UNIVERSITY OF CINCINNATI Date: 5-Aug-2010 I, Brian L Murphy , hereby submit this original work as part of the requirements for the degree of: Doctor of Philosophy in Neuroscience/Medical Science Scholars Interdisciplinary It is entitled: Aberrant hippocampal granule cell neurogenesis and integration in epilepsy Student Signature: Brian L Murphy This work and its defense approved by: Committee Chair: Steve Danzer, PhD Steve Danzer, PhD 8/18/2010 999 Aberrant hippocampal granule cell neurogenesis and integration in epilepsy A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in the Graduate Program in Neuroscience of the College of Medicine August 5, 2010 By Brian Liam Murphy Bachelor of Science, Hope College, 2004 Committee Chair: Kim Seroogy, Ph. D. Kenneth Campbell, Ph. D. Steve Danzer, Ph. D. Katherine Holland-Bouley , M.D., Ph. D. Neil Richtand, Ph. D. ii ABSTRACT The data from the present studies suggest that granule cells in different stages of neuronal differentiation may be differentially susceptible to changes in their cellular structure and survival following an epileptogenic stimulus. Recent work in the field has focused on the development of dendritic abnormalities with respect to immature neurons or newly generated neurons following exposure to an epileptogenic stimulus. In Chapter 2, we show for the first time that fully differentiated granule cells are capable of dendritic rearrangement. Specifically, we have shown that previously established apical dendrites shift to the basal portion of the cell as the somata of these cells radially migrate up an adjacent primary dendrite towards the dentate molecular layer. In doing so, dendritic branches on this dendrite become a new primary dendrite. We also propose that this migration underlies the dispersion of the granule cell layer, which was previously suggested by other laboratories; however, the utility of organotypic explant cultures made from Thy1-YFP mice allowed us for the first time to observe fully differentiated granule cell migration and their contribution to the dispersion of the granule cell layer. Granule cell dispersion and distortions to granule cell dendritic structure are common pathologies of the epileptic brain. Both phenomena also occur in adult animal models of epilepsy. Using bi-transgenic Gli-CreERT2+/-;Green fluorescent protein (GFP) reporter+/- mice, data from Chapter 3 suggests that the pool of hippocampal subventricular zone and/or subgranular cell layer neural progenitor cells active several days prior to a prolonged seizure become disrupted following early-life seizure activity. Specifically, fewer cells within the dentate gyrus were labeled with GFP, and associated with decreased numbers of progenitor cells, immature and mature granule cells. Additionally, we are the first laboratory to show that early-life seizures disrupt the integration of granule cells, which has been a ‘hot’ topic in studies using adult iii models of epilepsy. In Chapter 4, we used the same bi-transgenic mice, but in an adult model of epilepsy. From this study, we found that the pool of subgranular zone progenitor cells producing progeny following seizures become disrupted, and at later time points, give birth to fewer new granule cells. Additionally, this study implicates that granule cells born during the first week following a seizure may exhibit an accelerated rate of maturation. The results of these studies will hopefully spur further research into the underlying cellular signaling pathways involved in the formation of basal dendrites on dentate granule cells, etcopic localization of granule cells to the hilus and dentate molecular layers and granule cell layer dispersion. Additionally, future studies of cell-fate mapping in the early-life seizure and adult model of epilepsy should include the use of alternate tamoxifen injection time points to determine if progenitors active before or after an epileptogenic stimulus respond differently than the conditions tested here. iv ACKNOWLEDGMENTS I would like to thank Steve for the many lessons about thinking and doing science that he has provided me over the years – specifically when it comes to data mining. Additionally, I learned about the process of setting up a research lab, in particular management skills. This was a great surprise, and I think is important for any young scientist to see what their future career path entails. I know you did not plan on those lessons, but I am very grateful for being able to experience them with you. Additionally, I would like to thank you for giving me the opportunity to think and plan experiments independently during this last year. This has helped me feel ready for the next step in my scientific career. I would also like to thank the current (Ray, Hulian, Niki, Mike, Olagide and Victor) and past members (Cynthia and Stefanie) of the lab for making the laboratory a low stress and fun environement as well as all technical help when needed. I would also like to thank my committee members (Drs. Kim Seroogy, Kenneth Cambell, Katherine Holland-Bouley and Neil Richtand) for their guidance in data interpretation of the studies in this dissertation. Also, I am greatful for University of Cincinnati’s Biomedical Flex Program (Laura Hildreth, Dr. Robert Highsmith and Mary Joe Petersman) and Neuroscience graduate program (Deb Cummins, James Herman and Michael Lehman) faculty and staff for initially accepting me into their graduate programs. Also, thank you Susan Melhorn and Georgette Suidan for their company during the early years of graduate school…the late night study and drinking sessions have been missed over the last couple of years! I will also miss the friends that I made here, in and out of the program; for those of you that were or are in program, I will miss hanging out and sharing food, games, drinks and sciene talk (Jane/Peter A., Martine L./Dave Z., Amanda S., Matt L., Stefanie B., Katrina/Sean P., Anne/Kevin E.)! Lastly, none of this would have happened with out the encouragement of my undergraduate research advisor, Leah Chase, as well as the love and support of my parents, Maureen and Bill Murphy; sister, Eileen Murphy; aunt, Kathleen Killoughrey and my partner Jason Oscar. vi TABLE OF CONTENTS Table of Contents……………………………………………………………………..……………….. 1 List of Tables and Figures……………………………..…………………………..………………… 2 Abbreviations…………………………………………….…………………………...………………... 4 Chapter 1: Introduction……………………………………………………..………….…………….. 5 Chapter 2: Somatic translocation of mature hippocampal dentate granule cells as a novel mechanism of dendritic dysmorphogenesis and granule cell dispersion……………………………………………………………….…..…………… 42 Chapter 3: Reduced neurogenesis of hippocampal subventricular zone progenitor cells exposed to neonatal status epilepticus………………………………………….……… 84 Chapter 4: Genetic fate-mapping of adult-generated hippocampal dentate granule cells In the epileptic mouse brain………...……………..……………………………..……… 124 Chapter 5: Discussion……………………………...…………………………………………..….. 163 1 List of tables and figures Chapter 1 Figure 1. The “trisynaptic circuit.” pg. 18 Figure 2. Examples of aberrant dentate granule cells within the epileptic brain. pg. 19 Figure 3. Representative Thy1-YFP organotypic hippocampal explant culture. pg. 20 Chapter 2 Figure 1. Postmitotic age of YFP-expressing granule cells in vitro. pg. 65 Figure 2. Recurrent basal dendrite formation and granule cell layer dispersion. pg. 66 Figure 3. Branch to dendrite conversion. pg. 67 Figure 4. Extreme example of branch to dendrite conversion. pg. 68 Figure 5. Granule cells with normal morphology. pg. 69 Figure 6. Granule cell dispersion induced by IHpKA. pg. 70 Figure 7. Ectopic granule cells and recurrent basal dendrites occur in vivo. pg. 71 Figure 8. Model depicting granule cell dysmorphogenesis pg. 72 Supplemental Table 1. Recurrent basal dendrite formation and branch-to- dendrite conversion in control and kainic acid treated explants. pg. 73 Supplemental Figure 1. Dying YFP granule cell. pg. 74 Chapter 3 Figure 1. Cortical EEG recording from a seven day old Gli1-CreERT2 -/-;GFP+/- mouse. pg. 103 Figure 2. Cre-mediated recombination is efficacious and specific. pg. 104 Figure 3. GFP-expression in the adult mouse brain. pg. 105 Figure 4. GFP-expression in five cell types within the adult dentate gyrus. pg. 106 Table 1. Mean percentage of GFP-labeled cell types per dentate gyrus. pg. 107 Table 2. Mean number of GFP-labeled cell types per dentate gyrus. pg. 108 2 Figure 5. Early-life SE disrupts putative neural stem/progenitor cell fate. pg. 109 Figure 6. Scatter plot of Pearson correlation results. pg. 110 Figure 7. Examples of mature dentate granule cells with abnormal integraton following early-life SE. pg. 111 Supplemental figure 1. Cre-recombinase polymerase chain reaction. pg. 112 Supplemental table 1. Mean percentage of GFP-expressing dentate granule cells possessing basal dendrites (DGC+BD) or ectopically localized in the hilus (HEC). pg. 113 Chapter 4 Figure 1. Endogenous GFP expression in the adult brain. pg. 141 Figure 2. Specificity of induction and cell labeling in Gli1-CreERT2;GFP reporter mice. pg. 142 Figure 3. GFP-expression in cells of the SGZ, dentate gyrus and aberrantly integrated dentate granule cells. pg. 143 Table 1. Mean percentage of GFP-expressing cells per dentate gyrus five weeks following SE. pg. 144 Table 2. Mean percentage of GFP-expressing cells per dentate gyrus