THE IMPORTANCE OF DIURNAL CORTICOSTERONE RHYTHMS IN REGULATING MOOD A thesis submitted To Kent State University in partial Fulfillment of the requirements for the Degree of Master of Arts by DEVANSHI MITESH MEHTA August 2019 © Copyright All rights reserved Except for previously published materials Thesis written by Devanshi Mitesh Mehta B.S., Kent State University, 2017 M.S., Kent State University, 2019 Approved by John D. Johnson______________________, Advisor Ernest J. Freeman_____________________, Chair, Department of Biological Sciences James L. Blank_______________________, Dean, College of Arts and Sciences TABLE OF CONTENTS TABLE OF CONTENTS ............................................................................................ iii LIST OF FIGURES ...................................................................................................... v ACKNOWLEDGEMENTS ......................................................................................... vi CHAPTERS I. INTRODUCTION........................................................................................1 Depression....................................................................................................1 Stress……....................................................................................................2 HPA axis.......................................................................................................3 Glucocorticoids.............................................................................................4 Diurnal Rhythms of Corticosterone..............................................................5 Circadian rhythms and Circadian genes........................................................7 Corticosterone and Period genes...................................................................9 Hypothesis....................................................................................................10 II. METHODS AND MATERIALS.................................................................12 Study design..................................................................................................12 Animals…….................................................................................................14 Adrenalectomy..............................................................................................15 Corticosterone Replacement.........................................................................16 Blood and Tissue collection..........................................................................16 Quantitative PCR...........................................................................................18 Forced Swim test...........................................................................................18 Open Field test...............................................................................................19 iii Saccharin Preference test...............................................................................19 Statistics….....................................................................................................20 III. RESULTS…..................................................................................................22 IV. DISCUSSION…............................................................................................41 V. REFERENCES …………………….............................................................51 iv LIST OF FIGURES Figure 1. The hypothalamus-pituitary-adrenal axis............................................................. 4 Figure 2. Diurnal levels of Corticosterone............................................................................7 Figure 3. The hypothesis model……………..………………………..................................10 Figure 4. Circadian Rhythm across 24 hours in nocturnal animals......................................11 Figure 5. Timeline for study 1..............................................................................................13 Figure 6. Timeline for study 2..............................................................................................14 Figure 7. The rhythm of CORT after bilateral adrenalectomy (Study 1) ............................25 Figure 8. Sucrose-preference test.........................................................................................26 Figure 9. Total immobility time in 5-min forced swim test.................................................27 Figure 10. The rhythm of CORT after bilateral adrenalectomy (Study 2) ...........................32 Figure 11. Blood glucose test...............................................................................................33 Figure 12. PER2 mRNA expression in the PVN..................................................................34 Figure 13. PER2 mRNA expression in the BNST................................................................35 Figure 14. PER2 mRNA expression in the cAMY.............................................................. 36 Figure 15. Saccharin-preference test.....................................................................................37 Figure 16. Total immobility time in 5-min forced swim test................................................38 Figure 17. Total distance moved in 10 mins open field test..................................................39 Figure 18. Total duration in center zone in 10 mins open field test......................................40 v ACKNOWLEDGEMENTS I would like to thank my parents, Mitesh V Mehta and Parul Mehta, for their constant support and encouragement. I would like to thank my brother, Parth Mehta, for giving me strength and stability. I would like to thank Dr. John D Johnson for his continuous guidance throughout my academic career at Kent State University. Lastly, I would like to extend my gratitude to my senior lab members Adam Kulp, David Barnard and Amy Ionadi for contributing their time and efforts in my project. vi CHAPTER 1 INTRODUCTION DEPRESSION Major depressive disorder (MDD) is one of the most common disabling mental health disorders (1, 2), affecting up to 20% of the world population (3). Despite its widespread effect, it currently has limited therapeutic treatments. The core symptoms of depression include anhedonia (i.e. loss of interest in pleasurable activities), low mood, weight changes, sleep disturbances, retardation, loss of energy and concentration, and/or suicidal thoughts (4-6). This neuropsychiatric disorder can result in major alterations in emotional, motivational, neurovegetative and cognitive processes (7). MDD also shows strong associations with high mortality rates and other medical conditions such as heart diseases, diabetes, and stroke (4, 8). MDD often co-occurs with other psychiatric disorders, most common being anxiety, where 51% of those who have depression also suffer from anxiety disorders (9). Dysregulation of the hypothalamus-pituitary-axis (HPA) axis, which regulates cortisol levels, is often associated with MDD (10-14). Severely depressed patients that have high concentrations of cortisol (12) or are treated with synthetic glucocorticoid have greater risk of suffering from depressive episodes (15). Supporting this view, in depressed 1 patients, MDD is strongly correlated to elevated circulating cortisol levels (12), circadian dysregulation of cortisol (16), and/or impaired glucocorticoid receptor negative feedback of the HPA axis (17). Repeated stress exposure can result in hyperactivity and dysregulation of the HPA axis, which may be why stress is one of the major predisposing factors in the development of major depression. STRESS Stress can be defined as the behavioral and physiological response to a stimulus, crucial in adaptation to external demands and survival (18). This response requires proper engagement of the central and peripheral system, to produce an adequate response to the threat and then enable the body to return to biological equilibrium once the stressor is terminated (19). This response is primarily mediated by the HPA axis. The association between stress and depression was initially made from clinical observations of abnormalities of stress reactivity in depressed patients (10). These clinical cases primarily included dysfunction of the HPA axis caused by chronic or acute stress. Repeated activation of the HPA axis increases allostatic load on the body due to consequent activation of the different systems related to fight-flight responses (20, 21). As the HPA axis plays a major role in short-term and long-term effects of the stress on the body, it is expected that repeated activation of this system affects the neural adaptation of the stress-related circuits, which could then increase the risk of developing these mental health disorders (22, 23). Under a stressful situation, activation of brainstem catecholaminergic neurons and neurons in the hippocampus and amygdala contribute to the activation of the HPA axis. 2 HPA AXIS The hypothalamic-pituitary-adrenal axis is a biological system that serves as the major neuroendocrine mediator of stress responses (20). As shown in Figure 1, the activation of this system starts with the neurons in the paraventricular nucleus (PVN) of the hypothalamus. These neurons release corticotropin releasing hormone (CRH), which in turn act on the anterior pituitary to release adrenocorticotropic hormone (ACTH). ACTH is secreted into the circulation where it activates the production and release of glucocorticoids (GCs) by the adrenal gland (24). To reinstate homeostasis,