The Effects of Ovarian Hormones on Memory Bias and Progesterone Receptors in Female Rats
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The Effects of Ovarian Hormones on Memory Bias and Progesterone Receptors in Female Rats by Smita Patel A Thesis Submitted to the Department of Psychology Concordia University, Quebec In Partial Fulfillment of the Requirements for the Degree of Master of Arts (Psychology) August 2020 © Smita Patel, 2020 iii CONCORDIA UNIVERSITY School of Graduate Studies This is to certify that the thesis prepared By: Smita Patel Entitled: Effects of Ovarian Hormones on Memory Bias and Progesterone Receptors in Female Rats and submitted in partial fulfillment of the requirements for the degree of Master of Arts (Psychology) complies with the regulations of the University and meets the accepted standards with respect to originality and quality. Signed by the final examining committee: _____________________________________ Chair Andreas Arvanitogiannis _____________________________________ Examiner Andrew Chapman _____________________________________ Examiner Peter John Darlington _____________________________________ Thesis Supervisor Wayne Brake Approved by ____________________________________________________ Andreas Arvanitogiannis Pascale Sicotte Date ___________________________ iii ABSTRACT The Effects of Ovarian Hormones on Memory Bias and Progesterone Receptors in Female Rats Smita Patel, MA Concordia University, 2020 Ovarian hormones can bias female rats to use one memory system over another when navigating a novel or familiar environment, resulting in a memory bias. High levels of estrogen (E) promotes places memory while low level of E promotes response memory. However, little is known about the effects of progesterone (P) on memory bias. Experiment 1 determined whether P affects memory bias. Ovariectomized (OVX) female rats were trained in a plus- shaped maze, which assesses memory system bias, and received one of three hormonal treatments: Low 17β estradiol (E2), high E2 or high E2 + P. P did affect memory bias by reversing the effects of high E2 when rats receive P one hour prior to testing. To understand the mechanisms by which P affect memory bias in the hippocampus (HPC), antibodies directed at nPR, mPRβ and mPRδ were examined. The effects of low E2, high E2, and high E2+P were examined on immunoreactivity to these receptors in the HPC. All three receptor-types were found in the female rat HPC and were found to be insensitive to hormone administration. The presence of these receptors suggests that P can exert both genomic and non-genomic effects in the HPC. Other brain areas involved in memory bias remain to be examined further. iv Acknowledgements First and foremost, I would like to express my sincere gratitude to my supervisor Dr. Wayne Brake for the continuous support throughout my undergraduate and graduate years. I was a curious exercise science student looking to learn something about behavioral neuroscience. Thank you for taking me in, having faith in my skills and generously providing me with your time, patience and guidance. I could not have imagined having a better advisor and mentor. I thank my committee members, Dr. Chapman and Dr. Darlington, for taking the time to read this thesis and providing feedback. Many thanks to the CSBN staff members for their valuable technical support on this project. Special thanks Dr. Chris Law for his guidance in the realm of microscopy. This project would not have been possible without him. I thank you for your time, patience and dedication in helping every single one of us to optimize our experiments and making us better researchers. I thank my fellow lab mates: Czarina Evangelista, Conall Mac Connaith, Jesse Lacasse, Collin Gagne, Eamonn Gomez-Perales and Ariel Andrius Batallan for the stimulating discussions, for all the advice and help and for all the fun we have had over the years. I am also grateful to the hard working undergraduate students, Micheal Pileggi, Sehar Gul, Adamo Ghezzo and Alex Lestage without whom I would not have been able to complete this project. Finally, thank you Christopher Wong for being my rock, providing me with unfailing support, and continuous encouragement throughout my years of study. This accomplishment would not have been possible without you. Thank you. Smita Patel v Table of contents Abbreviations ............................................................................................................................ vi Figures and tables ..................................................................................................................... vii Introduction ................................................................................................................................ 1 Methods .................................................................................................................................... 14 Results ....................................................................................................................................... 24 Discussion .................................................................................................................................. 33 References ................................................................................................................................. 43 Appendix A: Effect sizes for Experiment 2 ................................................................................. 58 vi Abbreviations ALLO Allopregnanolone BSA Bovine serum albumin CA1-3 Cornu Ammonis layers 1, 2, and 3 of the hippocampus CNS Central nervous system DG Dentate gyrus region of the hippocampus dSTR Dorsal striatum E Estrogens E2 17β-estradiol EB Estradiol benzoate EC Entorhinal cortex ER Estrogen receptors GABA λ-aminobutyric acid GPER1 G-protein-coupled estrogen receptor 1 HPC Hippocampus IR Immunoreactivity IP Intraperitoneal mER Membrane-associated estrogen receptors mPFC Medial prefrontal cortex mPR Membrane progesterone receptors NDS Normal donkey serum nPR Nuclear progesterone receptors OR Odds ratio OVX Ovariectomy P Progesterone PAQR Progestin and adipoQ receptor PBS Phosphate buffered saline PFA Paraformaldehyde PFC Prefrontal cortex PGRMC1 Progesterone receptor membrane component 1 PR Progesterone receptor RT Room temperature SLM Stratum lacunosum-moleculare TBI Traumatic brain injury TBS Tris-buffered saline Tx Triton-X vii Figures and tables Figure 1. An example of a modified plus maze............................................................................. 3 Figure 2. Experiment 1 timeline……………………………………………………………………………………………….18 Figure 3. Experiment 1 Results.................................................................................................... 27 Figure 4. PR antibody verification via Western blot analysis....................................................... 28 Figure 5. nPR antibody verification in the hypothalamus……...................................................... 29 Figure 6. nPR distribution in the HPC ......................................................................................... 30 Figure 7. mPRβ distribution in the HPC....................................................................................... 31 Figure 8. mPRδ distribution in the HPC....................................................................................... 32 1 The ovaries primarily secrete estrogens (E) and progesterone (P). Traditionally, the primary functions of these ovarian hormones were thought to be only to regulate the female reproductive system, maintain pregnancy, and promote lactation. In naturally cycling females, ovarian hormone levels fluctuate across the menstrual cycle in women, which is called the estrous cycle in female rats. In addition to being important for reproductive and maternal behaviors, we now know that they have much more widespread effects in regions of the brain involved in cognition. Specifically, human and rodent research suggests that hormonal levels can account for major sex differences in the ability to navigate an environment (Brake & Lacasse, 2018). The effects of E and its receptors on spatial navigation have been studied extensively over the past few decades (for review, see; Hussain et al., 2014), yet less is known about the effects of P on spatial navigation despite that it co-fluctuates with P. Moreover, the distribution and function of progesterone receptors (PRs) in areas of the female brain outside the hypothalamus are virtually unknown. Multiple memory systems When navigating an environment, rats use one or more memory systems to reach a goal. Tolman and colleagues (1946) found that rats navigate through an elevated plus maze to find food by using different learning strategies. These types of memories are referred to as place (or spatial) memory and response (or habitual motor) memory. Response memory refers to an egocentric strategy that relies on internal cues by the body, such as habitual turns, to navigate an environment (Hussain et al., 2014). Place memory refers to an allocentric strategy that relies on multiple landmarks from the environment and creates a cognitive map to assist navigation (Hussain et al., 2014). These two types of memory rely on different regions of the 2 Figure 1. An example of a modified plus maze. (A), A rat is trained to find a reward in a baited arm. (B) Upon reaching criterion, the rat placed 180 degrees to the start arm. If the rat turns right, it was scored as using response memory and if it goes to the same spatial location, it was scored as using place memory. 3 brain. Impaired place memory has been observed in rats with hippocampal lesions