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Regulation of Dendritic Spine Proliferation Orenda Lyons Johnson

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Regulation of Dendritic Spine Proliferation Orenda Lyons Johnson Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2005 Regulation of Dendritic Spine Proliferation Orenda Lyons Johnson Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES REGULATION OF DENDRITIC SPINE PROLIFERATION By ORENDA LYONS JOHNSON A Dissertation submitted to the Department of Psychology Program in Neuroscience in partial fulfillment of the requirements for the degree of Doctor of Philosophy Degree Awarded: Summer Semester, 2005 The members of the Committee approve the Dissertation of Orenda L. Johnson defended on June 17, 2005. Charles C. Ouimet Professor Directing Dissertation Paul Q. Trombley Outside Committee Member Colleen M. Kelley Committee Member Cathy W. Levenson Committee Member Zuoxin Wang Committee Member Approved: Janet A. Kistner, Chair, Department of Psychology Joseph A. Travis, Dean, College of Arts and Sciences The Office of Graduate Studies has verified and approved the above named committee members. ii This body of work is dedicated to Carolyn Anagnos, my grandmother. iii ACKNOWLEDGEMENTS Very special thanks… To the following professors and staff members whose guidance, assistance and technical support have been invaluable during my tenure at Florida State: Charles Badland (Neuroscience Graphics), Karen Berkley, Ellen Berler, Laura Blakemore, Rani Dahnarajan (Molecular Cloning), Jon Ekman, Debbie Fadool, Jim Fadool, Tom Fellers, Cheryl Fitch, Mark Freeman, Chris Gillis, Dick Hagen, Tom Houpt, Rick Hyson, Frank Johnson, Colleen Kelley, Cathy Levenson, David Lowe, Mike Overton, Mike Rashotte, Kim Riddle (Biological Sciences Imaging Resources), Jim Smith, Paul Trombley, and Zuoxin Wang. Also to the administrative staff of the Psychology Department and the Program in Neuroscience: Cherie Dilworth, Sunsan Gay, Janice Parker, Sandy Saunders, Sharon Weaver and Earline Whitley. To my fellow students with whom I grew academically, professionally and personally: Brandon Aragona, Heather Bradshaw, Jesse Brann, Angie Cason, Brad Chambers, Beverly Colley, Mike Darcy, David Dietz, Karen Dietz, Christie Fowler, Glen Golden, Bumsup Kwon, Andrea Leishman, Denessa Lockwood, Micky Messina, Seth Norrholm, Chad Samuelsen, Mike Sellix, Patrick Smith, Derek Snyder, Carrie Stafstrom, Jake VanLandingham, Jennifer Wersinger, Jenne Westberry and Brandy Wilkinson. As Dr. Carter said, I feel like I grew up with you. To the Ouimet Lab technicians, who taught me many of the techniques used in this dissertation, who worked tirelessly by my side or in my place, and who all became dear friends: Katie Cavnar, Amanda Clark and Lisa Sander. To Dr. Ouimet, whose kindness, respect, intellect, encouragement, mentorship, direction, wit and friendship are unparalleled in a “boss,” and have likely spoiled me forever. iv To my wonderful devoted friends, for their tireless support and constant understanding: Bay Abreu, Sheyna Carroccio, Kristin Dozier, Kristen Holland, Gwen Hooper, Melanie Jones, LoriAnn Kehler, Eirin Lombardo and Shea Zahner. To my husband, Eric Johnson, my mother, Judith Lyons, my father, Tom Lyons, my sister, Halle Lyons, and to my extended family, whose amazing support and encouragement and faith in my abilities played an essential role in the completion of this dissertation, and in all of my academic, professional and personal accomplishments. Also to my son Logan Johnson, who has given me unparalleled perspective and incredible joy. And again to Eric, for his infinite patience. v TABLE OF CONTENTS List of Tables ................................................................................................viii List of Figures ................................................................................................ ix Abstract ...................................................................................................... x 1. Introduction ................................................................................................ 1 Composition of dendritic spines ................................................................. 1 Dendritic spine development ...................................................................... 3 Function of dendritic spines........................................................................ 4 Dendritic spine calcium dynamics.............................................................. 6 Local protein synthesis ............................................................................... 8 Plasticity of dendritic spines: an essential role for actin............................. 9 2. Protein synthesis is necessary for dendritic spine proliferation in adult brain slices ................................................................................................ 12 Introduction ................................................................................................ 12 Methods ................................................................................................ 13 Results ................................................................................................ 17 Discussion ................................................................................................ 27 Conclusions ................................................................................................ 30 3. A regulatory role for actin in dendritic spine proliferation............................. 31 Introduction ................................................................................................ 31 Methods ................................................................................................ 32 Results ................................................................................................ 37 Discussion ................................................................................................ 45 Conclusions ................................................................................................ 51 4. Discussion ................................................................................................ 52 Methodological considerations................................................................... 52 A common pathway for spine growth?....................................................... 54 Actin upregulation during spinogenesis ..................................................... 54 Proteins involved in spine growth .............................................................. 55 Possible involvement of dendritic protein synthesis................................... 59 Actin: a limiting resource for spine density................................................ 60 Actin treadmilling as a delivery system...................................................... 61 Conclusions ................................................................................................ 63 vi REFERENCES ................................................................................................ 64 BIOGRAPHICAL SKETCH .............................................................................. 81 vii LIST OF TABLES Table 1: Heterogeneous protein distribution in dendritic spines ........................ 2 Table 2: Degree of analysis for counting spines.................................................. 18 Table 3: Degree of analysis for measuring spine length and dendritic width...... 19 Table 4: Morphological consequences of protein overexpression or constitutive expression in neurons......................................................... 56 Table 5: Spine-localized proteins with actin binding domains or motifs ............ 62 viii LIST OF FIGURES Figure 1: Transfer of slices does not affect dendritic spine density .................... 15 Figure 2: Spine density increases on secondary apical dendrites of CA1 as a function of time (minutes in vitro)........................................................ 20 Figure 3: Protein synthesis inhibitors block spine proliferation ......................... 21 Figure 4: Representative dendritic twigs from each experimental group ........... 23 Figure 5: Early treatment with PUR does not prune spines ................................ 24 Figure 6: Mean spine length increases as a function of time, an effect that is blocked by protein synthesis inhibition ................................................ 25-26 Figure 7: Spine density as assessed by Golgi staining increases during the acute slice preparation in P21 rats.................................................................. 38 Figure 8: Spine density as assessed by phalloidin staining increases during the acute slice preparation in P21 rats ........................................................ 39 Figure 9: Actin expression increases during acute slice preparation................... 40 Figure 10: Spine density increases after actin-overexpression in organotypic cultures ............................................................................................... 42 Figure 11: Increase in density of elongated, filopodial and branched spines on apical dendrites of actin-EGFP-transfected pyramidal neurons ......... 43 Figure 12: Increase in density of elongated and filopodial spines on basal dendrites of actin-EGFP-transfected pyramidal neurons.................... 44 Figure 13: Elongated spines and filopodia are apposed by axon terminals ........ 46 Figure 14: Exogenous actin enters all spines ...................................................... 47 ix ABSTRACT
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