The Impact of Cognitive and Physical Enrichment on Contextual Fear Memory Retention & the Contribution of Oligodendrogenesis and Myelination by Jordan Mak A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Physiology University of Toronto © Copyright by Jordan Mak 2021 The Impact of Cognitive and Physical Enrichment on Contextual Fear Memory Retention & the Contribution of Oligodendrogenesis and Myelination Jordan Mak Master of Science Department of Physiology University of Toronto 2021 Abstract Memories are encoded in patterns of neural activity deemed “engrams” and are recalled when this network is reactivated. Physical and cognitive enrichment have been shown to increase neurogenesis and myelinogenesis which can alter the stability of the engram. We housed mice in both physical enrichment and a combination of physical plus cognitive enrichment and found that both caused forgetting of a contextual fear memory if housing occurred after conditioning, but improved memory recall if done beforehand. However, combining physical and cognitive enrichment caused a greater change in freezing behaviour relative to physical enrichment alone, only when housing was done after conditioning. Mice in which oligodendrogenesis was halted exhibited a trend of freezing less than mice with functioning oligodendrocytes regardless of enrichment, though this was not considered statistically significant. These results are consistent with the theory that enrichment exerts its effect on memory retention by altering engram stabilization during memory consolidation. ii Acknowledgements First and foremost, I would like to thank Dr. Anne Wheeler for all of her guidance, support, and mentorship as my supervisor. I cannot stress enough just how much your patience and kindness contributed to my success during this degree. You have always been there to further explain concepts, troubleshoot any issues, and provide input about future directions. I am always in awe at your level of excellence as a scientist, and I do not think I would have enjoyed my time during my MSc if it had not been for you and the lab environment you have fostered. I would also like to thank Dr. Lisa Gazdzinski, who was responsible for the vast majority of my training during my time in the lab. I am extremely appreciative of how you were patient with all of my questions and mistakes. You have been such an amazing role model with your knowledge, willingness to help, and overall efficiency with juggling a Cirque du Soleil level of tasks (I’m still not 100% certain you are not a robot…). It has also been a joy to get to know your family, as I always look forward to our lab socials to see what shenanigans your kids get into. I still do not fully understand your wizardry with our lab equipment, but I commend you for being the only one the cryostat respects and listens to. My project would not have been possible without the contributions of my advisory committee, Dr. Brian Nieman and Dr. Paul Frankland. From the beginning of our first meeting, I felt like we were on the same team and working together to shape this project to be the best it could be. Your advice and recommendations were second to none, and I always felt encouraged iii after our meetings. Thank you for helping me to develop as a scientist and honing my ability to think critically. I would also like to thank Percy Azzopardi, Nancy Simpson, and Igor Vukobradovic at The Centre for Phenogenomics. Percy, thank you so much for accommodating my complicated and hectic cage washing schedule with the custom cages. It was a pleasure working with you and your team. Nancy, thank you for helping me to set up more complicated mouse orders and answering my questions when I had other facility related questions. It was always reassuring when I would walk by your office knowing you had my back. Lastly, Igor I am so appreciative for all of your help with the behavioural testing. You were always supportive and ready to help whenever I needed it, and I enjoyed seeing you around the facility and joking around. Another huge “thank you” goes to my lab mates – both past and present. One of the best parts of coming to SickKids everyday was to see you guys. Perhaps my greatest contribution to the lab is the collection of Oreos kept above my desk. I’m proud to announce that we have successfully sampled all of the novelty flavours available in Canada and have even branched into flavours exclusive to the States. The friendships I have forged with you guys are very dear to me, and I look forward to continuing them even after I graduate. Lastly, I would like to thank my family and friends who were always behind me supporting me throughout this rollercoaster of a journey. You helped keep me on track and were there to prop me up during the rough patches. Though finishing my degree during a global pandemic was not how I envisioned my degree ending, being home with all of you really made this special and I hope I have made you proud. iv Table of Contents Table of Contents ................................................................................................ v List of Abbreviations: ....................................................................................... viii List of Figures: ..................................................................................................... x Chapter 1 1 Introduction .................................................................................................. 1 1.1 Overview of Memory Encoding, Recall, and Forgetting .................................................... 1 1.1.1 Memory & Engrams ................................................................................................... 1 1.1.2 Forgetting & Transience ........................................................................................... 3 1.1.3 Recent vs. Remote Contextual Fear Memory............................................................ 6 1.2 Myelin’s contribution to memory retention .................................................................... 8 1.2.1 Function of Myelin ..................................................................................................... 8 1.2.2 Development of Oligodendrocytes and Myelin ...................................................... 10 1.2.3 Adult Oligodendrogenesis, Myelin Plasticity, and Memory Retention ................... 12 1.3 Physical & Cognitive Enrichments’ Effects on Memory Retention ................................... 14 1.3.1 Brain Changes Associated with Physical & Cognitive Enrichment .......................... 14 1.3.2 Physical & Cognitive Enrichments’ Effect on Memory Recall ................................. 18 1.4 Rationale ...................................................................................................................... 19 1.5 Objectives & Hypotheses .............................................................................................. 20 Objective 1: ........................................................................................................................... 20 Objective 2: ........................................................................................................................... 20 Chapter 2 2 Materials & Methods .................................................................................. 21 2.1 Experimental Designs .................................................................................................... 21 2.1.1 Housing Before/After Conditioning Timeline & Details .......................................... 21 2.1.2 Recombination Efficiency & Toxicity Assessment ................................................... 22 2.1.3 MyRF:NG2-ERT:Cre Mice Enrichment Timeline & Details ....................................... 23 v 2.2 General Methods .......................................................................................................... 24 2.2.1 Mice ......................................................................................................................... 24 2.2.2 Tamoxifen & EdU Administration ............................................................................ 25 2.2.3 Housing Conditions .................................................................................................. 25 2.2.4 Contextual Fear Conditioning .................................................................................. 27 2.2.5 Perfusions ................................................................................................................ 29 2.2.6 Immunohistochemistry ........................................................................................... 29 2.2.7 Statistical Analysis.................................................................................................... 30 Chapter 3 3 Results ........................................................................................................ 33 3.1 Freezing when Enrichment Occurs Before & After Conditioning ..................................... 33 3.1.1 Enrichment After Conditioning Causes Forgetting of Contextual Fear Memories . 33 3.1.2 Timing of Enrichment Relative to Conditioning Causes Different Changes to Memory Retention .................................................................................................. 34 3.1.3 No Sex Effect When Comparing After, Before, or Combined Groups ..................... 36 3.2 Recombination Efficiency .............................................................................................