Photoperiodic properties of circadian rhythm in rat by Liang Samantha Zhang A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Neuroscience) in The University of Michigan 2011 Doctoral Committee: Associate Professor Jimo Borjigin, Chair Professor Theresa M. Lee Professor William Michael King Associate Professor Daniel Barclay Forger Assistant Professor Jiandie Lin © Liang Samantha Zhang 2011 To my loving grandparents, YaoXiang Zhang and AnNa Yu ii Acknowledgements To all who have played a role in my life these past four years, I give my thanks. First of all, I give my gratitude to the members of Borjigin Lab. To my mentor Dr. Jimo Borjigin whose intelligence and accessibility has carried me through in this journey within the circadian field. To Dr. Tiecheng Liu, who taught me all the technical knowledge necessary to perform the work presented in this dissertation, and whose surgical skills are second to none. To all the undergrads I have trained over the years, namely Abeer, Natalie, Christof, Tara, and others, whose combined hundreds if not thousands of hours in manually analyzing melatonin data have been an indispensible asset to myself and the lab. To Michelle and Ricky for taking care of all the animals over the years, which has made life much easier for the rest of us. To Alexandra, who was willing to listen and share her experiences, and to Sean, who has been a good friend both in and out of the lab. I would also like to thank my committee members for their help and support over the years. Both Dr. Terri Lee and Dr. Daniel Forger have guided me with their expert insights in the circadian field both during committee meetings as well as at Biological Rhythm Seminars and the annual Michigan Rhythm Meetings. I especially had a great time with the Lee Lab at the SRBR meeting, and it’s something I’ll always remember with fondness. I would like to thank Dr. Jiandie Lin for taking the time to be a part of my thesis committee, and providing his insights from a molecular perspective. Lastly, I would like to thank Dr. Mike King for all the kindness and support he has shown me over iii the years. Dr. King has been my neuroscience professor, my research mentor at my first lab rotation, my prelim committee chair, and my dissertation committee member for the last 3 years. If all goes well, soon he shall also be my postdoctoral mentor, and I truly look forward to working with him in the future. Finally, to my loving husband Michael Hypes, who has driven 4 hours round trip each and every single weekend to visit me in Ann Arbor these last few years. Thank you for taking such good care of me, and I’m so glad to have you in my life. iv Table of Contents Dedication ...................................................................................................................... ii Acknowledgements ....................................................................................................... iii List of Figures ............................................................................................................... ix Abstract ......................................................................................................................... xi Chapter 1 Introduction ................................................................................................... 1 1.1 The circadian system ............................................................................................ 1 1.2 Importance of entrainment ................................................................................... 3 1.3 Mechanisms of mammalian photic entrainment .................................................. 4 1.4 SCN outputs ......................................................................................................... 6 1.5 Pineal melatonin synthesis pathway .................................................................... 8 1.6 Role of melatonin ............................................................................................... 11 1.6.1 Circadian regulation .................................................................................... 11 1.6.2 Seasonal regulation ..................................................................................... 12 1.6.3 Melatonin as a circadian marker ................................................................. 12 1.7 Pineal microdialysis approach in circadian research ......................................... 14 1.8 Goals and Significance ....................................................................................... 17 Chapter 2 Experimental methods ................................................................................. 19 2.1 Long-term pineal microdialysis of freely moving rats ....................................... 19 2.1.1 System overview ......................................................................................... 19 2.1.2 Trans-pineal microdialysis probe implantation ........................................... 22 2.1.3 Sample acquisition ...................................................................................... 26 2.1.4 Data analysis ............................................................................................... 27 v 2.1.5 Experiments ................................................................................................. 30 2.2 Telemetry measurements ................................................................................... 30 2.2.1 Surgical Procedures ..................................................................................... 30 2.2.2 Data Collection and Analysis ...................................................................... 30 Chapter 3 Dissociation between phase angles of entrainment and free-running period ........................................................................................................................................... 31 3.1 Introduction ........................................................................................................ 31 3.1.1 Accepted relationship between entrained phase angle and free-running period ........................................................................................................................ 31 3.1.2 Published findings that contradict the relationship ..................................... 32 3.1.3 What this chapter offers .............................................................................. 32 3.2 Methods .............................................................................................................. 33 3.2.1 Animals ....................................................................................................... 33 3.2.2 Free-running period measurement .............................................................. 34 3.3 Results ................................................................................................................ 38 3.3.1 Interindividual differences in phase angle and free-running period of outbred rats ................................................................................................................ 38 3.3.2 Photoperiod manipulation of phase angles of inbred F344 rats .................. 40 3.4 Discussion .......................................................................................................... 45 3.4.1 Previous establishment of relationship between phase angle and period ... 45 3.4.2 Supporting evidence for a lack of direct correlation between phase angle and period. ................................................................................................................. 47 3.5 Conclusion ......................................................................................................... 51 3.6 Future directions ................................................................................................ 51 3.6.1 Wider range of phase angle and periods ..................................................... 51 3.6.2 After-effects ................................................................................................ 52 Chapter 4 Shorter melatonin onset phase angle predicts faster reentrainment rate ..... 54 vi 4.1 Introduction ........................................................................................................ 54 4.2 Method ............................................................................................................... 56 4.2.1 Animals ....................................................................................................... 56 4.2.2 Definition of reentrainment ......................................................................... 59 4.3 Results ................................................................................................................ 61 4.3.1 Reentrainment is a slow process ................................................................. 61 4.3.2 Interindividual differences in speed of reentrainment ................................ 63 4.3.3 Photoperiod manipulation ........................................................................... 66 4.3.4 Reentrainment to 2h advance or delay shift under different photoperiods . 68 4.3.5 Change in phase angle after phase shift ...................................................... 69 4.4 Discussion .......................................................................................................... 71 4.4.1 Preliminary data on interindividual differences of reentrainment .............