Estrogens in the bed nucleus of the stria terminalis: State-dependent modulation of inhibitory synaptic transmission in rats By James Gardner Gregory A thesis submitted to the Centre for Neuroscience Studies In conformity with the requirements of the degree of Doctor of Philosophy Queen’s University Kingston Ontario, Canada (October 2018) Copyright © James Gardner Gregory, 2018 Abstract Beyond their important role in reproduction, estrogens contribute in various adaptive and maladaptive physiological phenomena including, but not limited to, appetite, stress response, and the development of compulsive behaviours. There are estrogen receptors throughout the brain, including in regions and circuits contributing to maladaptive behaviours. Thus, pharmacological interventions targeting estrogens result in considerable undesirable side effects. Traditionally considered a female steroid hormone, estrogens are also potent neuromodulators in the male vertebrate brain. In comparison to female rats, peripherally administered estrogens have negligible effects as the male rat brain likely relies on brain-derived de novo estrogens synthesis. While the neurophysiological effects of estrogens are well characterized in the hippocampus of both sexes, nothing is known about their effects in the oval nucleus of the bed nucleus of the stria-terminalis (ovBNST), a key region that integrates sensory information within the appetite, anxiety, and compulsion circuits. The ovBNST is rich in estrogen receptors and has the capability of synthesising estrogens in both sexes. Using brain slice voltage-clamp electrophysiology, we characterized the effects of estrogens on inhibitory synaptic transmission within the rat ovBNST. These studies will demonstrate that Estradiol (E2) had a potent, but sexually dimorphic effects on both inhibitory synaptic transmission and plasticity at GABA synapses within the ovBNST. These effects of E2 were sensitive to changes in the rats metabolic-state and intriguingly, a form of compulsive behaviours in rats was associated with a dysfunction in the endogenous estrogenic system within the ovBNST. This dysfunction appears to be caused by a weakening of the paraventricular thalamus circuit with the ovBNST that may cause changes in E2 synthesis. Thus, we discovered that brain-derived estrogens are potent modulators of inhibitory synaptic transmission within the ovBNST and they gate a metabolic-state induced bi-directional plasticity ii that is associated with the development and maintenance of maladaptive compulsive behaviours in male rats. These studies lay the foundation in understanding how the endogenous estrogenic system within the male brain is integral in influencing state-dependent behaviour. iii Co-Authorship In all cases, I (James Gardner Gregory) contributed to the design of the experiments, the collection and analysis of all data and wrote the manuscripts. Dr. Eric C. Dumont contributed to the experimental design, data analysis and manuscript preparation. AIM 1: Emily Hawken contributed (~15%) electrophysiological data collection and (~5%) of data analysis. Staci Angelis contributed (~3%) to electrophysiological data collection and (~50%) to estrous cycle monitoring. Jean-Francois Bourchard provided the transgenic mice. AIM 2: Emily Hawken contributed (~45%) electrophysiological data collection and (~20%) of data analysis. Staci Angelis contributed (~75%) to estrous cycle monitoring. AIM 3: Staci Angelis shares co-first authorship on the manuscript and contributed (~40 %) to electrophysiological data collection and analysis and (~60%) of the behavioural data collection. Emily Hawken contributed to (~3%) of the electrophysiolgical data and provided invaluable insight into the experimental design. Calvin Sjaarda & Xudong Liu contributed to (~50%) of the experimental design, data collection and analysis of the rt-qPCR experiment. AIM 4: Francois Georges contributed to the experimental design and supervised the project. iv Acknowledgements I would like to thank first and foremost my wife Staci Angelis for the tremendous amount of support she has given me on my academic journey. She has been the best partner, colleague and collaborator imaginable. I would like to thank my cat Santi, who made sure I was awake every morning at 5 A.M to work on this thesis. I would like to thank my parents for their loving support and guidance. I would like to thank Eric Dumont for guiding me through my journey exploring molecular neuroscience and the countless hours spent editing my thesis and ongoing manuscripts. Ultimately, to all my friends and family, I would like to share my appreciation for the boundless support and encouragement throughout these last five years. v Table of Contents Abstract ........................................................................................................................................... ii Co-Authorship................................................................................................................................ iv Acknowledgements ......................................................................................................................... v List of Figures ................................................................................................................................ ix List of Tables .................................................................................................................................. x Abbreviation List ........................................................................................................................... xi Chapter 1: General Introduction ..................................................................................................... 1 Chapter 2 Literature Review ........................................................................................................... 5 2.1. Sex Steroid Hormones .......................................................................................................... 5 2.1.1. Estrogens ....................................................................................................................... 5 2.1.2. Estrogen receptors ......................................................................................................... 7 2.1.3. Estrogens in the brain .................................................................................................... 8 2.1.3.1. Effects of estrogens on synaptic transmission ........................................................ 8 2.1.3.1.1. Effects of estrogens on excitatory synaptic transmission................................. 8 2.1.2.1.2. Effects of estrogens on plasticity of excitatory synapses ............................... 10 2.2.3.1.2.1. Functional plasticity of excitatory synapses ............................................ 10 2.2.3.1.2.2. Morphological changes at dendritic spines ............................................. 12 2.1.3.2. Estrogens and Inhibitory Synaptic Transmission ................................................. 14 2.1.3. How sex steroid hormones influence food intake and energy balance ........................ 18 2.1.3.1. Estrogens ............................................................................................................... 18 2.1.3.2. Androgens ............................................................................................................. 20 2.1.4. How sex steroid hormones influence stress, fear, defense, and anxiety ...................... 21 2.1.4.1. Estrogens ............................................................................................................... 21 2.1.4.2. Androgens and Progestogens ................................................................................ 24 2.1.5. The role of E2 in compulsion: the case of compulsive drug-taking in animals and humans ................................................................................................................................... 25 2.2. E2 in the Bed Nucleus of the Stria Terminalis ................................................................... 27 2.2.1. Overview of The Bed Nucleus of the Stria Terminalis: structure and function .......... 27 2.2.2. Inputs and Outputs of the ovBNST ............................................................................. 30 2.2.3. The ovBNST: Hormone and peptide signaling ........................................................... 33 2.2.4. The ovBNST: Appetite and Metabolism ..................................................................... 36 2.2.5. The ovBNST: Stress and Anxiety ............................................................................... 40 vi 2.2.6. The ovBNST: Compulsivity ........................................................................................ 43 2.3. Research Aims and Hypotheses ......................................................................................... 47 2.3.1. AIM 1: Characterize the effects of E2 on inhibitory synaptic transmission in the ovBNST ................................................................................................................................. 47 2.3.2. AIM 2: Examine if estrogens are important for synaptic plasticity at GABA synapses within the ovBNST ................................................................................................................ 50 2.3.3. AIM 3: Determine if estrogen mediated synaptic plasticity at GABA synapses in the ovBNST is involved in the development of compulsive adjunctive behaviours