Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 2011 Effects of Neuronal Nogo-A on Properties of Excitatory Synapses of the Sensorimotor Cortex Alicia Marie Case Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Neuroscience and Neurobiology Commons Recommended Citation Case, Alicia Marie, "Effects of Neuronal Nogo-A on Properties of Excitatory Synapses of the Sensorimotor Cortex" (2011). Dissertations. 192. https://ecommons.luc.edu/luc_diss/192 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 2011 Alicia Marie Case LOYOLA UNIVERSITY CHICAGO EFFECTS OF NEURONAL NOGO-A ON PROPERTIES OF EXCITATORY SYNAPSES OF THE SENSORIMOTOR CORTEX A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY PROGRAM IN NEUROSCIENCE BY ALICIA M. CASE CHICAGO, ILLINOIS MAY 2011 Copyright by Alicia M. Case, 2011 All rights reserved ACKNOWLEDGEMENTS My thanks to the members of the Kartje lab, whose support has proved invaluable, especially Dr. Wendy Kartje, Melanie Bollnow, Becky Gillani, and Ian Vaagenes. I also owe a debt of gratitude to the neuroscience community at Hines and Loyola for moral backing, invigorating discussion, and scientific enthusiasm, particularly Cynthia Von Zee. Cynthia further aided this work with advice on qRT-PCR, as did Maggie Szymanska and Dr. Toni Pak. Thanks also to Dr. Ed Campbell, for generous use of his deconvolution microscope, and for patient instruction in the mechanics of this form of microscopy. I would be remiss without thanks to a network of support from the Neuroscience graduate students who began coursework with me, Maureen Petrunich-Rutherford, Michael Calik, and Sandra Rokosik. I would also like to thank my committee for their valuable guidance. Particular appreciation goes to Dr. Bill Wolf for his insightful scientific review and counsel on immunohistochemistry, Dr. Jody Martin for patient instruction on cloning, viral construction, and scientific writing, Dr. Evan Stubbs for recommendations on PCR and kind encouragement throughout the course of study, and Dr. E.J. Neafsey, an excellent instructor in the classroom and at the bench. iii Final thanks go to my family for their support and encouragement on this project, as with all things. iv For all the teachers who have inspired my love of science through the years, particularly Susan Osterlie, Susan Sarejko, Tom Bahl and James Doyle. I think, at a child's birth, if a mother could ask a fairy godmother to endow it with the most useful gift, that gift should be curiosity. -Eleanor Roosevelt TABLE OF CONTENTS ACKNOWLEDGEMENTS ...................................................................................... iii III LIST OF TABLES ..................................................................................................... x LIST OF FIGURES ................................................................................................. xi LIST OF ABBREVIATIONS ................................................................................. xiii ABSTRACT ........................................................................................................... xvii CHAPTER ONE: OVERVIEW AND HYPOTHESIS ................................................ 1 CHAPTER TWO: REVIEW OF THE LITERATURE .............................................. 5 Nogo-A ............................................................................................................... 5 Introduction .................................................................................................... 5 Axon Guidance ................................................................................................ 7 Nogo-A is a member of the Reticulon family .................................................. 8 Nogo-A Receptors ......................................................................................... 10 Nogo-A at the synapse .................................................................................... 13 Neocortical expression of Nogo-A ................................................................. 15 Dendritic spines ................................................................................................ 15 Postsynaptic molecules important for synaptic function ................................ 18 RNA Interference ...............................................................................................21 RNAi machinery ............................................................................................ 22 Synthetic RNAi as a tool for neuroscientists ................................................ 25 CHAPTER THREE: POSTNATAL KNOCKDOWN OF NEURONAL NOGO-A LEADS TO A DECREASE IN APICAL DENDRITIC SPINE DENSITY IN THE NEOCORTEX ............................................................................................. 29 Abstract ............................................................................................................ 29 Introduction ..................................................................................................... 30 Methods ........................................................................................................... 32 AAV production .............................................................................................. 32 Animals .......................................................................................................... 34 Verifying Nogo-A knockdown in vitro and in vivo ....................................... 35 Dendritic spine imaging via laser scanning confocal microscopy ................ 38 Statistical analysis ......................................................................................... 39 Results .............................................................................................................. 40 vii Recombinant virus, AAV-shNogo, mediated in vitro and in vivo Nogo-A gene knockdown ......................................................................................... 40 Neuronal Nogo-A knockdown in vivo results in reduced apical dendritic spine density and alterations in apical spine morphology ........................ 46 Reduced levels of postnatal, neuronal Nogo-A did not alter basilar spine density or morphology ............................................................................... 47 Discussion ........................................................................................................ 52 CHAPTER FOUR: POSTNATAL KNOCKDOWN OF NEURONAL NOGO-A LEADS TO A DECREASE IN MESSAGE LEVELS OF GENES IMPORTANT FOR SYNAPTIC PLASTICITY ..................................................... 57 Abstract ............................................................................................................ 57 Introduction ..................................................................................................... 58 Methods ........................................................................................................... 60 Quantitative RT-PCR (qRT-PCR) ................................................................. 60 Statistical analysis ......................................................................................... 62 Results .............................................................................................................. 65 AAV-shNogo leads to reduced levels of Nogo-A, with no changes to Nogo-B or Cofilin expression ..................................................................... 65 Postnatal loss of Nogo-A influences expression of genes important for synaptic plasticity ....................................................................................... 66 Discussion ........................................................................................................ 70 CHAPTER FIVE: POSTNATAL KNOCKDOWN OF NEURONAL NOGO-A LEADS TO A DECREASE IN THE APICAL COUNT OF AN EXCITATORY PRESYNAPTIC MARKER, VGLUT1, IN THE ADULT SENSORIMOTOR CORTEX ............................................................................................................. 75 Abstract ............................................................................................................ 75 Introduction ..................................................................................................... 76 Methods ........................................................................................................... 78 Immunohistochemical staining .................................................................... 78 Synaptic protein imaging via deconvolution microscopy ............................ 79 Statistical analyses ........................................................................................ 80 Results .............................................................................................................. 82 vGlut1 immunohistochemistry identifies potential presynaptic terminals . 82 AAV-shNogo leads to reduced number potential presynaptic connections onto apical dendrites .................................................................................. 82 Decreased postnatal