ELUDICATING TRIGGERS AND NEUROCHEMICAL CIRCUITS UNDERLYING HOT FLASHES IN AN OVARIECTOMY MODEL OF MENOPAUSE Lauren Michele Federici Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Medical Neuroscience Program, Indiana University April 2016 Accepted by the Graduate Faculty, Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. _____________________________ Anantha Shekhar, M.D., Ph.D., Chair _____________________________ Charles Goodlett, Ph.D. Doctoral Committee _____________________________ Philip L. Johnson, Ph.D. February 26, 2016 _____________________________ Gerry S. Oxford, Ph.D. _____________________________ Daniel E. Rusyniak, M.D. ii Acknowledgements The author would like to acknowledge, with sincere gratitude, the guidance of her mentor, Dr. Philip L. Johnson, and the support of her research committee: Drs. Charles Goodlett, Gerry Oxford, Daniel Rusyniak, and Anantha Shekhar. She would also like to thank all of the laboratory members that have provided assistance and training throughout her education, including Cristian Bernabe, Izabela Facco Caliman, Amy Dietrich, Stephanie Fitz, Dr. Andrei Molosh, Aline Abreu Rezende, and Dr. William Truitt. The author also gratefully acknowledges the staff and faculty of the Stark Neurosciences Research Institute, including Ms. Nastassia Belton and the graduate advisors, Drs. Andy Hudmon and Ted Cummins. The work described in this entity was supported in part by the National Institute of Aging (K01AG044466), Indiana Clinical and Translational Sciences Institute KL-2 Scholars Award (RR025760), Indiana Clinical and Translational Sciences Institute Project Development Team Pilot Grant (RR025761), Indiana University Simon Cancer Center Basic Science Pilot Funding (23-87597), all to Philip L. Johnson. Compound 56 and DORA-12 were donated by Janssen Research and Development, L. L. C., and Merck & Co, respectively. She would also like to thank her husband, Randy Julian, and family, Lee and Suzanne and Erinn and Taylor Federici, for their unwavering support through all the highs and lows of good and bad data. iii Lauren Michele Federici ELUCIDATING TRIGGERS AND NEUROCHEMICAL CIRCUITS UNDERLYING HOT FLASHES IN AN OVARIECTOMY MODEL OF MENOPAUSE Menopausal symptoms, primarily hot flashes, are a pressing clinical problem for both naturally menopausal women and breast and ovarian cancer patients, with a high societal and personal cost. Hot flashes are poorly understood, and animal modeling has been scarce, which has substantially hindered the development of non-hormonal treatments. An emerging factor in the hot flash experience is the role of anxiety and stress-related stimuli, which have repeatedly been shown to influence the bother, frequency, and severity of hot flashes. Causal relationships are difficult to determine in a clinical setting, and the use of animal models offers the ability to elucidate causality and mechanisms. The first part of this work details the development and validation of novel animal models of hot flashes using clinically relevant triggers (i.e., compounds or stimuli that cause hot flashes in clinical settings), which also increase anxiety symptoms. These studies revealed that these triggers elicited strong (7-9 °C) and rapid hot flash-associated increases in tail skin temperature in rats. In a surgical ovariectomy rat model of menopause, which typically exhibit anxiety-like behavior, hot flash provocation revealed an ovariectomy-dependent vulnerability, which was attenuated by estrogen replacement in tested models. An examination of the neural circuitry in response to the most robust flushing compound revealed increased cellular activity in key thermoregulatory and emotionally relevant areas. The orexin neuropeptide system was hyperactive and presented as a novel target; pretreatment with selective and dual orexin receptor antagonists significantly diminished or eliminated, respectively, the response to a hot flash provocation in ovariectomized rats. The insertion/deletion polymorphism of the serotonin transporter has been linked to increased anxiety- associated traits in humans, and subsequent studies prolonged hot flashes in SERT +/- rats , which also caused hot flashes in highly symptomatic women. These studies indicate the orexin system may be a novel non-hormonal iv treatment target, and future studies will determine the therapeutic importance of orexin receptor antagonists for menopausal symptoms. Anantha Shekhar, M.D., Ph.D., Chair v Table of Contents List of Tables vii List of Figures viii List of Abbreviations xi Outline of Parts I-IV xiv Parts I-IV 1 Materials and Methods 81 Chapter 1: Select Panicogenic Drugs and Stimuli Induce Increases in Tail Skin Temperature and Decreases in Core Body Temperature Introduction 94 Results 96 Discussion 97 Chapter 2: Hypothalamic Orexin’s Role in Exacerbated Cutaneous Vasodilation Responses to an Anxiogenic Stimulus in a Surgical Menopause Model Introduction 103 Results 105 Discussion 109 Chapter 3: Anxiogenic CO 2 Stimulus Elicits Exacerbated Hot Flash-like Responses in a Rat Menopause Model Introduction 122 Results 124 Discussion 125 Commentary 131 Future Directions 142 Bibliography 152 Curriculum Vitae vi List of Tables Table 1. Menopausal Symptoms Exacerbated by Oophorectomy 3 Table 2. Key Regions of Interest With Respect to Menopausal Symptoms 78 Table 3. Schematic of Clinical Study of Hot Flash Provocation with CO 2 135 Challenges in Highly Symptomatic Women and Participant Characteristics vii List of Figures Figure 1. Body Diagram of a Hot Flash 5 Figure 2. Physiological Recordings of a Hot Flash 7 Figure 3. Levels of Estradiol and Follicle Stimulating Hormone Across the 14 Lifespan Figure 4. Metabolic Pathway of Tamoxifen 20 Figure 5. Ambient Temperature-Induced Cutaneous Vasomotor 39 Response Preserves Normothermia Figure 6. Ambient Temperature-Induced Cutaneous Vasomotor 45 Responses Do Not Differ Between Ovariectomized and Sham- Ovariectomized Rats Figure 7. Ambient Temperature-Induced Cutaneous Vasomotor Response 50 Figure 8. Divisions and Neurochemicals of the Autonomic Nervous 51 System Figure 9. Reduction in Hot Flash Frequency by Study 63 Figure 10. Diurnal Variation in Temperature Between Ovariectomized 71 (OVEX) and Intact (Sham) Controls Figure 11. Accuracy of Thermal Data Acquisition 84 Figure 12. Panicogenic Drugs Elicit Profound Hot Flash- 100 viii Associated Cutaneous Vasomotor Responses in Male Rats (Chapter 1) Figure 13. Hypercapnic Gas Elicits Profound Hot Flash-Associated 101 Cutaneous Vasomotor Response in Male Rats (Chapter 1) Figure 14. Hot Flash Provocations Result in Increased Temperature 102 Along the Entire Length of the Tail (Chapter 1) Figure 15. Rats Modeling Surgical Menopause are Vulnerable to 114 Displaying Hot Flash-Associated Cutaneous Vasomotor Responses to a Low Dose of a Panicogenic Drug (Chapter 2) Figure 16. Alprazolam Pretreatment Attenuates Cutaneous Vasomotor 115 Responses to a Low Dose of a Panicogenic Drug (Chapter 2) Figure 17. Panicogenic Drug-Induced Cutaneous Vasomotor 116 Responses Precede a Decrease in Core Body Temperature in Rats with Surgical Menopause (Chapter 2) Figure 18. Rats with Surgical Menopause Display Hyperactive Cellular 117 Responses Following a Low Dose of a Panicogenic Drug in Neural Circuits Heavily Implicated in Menopausal Symptoms (Chapter 2) Figure 19. Neurochemical Circuits of Rats with Surgical Menopause that 118 Display Hyperactive Responses Following a Low Dose of a Panicogenic Drug (Chapter 2) Figure 20. Surgical Menopause Enhances Baseline Hypothalamic 119 Orexin/Glutamate Gene Expression (Chapter 2) ix Figure 21. Systemic Pretreatment with a SORA1 or Estrogen is Anxiolytic 120 in OVEX Rats (Chapter 2) Figure 22. Systemically Treating Ovariectomized Rats Treated with 121 Centrally Active Orexin 1 and/or 2 Receptor Antagonists Attenuates Hot Flash-Associated Cutaneous Vasomotor Responses to a Low Dose of a Panicogenic Compound (Chapter 2) Figure 23. Rats Modeling Surgical Menopause Have Exacerbated Hot 129 Flash-Associated Tail Skin Temperature Responses (Chapter 3) Figure 24. Rats with a Heterozygous Null Mutation of the Serotonin 130 Transporter (SERT +/-) Have Prolonged Hot Flash-Associated Tail Skin Temperature Responses to Hypercapnic Gas Infusion (Chapter 3) Figure 25. Schematic of Clinical Study of Hot Flash Provocation with CO 2 135 Challenges in Highly Symptomatic Women and Participant Characteristics Figure 26. The Elevated T-maze Test of Anxiety-Associated Behaviors in 141 Female Rats Across the Estrus Cycle x Abbreviations 3V third ventricle 5-HT 5-hydroxytryptophan/serotonin ACh acetylcholine AChE acetylcholinesterase AET adjuvant endocrine therapies AI aromatase inhibitor BNST bed nucleus of the stria terminalis BLA basolateral amygdala CBT core body temperature CeA central amygdala CO 2 carbon dioxide DMN dorsomedial hypothalamic nucleus DORA dual orexin receptor antagonist DRN dorsal raphe nucleus DRVL-VLPAG ventrolateral dorsal raphe nucleus, ventrolateral periaqueductal gray DSI Data Sciences International DSM-V Diagnostic and Statistical Manual of Mental Disorders-V E epinephrine E2 17-β estradiol EIT estrogen inhibition therapy EPM elevated plus maze ETM elevated T maze ER estrogen receptor ERα estrogen receptor α ERβ estrogen receptor β ERT estrogen replacement therapy FMP final menstrual period xi FSH follicle stimulating hormone g gram GABA γ-amino-butyric acid GnRH