CONTROL AND ANALYSIS OF SEIZURE ACTIVITY IN A SODIUM CHANNEL MUTATION MODEL OF EPILEPSY by KARA BUEHRER KILE Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Dominique M. Durand, Ph.D. Department of Biomedical Engineering CASE WESTERN RESERVE UNIVERSITY January 2009 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of _____________________________________________________ candidate for the ______________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. DEDICATION To my parents, Dr. Richard E. Buehrer and Maureen J. Buehrer, for the many ways they have enriched my life, one of which was instilling in me a passion for academia. To my husband, Warren R. Kile, for his unconditional love and limitless understanding. To my daughter, Rose Marie Kile, for enhancing every moment of my work with a greater perspective, motivation, and joy. iii TABLE OF CONTENTS DEDICATION………………………………………………………………………….III TABLE OF CONTENTS………………………………………………………………IV LIST OF TABLES…………………………………………………………………...VIII LIST OF FIGURES…………………………………………………………………….IX ACKNOWLEDGEMENTS……………………………………………………………XI ABSTRACT……………………………………………………………………………XII CHAPTER 1 ……………………………………………………………………………..1 DISSERTATION INTRODUCTION AND OBJECTIVES 1.1 EPILEPSY…………………………………………………………………….1 1.2 GENETIC ORIGINS OF EPILEPSY…………………………………………1 1.3 TRANSGENIC ANIMAL MODEL…………………………………………..3 1.4 CLINICAL TREATMENT OF EPILEPSY…………………………………..4 1.5 DEEP BRAIN STIMULATION………………………………………………6 1.6 THESIS OBJECTIVES AND ORGANIZATION……………………………9 1.6.1 Objective I…………………………………………………………..9 1.6.2 Objective II………………………………………………………...11 1.6.3 Objective III………………………………………………………..12 CHAPTER 2…………………………………………………………………………….16 SCN2A SODIUM CHANNEL MUTATION RESULTS IN HYPEREXCITABILITY IN THE HIPPOCAMPUS IN VITRO 2.1 ABSTRACT………………………………………………………………….16 2.2 INTRODUCTION……………………………………………………...……17 2.3 METHODS…………………………………………………………………..20 iv 2.3.1 Animals…………………………………………………………….20 2.3.2 Histology and cerebral spinal fluid extraction……………………..22 2.3.3 Hippocampal slice preparation and perfusion……………………...23 2.3.4 Electrophysiology and data analysis……………………….………23 2.4 RESULTS……………………………………………………………………25 2.4.1 Spontaneous activity in Q54 slices…………………...……………25 2.4.2 Evoked PS amplitude response curve in Q54 slices…………….…26 2.4.3 Cresyl violet histology of Q54 slices………………………………27 2.4.4 Cerebral spinal fluid of Q54 mice……………………………….…27 2.4.5 Paired pulse test of recurrent inhibition……………………………28 2.4.6 Response to high-frequency stimulus……………………………...28 2.5 DISCUSSION………………………………………………………………..30 CHAPTER 3…………………………………………………………………………….48 EFFECT OF LOW FREQUENCY DEEP BRAIN STMIULATION ON SEIZURE ACTIVITY IN VIVO 3. 1 ABSTRACT…………………………………………………………………48 3.2 INTRODUCTION…………………………………………………………...49 3.3 METHODS…………………………………………………………………..54 3.3.1 Animals…………………………………………………………….54 3.3.2 Electrode implantation……………………………………………..55 3.3.3 Stimulation and recording………………………………………….56 3.3.4 Data analysis……………………………………………………….57 3.4 RESULTS……………………………………………………………………57 3.4.1 Baseline seizure activity…………………………………………...57 v 3.4.2 High frequency oscillations during seizures……………………….59 3.4.3 Reduction of seizure frequency during LFS……………………….60 3.4.4 Effect of stimulation state on seizure frequency………..………….61 3.5 DISCUSSION………………………………………………………………..61 CHAPTER 4…………………………………………………………………………….81 A NOVEL MULTI-PRONGED ELECTRODE FOR DEEP BRAIN STMIULATION OF WHITE MATTER 4.1 ABSTRACT………………………………………………………………….81 4.2 INTRODUCTION…………………………………………………………...82 4.3 METHODS…………………………………………………………………..83 4.3.1 Bioelectric field model.…………………………………………….85 4.3.2 Electrode fabrication...……………………………………………..86 4.3.3 Impedance measurements………………………………………….87 4.3.4 Axonal stimulation by electrode prototypes……………………….87 4.3.5 Data analysis………………………….……………………………88 4.4 RESULTS……………………………………………………………………89 4.4.1 Bioelectric field model.…………………………………………….89 4.3.2 Electrode fabrication...……………………………………………..92 4.5 DISCUSSION………………………………………………………………..92 CHAPTER 5…………………………………………………………………………...111 CONCLUSIONS AND FUTURE DIRECTIONS 5.1 FULFILLMENT OF THESIS OBJECTIVES……………………………...111 5.1.1 Objective I………………………………………………………...111 5.1.2 Objective II……………………………………………………….112 vi 5.1.3 Objective III………………………………………………………113 5.2 CONCLUSIONS AND FUTURE DIRECTIONS…………………………114 REFERENCES………………………………………………………………………...117 vii LIST OF TABLES Table 1.1: Channelopathies: Voltage-gated sodium channel genes associated with human idiopathic epilepsy syndromes……………………………………………………….…..13 Table 1.2: Voltage-gated Na+ channel types and expression…………………………….14 Table 2.1: Quantitative histology………………………………………………………...38 Table 2.2: Slices with CA1 activity following tetanic stimulus…………………………39 Table 3.1: Signal power in frequency bands……………………………………………..68 Table 4.1: Effect of current injection on activation of target area…………………….....96 Table 4.2: Effect of electrode geometry on current density……………………………...97 Table 4.3: Effect of configuration on activation of target area………………………….98 Table 4.4: Electrode prototype impedance measurements………………………………99 viii LIST OF FIGURES Figure 1.1: Scn2a sodium channel mutation in Q54 mice………...……………………..15 Figure 2.1: Spontaneous activity in Q54 hippocampal slices……………………………40 Figure 2.2: Evoked CA1 population spike response curve………………………………41 Figure 2.3: Morphology of the hippocampus in Q54 hippocampal slices……………….42 Figure 2.4: Response to paired pulse stimuli in Q54 hippocampal slices……………….43 Figure 2.5: Response to tetanic stimulus in Q54 hippocampal slices……………………44 Figure 2.6: Duration of evoked after discharge in CA1………………………………….45 Figure 2.7: Frequency analysis of tetanically induced activity…………………………..46 Figure 2.8: CA1 population spike between tetanic stimulus trains……………………...47 Figure 3.1: Surgical targets………………………………………………………………69 Figure 3.2: Surgical implantation………..………………………………………………70 Figure 3.3: Q54 model seizures………………………………………………………….71 Figure 3.4: Chronic recording……………………………………………………………72 Figure 3.5: Daily seizure frequency...……………………………………………………73 Figure 3.6: Signal frequency analysis……………………………………………………74 Figure 3.7: Very high frequency band spectral density during seizure…………….……75 Figure 3.8: Changes in DPE signal power over frequency bands......……………………76 Figure 3.9: Low frequency stimulation (LFS) protocol…….……………………………77 Figure 3.10: Stimulation during chronic recording…...………………………………….78 Figure 3.11: Effect of low frequency stimulation (LFS) no seizure frequency….………79 Figure 3.12: Effect of stimulation state on seizure frequency…...………………………80 Figure 4.1: Deep Brain Stimulation (DBS) target……...………………………………100 ix Figure 4.2: Medtronic DBS electrodes………..………………………………………..101 Figure 4.3: Bioelectric field model design…..………………...………………………..102 Figure 4.4: Electrode prototype fabrication………………...…………………………..103 Figure 4.5: Model validation…………………….……...………………………………104 Figure 4.6: Stripped end (SE) current density profiles…………………………………105 Figure 4.7: Stripped middle (SM) current density profiles...…………………………...106 Figure 4.8: Current density along electrode edge………………...…………………….107 Figure 4.9: Electrode contact configurations…………………………………………...108 Figure 4.10: Electrode prototype impedance..………………...………………………..109 Figure 4.11: Evoked population spike……………………...…………………………..110 x ACKNOWLEDGEMENTS I would like to extend my sincerest gratitude and thanks to the numerous individuals without whom I could not have completed this project, including but not limited to the following. My advisor, Dominique M. Durand, provided extensive guidance and foresight as well as continuous, encouraging support in my becoming a competent research scientist. My committee members, David Friel, Joseph Nadeau, Dawn Taylor, and Mary Ann Werz, provided invaluable thoughts and ideas which contributed greatly to the development and assessment of this project. My lab mates, Alicia Jensen, Andrew Kibler, David Tang, and Nan Tian, were instrumental in their companionship, assistance, and intellectual support. My dear friends, Deborah Barkauskas, Rachel Maulucci, Jennifer Parker, and Christa Wheeler, enhanced my quality of life as a graduate student and continually showed me how to be a better person. My brothers, R. Michael Buehrer, Gregory T. Buehrer, Brian J. Buehrer, and Mathew W. Buehrer, gave thoughtful advice and set an example for me to follow. The faculty and staff within Departments of Biomedical Engineering, Neuroscience, and Genetics, created an atmosphere supportive of challenging research and great collaborations within Case Western Reserve University. Dr. Miriam Meisler and the Human Genetics Department at the University of Michigan, who graciously donated Q54 strain colony founders. Lastly, I would like to acknowledge the following institutions for their financial support of this project: The National Institutes of Health (R01-NS-40785), Ohio Board of Reagents (Innovation Incentive Fellowship), United States Department of Education (Neural Engineering Training Grant), and Walter H. Coulter Foundation.