University of South Carolina Scholar Commons Theses and Dissertations Fall 2019 Differential Cholinergic Modulation of Prelimbic and Thalamic Input to the Basolateral Amygdala Sarah Catherine Tryon Follow this and additional works at: https://scholarcommons.sc.edu/etd Part of the Exercise Science Commons Recommended Citation Tryon, S. C.(2019). Differential Cholinergic Modulation of Prelimbic and Thalamic Input to the Basolateral Amygdala. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/5521 This Open Access Dissertation is brought to you by Scholar Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. DIFFERENTIAL CHOLINERGIC MODULATION OF PRELIMBIC AND THALAMIC INPUT TO THE BASOLATERAL AMYGDALA by Sarah Catherine Tryon Bachelor of Science Furman University, 2012 Submitted in Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy in Exercise Science The Norman J. Arnold School of Public Health University of South Carolina 2019 Accepted by: David D. Mott, Major Professor Mark Davis, Committee Member Abbi Lane-Cordova, Committee Member Alexander J. McDonald, Committee Member Cheryl L. Addy, Vice Provost and Dean of the Graduate School © Copyright by Sarah Catherine Tryon, 2019 All Rights Reserved. ii DEDICATION This manuscript is dedicated to my family. To Hudson, thank you for your constant brotherly love and encouragement. While we don’t see each other as often now, I know that I can always count on you to listen and provide rational, thoughtful support. Mom and Dad, thank you for three decades of unwavering love and support. From early days of reading Bob Books, spending hours upon hours to flip through math flashcards and grammer lessons that never seemed to end, being at the sidelines of school and sports competitions, to later years of providing listening ears when I needed them and encouraging me to pursue my goals and never give up, you both have shaped who I am today. iii ACKNOWLEDGEMENTS Dr. Mott, thank you for giving me the opportunity to be in your lab and for the immeasurable amount of time you have dedicated towards mentoring and training me. I hope to one day exemplify your example of what it means to be a truly caring, dedicated mentor who so selflessly provides instruction, offers guidance, knows when to be critical and push students to challenge themselves, and fosters a positive lab environment of constant intellectual curiousity. To Dr. McDonald, thank you for all of the time and attention you have dedicated to my training. All of the detailed and thorough feedback you always provided, whether it was going over data in a lab meeting, a larger presentation for my committee, or a simple question regarding amygdalar anatomy, was deeply appreciated. To Dr. Davis, I have greatly enjoyed each and every one of our discussions about the connections between exercise and the brain. Thank you for your guidance and advice over the years as I navigated the intersection of neuroscience and exercise science. I am fortunate to have been able to learn from and receive guidance from someone who shared my passion for these two fields of research. To Dr. Lane-Cordova, I am so grateful for your generosity to join my committee and for the opportunity I had to be challenged to look at my research from different angles. Your readiness to dedicate time to further my training as a scientist, provide feedback and suggestions, and push me to make connections between concepts related to my work and concepts in other areas has been immensely appreciated. iv ABSTRACT The basolateral amygdala (BL) is critical for emotional memory acquisition and expression. It receives afferent projections from both cortical and subcortical regions that send glutamatergic transmission to the BL. This input conveys information necessary for survival, including information about one’s behavioral state and environment. How this information is integrated and processed by the BL, however, remains largely unknown. Interestingly, the BL receives the densest amount of cholinergic innervation from the basal forebrain. This acetylcholine (ACh) can modulate emotional memories, but how it modulates specific afferent inputs to the BL is unexplored. To answer this question, we used brain slice field and whole cell electrophysiology, optogenetics, and pharmacological tools to investigate how released endogenous ACh modulates afferent input to the BL. We found that endogenous ACh suppresses cortical input to the BL through muscarinic receptors. We then further explored this modulation by optogenetically activating prelimbic (PL) and thalamic (THAL) input to the BL and pharmacologically activating muscarinic ACh receptors to examine pathway-specific regulation of glutamatergic transmission from these inputs. Muscarine, by acting on M3 and M4 receptors at PL synapses and M3 receptors at THAL synapses, suppressed glutamatergic input from both regions. However, muscarinic receptor activation inhibited the prelimbic input to a significantly greater extent than the thalamic. Furthermore, in examining the mechanisms underlying this inhibition, it was found that muscarinic inhibition of these two pathways occurs through distinct mechanisms. At PL input muscarinic receptors inhibit v glutamatergic transmission through an endocannabinoid independent mechanisms whereas they inhibit thalamic input through an endocannabinoid-dependent mechanism. Additionally, muscarinic receptors displayed frequency-dependent regulation of glutamate transmission. When the PL and THAL inputs were stimulated at low frequency trains (1Hz), muscarinic inhibition was consistent throughout the train. However, when PL and THAL were stimulated at gamma frequency trains (40Hz), muscarinic inhibition remained intact throughout the train at THAL inputs, but was relieved at PL inputs. Taken together, these findings suggest differential modulatory mechanisms during enhanced cholinergic tone in the BL, such as during exercise or unexpected stimuli. However, this inhibition is frequency dependent. During behavioral states in which the PL is oscillating at gamma frequency, that muscarinic inhibition could be overcome. Together, these results indicate unique modulatory mechanisms conferred upon the PL and THAL input to the BL and could serve as rich avenues for pharmaceutical manipulations in future behavioral studies. vi TABLE OF CONTENTS DEDICATION ................................................................................................................... iii ACKNOWLEDGEMENTS ............................................................................................... iv ABSTRACT .........................................................................................................................v LIST OF FIGURES .............................................................................................................x LIST OF ABBREVIATIONS ........................................................................................... xii CHAPTER 1 GENERAL INTRODUCTION .....................................................................1 1.1 SIGNIFICANCE ................................................................................................1 1.2 ACETYLCHOLINE ..........................................................................................2 1.3 MUSCARINIC MODULATION OF GLUTAMATERGIC TRANSMISSION IN CORTICAL STRUCTURES .............................................15 1.4 ENDOCANNABINOID SYSTEM .................................................................19 1.5 BASAL FOREBRAIN .....................................................................................22 1.6 AMYGDALA: AN OVERVIEW ....................................................................25 1.7 SIGNIFICANCE REVISITED ........................................................................40 CHAPTER 2 GENERAL METHODS ..............................................................................41 2.1 NEURONAL SIGNALING: AN OVERVIEW ...............................................41 2.2 ELECTROPHYSIOLOGY: AN OVERVIEW ................................................46 2.3 OPTOGENETICS: AN OVERVIEW..............................................................50 2.4 FIELD RECORDINGS IN BASOLATERAL AMYGDALAR: AN OVERVIEW .......................................................................55 2.5 VALIDATION OF AMYGDALAR FEPSPS .................................................56 vii 2.6 ANIMAL CARE AND USE ............................................................................57 2.7 SURGICAL PROCEDURES FOR VIRAL DELIVERY................................57 2.8 SLICE PREPARATION ..................................................................................59 2.9 SLICE ELECTROPHYSIOLOGY RECORDINGS .......................................60 2.10 SPECIFICITY OF VIRAL VECTOR EXPRESSION IN CHAT+ BASAL FOREBRAIN NEURONS ...................................................61 2.11 MATERIALS .................................................................................................62 2.12 DATA ANALYSIS AND STATISTICS .......................................................62 CHAPTER 3 INHIBITION OF EXTERNAL INPUT TO THE BL BY ENDOGENOUS ACETYLCHOLINE ........................................................63 3.1 INTRODUCTION ...........................................................................................63 3.2 MATERIALS AND METHODS