Exploring novel treatment approaches for post-traumatic stress disorder Anthony Murkar Thesis submitted in partial fulfillment of the requirements of the degree of Doctor of Philosophy School of Psychology Faculty of Social Sciences University of Ottawa © Anthony Murkar, Ottawa, Canada, 2019 ii Acknowledgements I would like to express my sincerest thanks to my supervisor Zul Merali and to Pamela Kent for your continued support throughout these projects, and also for all of the amazing opportunities you made possible. Without your support I would not be where I am today. The success of these projects is a reflection of the tremendous support and supervision of you both. Your dedication to your work and love for research was an inspiration. From the day I set foot in the labs at The Royal until the day I left, never once did I feel unwelcome or unsupported. Likewise, to Jon James, Christian Cayer, Tony Durst, and John Arnason, my colleagues in the lab and outside of it who contributed to these published works: no man is an island. These projects would not have been possible without you. To the many mentors who contributed to my success, I would like to extend my thanks to you as well. To Stuart Fogel, Nafissa Ismail, and Claude Messier (the members of the thesis committee), and also to Joseph De Koninck (who was always available to lend his ear and his sage advice): your valuable input and guidance played a pivotal role in my success. Finally, to my mother, and to my friends and family throughout these years - there are too many of you to name. Your support, encouragement, and friendship uplifted me and meant more to me then you may ever realize. iii Abstract Post-traumatic stress disorder is a disorder characterized by an inability to extinguish traumatic memories and heightened reactivity to emotional stimuli. Due to the heightened resistance of traumatic memories to extinction, treatment for PTSD has been challenging and is limited to behavioral therapies targeted at reducing responsivity to threatening stimuli. Currently there are no standard pharmacological interventions that are specific to PTSD; rather, drugs used appear to target symptoms of some of the co-morbid conditions, such as anxiety (e.g. benzodiazepines) or depression (antidepressants) - which may also affect fear-memory. In this thesis, we explore the effects of natural health products (NHPs) including naturally occurring peptides and some medical botanicals on fear memory in order to explore the efficacy of natural products as potential pharmacological targets for fear-based disorders. Fear-conditioning has been used effectively in both rodents and humans to study fear- learning. Fear-conditioning is a learning paradigm during which an unconditioned aversive stimulus (such as foot shock) is paired with a neutral stimulus (such as light or tone), such that the neutral stimulus becomes associated with aversion. Fear-learning has several well- characterized stages, including acquisition, consolidation, reconsolidation, expression, and extinction that can be manipulated in order to study the pharmacological action(s) on the attenuation of learned-fear. Blockade of reconsolidation, the state during which formed memories are briefly rendered susceptible to change following recall, may provide a window of opportunity to pharmacologically diminish learned fear. In Chapter 1 of the thesis, we discuss fear-conditioning as a pre-clinical model of PTSD to explore the effects of novel pharmacological treatments on the reconsolidation process in rodents. We ultimately hope to iv provide a framework for translational work in humans for attenuating conditioned responses to trauma-related stimuli among humans with PTSD. In Chapter 2, we present evidence that systemic administration of gastrin-releasing peptide attenuates the reconsolidation of conditioned fear in rodents. Similarly, in chapter 3, we explore the effects of Δ9-Tetrahydrocannabinol (THC) and Cannabidiol (CBD) on the reconsolidation of learned-fear, and provide evidence that cannabinoid molecules may similarly prove effective at blocking the reconsolidation of conditioned fear memories. In chapter 4, we present evidence demonstrating that extracts of medical botanical Souroubea sympetala and its components may similarly block reconsolidation of conditioned fear-memory, and also exert more general anxiolytic-like activity in the elevated plus maze paradigm. Finally, in chapter 5 a general discussion considers the relative therapeutic potential for future human clinical trials of each of the three tested groups of compounds. v Co-Authorship In all cases, A. Murkar contributed to the study design, data collection, data analysis, and preparation of manuscripts. P. Kent contributed to the study design in chapters 2 and 3. J. James and C. Cayer contributed to data collection in chapters 2-4. Z. Merali contributed to the conceptualization of the study design and support to execution of all studies. Chapter 2 is published and can be cited as: Murkar, A., Kent, P., Cayer, C., James, J., & Merali, Z. (2018). Gastrin-releasing peptide attenuates reconsolidation of conditioned fear memory. Behavioural brain research, 347, 255-262. Chapter 3 is published and can be cited as: Murkar, A., Kent, P., Cayer, C., James, J., Durst, T. & Merali, Z (2019). Cannabidiol and the remainder of the plant extract modulate the effects of Δ9-Tetrahydrocannabinol on fear memory reconsolidation. Frontiers in behavioural neuroscience, 13, 174. Chapter 4 is in press and can be cited as: Murkar, A., Cayer, C., James, J., Durst, T., Arnason, J.T., & Merali, Z (in press). Extract and active principle of the neotropical vine Souroubea sympetala Gilg. block fear-memory reconsolidation. Frontiers in pharmacology. vi Table of Contents Acknowledgements ....................................................................................................................... ii Abstract ......................................................................................................................................... iii Co-authorship .................................................................................................................................v Table of contents .......................................................................................................................... vi List of figures ................................................................................................................................ ix List of abbreviations .................................................................................................................... xi Summary ...................................................................................................................................... xii 1.0 General Introduction ...............................................................................................................1 1.1 Onset and symptom clusters of PTSD ................................................................................1 1.2 Natural products as treatment ............................................................................................4 1.3 Rodent models of fear-learning and PTSD ........................................................................8 1.4 Consolidation, reconsolidation, and memory updating ..................................................10 1.5 Reconsolidation blockade ..................................................................................................16 1.6 Pathophysiology of PTSD: Role of the HPA axis and sympathetic nervous system ....18 1.7 Pathophysiology of PTSD: Fear Circuitry .......................................................................22 1.8 The role of sleep ..................................................................................................................27 1.9 Gastrin-releasing peptide ..................................................................................................29 1.10 The complex nature of cannabinoids ..............................................................................31 1.10.1 Role of CB1 in fear expression & extinction ...........................................................34 1.10.2 Role of CB1 in consolidation of learned fear ..........................................................37 vii 1.10.3 Differential effects of cannabinoid modulation at infralimbic versus prelimbic cortex .....................................................................................................................................39 1.10.4 The role of CB2 receptors .........................................................................................40 1.10.5 Summary: Cannabinoids & learned fear ................................................................41 1.11 S. Sympetala leaf extract and betulinic acid modulate learned fear ............................42 1.12 Thesis overview, objectives, & hypotheses .....................................................................44 2.0 Chapter 2: Gastrin-Releasing Peptide attenuates reconsolidation of conditioned fear-memory .................................................................................................................................47 2.1 Abstract ...............................................................................................................................48 2.2 Introduction ........................................................................................................................49