ESTROGENIC MODULATION OF FEAR GENERALIZATION A dissertation submitted To Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Joseph F. Lynch III August 2016 ©Copyright All rights reserved Except for previously published materials Dissertation written by Joseph Francis Lynch III B.A., SUNY Plattsburgh, 2011 M.A., Kent State University, 2013 Approved by David C. Riccio, Ph.D , Chair, Doctoral Dissertation Committee Aaron, M. Jasnow, Ph.D , Members, Doctoral Dissertation Committee Karin G. Coifman, Ph.D Stephen B. Fountain, Ph.D John D. Johnson, Ph.D Heather K. Caldwell, Ph.D Accepted By Manfred van Dulmen , Acting Chair, Psychological Sciences James L. Blank , Dean, College of Arts and Sciences TABLE OF CONTENTS………………………………...………………………………iii ACKNOWLEDGMENTS...……………………………………………………….........viii CHAPTERS I. Introduction ............................................................................................................... 10 1.1. PTSD .................................................................................................................. 10 1.2. Fear Generalization ............................................................................................ 12 1.3. Estrogens and Generalization ............................................................................. 15 1.4. General Scope .................................................................................................... 16 II. Experiment 1 – Sex Differences in Fear Generalization .......................................... 20 2.1. Introduction ........................................................................................................ 20 2.2. Methods .......................................................................................................... 20 2.3. Results and Discussion ................................................................................... 22 Figure 1. Sex Differences in the Generalization of Fear. ................................................. 27 III. Experiment 2 – Role of Estrogens in Fear Generalization .................................... 28 3.1. Introduction ........................................................................................................ 28 3.2. Methods .......................................................................................................... 29 3.3. Results and Discussion ................................................................................... 31 Figure 2. Chronic Exposure to Estradiol Induces Generalization in OVX Females ........ 36 IV. Experiment 3 - Chronic Hormone Exposure in GDX Males ................................. 37 4.1. Introduction ........................................................................................................ 37 4.2. Methods .......................................................................................................... 38 4.3. Results and Discussion ................................................................................... 40 Figure 3. Chronic Exposure to Testosterone or Estradiol Prevents Generalization in GDX Males ................................................................................................................................. 46 V. Experiment 4 - Effects of Estradiol on Memory Acquisition/Consolidation or Retrieval ............................................................................................................................ 47 5.1. Experiment 4A - Pre-Training Acute Hormone Exposure in OVX Females ..... 47 5.2. Methods .......................................................................................................... 48 5.3. Results and Discussion ................................................................................... 49 iii Figure 4. Acute Exposure to Estradiol Has a Transient Effect on Fear Generalization When Given 1 Hour Before Training in OVX Females ................................................... 53 5.4. Experiment 4B - Post-Training Acute Hormone Exposure in OVX Females ... 54 5.5. Methods .......................................................................................................... 54 5.6. Results and Discussion ................................................................................... 54 Figure 5. Estradiol has a Transient Effect on Memory Retrieval in OVX Females ......... 62 5.7. Experiment 4C - Post-Training Acute Hormone Exposure in GDX Males ....... 63 5.8. Methods .......................................................................................................... 63 5.9. Results and Discussion ................................................................................... 64 Figure 6. Acute Exposure to Estradiol Affects Memory Retrieval by Attenuating Generalization in GDX Males .......................................................................................... 67 5.10. Experiment 4D - Effects of Aromatase Inhibition on Generalization ............ 68 5.11. Methods....................................................................................................... 68 5.12. Results and Discussion ............................................................................... 69 Figure 7. Testosterone Prevents Generalization in Males Through Aromatization in Estradiol ............................................................................................................................ 73 VI. Experiment 5 - Activation of Specific Estrogen Receptor (ER) Subtypes ............ 74 6.1. Experiment 5A – Estrogen Receptor Agonist Injections to OVX Females ....... 74 6.2. Methods .......................................................................................................... 75 6.3. Results and Discussion ................................................................................... 76 6.4. Experiment 5B - Estrogen Receptor Agonist injections to GDX Males ............ 78 6.5. Methods .............................................................................................................. 78 6.6. Results and Discussion ....................................................................................... 79 Figure 8. Activation of ERβ Induces Generalization in Females, Activation of Either Subtype Prevents Generalization In Males ....................................................................... 82 VII. Experiment 6 - Assessing Cytosolic Versus Membrane-Bound Estrogen Receptors 83 7.1. Experiment 6A – Assessing Cytosolic ERs With Peripheral Estradiol Administration .............................................................................................................. 83 7.2. Methods .......................................................................................................... 85 7.3. Results and Discussion ................................................................................... 86 7.4. Experiment 6B - Assessing Cytosolic ERs With Central Infusions of Estradiol87 iv 7.5. Methods .......................................................................................................... 88 7.6. Results and Discussion ................................................................................... 89 7.7. Experiment 6C - Assessing Cytosolic ERs With Peripheral ERβ Activation .... 90 7.8. Methods .......................................................................................................... 90 7.9. Results and Discussion ................................................................................... 91 Figure 9. Estradiol Induces Generalization Through Activation of Cytosolic ERβ ......... 94 7.10. Experiment 6D - Assessing Membrane-Bound ERs ...................................... 94 7.11. Methods....................................................................................................... 95 7.12. Results and Discussion ............................................................................... 96 Figure 10. Activation of Membrane-Bound Receptors is Not Sufficient to Induce Generalization ................................................................................................................... 99 VIII. Experiment 7 - Circuitry Involved in Estradiol-Induced Fear Generalization . 100 8.1. Introduction ...................................................................................................... 100 8.2. Experiment 7A - Assessing Cytosolic ERs Within the Dorsal Hippocampus . 101 8.3. Methods ........................................................................................................ 102 8.4. Results and Discussion ................................................................................. 103 8.5. Experiment 7B - Assessing Cytosolic Estrogen Receptors With Dorsal Hippocampus Infusions of ER Agonists ..................................................................... 104 8.6. Methods ........................................................................................................ 105 8.7. Results and Discussion ................................................................................. 106 Figure 11. Activation of Cytosolic ERβ Within The Dorsal CA1 Results in Generalization ................................................................................................................. 109 8.8. Experiment 7C - Assessing Membrane-Bound Estrogen Receptors Within the Dorsal Hippocampus ................................................................................................... 110 8.9. Methods