in tactile sensitivity prior to autotomy. Following tail autotomy, the hindlimbs became significantly more sensitive, while the forelimbs did not. Golgi-Cox staining of Purkinje cells showed that tail autotomy had no significant effect on Purkinje cell structure. However, after 30 days of tail regeneration, there was evidence of dendritic remodeling corresponding to the interval where parallel fibers synapse with Purkinje cell dendrites. Together, these data provide support for short-term (transient) compensation of the peripheral nervous system, and long-term compensation of the central nervous system, in geckos following autotomy. iii ACKNOWLEDGEMENTS Time to acknowledge some cool people! First and foremost – Matt – for being an incredible P.I. A major reason I joined the lab was because of your friendly demeanor (as well as super cool science, of course). As an advisor, you’re especially hands-on, but also allow for independence in our research goals. I really appreciated your support when I wanted to branch into somewhat different territory for my project (i.e., poking geckos with nylon filaments). As a result of my time in the lab, I feel much more confident as a researcher and scientist. I also feel much more confident in assessing how “peaty” different whiskeys are (mostly so I can shoot them anyways). Whatever life skills you’re bestowing on us, the net result of your leadership is a unique and motivating environment in the lab. To Matt’s family - Jalene, for always being a gracious host, and always welcoming members of the lab with open arms (and amazing food). To honorary members of the lab, Isla and Piper, for their energy and gecko-centric artwork, which usually accompanies every lab milestone. Next – to the Eublephosphere. To Sarah, Rebecca, and Kathy who have been there since the beginning of my Master’s. From practice talks, to conferences, to bar nights - the dynamic in our lab is fun, scientifically collaborative, and genuinely passionate. To newer members of the lab: Laura and Yifan (also for helping with neuron tracings), and undergrad students as well. To my advisory committee - Craig and Leah. It was motivating to have a committee that was genuinely interested in the work that was being done. I think I lucked out in that you guys were actively involved in my project, always approachable, and always helpful in providing valuable feedback and hands-on expertise. I’d also like to thank my examination committee, Dr. iv MacLusky and Dr. LaMarre, for agreeing to participate in my defense on an end-of-semester Friday afternoon (ideal for celebrating but not ideal for everyone else). To Erika Howe from the Bent lab – my gargantuan monofilament data set may still remain a mystery if it had not been for you. I’m grateful for the many hours you spent helping me with statistical planning and analysis. To my parents - for being supportive of their offspring pursuing science instead of business. To my mom for listening to me recite my seminars approximately one million times, and always being a support system. To my dad for actually asking questions at my defense. To my brother Michael for being mildly interested in my research, and to my smaller brother Christopher for thinking stem cells and regenerative biology are the coolest things ever. To my friends – for listening to me talk about thesis writing for many months, and for putting up with me continually re-scheduling my defense date (and then still taking the day off work to make it to my defense). To the geckos and mice – for being super cute model organisms (besides the biting) and contributing to science. To the biomedical department at UofG – for providing a warm sense of community, and always being friendly. I’d also like to acknowledge Starbucks for providing endless amounts of caffeine and a makeshift office for many months of thesis writing. My Master’s project was definitely a collaborative effort. It allowed me to take science from a hobby/interest, to a field that I will most likely remain in for my career. If you’d asked me a few years ago where I’d be now, I don’t think I would ever have guessed this - so that’s pretty cool. v TABLE OF CONTENTS ABSTRACT ................................................................................................................................. ii ACKNOWLEDGEMENTS ....................................................................................................... iii TABLE OF CONTENTS ........................................................................................................... iv LIST OF FIGURES .................................................................................................................. viii LIST OF TABLES ...................................................................................................................... xi LIST OF APPENDICES .......................................................................................................... xiii LIST OF ABBREVIATIONS .................................................................................................. xiv DECLARATION OF WORK PERFORMED ........................................................................ xv CHAPTER 1: LITERATURE REVIEW .................................................................................. 1 1.1 Mass Change in Nature ................................................................................................ 1 1.2 Somatosensation .......................................................................................................... 3 1.3 The Cerebellum ............................................................................................................ 7 1.4 Dendritic Morphology ............................................................................................... 11 1.5 Appendage Loss and the Leopard Gecko .................................................................. 15 CHAPTER 1 FIGURES............................................................................................................. 19 RATIONALE ............................................................................................................................. 21 CHAPTER 2: SOMATOSENATION IN THE PNS: CHARACTERIZATION OF TACTILE SENSITIVITY FOLLOWING TAIL LOSS AND REGENERATION IN THE LEOPARD GECKO (EUBLEPHARIS MACULARIUS)........................................................ 23 2.1 INTRODUCTION ............................................................................................................... 23 2.2 MATERIALS AND METHODS......................................................................................... 28 2.2.1 Experimental Animals and Animal Care................................................................. 28 2.2.2 Experimental Design ............................................................................................... 29 vi 2.2.3 Tail Autotomy ......................................................................................................... 29 2.2.4 Monofilament Testing ............................................................................................. 30 2.2.5 Euthanasia, Tissue Collection, and Preparation....................................................... 31 2.2.6. Statistical Analysis.................................................................................................. 32 2.3 RESULTS ............................................................................................................................. 33 2.3.1 Experimental Geckos .............................................................................................. 33 2.3.2 Monofilament Testing ............................................................................................. 34 2.3.3 Regional Differences Prior to Autotomy ................................................................ 35 2.3.4 Sensitization of Control Group ............................................................................... 35 2.3.5 Hindlimb Sensitivity with Tail Loss and Regeneration .......................................... 36 2.3.6 Forelimb Sensitivity with Tail Loss and Regeneration ........................................... 37 2.3.7 Tail Base Sensitivity with Tail Loss and Regeneration .......................................... 37 2.3.8 Tail Tip Sensitivity with Tail Loss and Regeneration ............................................ 38 2.4 DISCUSSION ......................................................................................................................