Elucidating the Role of the Paraventricular Nucleus of the Thalamus in Cue-Motivated Behavior by Joshua Lewis Haight A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Neuroscience) in the University of Michigan 2016 Doctoral Committee: Assistant Professor Shelly B. Flagel, Co-Chair Professor Stanley J. Watson, Jr, Co-Chair Professor Kent C. Berridge Professor Terry E. Robinson Research Associate Professor Robert C. Thompson © Joshua L Haight 2016 DEDICATION This dissertation is dedicated to my wife, Emily, and my parents, Mark and Julie, for never letting me give up. ii ACKNOWLEDGEMENTS It would be impossible for me to acknowledge everyone who has had a significant impact on me, and my development as a person and scientist, these last five years. Nonetheless, I will try my best. First off, I have to acknowledge the entire Flagel lab, starting with my mentor, Dr. Shelly Flagel. When I joined Dr. Flagel’s lab in the Spring semester of 2012, I was a little worried about being ‘the first graduate student’. It turns out it was the best decision I could have made for my graduate school career. I would not be where I am today without her never-ending support, in science and in life. She encouraged me to explore my own ideas, and think critically yet positively about my work. Dr. Flagel’s contagious enthusiasm about science inspires me to this day, and will continue to inspire others for years to come. I know that, aside from a mentor, I have made a life-long friend. To all the wonderful people I have gotten to work with in the Flagel Lab: thank you for making this journey such a wonderful one! Your companionship, as colleagues and as friends, has influenced me in such positive ways throughout the years. Our lab managers Rebeca Kelly, Katie Long, Julia Boese and Marin Klumpner, have all helped in various ways on the projects contained in this thesis, and dealt with my tendency to order necessary items at the last minute with patience. The post-docs that I have gotten to work with, Aram Parsegian, Nacho Covelo- Rivera, and Paolo Campus, have patiently answered all my questions and provided advice on my projects and career path. My fellow Flagel Lab graduate students, Brittany Kuhn and Sofia Lopez, have shared (and commiserated at times) all the ups and downs with me. And most importantly, this thesis would not exist without the tireless commitment of two undergraduates: Kurt Fraser and Zack Fuller, who worked by my side on many nights and weekends to make this work possible. My dissertation committee also deserves special recognition. Drs. Kent Berridge, Terry Robinson, Robert Thompson, and Stanley Watson: thank you for your invaluable guidance and input on my thesis throughout the years, and for your patience with all the hiccups and iii complications along the way. While Dr. Huda Akil was not an official member of my committee, she, along with Dr. Watson, took a special interest in my work. As Co-Directors of the Molecular and Behavioral Neuroscience Institute, they opened up countless lab resources to me, and gave me the opportunity to present my work at their joint lab meetings, allowing me to grow as a scientist. Dr. Watson and Dr. Robinson also read many drafts of manuscripts that have since been published, and their feedback on the work in progress was invaluable. Last, when I was in one of many panicked states during my qualification exams, Dr. Thompson loaned me a book with a title akin to “Genetics for Dummies”. Without that book, I might never have passed those exams. The Neuroscience Graduate Program deserves recognition as well. Thank you for admitting me to the Ph.D. program in the first place, and allowing me to pursue this career. It is certainly a privilege. Special thanks to our Director, Edward Stuenkel, and Assistant Director, Audrey Seasholtz, for upholding the integrity of higher education in Neuroscience. And to my cohort: while we may have drifted apart as the years went by, the friendships we built during those first two grueling years will stay with me forever. Last, but certainly not least, I must acknowledge my family, for sticking with me over the last five years. To my parents, Julie and Mark: I would never have made it to graduate school in the first place, let alone completed my degree, without your endless love and encouragement. To my wife, Emily: you have seen me at my best, and also at my worst. Your strength and love have picked me up when times were rough, and inspired me to be the best husband and father that I could be. And to my son, Henry: even though you are young, and will not remember this time in Michigan, you have made everything worth it. iv TABLE OF CONTENTS DEDICATION ii ACKNOWLEDGEMENTS iii LIST OF FIGURES viii ABSTRACT xi CHAPTER 1. Introduction 1 A role for the paraventricular nucleus of the thalamus in reward processing and cue-motivated behaviors 2 Interactions between the paraventricular nucleus of the thalamus and the ventral striatum, with a focus on dopamine transmission 4 Exploiting individual variation in Pavlovian conditioned responses to parse the incentive from the predictive properties of reward-paired cues 7 The neurobiology underlying differences in Pavlovian conditioned approach behavior 10 A potential role for the paraventricular nucleus of the thalamus in mediating sign- vs. goal-tracking behaviors 13 Conclusions and hypothesis 14 References 17 Figures 29 2. Lesions of the paraventricular nucleus of the thalamus differentially affect sign- and goal-Tracking conditioned responses 33 Introduction 33 v Materials and methods 34 Results 40 Discussion 45 References 51 Figures 56 Appendix A 62 Appendix B 66 3. A food predictive cue engages sub-cortical efferents and afferents of the paraventricular nucleus of the thalamus if it is attributed with incentive salience 71 Introduction 71 Materials and methods 74 Results 81 Discussion 90 References 95 Figures 102 4. Investigating the Role of Orexin Receptor 2 in the paraventricular nucleus of the thalamus in cue-motivated behavior 114 Introduction 114 Materials and methods 118 Results 126 Discussion 131 References 136 Figures 144 vi 5. General Discussion 157 The role of the PVT in the propensity to attribute incentive salience to reward cues: Updating the model 157 The role of specific PVT afferents in sign- and goal-tracking behavior 160 The role of specific PVT efferents in sign- and goal-tracking behavior 169 Special note on anterior vs. posterior aspects of the paraventricular nucleus of the thalamus 173 Concluding remarks and future directions 175 References 178 Figure 186 vii LIST OF FIGURES Chapter 1 1.1. Dopamine D3 receptor mRNA expression in the PVT 29 1.2. Cartoon representation of sign-tracking and goal-tracking behaviors 30 1.3. ‘Functional connectivity’ in sign-trackers and goal-trackers 31 1.4. Schematic illustrating afferents and efferents of interest in the PVT 32 Chapter 2 2.1. Experimental Timelines 56 2.2. Histological analysis of lesion sites for Experiment 1 57 2.3. Effects of PVT lesion on the acquisition of (left) goal- and (right) sign-tracking conditioned responses 58 2.4. Histological analysis of lesion sites for Experiment 2 59 2.5. GTs learn to sign-track across sessions following PVT lesions 60 2.6. PVT lesions increase the tendency to sign-track in GTs 61 S2.1. Effects of PVT lesion on the acquisition of “off target” behaviors in bHR and bLR animals 66 S2.2. Activity during the inter-trial interval for bLR rats 67 S2.3. The acquisition of sign- and goal-tracking conditioned responses across 7 PCA training sessions 68 S2.4. The extent of PVT lesion underlies change in response bias score 69 S2.5. PVT lesions do not affect locomotor response to a novel environment 70 viii Chapter 3 3.1. Acquisition of the sign- and goal-tracking conditioned response following 5 Pavlovian conditioning sessions (afferent experiment) 102 3.2. Representative images of fluorogold (FG) injections into the PVT and immunohistochemical labeling 103 3.3. c-Fos expression in the medial prefrontal cortex following cue presentation 104 3.4. c-Fos expression in the central and medial amygdala following cue presentation 105 3.5. c-Fos expression in the hypothalamus following cue presentation 106 3.6. c-Fos expression in the ventral subiculum following cue presentation 108 3.7. Acquisition of the sign- and goal-tracking conditioned response following 5 Pavlovian conditioning sessions (efferent experiment) 109 3.8. Representative images of fluorogold (FG) injections into the NAc and immunohistochemical labeling 110 3.9. c-Fos expression in the PVT following cue presentation 111 3.10. Schematic demonstrating the efferent and afferent circuits of the PVT engaged by cue presentation in STs vs. GTs 113 Chapter 4 4.1. Timeline for Experiments 1, 2 and 3 144 4.2. Acquisition of goal-tracking behavior across 7 sessions of Pavlovian conditioned approach training 145 4.3 Cannula placement verification 147 4.4. Antagonism of OX-2 receptors in the PVT does not alter Pavlovian conditioned approach behavior in goal-trackers 148 4.5. Acquisition of sign-tracking behavior across 5 sessions of Pavlovian conditioned approach training 150 4.6. Antagonism of OX-2 receptors in the PVT attenuates Pavlovian conditioned approach behavior in sign-trackers 152 ix 4.7. Antagonism of OX-2 receptors in the PVT attenuates the escalation of sign-tracking behavior 154 4.8. Antagonism of OX-2 receptors in the PVT may alter the conditioned reinforcing properties of the lever-CS 155 4.9. Antagonism of OX-2 receptors in the PVT does not alter feeding behavior 156 Chapter 5 5.1.