REGULATION OF FOOD ANTICIPATORY ACTIVITY A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Jessica A. Krizo August, 2016 © Copyright All rights reserved Except for previously published materials i Dissertation written by Jessica A. Krizo B.S. Kent State University, 2007 Ph.D., Kent State University, 2016 Approved by Eric Mintz , Chair, Doctoral Dissertation Committee Colleen Novak , Members, Doctoral Dissertation Committee John Johnson Mary Ann Raghanti Stephen Fountain Accepted by Laura Leff , Chair, Department of Biological Sciences James Blank , Dean, College of Arts and Sciences ii TABLE OF CONTENTS Page TABLE OF CONTENTS……………………………...………………………………....iii LIST OF FIGURES………………………...…………………………………..…………v LIST OF ABBREVIATIONS……………………………..……………………………viii ACKNOWLEDGEMENTS………………………………………………………………ix CHAPTERS I. Introduction………………………………………………...……………………..1 Biological and Circadian Rhythms………………………………………………..1 The Mammalian Suprachiasmatic Nucleus……………………………………….3 SCN Afferents and Efferents ……………………………………………………..4 Molecular Mechanism of the SCN ……………………………………………….9 Entrainment to Photic and Non-photic Stimuli…………………………………..12 Extra-SCN and Peripheral Clocks……………………………………………….13 Food Anticipatory Activity………………………………………………………16 Biological Sex and Circadian Rhythms………………………………………….20 Overall Aims……………………………………………………………………..24 II. Role of tissue plasminogen activator in the locomotor response to food restriction ……...…………………………………………………………………26 Introduction………………………………………………………………………26 Materials and Methods……………………………………………………...……29 Results…………………………………………………………………...……….32 Discussion……………………………………………………………………..…46 iii III. Sex Differences in FAA in response to restricted feeding…………….….……..50 Introduction………………………………………………………………………50 Materials and Methods………………...…………………………………………53 Results…………………………...……………………………………………….55 Discussion……………………….……………………………………………….65 IV. Role of gonadal hormones in response to FAA ……….…………..………...…..68 Introduction………………………………………………………………………68 Materials and Methods………………………...…………………………………74 Results……………………………………...…………………………………….79 Discussion……………………………...……………………………………….110 V. Global Discussion………...…………………………………………………….118 Future Directions……………………………………………………………….119 REFERENCES…………………………………………………………………………121 iv LIST OF FIGURES Figure 1. The mouse suprachiasmatic nucleus …………………...…..…………………..3 Figure 2. Major projections to the SCN…………………..…………...………………….6 Figure 3. Molecular transcription/translation feedback loop….…………..……..……...10 Figure 4. Phase Response Curves…………………………………………...…..………13 Figure 5. The Fibrinolytic Pathway………………………………………………..……28 Figure 6. Food Restriction Protocol………………………………………………..……30 Figure 7. LD RF: representative actograms; 24 hour baseline activity profile….………33 Figure 8. LD RF: fast-induced 24 hour activity profiles…………………………..……35 Figure 9. LD RF: restricted feeding 24 hour activity profiles…………………….…….36 Figure 10. LDsk RF: representative actograms; 24 hour baseline activity profile………………………………………………………………………………….…38 Figure 11. LDsk RF: fast-induced 24 hour activity profiles…………….……………… 39 Figure 12. LDsk RF: restricted feeding 24 hour profile and weight change…….………40 Figure 13. Phase angle of entrainment representative actograms and period ……...……42 Figure 14. Phase angle of entrainment following RF in LD and LDsk ………….……….43 . Figure 15. Food intake analysis…………………………………………….……………45 Figure 16. Wild-type male vs female baseline locomotor activity………………...…….56 Figure 17. Wild-type male vs female fast-induced activity……………………………...58 Figure 18. Wild-type male vs female food restriction induced activity ………………..59 Figure 19. Knock-out male vs female baseline locomotor activity …………………….62 Figure 20. Knock-out male vs female fast-induced activity……………………...……..63 Figure 21. Knock-out male vs female food restriction induced activity ………..……..64 v Figure 22. Liver Clock gene expression ………………………………………..………65 Figure 23. Experimental protocol…………………………………..………………...…79 Figure 24. Male representative actograms ………………..……..…………...…………81 Figure 25. Female representative actograms……………………...………………….…82 Figure 26. Baseline total activity sex comparison………………………………...…….83 Figure 27. Male and female total baseline activity ……………………………………..84 Figure 28. Baseline 24 hour activity profile sex comparison …………………………..85 Figure 29. Baseline 24 hour activity profile male and female ………………..….……..86 Figure 30. Baseline weights in males and females …………………………….….……87 Figure 31. Fast day 1 male and female 24 hour activity profiles ……………..….….….89 Figure 32. Fast day 1 male vs female fasted groups 24 hour activity profiles ……...….90 Figure 33. Fast day 2 male and female 24 hour activity profiles ………………….……91 Figure 34. Fast day 2 male vs female fasted groups 24 hour activity profiles …..……..92 Figure 35. Fast-induced weight loss in males and females ………………………..……93 Figure 36. Food restriction total activity level sex comparison …………………..…….95 Figure 37. Food restriction male vs female food restricted 24 hour activity profiles …..96 Figure 38. Food restriction male and female 24 hour activity profiles…………...……..97 Figure 39. Food restriction total activity in males and females ………………...………98 Figure 40. Food anticipatory activity sex comparison ……………………….…………99 Figure 41. Food restriction-induced weight changes in males and females …………..100 Figure 42. Testosterone replacement baseline 24 hour activity profile ………...……..102 Figure 43. Testosterone replacement overall baseline and weight………………...…..103 Figure 44. Testosterone replacement fast day 1 ……………………………...………..104 vi Figure 45. Testosterone replacement fast day 2 ……………………………………….105 Figure 46. Testosterone replacement food restriction ………………………..………..106 Figure 47. Estrogen replacement baseline 24 hour activity profile ………………..….108 Figure 48. Estrogen replacement overall baseline and weight………………….……..109 Figure 49. Estrogen replacement fast day 1 ……………………………………….…..110 Figure 50. Estrogen replacement fast day 2 ……………………………………….…..111 Figure 51. Estrogen replacement food restriction ……………………………..………112 vii List of Abbreviations AL: ad libitum AR: androgen receptor AVP: arginine vasopressin Bmal: brain and muscle aryl hydrocarbon receptor nuclear translocator Clock: circadian locomotor output cycles kaput Cry1: cyrptochrome 1 Cry2: cyrptochrome 2 CT: circadian time DD: constant dark DHT: dihydrotestosterone DMH: dorsomedial hypothalamus DR: dorsal raphe ERα: estrogen receptor alpha ERβ: estrogen receptor beta FAA: food anticipatory activity FEO: food entrainable oscillator GDX: gonadectomy GHT: geniculohypothalamic tract IGL: intergeniculate leaflet KO: knock out LD: light dark viii LL: constant light MASCO: methamphetamine sensitive circadian oscillator mBDNF: mature brain derived neurotrophic factor MR: median raphe NPY: neuropeptide Y ORCH: orchiectomy OVX: ovariectomy Per1: period 1 Per2: period 2 PR: progesterone receptor PRC: phase response curve proBDNF: pro brain derived neurotropic factor PVN: paraventricular nucleus qRT-PCR: quantitative realtime polymerase chain reaction RF: restricted feeding RHT: retinohypothalamic tract RN: ralphe nuclei SCN: suprachiasmatic nucleus TP: testosterone proprionate tPA: tissue plasminogen activator WT: wild type ZT: zeitgeber time ix Acknowledgments Every journey starts with that first step that is nervous and unsteady, but throughout the years it is nourished and rewarded with a gentle hand and a proud word. I have been blessed to be surrounded by a village that has worked both directly and indirectly to allow me the resources and strength to achieve more than I ever could alone. These people are numerous but I will try to acknowledge those that have made an indelible impact on me both personally and professionally. First, I would like to thank my Ph.D. advisor, Dr. Eric Mintz, for his unwavering support and guidance through my career at Kent State University. He has helped me renew my joy of learning, even when things got rough, through his passion for learning. I have come to understand and deeply respect his manner of mentorship, and know that through this mentorship I have become the driver of my own career. I would also like to thank the members of my committee: Drs. Colleen Novak, John Johnson, and Mary Ann Raghanti for their scientific and personal help and guidance over the years. Additionally, I am forever thankful for the support and guidance from Drs. Heather Caldwell and Jennifer Marcinkiewicz. It is the breadth of knowledge presented to myself and students like me that allow us to develop a strong investigative background to take onward. I have been incredibly fortunate to work with these scientists in an open and collaborative environment. To the members of the Mintz lab, both past and present: Dr. Erin Paulus, who taught me the importance of attention to detail and helped me learn to think like a x scientist. Linley Moreland, who’s friendship and teaching provided and strong basis for my work at Kent and helped me to assist others in making the transition. To those I have had the privilege of working with over the years: Tracey Topacio, Jessica Vespoli, Ghada Nusair, and Amanda Klein: thanks for good company and companionship. A special thank you to William Huffman, who by virtue of friendship helped get me through many of the rough patches with little damage done. To Dr. Ashutosh Rastogi: thank you for your passion for your work, both academically and socially. I have appreciated