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Characterization of Neuronal Nicotinic Acetylcholine Receptors and Their Positive Allosteric Modulators Doris Clark Jackson Brigham Young University

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Characterization of Neuronal Nicotinic Acetylcholine Receptors and Their Positive Allosteric Modulators Doris Clark Jackson Brigham Young University Brigham Young University BYU ScholarsArchive All Theses and Dissertations 2017-06-01 Characterization of Neuronal Nicotinic Acetylcholine Receptors and their Positive Allosteric Modulators Doris Clark Jackson Brigham Young University Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Organismal Biological Physiology Commons BYU ScholarsArchive Citation Jackson, Doris Clark, "Characterization of Neuronal Nicotinic Acetylcholine Receptors and their Positive Allosteric Modulators" (2017). All Theses and Dissertations. 6856. https://scholarsarchive.byu.edu/etd/6856 This Dissertation is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in All Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Characterization of Neuronal Nicotinic Acetylcholine Receptors and Their Positive Allosteric Modulators Doris Clark Jackson A dissertation submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Neuroscience Sterling N. Sudweeks, Chair Arminda Suli Jonathan J. Wisco R. Paul Evans Scott C. Steffensen Department of Physiology and Developmental Biology Brigham Young University Copyright © 2017 Doris Clark Jackson All Rights Reserved ABSTRACT Characterization of Neuronal Nicotinic Acetylcholine Receptors and Their Positive Allosteric Modulators Doris Clark Jackson Department of Physiology and Developmental Biology, BYU Doctor of Philosophy Neuroscience Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that are necessary in memory and cognition. They are pentameric and consist of α and β subunits. They are most commonly heteromeric but, can sometimes be homomeric. nAChRs are activated by many ligands including nicotine (exogenous) and acetylcholine (endogenous). nAChRs are located on hippocampal interneurons. The interneurons, although sparse, control the synchronous firing of the pyramidal cells. However, the hippocampal interneuron structure and function is quite diverse and not fully characterized. Therefore, we sought to quantify nAChR subunit mRNA levels using real-time PCR of CA1 hippocampal interneurons. Surprisingly we found that the α3 and β2 mRNA subunits were the highest expressed and highest co-expressed subunits. Additionally, the α4 mRNA subunit was the lowest expressed of the subunits detected. The α4 subunit is one of the most pharmacologically targeted nAChR subunits and is found throughout the rest of the brain at much higher levels than the α3 mRNA subunit. Upon PCR analysis two subpopulations of the α3 and β2 subunits emerged: those that contained 3X more α3 than β2 and those that contained 3X more β2 than α3. Therefore, we hypothesized that two likely α3β2 nAChR stoichiometries are present in hippocampal interneurons. We differentiated their kinetic properties using electrophysiology. Additionally, like the α4 subunit, the α7 subunit is highly targeted in cognitive therapeutics. Since, the α7 subunit is the most characterized nAChR subunit, there are current efforts to develop allosteric modulators of the α7 subunit. The α7 subunit is found at moderate levels within hippocampal interneurons and remains a valid target. Current treatment options for Alzheimer’s disease, and other dementias are limited and only mildly effective. Therefore, we sought to characterize the effect of 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2) on α7. Furthermore, there are no current methods to distinguish the α7 from the α7β2 nAChRs during whole cell electrophysiological recordings. Therefore, we also characterized the PAM-2 effect on α7β2 nAChRs. Our results highlight at least 2 ways PAM-2 can be used to differentiate α7 from the α7β2 during whole-cell recordings. Keywords: nicotinic acetylcholine receptors (nAChRs), hippocampus, interneurons, electrophysiology, allosteric modulators, real-time PCR ACKNOWLEDGMENTS I am more than grateful for the support of all my committee members. I have received valuable mentoring and research advice from all members. I am grateful for Dr. Hugo Arias and his invitation to research using positive allosteric modulators. It opened a new path of research that I found very rewarding and enjoyable. I am particularly grateful for the years of research and mentoring in Dr. Sudweeks’ lab. I am appreciative for all of the funding from the Physiology and Developmental Biology Department and would not have been able to complete my degree without sufficient funding. In addition, Connie Provost was a vital asset to my graduate experience at BYU and in the submission of my dissertation. I am particularly grateful for the support of my husband, daughter, parents, and siblings. All have played a vital role in my success. The opportunity to study at BYU has strengthened my faith and increased my capacities. TABLE OF CONTENTS TITLE PAGE .................................................................................................................................. i ABSTRACT ................................................................................................................................. ii ACKNOWLEDGMENTS ............................................................................................................. iii TABLE OF CONTENTS ............................................................................................................... iv LIST OF TABLES ......................................................................................................................... vi LIST OF FIGURES ...................................................................................................................... vii LIST OF SYMBOLS AND ABBREVIATIONS .......................................................................... ix INTRODUCTION .......................................................................................................................... 1 References ........................................................................................................................... 9 CHAPTER 1: Expression of nAChR mRNA in Rat Hippocampal Interneurons ......................... 12 Abstract ............................................................................................................................. 13 Introduction ....................................................................................................................... 14 Methods............................................................................................................................. 15 Slice Preparation ................................................................................................... 15 Cytoplasm Aspiration ........................................................................................... 16 Electrophysiology ................................................................................................. 16 Primers and Probes ............................................................................................... 16 RT Reaction .......................................................................................................... 17 Multiplex Reaction and Real-time Quantitative PCR ........................................... 17 Data and Statistical Analysis ................................................................................ 18 Equation 1: ............................................................................................................ 19 Equation 2: ............................................................................................................ 19 Equation 3: ............................................................................................................ 19 Materials ............................................................................................................... 20 Results ............................................................................................................................... 20 Discussion ......................................................................................................................... 23 References ......................................................................................................................... 38 CHAPTER 2: The Human Alpha 3 Beta 2 Neuronal Nicotinic Acetylcholine Receptor Forms Two Distinguishable Subtypes .................................................................. 40 Abstract ............................................................................................................................. 41 Introduction ....................................................................................................................... 42 iv Methods............................................................................................................................. 43 Data and Statistical Analysis ................................................................................ 45 Materials ............................................................................................................... 46 Results ............................................................................................................................... 47 Discussion ......................................................................................................................... 49 Supporting Information ....................................................................................................
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