SELF-PROPAGATING, NON-SYNAPTIC HIPPOCAMPAL WAVES RECRUIT NEURONS BY ELECTRIC FIELD COUPLING By RAJAT SHAMACHAR SHIVACHARAN Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Advisor: Dominique M. Durand, PhD Department of Biomedical Engineering CASE WESTERN RESERVE UNIVERSITY May 2019 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis dissertation of Rajat Shamachar Shivacharan candidate for the degree of Doctor of Philosophy*. Committee Chair Jeffrey R. Capadona Thesis Advisor/Committee Member Dominque M. Durand Committee Member Cameron C. McIntyre Committee Member Hillel J. Chiel Date of Defense: April 8th, 2019 *We also certify that written approval has been obtained for any proprietary material contained therein. Dedication To my great grandfathers, Mr. Doddappa Acharya and Mr. Shilipi Shamachar; to my grandparents Mr. Y.S. Shamachar & Mrs. Lalitha, and Mr. H.D. Ramakrishna Sastry & Justice B.S. Indrakala; for being exemplary role models for me, and for being the luminaries of my family. To my Mom & Dad, my brother Manju, our loving, four-legged pup Rocky, and the rest of my family and friends near and far; for your unwavering love and support throughout my adventures. Table of Contents Contents Table of Contents ............................................................................................................... i List of Figures .................................................................................................................. vii List of Tables ......................................................................................................................x Acknowledgments ............................................................................................................ xi List of Abbreviations ..................................................................................................... xiv Abstract ........................................................................................................................... xvi Overview ...........................................................................................................................18 Chapter 1: Introduction and Thesis Organization .......................................................21 1.1 Brain Wave Propagation ............................................................................................. 22 1.2 Hippocampus .............................................................................................................. 23 1.3 Pathological Waves – Epilepsy ................................................................................... 24 1.4 Physiological Waves – Slow Oscillation Sleep Waves .............................................. 26 1.5 Mechanisms of Neural Propagation ............................................................................ 27 1.6 Ephaptic, or Electric Field, Coupling ......................................................................... 29 1.7 Thesis Organization: Specific Objectives Overview and Hypothesis ........................ 30 1.7.1 - Objective 1: To determine if epileptiform waves self-propagate in the hippocampus by electric field coupling. ..................................................................................................31 i 1.7.2 - Objective 2: To determine if spontaneous waves can recruit neurons via electric fields across a physical cut. ................................................................................................32 1.7.3 - Objective 3: To determine if hippocampal waves under physiological conditions can self-propagate, non-synaptically by electric field coupling. .......................................32 1.8 Figures......................................................................................................................... 34 Chapter 2: Self-propagating epileptiform activity recruits neurons by endogenous electric fields .....................................................................................................................37 2.1 Abstract ....................................................................................................................... 38 2.2 Introduction ................................................................................................................. 39 2.3 Material and Methods ................................................................................................. 42 2.3.1 Longitudinal hippocampal slice preparation .............................................................42 2.3.2 Solution preparation to elicit spontaneous epileptiform activity ..............................42 2.3.3 Electrophysiology recording setup ...........................................................................43 2.3.4 Optical recording setup .............................................................................................44 2.3.5 In vitro osmolarity to study the effect of cell distance on propagation speeds under 4- AP conditions .....................................................................................................................44 2.3.6 Extracellular electric field clamp ..............................................................................45 2.3.7 Isopotential experiment setup ...................................................................................46 2.3.8 Data and statistical analysis ......................................................................................46 2.4 Results ......................................................................................................................... 47 ii 2.4.1 4-AP-induced activity propagates along the longitudinal direction of the hippocampus ......................................................................................................................47 2.4.2 Modifying the extracellular space changes propagation speed of 4-AP-induced activity in vitro ...................................................................................................................48 2.4.3 Canceling the endogenous electric field with an extracellular electric field clamp blocks propagation of epileptiform activity .......................................................................49 2.4.4 Increasing the conductivity along the cell layer decreases the magnitude and propagation speed of epileptiform activity ........................................................................51 2.4.5 Applied electric fields can trigger self-propagating waves in the longitudinal hippocampal slice...............................................................................................................52 2.5 Discussion ................................................................................................................... 54 2.6 Figures......................................................................................................................... 59 Chapter 3: Epileptiform waves propagate through a physical cut solely by self- sustaining electric fields ...................................................................................................69 3.1 Abstract ....................................................................................................................... 70 3.2 Introduction ................................................................................................................. 71 3.3 Materials and Methods ................................................................................................ 74 3.3.1 Acute in vivo transection and recording experimental design ..................................74 3.3.2 Longitudinal hippocampal slice preparation .............................................................75 3.3.3 Electrophysiology recording setup in vitro ...............................................................76 iii 3.3.4 Optical recording setup and image processing .........................................................77 3.3.5 Statistical analysis .....................................................................................................78 3.4 Results ......................................................................................................................... 79 3.4.1 Seizure-like activity propagates through a physical hippocampal transection in vivo acutely ................................................................................................................................79 3.4.2 Spontaneous epileptiform activity propagates non-synaptically through a cut in vitro. ............................................................................................................................................80 3.4.3 Spatio-temporal dynamics of the epileptiform remain unchanged through a cut in the hippocampus in vitro..........................................................................................................81 3.4.4 Endogenous electric field can cross a cut and are self-sustaining activity in vitro ...81 3.5 Discussion ................................................................................................................... 83 3.6 Figures......................................................................................................................... 87 Chapter 4: Slow oscillation sleep waves can self-propagate non-synaptically by a mechanism consistent with ephaptic coupling ..............................................................95