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The role of the NMDA receptor subunit GluN2A in synaptic and structural plasticity in the mouse dentate… Kannangara, Timal Saman 2012

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THE ROLE OF THE NMDA RECEPTOR SUBUNIT GLUN2A IN SYNAPTIC AND STRUCTURAL PLASTICITY IN THE MOUSE DENTATE GYRUS by TIMAL SAMAN KANNANGARA B.Sc., Integrated Science, The University of British Columbia, 2006  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA (VANCOUVER) June 2012 ©Timal Saman Kannangara, 2012  Abstract The N-methyl-D-aspartate (NMDA) receptor has been closely associated with learning and memory process in the hippocampus. The different subunits that comprise the NMDA receptor convey unique biophysical properties to these receptors. The GluN2A and GluN2B subunits (formerly NR2A and NR2B, respectively) are particularly abundant in the hippocampus, however their role in synaptic, structural and behaviour plasticity remains unclear. In this thesis, we use mice lacking GluN2A subunit expression to fully examine the role of the GluN2A subunit in the first region of the hippocampus, the dentate gyrus (DG). We reveal a significant deficit in the ratio of NMDA to AMPA (!-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors in mature dentate granule cells (DGCs). At a synaptic level, both long-term potentiation (LTP) and long-term depression (LTD) are significantly impaired in the DG, but not the neighboring CA1 region of GluN2A-/- mice. In addition to this decrease in bidirectional synaptic plasticity, GluN2A-/- animals show impairments in spatial pattern separation tasks that have been reported to be DG-specific. In contrast, GluN2A-/- mice showed no deficits in temporal pattern separation, a process associated with CA1 functioning. At a structural level, quantitative immunohistochemistry revealed that GluN2A deletion did not alter the levels of cell proliferation and neuronal differentiation in the adult DG. However, there were significant alterations in the morphology of late immature DGCs. Specifically, GluN2A deletion significantly decreased total dendritic length and dendritic complexity in late immature DGCs. Furthermore, late immature GluN2A-/- DGCs also showed a localized increase in spine density in the middle molecular layer, a region innervated by the medial perforant path. Interestingly, alterations in dendritic  !  ""!  morphology and spine density were no longer seen in mature cells. Our data indicates that the GluN2A subunit plays a critical role in bidirectional synaptic plasticity, neuronal morphology and spatial pattern separation in the DG.  !  """!  Preface A version of Chapter 3 has been published. Kannangara, T.S., Webber, A., GilMohapel, J., and Christie, B.R. (2009). Stress differentially regulates the effects of voluntary exercise on cell proliferation in the dentate gyrus of mice. Hippocampus. Oct; 19(10): 889-97. I performed half of the experiments and analysis, and wrote half of the manuscript.  UBC Research Ethics Board: UBC Animal Care Committee Application Number: A04-0296 Application Name: Glutamate receptor function in the aging brain. (Previously called: Role of kainite receptors in hippocampal synaptic plasticity) Investigator: Brian R. Christie Department: Psychology Animals: Mice Online animal care training program certificate number: 1804 Practical animal care training program certificate number: RBH-172-07  !  "#!  Table of Contents Abstract....................................................................................................................... ii Preface ........................................................................................................................ iv Table of Contents ........................................................................................................v List of Tables ...............................................................................................................x List of Figures............................................................................................................ xi List of Symbols and Abbreviations ........................................................................ xii Acknowledgments ................................................................................................... xiv Dedication ................................................................................................................ xvi 1 Introduction ..............................................................................................................1 1.1 The Hippocampus ...................................................................................................1 1.1.1 The Basic Anatomy and Major Cell Types of the Hippocampus ....................... 2 1.1.1.1 The Dentate Gyrus .............................................................................. 3 1.1.1.1.1 The Basic Anatomy of the Dentate Gyrus .......................... 3 1.1.1.1.2 The Principal Cell of the Dentate Gyrus: The Dentate Granule Cell ...................................................................................... 3 1.1.1.2 The CA Regions ................................................................................. 4 1.1.1.2.1 The Basic Anatomy of the CA Regions ............................. 4 1.1.1.2.2 The Principal Cell of the CA Regions: The Pyramidal Cell ............................................................................ 5 1.1.2 The Connections and Projections of the Hippocampus ...................................... 7 1.1.2.1 Synapse I: The Perforant Path-Dentate Granule Cell Synapse .......... 8 1.1.2.2 Synapse II: The Mossy Fiber-CA3 Synapse ...................................... 8 1.1.2.3 Synapse III: The Schaffer Collateral-CA1 Synapse ........................... 9 1.1.2.4 Efferent Projections From the Hippocampus ................................... 10 1.1.3 Proposed Functions of the Hippocampus ......................................................... 10 1.1.4 Region-specific Contributions to Hippocampal Function ................................ 12 1.1.4.1 The Dentate Gyrus and Spatial Pattern Separation .......................... 12 1.1.4.2 The CA3 and Spatial Pattern Completion ........................................ 13 1.1.4.3 The CA2 and Possible Functions ..................................................... 13 1.1.4.4 The CA1 and Temporal Pattern Separation ...................................... 14 1.1.5 Summary ........................................................................................................... 15  1.2 The Hippocampus as a Site for Neuronal Plasticity ............................................16 1.2.1 Synaptic Plasticity ............................................................................................ 16 1.2.1.1 Long-term Potentiation ..................................................................... 17 1.2.1.1.1 Mechanisms of Long-term Potentiation ........................... 17 1.2.1.1.2 Long-term Potentiation as a Model for Learning and Memory .................................................................................... 19 1.2.1.2 Long-term Depression ...................................................................... 20 1.2.1.2.1 Mechanisms of Long-term Depression ............................ 20 1.2.1.2.2 Links Between Long-term Depression and Learning and Memory ........................................................................................... 22  !  #!  1.2.1.3 Unique Synaptic Plasticity in the Dentate Gyrus ............................. 23 1.2.1.3.1 The Medial Perforant Path-Dentate Granule Cell Synapse ...................................................................... 23 1.2.1.3.2 The Lateral Perforant Path-Dentate Granule Cell Synapse ...................................................................... 24 1.2.2 Structural Plasticity........................................................................................... 25 1.2.2.1 Mechanisms of Hippocampal Adult Neurogenesis .......................... 26 1.2.2.2 Links Between Adult Neurogenesis in the Dentate Gyrus and Learning and Memory .................................................................................. 28 1.2.2.3 Alternatives to Neurogenesis: Could Dendritic Morphology Contribute to Learning and Memory? .......................................................... 30 1.2.3 Summary ........................................................................................................... 31  1.3 The NMDA receptor – a Molecular Substrate for Synaptic Plasticity .................32 1.3.1 The Overall Structure of the NMDA Receptor ................................................ 32 1.3.2 The Different Subunit Types of the NMDA Receptor ..................................... 34 1.3.2.1 The Obligatory GluN1 Subunit ........................................................ 34 1.3.2.2 The Regulatory GluN2 Family ......................................................... 34 1.3.2.3 The Regulatory GluN3 Family ......................................................... 36 1.3.3 The Temporal Expression Patterns of the NMDA Receptor Subunits in the Hippocampus ............................................................................................................. 37 1.3.4 Summary of the NMDA Receptor and its Subunits ......................................... 38  1.4 The GluN2A-/- Mouse Model ................................................................................39 1.4.1 Generation of the GluN2A-/- Mouse Model ...................................................... 39 1.4.2 Altered Long-term Potentiation Threshold in the CA1 of GluN2A-/- Mice ..... 40 1.4.3 Impaired Long-term Potentiation in Other Brain Regions ............................... 41 1.4.4 Unknown Capacity for Structural Plasticity in the GluN2A-/- Mouse .............. 41 1.4.5 Compromised Hippocampal-Related Behaviour in GluN2A-/- Mice ................ 42 1.4.6 Summary of the GluN2A-/- Mouse Model ........................................................ 43  1.5 Summary ...............................................................................................................44 1.6 Hypothesis.............................................................................................................45 2 Requirement of GABAA Receptor Antagonism for the Induction of in vitro Long-term Potentiation in the Mouse Dentate Gyrus ...........................................46 2.1 Introduction ...........................................................................................................46 2.1 Materials and Methods ..........................................................................................47 2.3 Results ...................................................................................................................49 2.4 Discussion .............................................................................................................51 3 Voluntary Exercise and Stress, but no Housing Conditions, Regulate Adult Neurogenesis in the Adult Mouse Dentate Gyrus ..................................................55 3.1 Introduction ...........................................................................................................55 3.2 Materials and Methods ..........................................................................................57 3.2.1 Animals and Housing Conditions ..................................................................... 57  !  #"!  3.2.2 BrdU Administrations and Tissue Preparation ................................................. 58 3.2.3 Restraint Stress ................................................................................................. 58 3.2.4 Immunohistochemistry ..................................................................................... 59 3.2.5 Quantification ................................................................................................... 61 3.2.6 Serum Corticosterone ....................................................................................... 62 3.2.7 Statistical Analysis ........................................................................................... 62  3.3 Results ...................................................................................................................63 3.3.1 Voluntary Exercise Increases Cellular Proliferation in Socially and Individually House Mice .................................................................................... 63 3.3.2 Social Housing Condition Differentially Affects Cell Proliferation in Exercising Mice Subjected to Short-term Restraint Stress.................................... 66 3.3.3 Social Isolation has No Effect on the Production of New Neurons in Response to Voluntary Exercise ............................................................................ 68  3.4 Discussion .............................................................................................................70 4 Deletion of GluN2A-containing NMDA Receptors Impairs Bidirectional Synaptic Plasticity and Spatial Pattern Separation in the Adult Dentate Gyrus ...............75 4.1 Introduction ...........................................................................................................75 4.2 Materials and Methods ..........................................................................................77 4.2.1 Animals and Housing Conditions ..................................................................... 77 4.2.2 Electrophysiology ............................................................................................. 77 4.2.2.1 Electrophysiological Tissue Preparation .......................................... 77 4.2.3.2 Field Electrophysiology.................................................................... 78 4.2.2.3 Whole Cell Electrophysiology ......................................................... 80 4.2.2.4 Electrophysiology Dugs ................................................................... 81 4.2.3 Golgi Staining ................................................................................................... 81 4.2.3.1 Golgi Impregnation and Slice Processing ........................................ 81 4.2.3.2 Dendritic Analysis ............................................................................ 82 4.2.4 Hippocampal-dependent Behavioural Tasks .................................................... 83 4.2.4.1 Behavioural Apparatus ..................................................................... 83 4.2.4.2 Metric Spatial Change Task ............................................................. 83 4.2.4.3 Temporal Ordering Task .................................................................. 84 4.2.4.4 Behavioural Analysis........................................................................ 85 4.2.5 Statistical Analysis ........................................................................................... 86  4.3 Results ...................................................................................................................86 4.3.1 Reduced NMDA:AMPA Ratio in Dentate Granule Cells of GluN2A-/- Mice ..................................................................................................... 86 4.3.2 Abolished LTP in the DG and Reduced LTP in the CA1 of Adult GluN2A-/- Mice ............................................................................................ 90 4.3.3 Abolished LTD in the DG and Intact LTD in the CA1 of Adult GluN2A-/- Mice .......................................................................................... 92 4.3.4 Intact Dendritic Structure of Dentate Granule Cells in Glun2A-/- Mice ........... 93 4.3.5 Compromised Spatial Pattern Processing in GluN2A-/- Mice .......................... 95  !  #""!  4.3.6 Intact Temporal Pattern Processing in GluN2A-/- Mice .................................... 96  4.4 Discussion .............................................................................................................97 4.4.1 Alterations in Synaptic NMDA Receptor Response in the Dentate Gyrus ...... 98 4.4.2 Role of GluN2 subunits in LTP ........................................................................ 99 4.4.3 Role of GluN2 subunits in LTD ..................................................................... 100 4.4.4 Deletion of GluN2A Does Not Alter Dendritic Morphology ......................... 101 4.4.5 GluN2 Subunit Contribution to Pattern Separation ........................................ 102  5 NMDA Receptor GluN2A Subunits are Required for Normal Dendritic Development in Dentate Granule Neurons ...........................................................104 5.1 Introduction .........................................................................................................104 5.2 Materials and Methods ........................................................................................106 5.2.1 Animals and Housing Conditions ................................................................... 106 5.2.2 Genotyping ..................................................................................................... 107 5.2.2.1 DNA Isolation ................................................................................ 107 5.2.2.2 Polymerase Chain Reaction ............................................................ 108 5.2.3 Immunohistochemistry ................................................................................... 108 5.2.3.1 Tissue Preparation .......................................................................... 108 5.2.3.2 Immunohistochemistry ................................................................... 109 5.2.3.3 Cell Quantification ......................................................................... 110 5.2.4 Golgi Staining ................................................................................................. 111 5.2.4.1 Golgi Impregnation ........................................................................ 111 5.2.4.2 Slice Preparation and Processing .................................................... 111 5.2.4.3 Dendritic Analysis .......................................................................... 112 5.2.4.4 Spine Analysis ................................................................................ 113 5.2.5 Statistical Analysis ......................................................................................... 114  5.3 Results .................................................................................................................114 5.3.1 Cell Proliferation and Neurogenesis is Intact in GluN2A-/- Mice ................... 114 5.3.2 Impaired Dendritic Morphology in Immature Neurons of GluN2A-/- Mice ... 116 5.3.3 Immature Neurons of GluN2A-/- Mice show Localized Increases in Spine Density....................................................................................................... 119  5.4 Discussion ...........................................................................................................120 5.4.1 Role of the GluN2A Subunit in Early Stages of Adult Neurogenesis ............ 121 5.4.2 Role of the GluN2A Subunit in the “Late Immature” Stage of Adult Neurogenesis ............................................................................................. 123 5.4.3 Role of the GluN2A Subunit in Spinogenesis ................................................ 124  5.5 Conclusions .........................................................................................................126 6 General Discussion ...............................................................................................128 6.1 Summary of Findings..........................................................................................128 6.1.1 Establishment of Methodology ....................................................................... 129 6.1.2 Synaptic Plasticity .......................................................................................... 129 6.1.3 Structural Plasticity......................................................................................... 129  !  #"""!  6.1.4 Behavioural Plasticity ..................................................................................... 130  6.2 Hippocampal Plasticity in the GluN2A-/- Mouse ................................................131 6.2.1 Synaptic Plasticity and GluN2 Subunits ......................................................... 131 6.2.1.1 The Subunit Theory of Hippocampal Synaptic Plasticity .............. 131 6.2.1.2 The Charge Transfer Theory of Hippocampal Synaptic Plasticity ..................................................................................................... 133 6.2.1.3 The GluN2 Structure Theory of Hippocampal Synaptic Plasticity ..................................................................................................... 134 6.2.1.4 The GluN2 Ratio Theory of Hippocampal Synaptic Plasticity ...... 135 6.2.2 Structural Plasticity......................................................................................... 136 6.2.2.1 The Role of the NMDA Receptor in Adult Neurogenesis ............. 136 6.2.2.2 The Role of GluN2 Subunits in Adult Neurogenesis ..................... 137 6.2.2.3 Dendrite Formation and the NMDA Receptor ............................... 138 6.2.2.4 Spine Density and the NMDA Receptor ........................................ 141 6.2.3 Hippocampal-dependent Behaviour ............................................................... 142  6.3 Current Model .....................................................................................................145 6.3.1 Neuronal Morphology of Late Immature Dentate Granule Cells ................... 145 6.3.2 Role of the GluN2A Subunit in Specific Hippocampal Regions ................... 146 6.3.3 Impairments in the Dentate Gyrus that Underlies Behavioural Deficits ........ 147  6.4 Limitations and Pitfalls .......................................................................................150 6.4.1 Using a Global Mutant Mouse Model ............................................................ 150 6.4.2 Synaptic Plasticity Deficits due Loss of GluN2A or the NMDA Receptor?.. 150 6.4.3 Performance of the Temporal Ordering Task in Mice ................................... 151 6.4.4 Limitations of the Golgi Impregnation Technique ......................................... 152  6.5 Future Directions ................................................................................................153 6.5.1 Contribution of the GluN2A Subunit to Late Immature Dentate Granule Cell Physiology ............................................................................ 153 6.5.2 Role of the GluN2A Subunit in Exercise-Induced Changes in Synaptic Plasticity in the Dentate Gyrus ............................................................. 154 6.5.3 Role of the GluN2A Subunit in Activity-dependent Increases in Cell Proliferation in the Dentate Gyrus ............................................................... 155  6.6 Conclusions .........................................................................................................157 References ................................................................................................................158  !  "$!  List of Tables Table 1.1 Kinetics of GluN2 Subunits .......................................................................35  !  $!  List of Figures Figure 1.1 Anatomy of the hippocampus .....................................................................1 Figure 1.2 The hippocampal trisynaptic circuit ...........................................................7 Figure 1.3 Long-term potentiation .............................................................................18 Figure 1.4 Long-term depression ...............................................................................21 Figure 1.5 Mechanisms of adult neurogenesis in the dentate gyrus ..........................26 Figure 1.6 Structure of the NMDA receptor ..............................................................33 Figure 1.7 Differential NMDA receptor current attributed to GluN2 subunits .........36 Figure 2.1 Changes in post-tetanic and long-term potentiation with different bicuculline methiodide concentrations in the mouse dentate gyrus............................50 Figure 3.1 Voluntary exercise promotes cellular proliferation, irrespective of social condition. ......................................................................................................64 Figure 3.2 Acute restraint stress selectively modulates proliferation in exercising mice. ......................................................................................................67 Figure 3.3 Neurogenesis and branching of new neurons is induced by exercise, irrespective of social condition ...................................................................................69 Figure 4.1 Synaptic transmission and presynaptic properties are normal in GluN2A-/- mice. ......................................................................................................87 Figure 4.2 Reduced NMDA:AMPA ratio and increased NMDA receptor decay rate in GluN2A-/- mice. .....................................................................................89 Figure 4.3 GluN2A-/- mice have abolished LTP in the DG, altered LTP in the CA1. ..................................................................................................................91 Figure 4.4 GluN2A-/- mice have abolished LTD in the DG, intact LTD in the CA1. ..................................................................................................................93 Figure 4.5 Normal dendritic morphology of dentate granule cells in GluN2A-/- mice. ......................................................................................................94 Figure 4.6 Compromised spatial pattern separation in GluN2A-/- mice. ...................96 Figure 4.7 Normal temporal pattern separation in GluN2A-/- mice ...........................97 Figure 5.1 Intact cell proliferation in the adult dentate gyrus of GluN2A-/- mice. ..115 Figure 5.2 Intact differentiation in the adult dentate gyrus of GluN2A-/- mice. .......116 Figure 5.3 GluN2A does not alter the number of primary dendrites in immature and mature cells in the adult dentate gyrus. .........................................117 Figure 5.4 Altered dendritic morphology in immature dentate granule cells from GluN2A-/- mice. ................................................................................................118 Figure 5.5 Immature cells have localized increases in spine density in the adult dentate gyrus of GluN2A-/- mice. ...........................................................120 Figure 5.6 Summary schematic of the results. .........................................................121  !  $"!  List of Symbols and Abbreviations GluN2C  NMDA receptor subunit 2C  GluN2D  NMDA receptor subunit 2D  GluN3A  NMDA receptor subunit 2A  GluN3B  NMDA receptor subunit 3B  GSK-3  %&'()*+,!-',./01+!2",01+!3  HFS  High Frequency Stimulation Isolated Controls Isolated Runners Inner Granular Zone Intraperitoneal  ACSF  Artificial Cerebrospinal Fluid  AMPA  BMI  !-amino-3-hydroxy-5methyl-4-isoxazolepropionic Acid Analysis of Variance 2-Amino-5phosphonopentanoic acid Bicuculline Methiodide  BrdU CA CA1  Bromodeoxyuridine Cornu Ammonis Cornu Ammonis Region 1  I-CON I-RUN IGZ i.p.  CA2  Cornu Ammonis Region 2  LFS  Low Frequency Stimulation  CA3  Cornu Ammonis Region 3  LPP  Lateral Perforant Path  CaMKII  Calcium-CalmodulinDependent Kinase-II  LTD  Long-term Depression  LTP  Long-term Potentiation  CON CORT CRE  MAPK MPP  Mitogen-activated Protein Kinase Medial Perforant Path  CREB  Control Corticosterone Cyclic-AMP Response Element Cyclic-AMP Binding Protein  mRNA  Messenger Ribonucleic Acid  DAB  3,3-diaminobenzidine  NeuroD  DCX DG DGC fEPSP  Doublecortin Dentate Gyrus Dentate Granule Cell Field Excitatory Postsynaptic Potential  Neurogenic Differentiation Protein N-methyl-D-aspartate  GABA  "-aminobutyric Acid  GABAA  "-aminobutyric Acid Type A  ANOVA APV  NMDA NeuN OGZ PCNA  Neuronal Nuclei Outer Granular Zone Proliferating Cell Nuclear Antigen Paraformaldehyde Protein Kinase A Protein Kinase C Protein Phosphatase 1  GCL GFAP  PFA PKA Granule Cell Layer PKC Glial Fibrillary Acidic Protein PP1  GluN1  NMDA receptor subunit 1  PP2A  Protein Phosphatase 2A  GluN2A  NMDA receptor subunit 2A  PP2B  Protein Phosphatase 2B  GluN2A-/- GluN2A Knock-out  PTP  Post-tetanic Potentiation  GluN2B  S-CON S-RUN  Social Controls Social Runners  !  NMDA receptor subunit 2B  $""!  S.E.M.  Standard Error of the Mean  wHFS  sHFS  Strong High Frequency Stimulation  WT  SGZ TBS RUN  Subgranular Zone Tris-buffered Saline Voluntary Exercise  !  Weak High Frequency Stimulation Wild-type  $"""!  Acknowledgements First, I would like to thank my supervisor and mentor, Dr. Brian Christie. When you met me, I was just a naïve undergrad with an interest in how the brain works. You took a chance on me, which was quite the career risk. I couldn’t have asked for a better graduate school experience, or a better supervisor. Thank you. Additional thanks goes to Dr. Jeremy Seamans, Dr. Yu-Tian Wang, and Dr. Liisa Galea for your thoughtful critical feedback on my research, and Dr. Tim O’Connor, who provided one of my first research experiences at UBC. I would also like to thank NSERC for the financial support. I would also like to thanks all the postdoctoral fellows, graduate students and laboratory technicians that I had the privilege working with, especially: Dr. Patricia Brocardo, Eu realmente sinto muito por chamar-lhe a gordura; Anna Patten, who should start a Vegas show with an hour of prop comedy followed by an hour and a half monologue about her favorite bands; Jennifer Helfer, whose love for Britney Spears must be ironic; James Shin, who taught me that predators are not the only people who live in vans (although the jury may still be literally out on that one); Ross Peterson, for the court drama story; Jessica Nathan, who needs a mustache like I don’t need pickles; Fanny Boehme for confirming several German stereotypes; Pam Parkinson, for dragging me into the not-so-fruitful world of ABCG1; Crystal Rodriguez Bostrom, for telling me about how cheap your pants are; Jennifer Graham, whose horse mating knowledge and erotic baked goods hobby is going to make some psychoanalyst very rich; and Evelyn “Evilyn Deed” Wiebe, for introducing me to roller derby. Thanks to honourary Christie lab members as well: Fiona Zeeb, for your gigantic office furniture; Graeme “Fun fact! Blah, blah blah Can-con…” Taylor, and Mr. Cheap beer and sports - Sir Jon LeBlanc.  !  $"#!  I would also like to single out a few people from the laboratory who personally took me under their collective wings. Dr. Joana Gil-Mohapel – your amazing amount of help changes nothing. We are still enemies. Dr. Andrea Titterness – I often turned to you for advice about grad school, and you always provided me with help. Thank you. By the way, you “mustard” never use a condiment joke again. Brennan Eadie, BA, MSc, PhD, MD, HD – perfect stranger, big brother, mentor. I can’t imagine what grad school would have been like without you sitting across from me. Thank you. Thanks to Aaron and Elanor Franks, Cheryn Wong, and Jason Kim: You’ve all been a pretty amazing support system for me. I will repay you all in high-fives. Thank you to my soon- to- be-sister-in-law Trisha. Your cooking kept me nourished, and your drama allowed me to not pay for cable. Thank you to my sister Tara Kannangara, who I’m proud to say is a better version of me in every way. Thank you Grandma, Aslin Dharmawardana, for reminding me what is truly important in life. Thank you Metro Kannangara, my now-passed dog, woof woof, grrr, woof woof. Thank you to my soon-to-be wife, Elizabeth Bonham. You supported me during the toughest times in my life, and always reminded me that science is not everything – that alone might have kept me sane. There are only so many ways to say that you are the best thing to ever happen to me. I could never have imagined finding someone so amazing, and cannot wait to spend the rest of my life with you. Finally, thank you to my parents, Dr. Tissa Kannangara and Mrs. Mali Kannangara. You’ve given me everything, without asking for anything. Both of you have been unwavering pillars of support and I will be forever grateful.  !  $#!  To Liz and my family  !  $#"!  1 Introduction 1.1 The Hippocampus The hippocampus is a bilateral structure contained in the medial temporal lobe of the mammalian brain (Fig. 1.1). The term hippocampus originates from the Latin word for “seahorse”, coined by the 16th century anatomist Giulio Cesare Aranzi, who likened the unique curvature of this brain structure to that of the marine fish (Andersen, 2007, pg 9). The hippocampus is often considered to be part of a larger collection of brain regions called the hippocampal formation, however there is currently little consensus regarding which brain regions are encompassed by the term; at different points in time, the hippocampal formation has been defined to include the dentate gyrus (DG), three Cornu  ! !  !  Figure 1.1 Anatomy of the Hippocampus A, Threedimensional rendering of the rodent brain (grey), surrounding the two hemispheres of the hippocampus (green) B, Three-dimensional rendering of the hippocampus (green), with a transverse crosssection overlay (Orientation of the entire brain shown in top right) C, Nissl stain of coronal hippocampal section demonstrating the different regions and layer of the hippocampus. CA: Cornu Ammonis, DG: Dentate Gyrus, GCL: Granule Cell Layer, ML: Molecular Layer, PCL: Pyramidal Cell Layer, SL: Stratum Lucidum, SL-M: Stratum Lacunosum Moleculare, SO: Stratum Oriens, SR: Stratum Radiatum (© Allen Mouse Brain Atlas (2009), adapted by permission)  !  1  Ammonis (CA) regions (CA1-3), subiculum, presubiculum, parasubiculum and entorhinal cortex. In this thesis, the hippocampus will be defined as comprising of the DG and the three CA regions. The following section will provide a brief overview of this fascinating structure, examining both its functional anatomy and its proposed role in the mammalian brain. The main focus of this thesis centers on the DG; as such, sections pertaining to the DG will be emphasized.  1.1.1 The Basic Anatomy and Major Cell Types of the Hippocampus The hippocampus is differentiated from other cortical areas in the brain due to its archicortical architecture and highly laminar organization. The basic anatomy of the hippocampus will be discussed here. It should be noted that the three-dimensional structure of the hippocampus presents difficulties when describing the location of hippocampal subregions. For the purpose of this thesis, a dorso-ventral axis in relation to a coronal cross-section of the hippocampus will be used to describe anatomical locations. Contained within the hippocampus are a variety of cell types, ranging from the excitatory principal neurons, several classes of inhibitory interneurons, glial cells and precursor cells. For the purpose of this thesis, the excitatory principal cells of the hippocampus will be further discussed, focusing on the physiology of dentate granule cells (DGCs) and CA pyramidal cells.  !  2  1.1.1.1 The Dentate Gyrus 1.1.1.1.1 The Basic Anatomy of the Dentate Gyrus The DG is a C-shaped, trilaminar structure, composed of the molecular layer, granule cell layer (GCL), and hilus (Fig. 1.1C). The most dorsal layer of the DG, bordering the hippocampal fissure, is the molecular layer. The molecular layer is occupied by the primary afferent input into the hippocampus, the perforant path, which has a laminar distribution along the outer two thirds of the layer (discussed in 1.1.2.1). The molecular layer also contains the dendrites of the principal cells in the DG, the DGCs, as well as several classes of interneurons. Ventral to the molecular layer is the GCL. The GCL is a tightly compacted cell layer, predominantly composed of the somas of DGCs. The GCL encapsulates the third layer of the DG, the hilus. It is in the hilus that the other principal neurons of the DG, the mossy cells, and interneurons are located. Traversing through the hilus are the DGC axon bundles called the mossy fibers (discussed in 1.1.2.2). Located between the hilus and the GCL is a small subregion called the subgranular zone (SGZ). This 20 µm wide layer is the location of neural precursor cells (discussed in 1.2.2.1) and interneurons.  1.1.1.1.2 The Principal Cell of the Dentate Gyrus: The Dentate Granule Cell The DGCs are one of the predominant types of excitatory neurons in the DG (the other type, the mossy cells, will not be discussed in this thesis). DGCs have relatively small, elliptical somas, and multiple primary apical dendrites that extend a dense arbour into the adjacent molecular layer. Dendrites are often heavily covered with small  !  3  dendritic protrusions called spines (Yuste, 2011). Basal dendrites are also present in immature DGCs, but disappear in mature neurons (Schmidt-Hieber et al., 2004). The physiological properties of DGCs are unique in comparison to other principal neurons in the hippocampus. First, DGCs show little signal attenuation – the strength of excitatory potentials elicited in distal dendrites show little decay as they travel towards the soma (Schmidt-Hieber et al., 2007). This property has been attributed to two physical characteristics of the DGC dendrites: dendrite diameter is inversely proportional to the distance from its soma, and DGCs do not have a single large apical dendrite, which would greatly increase the decay rate of the excitatory potential (Schmidt-Hieber et al., 2007). Second, DGCs have a hyperpolarized resting membrane potential (~-80 mV), while maintaining a stereotypical action potential threshold (Spruston and Johnston, 1992); these two attributes decrease the likelihood of spontaneous activation. Third, action potentials in DGCs are brief, and trains of action potentials have accommodating properties, primarily due to the increased amount of calcium-activated potassium channels in comparison to other principal cells (Staley et al., 1992). Together, these properties enable DGCs to functionally act as an information filter: highly capable of receiving information, but conveying this information to other hippocampal regions in a selective way.  1.1.1.2 The CA Regions 1.1.1.2.1 The Basic Anatomy of the CA Regions Overall, the layers that make up the CA1, CA2 and CA3 regions are fairly consistent (Fig 1.1C). The pyramidal cell layer contains the somas of the principal cells  !  4  in the CA regions, the pyramidal cells. Adjacent, but dorsal to the pyramidal cell layer is the stratum oriens, which contains the basal dendrites from pyramidal cells, as well as interneurons. Encompassing the stratum oriens is the alveus and fimbria, a layer of myelinated fibers going to and from the hippocampus. Adjacent, but ventral to the pyramidal cell layer lies the stratum radiatum, a region containing the apical dendrites of pyramidal cells as well as the axons from CA3 pyramidal cells, the Schaffer Collaterals (discussed in 1.1.3.3). Between the stratum radiatum and the hippocampal fissure is the stratum lacunosum moleculare, where inputs from the entorhinal cortex terminate. While similar, the three CA regions show several anatomical differences. For example, the CA1 region is larger than the CA3 region, which in turn is much larger than the CA2 region. In addition, the cell layer is tightly compacted in the CA1 region and more dispersed in the CA2 (Wyszynski et al., 1998; Chevaleyre and Siegelbaum, 2010). Another difference is the stratum lucidum, a thin layer located between the stratum radiatum and the pyramidal cell layer that is only found in the CA3 region.  1.1.1.2.2 The Principal Cells of the CA Regions: The Pyramidal Cells The morphology of pyramidal cells is largely uniform across CA regions. Each pyramidal cell type has a soma located in the pyramidal cell layer, spiny basal dendrites extending into the stratum oriens, and spiny apical dendrites that extend through the ventral molecular layers of the CA region, to the hippocampal fissure. However, several region-specific properties can be observed. Pyramidal cell somas are highly variable in size, ranging from the smaller CA1 somas (~180 µm2) to the larger CA3 somas (~300700 µm2) (Ishizuka et al., 1995; Chevaleyre and Siegelbaum, 2010). The locations of  !  5  dendritic arbors, which reflect sites of pyramidal cell innervation, are also distinct. CA1 pyramidal cells demonstrate significant dendritic branching in both the stratum radiatum, where they receive input from the Schaeffer Collaterals, and the stratum oriens, where they receive additional input from the CA3 pyramidal cells (Ishizuka et al., 1995). In contrast, CA2 cells have extensive dendritic branching in the stratum lacunosum moleculare (Ishizuka et al., 1995; Chevaleyre and Siegelbaum, 2010). CA3 pyramidal cells are structurally similar to CA1 cells, but have a short apical trunk in the stratum lucidum, where they receive input from the DGC mossy fibers (discussed in 1.1.3.2), and an apical dendritic tuft in the stratum lacunosum-moleculare, where they receive input from the entorhinal cortex (Ishizuka et al., 1995). Additionally, CA3 pyramidal cells are unique due to their prominent collateral connections formed between neighboring CA3 pyramidal cells. Pyramidal cells from each region also show distinct physiological properties, including input resistance and cell capacitance (Chevaleyre and Siegelbaum, 2010). The resting membrane potential of CA1 pyramidal cells is stereotypical (~-70 mV), and slightly more hyperpolarized in CA2 and CA3 cells (~-75 mV), indicating that stronger depolarization is required for activation in the CA2 and CA3 (Chevaleyre and Siegelbaum, 2010). Action potentials are also distinct. Action potentials in CA1 pyramidal cells are followed by a characteristic slow after-hyperpolarization that is not present in CA2 cells (Chevaleyre and Siegelbaum, 2010). Furthermore, both CA1 and CA2 pyramidal cells fire action potentials throughout depolarizations, while CA3 cells respond to depolarizations by firing an initial burst of action potentials (Chevaleyre and Siegelbaum, 2010).  !  6  1.1.2 The Connections and Projections of the Hippocampus The hippocampus itself is unique to other areas of the neocortex in that it is interconnected via a predominantly unidirectional excitatory pathway. This circuit first described by Anderson et al. (1971), is referred to as the hippocampal trisynaptic circuit, and consists of three major synapses (Fig. 1.2).  Figure 1.2 The hippocampal trisynaptic circuit An illustration of the circuitry in the hippocampus (Upper Panel) and schematic of the hippocampal network (Lower Right) demonstrating the three synapses of the trisynaptic circuit. Layer II neurons from the entorhinal cortex project to the dentate gyrus via the perforant path (Synapse I), itself composed of the medial perforant path, and the lateral perforant path. Dentate granule cells project to CA3 pyramidal cells (Synapse II), via the mossy fibers. CA3 pyramidal cells send recurrent collaterals to neighboring CA3 pyramidal cells, as well as project to the CA1 pyramidal cells (Synapse III), via the Schaffer Collaterals. CA1 pyramidal cells send projects to the subiculum (not shown) and back to the entorhinal cortex, to Layers V and VI. CA: Cornu Ammonis, DG: Dentate Gyrus, EC: Entorhinal Cortex, LPP: Lateral Perforant Path, MPP: Medial Perforant Path (© Nature Publishing Group, Deng et al. (2010), adapted by permission)  ! !  7  1.1.2.1 Synapse I: The Perforant Path-Dentate Granule Cell Synapse The input for the first connection in the trisynaptic circuit originates from Layer II cells in the entorhinal cortex, which project to the DG via a group of fibers called the perforant path. The perforant path, which delivers the majority of the cortical information into the hippocampus, passes through the dentate molecular layer and innervates dendritic spines of DGCs, along with !-aminobutyric acid (GABA)-ergic spines of interneurons. The perforant path can be subdivided into two distinct inputs: the medial perforant path (MPP) and the lateral perforant path (LPP). The MPP originates from the medial entorhinal cortex and passes through the middle one-third of the molecular layer, while the LPP originates from the lateral entorhinal cortex and passes through the outer one-third of the molecular layer. While both provide excitatory input onto DGCs, the MPP and LPP appear to have different short-term and long-term plasticity (McNaughton, 1980; Bramham et al., 1991b, a; Colino and Malenka, 1993) (discussed in 1.2.1.3.1). As the perforant path delivers the majority of cortical information into the hippocampus, DGCs (and the entire DG by extension) are uniquely positioned to regulate and filter information before other hippocampal regions receive it.  1.1.2.2 Synapse II: The Mossy Fiber-CA3 Synapse The second major component in the trisynaptic circuit connects the DG to the CA3. DGCs project bundles of unmyelinated axons, also known as mossy fibers, into the hilus. In the hilus, mossy fibers have small, en passant (located on axonal shafts, rather  !  8  than terminals) synapses to mossy cells and interneurons (approximately seven collaterals per mossy fiber) en route to the CA3 region. Once there, mossy fibers produce large en passant synapses to CA3 pyramidal cells within the stratum lucidum. The mossy fiber-CA3 pyramidal cell synapses provide a powerful connection between the DG and the CA3, originating from two properties: a large (4-10 µm) presynaptic bouton located within close proximity of the CA3 soma and the multiple contacts a single mossy fiber has on a single CA3 dendrite (Acsady et al., 1998; Galimberti et al., 2006). Interestingly, DGCs are only sparsely connected to CA3 neurons overall; each DGC may only synapse with 15 CA3 pyramidal cells in total (Galimberti et al., 2006). This characteristic of strong, yet sparse connectivity with the CA3 is congruent with several of the proposed functions of the DG (discussed in 1.1.4.1).  1.1.2.3 Synapse III: The Schaffer-Collateral-CA1 Synapse The third component of the trisynaptic circuit is between the CA3 and CA1 regions. CA3 pyramidal cells send axons into both the stratum radiatum and stratum oriens of the CA1 region via a pathway known as the Schaffer-Collaterals, innervating both the apical and basal dendrites of CA1 pyramidal cells. The Schaffer-Collateral-CA1 pyramidal cell synapse is one of the most studied synapses in the entire central nervous system, and has contributed much to our knowledge of synaptic transmission and plasticity (discussed in 1.2.1). In summary, the trisynaptic circuit is a connection between three major regions of the hippocampus: the DG, the CA3 and the CA1. While this circuit appears to function as the strongest conduit of cortical information, it should be noted that other pathways are  !  9  present. These pathways include a disynaptic circuit consisting of the entorhinal cortex bypassing the DG to send direct input into the CA2 (Chevaleyre and Siegelbaum, 2010), or a pathway showing direct subcortical output from the CA3 to the lateral septum (Luo et al., 2011). Nevertheless, the trisynaptic circuit has been demonstrated to act as the primary circuit for information traveling through the hippocampus, with the DG importantly situated as the circuit’s first region.  1.1.2.4 Efferent Projections From the Hippocampus The CA1 region projects to the adjacent subiculum and the entorhinal cortex (Naber et al., 2001). The subiculum, which has a similar anatomical layout to the CA regions, receives input in its pyramidal cell layer and dorsal region of its molecular layer (equivalent to the stratum radiatum of the CA regions). This input originates from CA1 pyramidal cells, whose axons enter the stratum oriens or the alveus and innervate the subiculum topographically. Both the CA1 and subiculum sent projections to Layers V and VI of the entorhinal cortex (Naber et al., 2001). Notably, the return of excitatory input into the entorhinal cortex completes a cortical information loop, as the entorhinal cortex is both the main source for, and main recipient of hippocampal information.  1.1.3 Proposed Functions of the Hippocampus Early theories regarding hippocampal function suggested that the human hippocampus might be involved in olfaction (reviewed in Brodal, 1947; Compston, 2010) or emotion (Papez, 1937, republished in Papez, 1995). However, most evidence supporting these theories at the time was largely circumstantial and lacked  !  10  neuroanatomical accuracy. More than a decade later, the first direct evidence for the function of the hippocampus was reported. In the late 1950s, Dr. William Scoville performed a bilateral limbic surgery to treat the frequent and severe seizures of Henry Molasion, commonly known as H.M. (Scoville and Milner, 1957). The brain regions removed included the hippocampus, amygdala, collateral sulcus, perirhinal cortex, entorhinal cortex and the medial mammillary nucleus (Corkin et al., 1997). Following the surgery, it was discovered that H.M. was afflicted with severe memory deficits; specifically, H.M. lacked the ability to form and retain long-term memories of new facts (semantic memory) and events (episodic memory). The surgery did not alter intellect, perception or working memory. This discovery of H.M.’s memory performance was the first to suggest that the human hippocampus is involved in semantic and episodic memory. Since then, numerous studies have implicated the hippocampus with the formation and maintenance of explicit (i.e., intentional and conscious) spatial memory. An example of one such studies was conducted by Morris et al. (1982) who trained rodents to learn the spatial location of a hidden platform submerged in opaque water, in a task known as the Morris water maze. Hippocampal lesions disrupted learning of this task. Another example involves the discovery that rats have place cells, hippocampal cells whose activity is associated with a rodent’s specific location in a specific environment, irrespective of local cues (O'Keefe and Dostrovsky, 1971). Importantly, the presence of place cells has also been observed in humans. Ekstrom et al. (2003) recorded hippocampal activity from implanted depth electrodes as patients navigated a virtual town as a taxi driver. They found that a significant population of hippocampal cells  !  11  demonstrated activity related to the subject’s virtual spatial location. Taken together, human and rodent studies implicate the involvement of the hippocampus in spatial memory.  1.1.4 Region-specific Contributions to Hippocampal Function It has recently become clear that the hippocampus acts as a network that automatically encodes attended events and their contexts, with each hippocampal region playing a complementary, but unique part in the processing of spatial information.  1.1.4.1 The Dentate Gyrus and Spatial Pattern Separation Spatial pattern separation is the enhancement of contrast between two spatial patterns or events. This is hypothesized to occur, at a neuronal level, through either a change in neuronal firing rate, or through the firing of different neuronal sets (Leutgeb et al., 2007; Treves et al., 2008). The DG is optimally structured for spatial pattern separation due to the aforementioned physiology of DGCs: the sparse firing (discussed in 1.1.1.1.2), the few connections between DGCs and CA3 pyramidal cells, and the powerful mossy fiber boutons (discussed in 1.1.2.2). Spatial pattern separation in the DG has been assessed using two different tasks: contextual fear conditioning and metric spatial change tasks. The contextual fear conditioning task, in which animals are assessed regarding their ability to differentiate between a context where a shock is administered and a non-shock context, has been recently linked to the DG function of the N-methyl-D-aspartate (NMDA) receptor, an ionotropic glutamate receptor highly expressed in the hippocampus (McHugh et al., 2007;  !  12  Eadie et al., 2012) (the NMDA receptor is discussed in 1.3). The metric spatial change task, which involves the discrimination of distances between objects, has also been associated with DG function (Gilbert et al., 2001; Goodrich-Hunsaker et al., 2008; Clelland et al., 2009)(described in 4.2.4), but the contribution of the NMDA receptor in this task has not yet been examined. Nevertheless, evidence supports a link between intact function of the DG (perhaps through NMDA receptors) and spatial pattern separation.  1.1.4.2 The CA3 and Spatial Pattern Completion Spatial pattern completion refers to the capacity to retrieve previously stored information when presented with partial or incomplete inputs (Kesner, 2007a). The high number of recurrent collaterals observed in CA3 pyramidal cells has indicated that the CA3 region may be optimally organized for this task (Marr, 1971). Supporting this hypothesis is a report by Nakazawa et al. (2002), who trained mice that lacked NMDA receptor expression in the CA3 on the Morris water maze. These mutant mice demonstrated normal performance on the task, however, deficits were observed if the external environment was altered. This result indicates that the ability to use partial spatial input was compromised, and implicates the CA3 region in spatial pattern completion.  1.1.4.3 The CA2 and Possible Functions The function of the CA2 has been quietly ignored, with no behaviour experiments explicitly targeting this particular hippocampal region published to date (Jones and  !  13  McHugh, 2011). The inputs to the CA2 region, via the CA3 and the entorhinal cortex, suggest that the CA2 region may integrate several types of spatial information, but direct experimentation is warranted in order to determine the functional impact of the CA2.  1.1.4.4 The CA1 and Temporal Pattern Separation The CA1 has been associated with processing the temporal order of spatial patterns or events, which would critically ensure that spatial events separated by time were encoded with minimal overlap (Kesner et al., 2004). This role of the CA1 has been supported by Gilbert et al. (2001), who trained rats to visit the arms of a radial arm maze in a specific sequence. After training, rats were given the choice of two such arms. Importantly, the two different arms were temporally separated in the previously learnt sequence (e.g. access would be given to the second arm in the sequence, and the sixth). Unlike control groups, rats with targeted CA1 lesions were unable to determine which arm had been presented first in the sequence. Another CA1-associated task, termed the temporal ordering task (described in 4.2.4), presents rodents with three pairs of unique objects, in sequence. Rodents are then presented with one object from the first pair, and one object from the third. Control rodents explored the object from the first pair as it was presented first temporally; however, rodents with CA1 lesions showed no preference to either object (Hoge and Kesner, 2007). Taken together, these studies highlight the role of the CA1 in temporal processing.  !  14  1.1.5 Summary The hippocampus, composed of the DG and CA regions, is one of the most studied regions in the brain. Each region has distinct principal cells that interact via the trisynaptic circuit. The major function associated with the hippocampus is the encoding of spatial memory. While each hippocampal region works in concert to process spatial information, recent evidence have highlighted individual roles for each region. This thesis focuses on the DG, which plays a distinct role in spatial pattern separation. The capacity for this role is heavily dictated by the physiology of DGCs, which are capable of receiving input from distal and proximal dendrites, but are generally inactive and fire few action potentials. Despite this sparse activity, excitation of DGCs is highly coupled to CA3 activity due to powerful synapses on CA3 pyramidal cells. Taken together, the physiological properties of DGCs suggest a unique role for the DG in spatial pattern separation.  !  15  1.2 The Hippocampus as a Site for Neuronal Plasticity The evidence associating hippocampal function with spatial memory has spurred researchers to examine this brain region for potential neuronal correlates for learning and memory. The resulting studies have highlighted the fact that the hippocampus is one of the most plastic regions in the brain, both at the synaptic and structural level. The following sections will further describe synaptic and structural plasticity, as well as evidence linking these forms of plasticity to learning and memory.  1.2.1 Synaptic Plasticity The term “synaptic plasticity” was first coined by the Polish neurophysiologist Jerzy Konorski, who hypothesized of activity-dependent, morphological changes at synapses being the basis for learning and memory (Konorski, 1948). Others later proposed the idea that activity-dependent changes between neurons may be the result of altering their strength of communication (reviewed in Markram et al., 2011); arguably, this concept was most eloquently stated in the book “The Organization of Behaviour.” In this book, the Canadian neuropsychologist Donald Hebb formulated a profoundly influential postulate regarding the neuronal mechanisms underlying learning and memory processes:  “When an axon of cell A is near enough to excite a cell B and repeatedly or persistently takes part in firing it, some growth process or metabolic change takes place in one or both cells such that A's efficiency, as one of the cells firing B, is increased.” - Hebb (1949)  !  16  The first experimental evidence of sets of neurons producing long-lasting changes to co-active neurons was demonstrated more than 30 years later. In 1973, Bliss and Lomo discovered that a brief amount of electrical stimulation, applied at a high frequency (high frequency stimulation; HFS), could induce a persistent increase in synaptic efficacy in the rabbit DG (Bliss and Lomo, 1973). The following sections will briefly discuss this form of synaptic plasticity, known as long-term potentiation (LTP), in addition to its close relative, long-term depression (LTD).  1.2.1.1 Long-term Potentiation 1.2.1.1.1 Mechanisms of Long-term Potentiation LTP can be defined as a persistent increase in synaptic efficacy, as a result of coactivation of synaptically connected neurons. Various forms of LTP exist in the hippocampus, but the most well characterized form employs HFS and involves two ionotropic glutamate receptors: the "-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and the NMDA receptor (Collingridge et al., 1983; Bliss and Collingridge, 1993). Excitatory synaptic transmission predominantly occurs via AMPA receptors (Andreasen et al., 1989). Most AMPA receptors are highly permeable to sodium and potassium (notably, a subset of AMPA receptors are also permeable to calcium) (Hollmann et al., 1991). In non-potentiated synapses, presynaptically released glutamate binds to postsynaptic AMPA receptors, temporarily depolarizing the postsynaptic neuron. This temporary depolarization, however, is insufficient to induce LTP.  !  17  The induction of LTP is tightly linked to the activity of the second type of ionotropic glutamate receptors, the NMDA receptor. This receptor is distinct from the AMPA receptor due to three main properties. First, the NMDA receptor requires a coagonist apart from glutamate; this can be either glycine, or the endogenous d-serine. Second, the pore of the NMDA receptor is blocked by magnesium ions under hyperpolarized conditions, but becomes unblocked when the postsynaptic neuron is depolarized. Third, in addition to sodium and potassium, the NMDA receptor is highly permeable to calcium (MacDermott et al., 1986; Cull-Candy and Leszkiewicz, 2004). The NMDA receptor and its kinetics will be extensively discussed in the following section (Section 1.3). LTP induction occurs when strong afferent stimulation, often HFS, sufficiently depolarizes the postsynaptic neuron, unblocking the NMDA receptor. Depolarization concurrent with glutamate release allows the NMDA receptor pore to open, permitting the sharp rise in postsynaptic intracellular calcium concentration that is necessary for LTP (Lynch et al., 1983; Yang et al., 1999) (Fig. 1.3). The expression and maintenance of LTP is ! Figure 1.3 Long-term potentiation An illustration outlining the molecular mechanisms underlying long-term potentiation (LTP).  !  !  complex, involving the activation of several protein kinases under various conditions; these kinases include protein kinase A (PKA) (Yasuda et al., 2003), protein kinase C (PKC) (Malinow et al., 1989; but  18  see Bortolotto and Collingridge, 2000), mitogen-activated protein kinase (MAPK) (English and Sweatt, 1997), and calcium-calmodulin-dependent kinase-II (CaMKII) (Giese et al., 1998). While the description of each kinase’s activity is beyond the scope of this thesis, many of these pathways appear to result in an increase in the number of AMPA receptors at the synapse (reviewed in Derkach et al., 2007). It should also be noted that NMDA receptors might also be trafficked to the synapse in response to LTP induction, albeit on a slower time scale. Evidence both supporting (Xiao et al., 1995; Watt et al., 2004) and refuting (Kauer et al., 1988; Muller and Lynch, 1988) this notion have been reported; therefore the role and mechanisms of NMDA receptor trafficking during synaptic plasticity remain unknown. The later phases of LTP are characterized by their dependency on protein synthesis (Frey et al., 1996; Nguyen and Kandel, 1996). A target for most intracellular cascades in synaptic plasticity at this stage is the phosphorylation (and activation) of cyclic-AMP response element binding protein (CREB). CREB binds to specific DNA sequences called cAMP response elements (CRE) (Lonze and Ginty, 2002), initiating the transcription of numerous synaptic plasticity-related genes (Dragunow, 1996; Davis et al., 2000). Late, protein synthesis-dependent forms of LTP were not examined in this thesis, and will not be further discussed.  1.2.1.1.2 Long-term Potentiation as a Model for Learning and Memory LTP has become an attractive neuronal mechanism for learning and memory processes, mostly because it shares many characteristics that we would expect of a neuronal candidate for these processes (reviewed in Shors and Matzel, 1997). The first  !  19  evidence that LTP may underlie learning and memory was produced by Richard Morris and colleagues, who demonstrated that the NMDA receptor antagonist 2-amino-5phosphonopentanoic acid (APV) was able to block the induction of LTP, as well as performance on the Morris Water Maze (Morris et al., 1986). Since then, hundreds of reports have provided correlative evidence for the role of LTP in learning and memory, with two of the most convincing studies produced in 2006. In the first, Whitlock et al. (2006) implanted a microelectrode array into the CA1 of awake rodents. After a single trial in an inhibitory avoidance task, rodents produced a 10% potentiation in a significant proportion of the microelectrode array; this was accompanied by phosphorylation of AMPA receptors at sites associated with synaptic AMPA receptor insertion. In the second study, Pastalkova et al. (2006) associated LTP with memory by using an inhibitor for the PKC isoform PKMzeta to reverse both long-term LTP and place avoidance memory, several hours after initially induced. Together, these results strongly implicate a link between LTP and learning and memory.  1.2.1.2 Long-term Depression 1.2.1.2.1 Mechanisms of Long-term Depression LTD, defined as a persistent decrease in synaptic efficacy as a result of coactivation of synaptically connected neurons, was first discovered by Gary Lynch and colleagues (Dunwiddie and Lynch, 1978). Two forms of LTD have been reported in the hippocampus, termed homosynaptic and heterosynaptic LTD (Christie et al., 1994; Bear and Abraham, 1996). Homosynaptic LTD occurs when synapses are directly activated, often using a prolonged amount of electrical stimulation, applied at a low frequency (low  !  20  frequency stimulation; LFS) (Dunwiddie and Lynch, 1978; Dudek and Bear, 1992). In contrast, heterosynaptic LTD occurs at inactive synapses. It is often induced when HFS, at a strength that can elicit LTP, is applied to a neighbouring pathway that converges on a single synaptic input (Levy and Steward, 1979). This form of LTD can be regularly induced in the DG, but is less prominent in the CA1 (Abraham and Goddard, 1983; Christie and Abraham, 1992a; Bear and Abraham, 1996) (discussed in 1.2.1.3.2). Heterosynaptic LTD was not investigated in this thesis. Much like LTP, various forms of LTD exist in the hippocampus and the most well characterized form also employs the AMPA receptor and calcium influx via the NMDA receptor. However, LTD is differentiated from LTP in several ways. First, calcium influx kinetics is critically different: a large and rapid rise in intracellular calcium results in LTP, while a small and prolonged rise in intracellular calcium results in LTD (Mulkey and Malenka, 1992; Christie et al., 1997; Yang et al., 1999). Second, LTD expression and maintenance does not involve kinases (Peineau et al., 2009). Rather, protein phosphatases, which have a higher calcium affinity in comparison to kinases, are preferentially activated in response to the small and prolonged rise in calcium (Lisman, 1989; Mulkey et al., 1993) (Fig. 1.4). During LTD, calcium binds protein phosphatase 2B (PP2B; also known as calcineurin), which in turn activates protein  !  ! Figure 1.4 Long-term depression An illustration outlining the molecular mechanisms underlying long-term depression (LTD).  !  21  phosphatase 1 (PP1) and 2A (PP2A). Both PP2B and PP1 are necessary for LTD (Mulkey et al., 1993; Mulkey et al., 1994). PP2B also has a number of other activities, which include dephosphorylation of AMPA receptors (Thiels et al., 1998) and the serine/threonine kinase, glycogen synthase kinase 3 (GSK-3) (Peineau et al., 2007). Third, LTD expression is accompanied by a reduction in the number of synaptic AMPA receptors (Collingridge et al., 2004; Fox et al., 2007). The low concentration of calcium has been found to induce AMPA receptor endocytosis via hippocalcin (Palmer et al., 2005) and PICK1 (Lu and Ziff, 2005). In summary, LTD induction via LFS produces a slow, prolonged influx of calcium, activating protein phosphatases and intracellular cascades that lead to AMPA receptor endocytosis.  1.2.1.2.2 Links Between Long-term Depression and Learning and Memory The role of LTD in spatial learning and memory processes is still unclear. Some have suggested that LTD could reset potentiation after exploration, novelty or stress. This idea is supported by the observation that behavioural stress promotes LFS-induced LTD (Xu et al., 1997). LTD may also be involved in novel object exploration when entering new environments, as LFS-induced LTD was facilitated when delivered during the active exploration of a novel object in an unfamiliar environment, but not during active exploration of the unfamiliar environment alone (Kemp and Manahan-Vaughan, 2004). Studies in mutant mice also associate LTD with behavioural flexibility: the ability to change a learned behaviour based on task variations (Collingridge et al., 2010). For example, mice lacking forebrain expression of PP2B show impaired LTD in the CA1; these mice could learn the location of a hidden platform in the standard version of the  !  22  Morris water maze, but were unable to learn the location of a newly relocated hidden platform (Zeng et al., 2001). While the specific contribution of LTD to learning and memory is still unknown, converging evidence suggests that LTD is indeed associated with these cognitive processes.  1.2.1.3 Unique Synaptic Plasticity in the Dentate Gyrus Much of the work described in the preceding sections was conducted in the CA1. While this work has contributed to our understanding of synaptic plasticity, several reports have suggested that the mechanisms governing synaptic plasticity in the DG may be significantly different.  1.2.1.3.1 The Medial Perforant Path-Dentate Granule Cell Synapse The DG demonstrates significant levels of synaptic plasticity at both the MPP and LPP synapses (MPP-DGC and LPP-DGC synapses, respectively). The MPP-DGC synapses show the most similarities in synaptic plasticity with the CA1, but numerous differences are present. First, the MPP-DGC synapse demonstrates a short-term form of synaptic plasticity called paired pulse depression, which is observed when a pathway is stimulated twice in quick succession, and the size of the second response is significantly smaller than the first (McNaughton, 1980; Colino and Malenka, 1993; but see Petersen, 2009). This is in contrast to paired pulse facilitation (where the second response is larger than the first), which is observed in the LPP-DGC and CA1 synapses (McNaughton, 1980; Colino and Malenka, 1993). Second, like the CA1, HFS-induced LTP at the MPPDGC synapse has been shown to be dependent on NMDA receptors, although unlike the  !  23  CA1, additional HFS can result in increased potentiation that is dependent on metabotropic glutamate receptors and intracellular calcium stores (Wu et al., 2008). Third, while LTP expression in the CA1 requires active CaMKII, LTP at the MPP-DGC synapse uses independent, but parallel signaling cascades involving CaMKII and PKA/MAPK (Cooke et al., 2006; Wu et al., 2006). Forth, LFS-LTD at the MPP-DGC synapse may not be solely dependent on NMDA receptors, as voltage-gated calcium channels have also been shown to contribute to LTD in some studies (Wang et al., 1997), but not others (Vasuta et al., 2007; Eadie et al., 2012). Fifth, in vitro LTP induction at MPP-DGC (and LPP-DGC) synapses requires a GABAA antagonist, suggesting that the DG is more extensively inhibited by GABAergic interneurons than the CA1 (Wigstrom and Gustafsson, 1983) (discussed in Chapter 2). The MPP-DGC synapse is the primary focus of this thesis.  1.2.1.3.2 The Lateral Perforant Path-Dentate Granule Cell Synapse The LPP-DGC synapse demonstrates synaptic plasticity that is distinct from that observed in both the CA1 and the MPP-DGC. For example, opioid receptors contribute to LTP at the LPP-DGC synapses, but not in the CA1 (Matthies et al., 2000), or the MPPDGC (Bramham et al., 1988). In addition, less LTP is observed in the LPP-DGC synapse in vitro, in comparison to the MPP-DGC (Colino and Malenka, 1993) – this may in part be due to the lower AMPA to NMDA receptor ratio observed at the LPP-DGC synapse (Laplagne et al., 2006). Unlike other hippocampal synapses, heterosynaptic LTD is regularly observed at LPP-DGC synapses (Abraham and Goddard, 1983; Christie and Abraham, 1992a), while homosynaptic LTD at LPP-DGC synapses requires a  !  24  priming stimulation paradigm, followed by an associative stimulation protocol (where single stimuli applied to the LPP, interleaved between MPP stimulation) (Christie and Abraham, 1992b). Taken together, these observations highlight the unique physiology of the DG and caution against generalizing synaptic plasticity mechanisms discovered in the CA1.  1.2.2 Structural Plasticity One well-recognized property of memory is that it is long lasting. While changes in synaptic plasticity persist for prolonged periods of time (Abraham et al., 2002), it is now believed that long-term modifications of the hippocampal circuitry must at least accompany synaptic changes in order to produce long-term memory. One form of structural plasticity is neurogenesis, the process by which functional neurons are generated from neural stem cells or progenitor cells. Although initially thought to occur only during development, it is now well established that neurogenesis continues to take place in certain restricted regions of the mammalian brain, including the DG (Aimone et al., 2006). Since its initial discovery in rodents (Altman and Das, 1965; Kaplan and Hinds, 1977) and later in humans (Eriksson et al., 1998), adult neurogenesis has redefined how researchers picture the adult brain. The following section will introduce the mechanisms underlying hippocampal adult neurogenesis, as well as its association with learning and memory. Furthermore, other forms of structural plasticity will also be presented.  !  25  1.2.2.1 Mechanisms of Hippocampal Adult Neurogenesis Significant research has been conducted to characterize the development of adultborn DGCs; for the purpose of this thesis, adult neurogenesis has been grouped into four distinct stages: the precursor stage, immature stage, late immature stage, and mature stage (Fig. 1.5). The precursor stage is marked by the transition between four types of proliferating precursor cells: the radial glia-like neural stem cells (Type 1), the transiently amplifying cells (Type 2a and 2b), and the transitionary cells (Type 3). These cells, with the exception of some Type 3 cells, reside in the SGZ. While each cell has the capacity for proliferative activity, the Type 2 cells make up the majority of the dividing cells in the DG (Ehninger and Kempermann, 2008). All cells at the precursor stages can be easily  ! Figure 1.5 Mechanisms of adult neurogenesis in the dentate gyrus Stages of DG adult neurogenesis (Upper Panel) and approximate expression pattern of endogenous markers (Lower Panel). The first stage of adult neurogenesis is the precursor stage, comprised of radial glia-like stem cells (Type 1), transiently amplifying cells (Type 2a and 2b) and transitionary cells (Type 3); these precursor cells reside in the subgranular zone (SGZ). Many precursor cells differentiate into immature neurons during the immature stage, and begin to migrate into the inner granular zone (IGZ). Immature cells transition into late immature cells, where they extend dendrites into the molecular layer and undergo spinogenesis. As late immature cells mature into mature cells, they migrate into the outer granular zone (OGZ) and undergo further dendritic arbourization. DCX: Doublecortin, IGZ: Inner Granular Zone, NeuN: Neuronal Nuclei, NeuroD: Neurogenic Differentiation Protein, OGZ: Outer Granular Zone, PCNA: Proliferating Cell Nuclear Antigen, SGZ: Subgranular Zone  ! !  26  identified using immunohistochemistry for common proliferative markers, including the exogenous mitotic marker bromodeoxyuridine (BrdU), and the endogenous cell cycle markers Ki-67, and proliferating cell nuclear antigen (PCNA) (Kee et al., 2002; Christie and Cameron, 2006). The immature stage is characterized by the acquisition of a neuronal phenotype. The vast majority (>80%) of adult-born cells take on this neuronal phenotype (Cameron et al., 1993), and, approximately one or two weeks post-mitosis, these new neurons migrate from the SGZ to the most adjacent regions of the GCL, called the inner granular zone (IGZ). At this stage, neurons sprout axons to the CA3 regions (Faulkner et al., 2008), followed by the extension of a single primary apical dendrite into the DG molecular layer (Zhao et al., 2006). These neurons can be identified due to their strong expression of differentiation markers, including doublecortin (DCX) (Brown et al., 2003b; Rao and Shetty, 2004) and neurogenic differentiation protein (NeuroD) (Brunet and Ghysen, 1999; Miyata et al., 1999), and weak expression of the neuronal marker Neuronal Nuclei (NeuN) (Mullen et al., 1992; Esposito et al., 2005). The late immature stage marks a critical time window of approximately three to eight weeks post-mitosis. Late immature neurons, with somas still located in the IGZ, form dendritic arbours that span the full extent of the molecular layer (Zhao et al., 2006). Approximately 16 days post-mitosis, spine formation commences (Zhao et al., 2006). Neurons at this stage are highly excitable (Mongiat et al., 2009), have a lower threshold for LTP (Schmidt-Hieber et al., 2004), and have greater potentiation in comparison to fully mature cells (Ge et al., 2008). Immunohistochemically, these cells can be identified  !  27  due to their high expression of NeuN (Snyder et al., 2009), while still expressing differentiation markers (Schmidt-Hieber et al., 2004). The final stage in adult neurogenesis is the mature stage. At this stage, neurons have migrated to the outer region of the granule cell layer, named the outer granular zone (OGZ). Neurons extend the full extent of their dendritic arbors and may undergo spine pruning (discussed in 1.2.2.3). Adult born DGCs at this stage are indistinguishable, both morphologically and physiologically, from DGCs born during development (Zhao et al., 2008).  1.2.2.2 Links Between Adult Neurogenesis in the Dentate Gyrus and Learning and Memory Adult neurogenesis in the DG has been tightly linked to learning and memory processes. Many factors that positively influence learning and memory, such as environmental enrichment and voluntary exercise, also increase adult neurogenesis (Kempermann et al., 1997; van Praag et al., 1999b). Conversely, factors that negatively influence learning and memory, such as stress, decrease adult neurogenesis (Cameron and Gould, 1994; Gould et al., 1997). Interestingly, behavioural examinations of the adult neurogenesis function have been largely inconsistent. This has been particularly true for studies using the Morris water maze and contextual fear conditioning (Shors et al., 2002; Snyder et al., 2005; Meshi et al., 2006; Saxe et al., 2006; Winocur et al., 2006). Several factors seem to underlie these contradictory findings. One factor appears to be the behavioural task itself. Performance in the Morris water maze task may not be sensitive enough to reveal impairments in DG adult neurogenesis. In fact, recent studies  !  28  have demonstrated that the traditional parameters examined during the Morris water maze (i.e., latency to find the hidden platform) may not reflect changes in adult neurogenesis, and that a detailed analysis of the path strategies the animals adopt in order to find the platform is a more sensitive approach to detect neurogenesis-related alterations in learning and memory (Garthe et al., 2009; Stone et al., 2011, unpublished work from our laboratory). Another important factor is the specific age of the adult-born neurons being examined. Two elegant studies have recently demonstrated that the Morris water maze task selectively promotes the survival of new cells that are approximately one week old, while inducing apoptosis in younger cells (Dupret et al., 2007; Epp et al., 2007). What function do newborn neurons have in the DG? One theory proposes that adult neurogenesis may be critical for pattern integration, which refers to the act of associating differing patterns, or events. This may seem counterintuitive, due to the role of the DG in pattern separation (discussed in 1.1.4.1). However, the unique physiology of late immature DGCs appears to be the key. These highly excitable neurons are preferentially activated during the acquisition of new patterns (Kee et al., 2007), producing a population of neurons that is encoded at the same time (Aimone et al., 2010). However, since the number of newborn neurons is small in comparison to the total number of DGCs (Cameron and McKay, 2001; Snyder et al., 2009), the process of pattern integration provides minimal interference in pattern separation. Another proposed role for adult neurogenesis is hippocampal memory displacement. Specifically, this refers to the theory that adult-born neurons may help displace memories that are initially formed in the hippocampus to other, nonhippocampal regions. This theory was recently supported by Kitamura and colleagues,  !  29  who assessed the contribution of adult neurogenesis in the retrieval of recent and old memories (Kitamura et al., 2009). They discovered that reducing neurogenesis extended the amount of time a contextual fear memory was dependent on the hippocampus, while the opposite was true when neurogenesis was increased. This suggests that adult neurogenesis may function to transfer memories out of the hippocampus.  1.2.2.3 Other Forms of Structural Plasticity: Could Dendritic Morphology Contribute to Learning and Memory? Dendrites and dendritic spines receive excitatory information and propagate synaptic potentials to the soma. These neuronal structures, however, are not simple conduits. Instead, integration of synaptic potentials also occurs at this level, and this integration is greatly influenced by spine density and the morphology of the dendritic tree (Tsay and Yuste, 2004; Gulledge et al., 2005). Modulation of dendritic morphology is therefore a unique form of structural plasticity. In the adult DG, immature DGCs first extend single, non-spiny apical dendrites into the molecular layer. Dendritic arbours become significantly more elaborate as cells age, showing increases in the number of primary dendrites and dendritic complexity (Redila and Christie, 2006; Zhao et al., 2006). Spinogenesis occurs soon after the commencement of dendritic arbourization. While one study has suggested that spine density steadily increases in DGCs as they mature (Zhao et al., 2006), recent models of spinogenesis indicate that an excessive number of spines are initially produced, followed by a reduction, or pruning, of spine density (Yuste, 2010, pg 85-87).  !  30  How does dendritic morphology contribute to learning and memory in the DG? This has been difficult to determine as factors that induce changes in dendritic morphology often produce concurrent changes in adult neurogenesis. For example, both voluntary exercise and Morris water maze training increase dendritic complexity and spine density in DGCs, but both also increase cell proliferation (Eadie et al., 2005; Tronel et al., 2010). Nevertheless, due to their ability to modify synaptic potential integration prior to reaching the soma, changes in dendritic morphology have the potential to contribute to learning and memory.  1.2.3 Summary of Hippocampal Plasticity The hippocampus is a highly plastic structure, with the capacity to change the strength of synaptic transmission, and the structure of its circuitry. Both forms of plasticity can profoundly impact how the hippocampus functions and contribute to learning and memory. This thesis examines synaptic and structural plasticity in the adult DG.  !  31  1.3 The NMDA Receptor – a Molecular Substrate for Hippocampal Plasticity The NMDA receptor has long garnered interest due to its close association with hippocampal synaptic plasticity and learning and memory (Morris et al., 1986). Other neuronal mechanisms involving this receptor have also been revealed, including roles in hippocampal structural plasticity (Cameron et al., 1995). Together, these findings place the NMDA receptor as a molecular substrate for plasticity mechanisms in the hippocampus. The following sections will describe the structure and expression of the NMDA receptor in detail.  1.3.1 The Overall Structure of the NMDA Receptor The NMDA receptors are a family of ionotropic glutamate receptors composed of four subunits. Each subunit has four major functional domains (Fig 1.6). The first domain, known as the N-terminus domain, is an extracellular, clam-shaped region that contains a leucine/isoleucine/valine-binding protein (LIVBP)-like segment; this segment acts as the action site for several non-competitive NMDA receptor antagonists (PerinDureau et al., 2002). The second domain, the agonist binding domain, is another clamshaped region that is formed by the S1 and S2 extracellular segments and binds either glutamate or glycine. The third domain is the transmembrane domain, composed of three transmembrane regions (TM1, TM3, TM4), and one re-entry loop (M2). The re-entry loop of each subunit forms a channel pore structurally similar to an inverted potassium channel. In contrast to potassium channels, the pore region of the NMDA receptor lacks an inherent selectivity filter; instead a Q/R/N site at the apex of the M2 loop determines  !  32  !  ! Figure 1.6 Structure of the NMDA receptor The heterodimeric complex of the NMDA receptor (Left) is composed of two obligatory GluN1 subunits (grey) and two regulatory GluN2 or GluN3 subunits (red). Diagram of individual GluN subunits (Right).  ! the receptor’s cation selectivity. The forth domain is an intracellular C-terminus domain, that has the ability to bind post-synaptic density proteins (Paoletti and Neyton, 2007). While sharing many similarities with other ionotropic glutamate receptors, the NMDA receptor has several unique properties (reviewed in Cull-Candy and Leszkiewicz, 2004; Paoletti and Neyton, 2007). First, the receptor requires con-current binding of glutamate and glycine (or d-serine) prior to activation. Binding of these neurotransmitters to the agonist binding domain produces a conformational change in the TM3 region that opens the channel pore. A second unique property is that the NMDA receptor is not intrinsically voltage sensitive, but exhibits voltage dependence due to extracellular magnesium ion blocking of the channel pore under basal (hyperpolarized) conditions. Third, the NMDA receptor is unique in that it is more than ten times more permeable to calcium than sodium or potassium (Sharma and Stevens, 1996).  !  33  1.3.2 The Different Subunit Types of the NMDA Receptor The NMDA receptor itself is a heterodimer complex, composed of two obligatory GluN1 (formerly NR1) subunits, and two regulatory GluN2 (formerly NR2) or GluN3 (formerly NR3) subunits. A concise description of each subunit type is provided. The focus of this thesis is on the role of the GluN2 subunits; therefore, the GluN1 and GluN3 subunit families will only be briefly described.  1.3.2.1 The Obligatory GluN1 Subunit The GluN1 subunit is a necessary component of every NMDA receptor, required for the assembly and trafficking of functional NMDA receptors to the cellular membrane (Cull-Candy and Leszkiewicz, 2004). GluN1 contains a small agonist binding domain that binds glycine (or the endogenous ligand d-serine) (Paoletti and Neyton, 2007). While GluN1 has only one form and is encoded by a single gene, it obtains diversity via eight splice variants (Paoletti and Neyton, 2007). GluN1 subunits can vary at three different regions (one site in the N-terminus domain, and two sites in the C-terminus domain), which can induce subtle changes to the NMDA receptor’s agonist potency and sensitivity to protons, zinc, and polyamines (Cull-Candy and Leszkiewicz, 2004).  1.3.2.2 The Regulatory GluN2 Family The GluN2 subunit family is comprised of four members (GluN2A-D, previously NR2A-D), each encoded by its own gene. Unlike GluN1, these regulatory subunits bind glutamate to their agonist binding domain (Cull-Candy and Leszkiewicz, 2004). This family of subunits has been of particular interest to researchers as each subunit imparts  !  34  distinct physiological properties to the receptor; some of these properties are summarized in Table 1.1. NMDA Receptor Properties Conductance (Main/Sub) (pS) Glutamate Affinity (Kd; nM) Glutamate Potency (EC50 of NMDAR response, µM) Peak Open Probability (ms) Calcium Permeability (normalized to Cesium Permeability) Mg2+ Unblock Time Constant (ms) Mg2+ Block Potency (IC50, µM) Decay Time Constant (ms)  GluN2A 50/38 102 3.3  GluN2B 50/38 51 1.3  GluN2C 38/18 208 0.8  GluN2D 38/18 117 0.35  Ref. 1-3 4 5  0.35-0.5 7.6  0.01-0.07 7.2  0.01 5.1  0.01 4.6  5,6 7  0.46 2.4 50-100  0.69 2.1 250-420  0.28 14.2 250-310  0.25 10.2 1500-4000  8 9 10  Table 1.1 Kinetics of GluN2 subunits. [1] Cull-Candy and Leszkiewicz (2004), [2] Brimecombe et al. (1997), [3] Momiyama et al. (1996), [4] Laurie and Seeburg (1994), [5] Yuan et al. (2009), [6] Chen et al. (1999), [7] Retchless et al. (2012), [8] Clarke and Johnson (2006), [9] Kuner and Schoepfer (1996), [10] Vicini et al. (1998)  Each subunit is structurally very similar, although differences have been observed in three key regions: 1) a segment that spans the N-terminus domain and linker region (between the N-terminus domain and the agonist binding domain) determines open probability (Gielen et al., 2009), 2) a single residue at the third transmembrane region which determines calcium permeability, channel conductance and magnesium block potency (Retchless et al., 2012), and 3) the C-terminus region which binds different intracellular proteins (Yashiro and Philpot, 2008) but generally does not alter receptor kinetics (Punnakkal et al., 2012). The unique subunit properties listed in Table 1.1 allude to distinct characteristics for GluN2 subunit-containing receptors. For example, GluN2A-containing receptors have a low affinity for glutamate, but a higher glutamate potency, and faster overall kinetic properties; these receptors appear to be optimally designed for quick responses to synaptic activity (Fig. 1.7). In contrast, GluN2B-containing receptors have a high affinity to glutamate, but a lower glutamate potency and significantly slower kinetics; this  !  35  Figure 1.7 Differential NMDA receptor current attributed to GluN2 subunits Diheteromeric NMDA receptors containing GluN1 and either GluN2A (blue), GluN2B (green), GluN2C (orange) or GluN2D (purple) subunits, with corresponding NMDA receptor-mediated currents (1 ms pulse of 1 mM glutamate applied to HEK-293 cells transfected with distinct NMDA receptor subunit cDNAs ) Approximate decay time constants for receptors: GluN1/2A: 50 ms, GluN1/2B: 250 ms, GluN1/2C: 250 ms, GluN1/2D: 1.7 s (© The American Physiological Society, Vicini et al. (1998), adapted by permission)!  ! !  would indicate that GluN2B-containing receptors might be preferentially activated during slower, prolonged synaptic activity (Erreger et al., 2005). These properties suggest that GluN2 subunits may have distinct functional roles in various neuronal mechanisms, including synaptic plasticity (discussed in Chapter 4).  1.3.2.3 The Regulatory GluN3 family The GluN3 family (GluN3A-B, previously NR3A-B) is a relatively newly explored group of NMDA receptor subunits. Like GluN1, GluN3 subunits bind to glycine (with a lower affinity for D-serine) at their agonist binding domain, and functionally transform NMDA receptors to glycine-activated receptors (Cavara and Hollmann, 2008). The subunit itself is unique at two key amino acid sites, and this difference is thought to lower magnesium sensitivity and calcium permeability in GluN3-  !  36  containing receptors (Cavara and Hollmann, 2008). The role of GluN3 subunits was not investigated in this thesis, and is only minimally discussed further.  1.3.3 The Temporal Expression Patterns of the NMDA Receptor Subunits in the Hippocampus NMDA receptor expression begins at approximately the same time as formation of the hippocampus. From embryonic day 17 (E17), and for the remainder of the embryonic period, GluN1 mRNA is strongly expressed in the hippocampus and GluN2B mRNA is expressed shortly after (E19) (Monyer et al., 1994). In contrast, mRNA for GluN2A and GluN2D are expressed during postnatal stages (approximately postnatal day zero (P0) and P7, respectively) (Monyer et al., 1994). In regards to protein expression, the GluN1 levels are strong at P0 (Wenzel et al., 1997). GluN2B protein is found at moderate levels at P0, with expression peaking and remaining constant at P10 (Wenzel et al., 1997). In contrast, GluN2A protein levels are weakly detected at P0, but steadily increase until it shares equally strong expression with GluN2B at P21 (Wenzel et al., 1997). Other subunits that are expressed in the hippocampus include GluN2D and GluN3A, which show weak and moderate expression in the adult hippocampus, respectively (Wenzel et al., 1997). The temporal expression pattern of GluN2 subunits during DG adult neurogenesis is discussed in Chapter 5.  !  37  1.3.4 Summary of the NMDA Receptor and its Subunits The NMDA receptor is a heteromeric complex composed of several different subunit families that have the capacity to modify the function of the receptor. The GluN2 subunits in particular drastically alter the kinetics of the NMDA receptor. This observation, in conjunction with the unique temporal expression patterns of these subunits, suggests that each GluN2 subunit may play its own unique role in different NMDA receptor-dependent mechanisms.  !  38  1.4 The GluN2A-/- Mouse Model The experiments described in this thesis utilize the GluN2A-/- mouse model, a mutant mouse lacking global expression of the GluN2A subunit. A brief overview of literature utilizing this mouse model is provided below, focusing on research pertaining to the hippocampus.  1.4.1 Generation of the GluN2A-/- Mouse Model The GluN2A-/- mouse model was first generated in the laboratory of Dr. Masayoshi Mishina (University of Tokyo) in 1995. Homogenous GluN2A mutant mice were developed by transfecting embryonic stem cells with a targeting vector directed at a segment of the Grin2A gene that encodes the second and third transmembrane region of the GluN2A subunit (Sakimura et al., 1995). The first report of these mice used in situ hybridization to demonstrate the absence of GluN2A mRNA without disrupting the mRNA of other NMDA receptor subunits (Sakimura et al., 1995). Furthermore, CA1 pyramidal cells in mutant mice demonstrated a 50% reduction in NMDA receptormediated current (Sakimura et al., 1995; Ito et al., 1996). While disruption of other NMDA receptor subunits produced lethal phenotypes in mutant mice (Li et al., 1994; Kutsuwada et al., 1996), GluN2A-/- mice demonstrated normal development and breeding, as well as intact brain morphology (Sakimura et al., 1995). The viability of the GluN2A-/- mice has been attributed to the late expression pattern of the subunit: as previously discussed, the GluN2A subunit is marginally expressed prenatally, and therefore may produce minimal effects during development.  !  39  1.4.2 Altered Long-term Potentiation Threshold in the CA1 of GluN2A-/- Mice The first characterizations of the GluN2A-/- mouse examined LTP in the CA1 region. HFS-induced LTP was significantly reduced by approximately 75% in mutant mice (Sakimura et al., 1995), despite the retention of synaptic GluN2B-containing receptors (Thomas et al., 2006). It was later demonstrated that this deficit in LTP could be restored by applying stronger HFS (Kiyama et al., 1998), indicating that GluN2A-/mice have a disrupted threshold for LTP. Surprisingly few reports have examined hippocampal LTD in the GluN2A-/- mouse line, however a recent study observed normal LTD in mutant mice (Longordo et al., 2009). Overall, these experiments suggest that the GluN2A subunit may be preferentially involved in LTP in the CA1. It should be noted that the disrupted LTP threshold in the CA1 of GluN2A-/- mice could be attributed to alterations in presynaptic mechanisms. While early studies demonstrated intact basal excitatory transmission and paired-pulse facilitation in GluN2A-/- mice (Sakimura et al., 1995), presynaptic GluN2A-containing receptors may be present at these synapses (Suarez and Solis, 2006). Presynaptic GluN2A-containing receptors have the capacity to increase axon excitability and glutamate release; therefore, a loss of presynaptic receptors in GluN2A-/- mice could lower axonal excitability and raise the threshold for LTP. Other synapses in the hippocampus have also been investigated in the GluN2A-/mouse. Reduced LTP has been observed at the synapse between the commissural/associational fibers and the CA3 pyramidal cells, while LTP is intact at the synapse between the fimbria and the CA3 pyramidal cell (Ito et al., 1997; Ito et al., 1998). These results present the possibility that the role of GluN2A at the CA1 may not  !  40  be identical at other hippocampal synapses. To date, there have been no studies investigating synaptic plasticity in the DG of GluN2A-/- mice.  1.4.3 Impaired Long-term Potentiation in Other Brain Regions In addition to the hippocampus, synaptic plasticity in the GluN2A-/- mouse model has been investigated in the visual superior colliculus. Superior colliculus neurons from GluN2A-/- mice had significantly smaller NMDA receptor-mediated currents, impaired LTP, and intact LTD (Zhao and Constantine-Paton, 2007). Overall, results in the superior colliculus correspond with those seen at the CA1, indicating a prominent role for GluN2A-containing receptors in LTP, but not LTD.  1.4.4 Unknown Capacity for Structural Plasticity in the GluN2A-/- Mouse While numerous studies have examined synaptic plasticity in this GluN2A-/mouse, only a single study has examined hippocampal structural plasticity: a report examining DG adult neurogenesis (Kitamura et al., 2003). This report used the exogenous marker BrdU, and demonstrated no alterations in cellular proliferation in GluN2A-/- mice. It was also shown that the proportion of BrdU cells (quantified four weeks after BrdU injection) co-expressing either the neuronal marker NeuN, or the glial marker glial fibrillary acidic protein (GFAP) was unchanged in mutant mice, suggesting that neuronal maturation and survival was also intact. Despite these results, several questions remain unanswered. First, the use of BrdU in adult neurogenesis experiments has often been questioned due to its capacity to label cells undergoing DNA repair or apoptosis and concerns over BrdU toxicity (reviewed in Kempermann, 2006). While  !  41  most of these concerns have been addressed, it has become common for adult neurogenesis experiments to employ additional endogenous markers of cellular proliferation. Endogenous markers have the additional advantage of being expressed throughout all active phase of the cell cycle, and therefore offer a more complete picture of the entire progenitor pool at any given time; this is in contrast to BrdU, which is only incorporated during the S-phase (Christie and Cameron, 2006). Second, the dendritic structure of new neurons has not been examined. Third, it is unclear if GluN2A plays a role in spinogenesis in these newly generated neurons.  1.4.5 Compromised Hippocampal-Related Behaviour in GluN2A-/- Mice Overall, GluN2A-/- mice are physically healthy. They show normal behavioural habits in empty cage conditions, intact sensory reflexes, and standard neurological motor functions (Sakimura et al., 1995; Boyce-Rustay and Holmes, 2006). Locomotor activity in home cage, open field and elevated plus maze conditions is also unaffected in mutant mice (Boyce-Rustay and Holmes, 2006). However, several reports have indicated that mutant mice demonstrate deficits in hippocampal-dependent tasks. The first study characterizing the GluN2A-/- mouse reported reduced performance during the Morris water maze (Sakimura et al., 1995). Furthermore, mutant mice demonstrated less freezing during the hippocampal dependent short-term contextual fear task, while performance on the hippocampal-independent tonedependent fear conditioning task was normal (Kiyama et al., 1998). GluN2A-/- mice also show a significant emotion-related phenotype. Specifically, mutant mice demonstrate reduced anxiety-like behaviour in the elevated plus-maze, light-  !  42  dark box exploration, and open field tasks (Boyce-Rustay and Holmes, 2006). Mutant mice also demonstrate antidepressant-like behaviours, as assessed by the forced swim and tail-suspension tests (Boyce-Rustay and Holmes, 2006). Notably, it is unclear whether these behavioural changes are due to the absence of the GluN2A subunit specifically, as GluN2A-/- mice have increased serotonergic and dopaminergic activity in the frontal cortex (Miyamoto et al., 2001).  1.4.6 Summary of the GluN2A-/- Mouse Model Since its generation in 1995, the Glun2A-/- mouse model has provided extensive insight into the role of the NMDA receptor subunits in synaptic plasticity, supporting the role of GluN2A in LTP, but not LTD. However, most experiments to date have focused on the CA1 region and evidence from other hippocampal synapses suggests that the specific contribution of GluN2A to LTP may not be uniform across the hippocampus. In contrast to synaptic plasticity, very little is known about the role of the GluN2A subunit in structural plasticity. In the DG, adult neurogenesis appears to be unaltered, although it is unknown whether the GluN2A subunit alters the later stages of adult neuronal development, specifically dendritic formation and spinogenesis. This thesis will address several of these questions by examining synaptic, structural and behavioural plasticity in the DG of the GluN2A-/- mouse.  !  43  1.5 Summary The hippocampus, a brain region associated with learning and memory processes, is both anatomically and functionally divided into several regions. One region of particular interest is the DG, the first region of the hippocampal trisynaptic circuit. The hippocampus, and by extension the DG, is highly plastic. The DG in particular demonstrates robust synaptic plasticity, in addition to two separate but related forms of structural plasticity: adult neurogenesis and dendritic morphological plasticity. One molecular candidate that appears to be involved in each of these forms of plasticity is the NMDA receptor. This receptor is composed of several obligatory and regulatory subunits. The most highly expressed regulatory subunits in the hippocampus are GluN2A and GluN2B, each of which can distinctly modify the physiology of the receptor itself. Differences in kinetics, temporal expression and spatial location have contributed to the hypothesis that GluN2A- and GluN2B-containing receptors may uniquely contribute to different forms of plasticity. This thesis examines the role of the GluN2A subunit in synaptic and structural plasticity by using the GluN2A-/- mouse model. Previous work has shown that GluN2A-/mice have an altered LTP threshold and intact LTD in the CA1; however, it is unknown if synaptic plasticity is altered in the DG of these mice. In addition, it is unclear whether the neuronal development of adult-born neurons is intact in the DG of GluN2A-/- mice. Finally, while overall deficits in hippocampal function have been observed in GluN2A-/mice, it is unknown whether behavioural deficits associated with individual hippocampal regions are affected.  !  44  1.6 Hypothesis Our overall hypothesis is: Loss of GluN2A-containing NMDA receptors produces synaptic and structural plasticity deficits, and impairs information processing in the adult DG.  Our specific hypotheses are as followed: Synaptic Plasticity 1. Mature DGCs from GluN2A-/- mice have normal AMPA-mediated currents, but reduced NMDA currents, resulting in a decrease NMDA:AMPA ratio. 2. Mature DGCs from GluN2A-/- mice have NMDA receptor-mediated currents with a prolonged decay rate time constant. 3. GluN2A-/- mice retain intact presynaptic properties in the adult DG. 4. GluN2A-/- mice show impairments in LTP in the adult DG. 5. GluN2A-/- mice retain intact LTD in the adult DG. Structural Plasticity 1. GluN2A-/- mice have intact cell proliferation and neuronal differentiation in the adult DG. 2. Mature DGCs from GluN2A-/- mice show decreases in dendritic length. 3. Mature DGCs from GluN2A-/- mice show decreases in dendritic complexity. 4. Mature DGCs from GluN2A-/- mice show increases in dendritic spine density. Behavioural Plasticity/Information Processing 1. Adult GluN2A-/- mice show impairments in spatial pattern separation. 2. Adult GluN2A-/- mice show impairments in temporal pattern separation.  !  45  2 Requirement of GABAA Receptor Antagonism for Induction of in vitro Long-term Potentiation in the Mouse Dentate Gyrus1 2.1 Introduction The dentate gyrus (DG) was the first region in the hippocampus to demonstrate long-term potentiation (LTP), a leading candidate mechanism for learning and memory processes (Bliss and Lomo, 1973; Martin et al., 2000). Following the discovery of LTP, several experimental techniques were developed and refined to investigate synaptic plasticity; the most prominent of which was the in vitro hippocampal slice method. This in vitro method allows for the reliable induction of LTP in the CA regions of the hippocampus. The DG, however, is suggested to be more heavily inhibited by interneurons than its neighboring hippocampal regions, and is also subject to complex feed-forward and feedback circuitry (Coulter and Carlson, 2007). Correspondingly, an early study by Wigstrom and Gustafsson suggested that the successful induction of LTP in the DG required inhibition of the GABAA receptor (Wigstrom and Gustafsson, 1983). It has since been common to block GABA inhibition with a GABAA receptor antagonist during studies of in vitro LTP in the DG, however there is little consensus between laboratories as to the optimal degree of disinhibition required. Studies either use minimal (i.e. 1 µM (Vasuta et al., 2007)), moderate (i.e. 3-5 µM (Garthe et al., 2009; Eadie et al., 2012)), or maximal (i.e. >10 µM (Yu et al., 2003)) concentrations of the specific GABAA receptor antagonist bicuculline methiodide (BMI) to block the GABAA receptor for LTP studies in the DG. This is an important methodological setting to consider as using a !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 1 A version of this chapter has been submitted for publication. Kannangara, T.S., Helfer, J.L., Cater, R.M., and Christie, B.R. (2012). Requirement of GABAA receptor antagonism for induction of in vitro long-term potentiation in the rodent dentate gyrus. !  46  suboptimal amount of disinhibition may mask subtle differences in LTP between experimental groups. In the current study, we systematically altered the concentration of GABAA receptor antagonism and examine the effects of disinhibition on post-tetanic potentiation (PTP) and LTP in the C57/Bl6 mouse, a species widely used for electrophysiological studies.  2.2 Material and Methods Adult (2-4 month) male C57/Bl6 mice were socially housed in standard cages and maintained on a 12-hour light/dark cycle with access to food and water ad libitum. All procedures were approved by the University of Victoria Animal Care Committee and in accordance with the Canada Council on Animal Care. Transverse hippocampal slices (350 µm) were generated as previously described (Eadie et al., 2012). Briefly, animals were deeply anesthetized and brains were removed and immersed in ice-cold, oxygenated artificial cerebrospinal fluid (ACSF) containing (in mM): 125.0 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25.0 NaHCO3, 2 CaCl2, 1.3 MgCl2, and 10 dextrose. Slices generated from a Vibratome Sectioning System 1500 (Ted Pella, Redding, CA) were gently place in incubation chambers filled with oxygenated ACSF and maintained at 30oC for a minimum of 1 hour. Hippocampal slices were transferred to recording chambers and superfused with oxygenated ACSF (1-2 mL/min). Using an upright Olympus microscope (BX50W1 or BX51W1), concentric bipolar stimulating electrodes (FHC, Bowdoin, ME) and borosilicate recording electrodes (0.7-1.5 M#) filled with ACSF were placed in the medial aspect of the DG molecular layer. Field excitatory postsynaptic potentials  !  47  (fEPSPs) were evoked using monophasic negative current pulses supplied via a digital stimulus isolation unit (Getting Instruments, San Diego, CA). Responses were acquired at 100 kHz using either a MultiClamp 700B (Molecular Devices) or BVC-700A (Dagan Corporation) amplifier and collected using pClamp software (Molecular Devices) for offline analysis. For each slice, stimulus intensity was adjusted to yield 50-55% of the maximal response slope (without population spikes) and paired pulse plasticity at a 50 ms inter-stimulus interval was assayed to confirm stimulation of the medial perforant path. We prepared BMI (Sigma, St. Louis, MO) as concentrated stock solutions and diluted them with ACSF to the specified concentrations prior to each experiment. BMI concentrations were categorized as low (0, 1 µM), moderate (2.5, 5 µM), high (7.5, 10, 12.5 µM). All the following experimental recordings were conducted in the presence of ACSF at the specified BMI concentration. Baseline measurements were collected using individual fEPSPs evoked every 15 seconds. A steady baseline of 20 min was required for all responses. Following baseline acquisition, a high frequency stimulation (HFS) conditioning protocol consisting of four trains of 50 pulses at 100 Hz (30 s inter-train intervals) was used to induce LTP. Immediately following the conditioning protocol, baseline measurements resumed for 1 hour. For analysis purposes, recordings were normalized to the average value of the 20 min baseline. PTP was measured by averaging the first four traces (i.e., first minute) after the conditioning protocol, whereas LTP was measured by averaging the last 40 traces (i.e., 50-60 min) of the post-conditioning baseline. Computed results were processed for statistical analysis using Clampfit (Molecular Devices), Excel 2007 (Microsoft) and Statistica 7.0 (Statsoft). For all studies,  !  48  data was presented as means ± standard error of the mean (SEM). Student t-tests were used to examine if LTP was present, comparing the average of the last 40 traces (i.e., -100 min) prior to the conditioning protocol with the average of the last 40 traces of the postconditioning baseline. Comparisons between BMI concentrations were conducted using a one-way ANOVA, followed by Tukey post hoc tests as appropriate. Differences were considered significant when p < 0.05.  2.3 Results An HFS protocol was applied to the mouse DG to examine amounts of in vitro PTP and LTP facilitated under different concentrations of BMI (Fig. 2.1A). BMI concentrations were categorized as being low (0, 1 µM), moderate (2.5, 5 µM) or high (10, 12.5 µM). PTP was observed both in the absence and presence of BMI (Fig. 2.1B). A main effect was observed for BMI concentration (F(5,48) = 36.36, p < 0.000001). The low BMI concentrations produced significantly less PTP than all other concentrations ([0 µM] = 34.12 ± 14.03%, n = 8; [1 µM] =40.59 ± 5.07%, n = 14). Both moderate and high BMI concentrations produced similar PTP ([2.5 µM] = 143.65 ± 11.87%, n = 8; [5 µM] = 169.11 ± 8.96%, n = 7; [10 µM] = 198.25 ± 14.50%, n = 8; [12.5 µM] = 154.22 ± 20.01%, n = 9). Notably, the 2.5 µM BMI exposure produced significantly less PTP than the 10 µM BMI exposure (p > 0.04), but the 5 µM concentration did not produce statistically different amounts of PTP  !  49  in comparison to 10 µM. A moderate BMI concentration of 5 µM is therefore the lowest concentration that can induce maximal PTP in the mouse DG.  Figure 2.1 Changes in post-tetanic and long-term potentiation with different bicuculline methiodide concentrations in the mouse dentate gyrus. A, Data showing changes in potentiation induced using a high frequency stimulation conditioning protocol (4x100 Hz). B, Posttetanic potentiation, (first minute following conditioning protocol) is increased with increased BMI concentrations. A significant difference is seen between 2.5 µM and 10 µM (p < 0.05), but not between 5 µM and 10 µM. C, Long-term potentiation (50-60 minutes following conditioning protocol) is not observed with 0 and 1 µM concentrations, but is observed at higher concentrations (* indicate p < 0.05 in comparison to low concentrations ).  !  !  !  50  LTP was not observed at low BMI concentrations ([0 µM] = -2.34 ± 5.26%, n = 8; [1 µM]: -7.61 ± 2.37%, n= 14, Fig. 2.1C). In contrast, all moderate and high concentrations were able to reliably produce LTP ([2.5 µM] = 51.34 ± 6.97%, n = 8; [5 µM] = 57.77 ± 12.76%, n = 7; [10 µM] = 68.55 ± 10.74%, n = 8; [12.5 µM] = 44.70 ± 9.31%, n = 9). Again, a main effect was observed for BMI concentration (F(5,48) = 22.53, p < 0.000001). Both moderate and high concentrations produced similar amounts of LTP (p > 0.1), therefore the smallest BMI concentration of these, 2.5 µM, can be used to induce maximal LTP in the mouse DG.  2.4 Discussion In this report, we demonstrate the optimal concentration of BMI that can be used to facilitate LTP in the mouse DG in an in vitro slice preparation method. In the mouse DG, a BMI concentration of 5 µM appears to be optimal for inducing in vitro LTP, as it is the lowest concentration that can induce both maximal PTP and LTP. GABAA receptors contribute to both phasic (synaptically located) and tonic inhibition (extrasynaptically located) within the hippocampal formation. Unlike GABAB receptors, GABAA receptors are responsible for mediating fast inhibition resulting from the influx of chloride into the cell. During HFS application to the DG, GABA is released causing an increase in granule cell membrane conductance and holding the resting membrane potential at hyperpolarizing levels. These actions of GABA therefore block the production of LTP.  !  51  In the mouse DG, we were unable to induce LTP at low BMI concentrations, a finding that is in agreement with several in vitro rodent studies (Wigstrom and Gustafsson, 1983; Hanse and Gustafsson, 1992). However, our results are in contrast to previous work showing that a small (~10%) amount of LTP could be induced without suppression of GABAergic inhibition in Wistar rats (Snyder et al., 2001). The authors suggest that this form of LTP stems from stimulation of immature dentate granule cells. The DG is one of two regions in the adult brain that retains the capacity to continuously produce new neurons (Altman and Das, 1965). During granule cell development, GABA facilitates the depolarization of immature neurons, whereas GABA acts to hyperpolarize mature neurons (Karten et al., 2006). Mice demonstrate significantly less cell proliferation than rats (Snyder et al., 2009); therefore the lack of LTP observed in our mouse experiments may reflect these species differences. In addition, a study has recently demonstrated that GABAergic signaling can result in both excitatory and inhibitory mediation of dentate granule cells, depending on its precise timing and location (Chiang et al., 2012). These new findings may also explain the discrepancies between our study and others. The GABAA receptor antagonist chosen for this study was BMI, an N-methyl derivative of bicuculline commonly used as an alternative to bicuculline due to its increased aqueous solubility and stability at physiological pH (Olsen et al., 1976). While BMI has been shown to be highly selective for GABAA receptors, reports have also show that BMI can block small-conductance Ca2+-activated K+ (SK) channels. SK channels underlie the medium kinetic current of afterhyperpolarizations (Bond et al., 2004), which in turn can alter neuronal excitability. SK channels can also reduce the amplitude of  !  52  calcium influx through the NMDA receptor (Ngo-Anh et al., 2005). In the CA1, the SK channel blocker aparmin converts short-term LTP to long-lasting LTP (Ris et al., 2007), and potentiates LTP induced by weak high frequency conditioning protocols (Stackman et al., 2002). While most experiments investigating SK-channel function and bicuculline derivatives utilize BMI at high concentrations (~100 µM), it is unknown to what degree SK-channel function has on BMI-induced in vitro LTP in the DG and further experiments employing genetic manipulation of SK-channels may help to resolve this issue. The use of GABAA receptor antagonists to facilitate in vitro LTP in the DG has been used for some time. Dentate granule cells are heavily inhibited by local interneurons, which cause granule cells to fire at low frequencies despite strong afferent input. It is still unclear as to why GABAA receptor antagonists are required for LTP induction in vitro, but not in dentate gyrus recordings conducted in vivo. It is possible that this could be attributed to a decrease or abolishment of inhibition originating from the medial septum. In the intact brain, the medial septum sends inhibitory input onto GABAergic interneurons in the hilus and subgranular zone of the hippocampus (Freund and Antal, 1988). It is hypothesized that this septal inhibition indirectly facilitates the firing of dentate granule cells by reducing the tonic, feed-forward inhibition produced by hippocampal interneurons. Consistent with this idea, it has been shown that medial septum stimulation, in conjunction with stimulation to the perforant path, can increase the observed population spike without altering the size of fEPSP (Bilkey and Goddard, 1985). It is possible that during the preparation of the in vitro hippocampal tissue slices, inhibitory medial septal inputs may be disrupted. Indeed, in specific hippocampal slice preparations that spare the septal region, stimulation of the medial septum suppresses  !  53  spontaneous inhibitory currents in hippocampal pyramidal neurons (Toth et al., 1997). Further investigations into the role of the medial septum on hippocampal plasticity would be advantageous.  !  54  3 Voluntary Exercise and Stress, but not Housing Conditions, Regulate Adult Neurogenesis in the Mouse Dentate Gyrus.2 3.1 Introduction Exercise has long been associated with a number of positive benefits for the body, including weight management, improving stamina, and even combating chronic diseases such as high blood pressure and cholesterol. The benefits of exercise are also apparent in the central nervous system, where exercise can protect against neurodegenerative disorders, facilitate learning and memory, and alleviate symptoms of psychiatric disorders such as depression and anxiety (Barbour et al., 2007; Cotman et al., 2007). One region of the brain that is dramatically affected by exercise is the dentate gyrus (DG) subfield of the hippocampus. The DG is known to be intimately involved in learning and memory processes and is one of the few regions of the brain that maintains the ability to produce a substantial number of new neurons well into adulthood (Cameron and McKay, 2001). Indeed, the very existence of adult neurogenesis indicates that the hippocampus may possess an innate regenerative capacity that, once understood, could be harnessed for other regenerative purposes. In corroboration of this hypothesis that exercise can benefit neuronal function, adult neurogenesis can be enhanced markedly in rats and mice if they engage in exercise (van Praag et al., 1999b; van Praag et al., 1999a; Farmer et al., 2004; Eadie et al., 2005). Interestingly, both forced and voluntary exercise can increase hippocampal neurogenesis (van Praag et al., 1999a; Farmer et al., 2004; Overstreet et al., 2004; Llorens-Martin et al., 2006; Redila et al., 2006; Uda et al., 2006), although a recent !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 2 A version of this chapter has been published. Kannangara, T.S., Webber, A., GilMohapel, J., and Christie, B.R. (2009). Stress differentially regulates the effects of voluntary exercise on cell proliferation in the dentate gyrus of mice. Hippocampus. 19 (10). 889-97 !  55  study conducted in rats have raised the question as to whether social and/or environmental factors might modulate the ability of exercise to enhance neurogenesis in the adult DG (Stranahan et al., 2006). This controversy may, in part, reflect differences in the social habits between rodent species. Social preferences in rats differ from those in mice, a more common tool for biological research where genetic manipulations are required. To date, the effects of voluntary exercise on neurogenesis has been examined in group housed male mice (Fabel et al., 2003; Kitamura et al., 2003; Kronenberg et al., 2005), group housed female mice (van Praag et al., 1999b; van Praag et al., 1999a; Brown et al., 2003a), as well as isolated mice (Holmes et al., 2004; van Praag et al., 2005). Male mice prefer to be socially housed when given preference tests; furthermore, the preference to be socially housed is stronger than a preference for more standard environmental enrichment (Van Loo et al., 2003; Van Loo et al., 2004). While rats also show a preference towards social housing, this preference does not appear to be as strong in comparison to other types of enrichment (Patterson-Kane et al., 2001; Patterson-Kane et al., 2004). The strong social preference in mice, but not rats, suggests that exercise-induced effects on the mouse DG may differ from those in the rat DG. Differences in housing preference between rodent species may also reflect how stressful the animals perceive the housing condition to be. Stress is a complex response that can alter a number of physiological processes, including levels of corticosterone (CORT), a hormone commonly associated with the stress response. Elevated CORT levels stimulated through either stress (Gould et al., 1997; Gould et al., 1998) or directly via corticosterone application (Wong and Herbert, 2006) can produce a dramatic  !  56  decrease in cell proliferation and neurogenesis in the DG. Interestingly, voluntary exercise enhances cell proliferation and neurogenesis (van Praag et al., 1999a; van Praag et al., 2005) while also increasing CORT levels. This suggests that voluntary exercise might be protective against some deleterious effect of CORT on neurogenesis. In the current study, we examined how the effects of voluntary exercise on neurogenesis in the mouse dentate gyrus are influenced by both a mild stressor (social isolation), and a more pronounced stressor (acute restraint stress).  3.2 Materials and Methods 3.2.1 Animals and Housing Conditions Male C57BL/6J mice (2 months old, 23-25g; Charles River, QC, Canada) were individually/group housed in standard cages in a colony maintained at 21oC. Mice were maintained on a 12-hour light/dark cycle with access to food and water ad libitum. Following a one week acclimatization period, mice were assigned to one of two housing conditions: isolated (I) or social (S) containing three animals per cage. Following a one week period in these respective housing conditions, animals were assigned to one of two housing conditions: voluntary exercise (RUN) that contained a running wheel or sedentary control (CON) that did not contain a running wheel. To accommodate for the size of the running wheel, all mice were housed in large cages (46 $ 24 $ 20 cm). In total, this yielded four groups: isolated controls (I-CON); isolated runners (I-RUN); social controls (S-CON); and social runners (S-RUN). Group-housed mice were carefully monitored for signs of aggression throughout the experiment. If any mice displayed aggression and/or signs of injury, the cohort of animals in the cage was  !  57  removed from the study: in the present study, a group of three mice were excluded. All procedures were approved by the University of British Columbia Animal Care Committee and in accordance with the Canada Council on Animal Care.  3.2.2 BrdU Administration and Tissue Preparation Mice were maintained in running/control housing conditions for 12 days, as outlined in Figure 3.1A. On day 12, animals received a single intraperitoneal (i.p.) injection of bromodeoxyuridine (BrdU; 200 mg/kg), a thymidine analog that is incorporated into the DNA of cells during S-phase. Mice were removed from their cage for an average of 30 seconds for BrdU injection. Two hours following BrdU administration, subjects were deeply anesthetized with urethane and trunk blood samples were collected (2-3 hours after the onset of the night cycle). An additional set of mice used for PCNA immunohistochemistry were not administered BrdU injections. Mice were perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde. Brains were postfixed in 4% paraformaldehyde for 24 hours before being transferred to 30% sucrose. Twelve series of coronal sections were cut throughout the entire hippocampus using a Leica VT1000 vibratome at a thickness of 40 µm and stored in 0.1M Tris-buffered saline (TBS; 0.15 M NaCl, 0.1 M Tris-HCl, pH 7.5) at 4°C.  3.2.3 Restraint Stress In restraint stress experiments, mice were placed in restraint tubes for 15 minutes immediately after BrdU administration, as outlined in Figure 3.2A. Restraint tubes were  !  58  made from plastic 50mL Falcon tubes with small holes drilled in them to facilitate gas exchange. Tissue preparation was carried out as described above.  3.2.4 Immunohistochemistry A 1:12 series of brain sections were processed for immunohistochemical detection of BrdU-labeled cells as previously described (Farmer et al., 2004; Christie et al., 2005; Eadie et al., 2005; Redila et al., 2006). Briefly, free floating brain sections were first washed in 0.1M TBS. To block endogenous peroxidase activity, tissue was immersed in 0.6% H2O2 in TBS for 30 minutes. Sections were then placed in 50% formamide/2x SSC (0.3M NaCl and 0.3M sodium citrate) at 65°C for 2 hours, rinsed for 5 minutes in 2x SSC, then incubated in 2N HCl at 37°C for 30 minutes. Sections were placed in 0.1 M boric acid (pH 8.5) for acid neutralization. The tissue was then rinsed six times in TBS for a total of 90 minutes and incubated in TBS++ (0.1% Triton X-100 and 3% donkey serum in 0.1M TBS). Sections were incubated with a biotinylated monoclonal anti-mouse BrdU antibody (diluted 1:100; MAB3262B, Chemicon, Temulcula, CA) overnight at 4°C. Following antibody incubation, tissue was rinsed in TBS and an avidin-biotinperoxidase solution (ABC Elite Kit, Vector Laboratories, Burlingame, CA, USA) was applied for 2 hours. Tissue was rinsed and treated with a peroxidase detection kit using 3,3-diaminobenzidine (DAB) as the chromogen (DAB Kit, Vector Laboratories) according to manufacturers’ directions. The sections were then mounted on 2% gelatin coated slides. A cresyl violet background stain was applied prior to samples being coverslipped for microscopic analysis.  !  59  From a separate set of mice not subjected to BrdU injection, an additional series of free-floating brain sections were processed for immunohistochemistry against the endogenous cell cycle marker proliferating cell nuclear antigen (PCNA), using a similar protocol to one previously described (Gil et al., 2005). Antigen retrieval was achieved by incubating brain sections in 10 mM sodium citrate buffer (in TBS, pH 6.0) at 95°C for 5 minutes. To completely unmask the antigens, this step was repeated twice (sections were allowed to cool for approximately 20 min in between). Sections were then quenched in 3% H2O2/10% methanol in 0.1 M TBS for 15 minutes. After preincubation with 5% normal goat serum (NGS) for 1 hour, the sections were incubated with a rabbit polyclonal antibody against PCNA (diluted 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 24 hours at room temperature. The sections were then incubated with the secondary antibody biotinylated goat anti-rabbit IgG (diluted 1:200; Vector Laboratories), and visualized with an avidin-biotin–peroxidase complex system (Vectastain ABC Elite Kit, Vector Laboratories) using DAB as the chromogen (DAB Kit, Vector Laboratories). The sections were then mounted on 2% gelatin coated slides, dehydrated, and coverslipped. To determine the rate of neurogenesis in these animals, we performed immunohistochemistry against Doublecortin (DCX), a cytoskeletal protein that is expressed exclusively in migrating immature neurons or neuroblasts (Rao and Shetty, 2004). DCX is thought to be expressed in immature hippocampal granule cells for approximately 3 weeks after cell division (Phillips et al., 2005). A 1:6 series of brain sections were processed for DCX immunohistochemistry as previously described (Spanswick et al., 2007). Sections were washed in 0.1M TBS and incubated at room  !  60  temperature for 16 hours in goat anti-DCX polyclonal primary antibody (diluted 1:1000; Jackson ImmunoResearch Labs, Baltimore, MD) in 0.1 PBS, 0.5% Triton-X, and 2% normal rabbit serum at 4°C. Sections were washed three times for 10 minutes in 0.1M PBS, then incubated in secondary antibody biotinylated rabbit anti-goat IgG (Chemicon; diluted 1:1000) for 1 hour at room temperature. Tissue was then rinsed and the bound antibodies were visualized with an avidin-biotin–peroxidase complex system using DAB as the chromogen (Vector Laboratories). The sections were then mounted on 2% gelatin coated slides, dehydrated, and coverslipped.  3.2.5 Quantification All morphological analyses were performed using coded slides with the experimenter kept blind as to the animal experimental group. The number of BrdU- and PCNAimmunopositive cells in the entire hippocampus was assessed by manually counting all positive cells in the granular cell layer (GCL) of the DG in both the left and right hemispheres using an Olympus BX50 microscope with a 100x objective lens. Cells within the subgranular zone, defined as the area within two cell bodies (~20 µm) of the inner edge of the GCL, were combined with the GCL for quantification. Split nuclei cells that had not finished cytokinesis were counted as one cell for a conservative estimate. The distance between the sections was approximately 440 µm. A modified stereological approach, which has been shown to demonstrate similar results to other stereological methods in the dentate gyrus, was employed to estimate the total number of BrdU-positive cells/hippocampus as previously described (Nixon and Crews, 2004; Eadie et al., 2005; Kempermann, 2006).  !  61  DCX-immunopositive cells were also counted manually using the same equipment and procedures. Cells were counted only if individual membranes could be observed at 100x, thus resulting in a conservative estimate in animals with many DCX-immunopositive cells. Thirty cells per animal were chosen quasi-randomly for measures of nodes. Nodes were defined as the branch points of the immature dendrites. Neurons were not counted if dendrites were broken or if they were obscured by a neighboring cell body. Images were captured with a CoolSnap CCD camera and processed using Image-Pro Plus (Media Cybernetics, Bethesda, MD).  3.2.6 Serum Corticosterone Total corticosterone levels (bound plus free) were measured by radioimmunoassay (RIA). A tritiated corticosterone RIA kit (MP Biomedicals, Solon, OH), was used to determine plasma corticosterone levels using an adapted protocol previously described (Weinberg and Bezio, 1987; Gabriel et al., 2002). Dextran-coated charcoal was used to absorb and precipitate free steroids after incubation. Samples were analyzed using a Scintisafe Econo2 analysis kit (Fisher Scientific Ltd, Ottawa, Canada).  3.2.7 Statistical Analysis For all studies, data are presented as means ± standard error of the mean (S.E.M.). Differences between mean values of experimental groups were compared using two-way analysis of variance (ANOVA), followed by Fisher post hoc tests as appropriate. Differences were considered significant when P < 0.05.  !  62  3.3 Results 3.3.1 Voluntary Exercise Increases Cellular Proliferation in Socially and Individually Housed Mice We initially compared the rates of cellular proliferation between individually or socially housed mice with and without access to a running wheel. Both groups of mice were injected once with the thymidine analog BrdU (200 mg/kg; i.p.) and then sacrificed two hours later (Fig. 3.1A). As shown in Figure 3.1F, social housing did not increase the number of BrdU-positive (BrdU+) cells compared to individually housed mice (S-CON: 1156 ± 146.3, n=5; I-CON: 1227 ± 69.05, n=10). Free access to a running wheel increased the number of BrdU+ cells in both social and individual housing conditions compared to their respective non-running controls (F(1,28) = 25.0, P=0.00003). These differences were apparent in both individually (I-RUN: 1876 ± 146.5, n=13 vs I-CON: 1227 ± 69.05, n=10) and socially (S-RUN: 2235 ± 411.1, n=4 vs S-CON: 1156 ± 146.3, n=5) housed mice. No significant differences were observed between I-CON and S-CON mice (P=0.77), indicating that socialization did not have an additive effect on hippocampal cell proliferation in exercising mice. In order to examine the effect of an intermediate housing condition, a separate group of animals was housed in a social environment for one week followed by one week of isolation prior to access to a running wheel. However, we did not observe any significant differences between this group and the I-RUN and S-RUN groups (data not shown). We also sampled levels of corticosterone, as an index of adrenal-cortical activity, to investigate if the two housing conditions differentially affected this system. An ANOVA revealed there was not a significant effect for either exercise or social condition  !  63  ! !  64  Figure 3.1 Voluntary Exercise Promotes Cellular Proliferation, Irrespective of Social Condition. A, Schematic drawing of the experimental paradigm and BrdU injection protocol. Exercising mice were allowed access to running wheels for 12 days. On day 12, mice were administered BrdU and perfused two hours later. (d:day) B-E, Representative pictures of BrdUlabeled cells in the mouse dentate gyrus of isolated controls (I-CON; B), isolated runners (I-RUN; C), social controls (S-CON; D), and social runners (S-RUN; E). Scale bar: 100 %m F, Both isolated and social running groups (black bars) demonstrate increases in the amount of BrdU-labeled cells in the mouse dentate gyrus in comparison to their sedentary controls (white bars). G, Corticosterone levels obtained from trunk blood of animals administered BrdU shows no effects of exercise (black bars = running; white bars = sedentary controls) or social condition. H, Labeling of the endogenous marker PCNA confirms that cellular proliferation is promoted in isolated and social running groups (black bars) in comparison to sedentary controls (white bars). I, Corticosterone levels of animals not subjected to BrdU injection demonstrate no main effects of exercise (black bars = running; white bars = sedentary controls) or social condition. Error bars represent S.E.M. (*) denotes significance (P < 0.05) in comparison to controls (two-way ANOVA and Fisher post-hoc test).  on corticosterone levels (P>0.05). However, there was a trend for mice in social housing to show higher corticosteroid levels (P=0.06), irrespective of whether or not they were given free access to a running wheel (Fig. 3.1G). While BrdU has been well-established as a proliferation marker in the DG, there are always concerns when using an exogenous marker in an exercising paradigm. Exercise increases cerebral blood volume and blood flow to the DG (Pereira et al., 2007), and it is possible that this might increase the ability of BrdU to access this region of the brain, resulting in increased staining in animals that exercise. To resolve this issue, we replicated the above experiment using PCNA, an endogenously expressed marker for cells engaged in mitotic activity, in a separate group of mice that were not submitted to BrdU injections. This experiment also indicated that cell proliferation was not significantly different in either the individually housed (1706 ± 52.65) or socially housed (1665 ± 125.3) mice (Fig. 3.1H). Also, in agreement with the results obtained with BrdU, quantitative analysis of PCNA-labeled cells demonstrated a significant increase in cellular proliferation (F(1,18) = 12.0, P=0.003) in mice that exercised, irrespective of their social housing condition (I-CON: 1706 ± 52.65, n=4; I-RUN: 2427 ± 243.6, n=7; S-  !  65  CON: 1665 ± 125.3, n=5; S-RUN: 2403 ± 231.8, n=6). In addition, there were also no significant differences in corticosterone levels between any of these groups (Fig. 3.1I). Thus, these results further confirm that voluntary exercise, but not social condition, increases cellular proliferation in the mouse DG.  3.3.2 Social Housing Condition Differentially Affects Cell Proliferation in Exercising Mice Subjected to Short-term Restraint Stress. Activation of the adrenal-cortical axis leads to an increase in the levels of corticosteroids, which can in turn be deleterious to neurogenic activity (Cameron and Gould, 1994). Since previous studies have proposed that exercise may ameliorate this effect (Adlard and Cotman, 2004; Duman, 2005), we investigated whether voluntary exercise can prevent stress-induced reductions in neurogenesis. In these experiments, mice from the different groups were placed in a restraint tube for 15 minutes immediately after receiving an injection of BrdU (200 mg/kg; i.p.; Fig. 3.2A). Mice were sacrificed 2 hours after the BrdU injection. Mice from the stressed I-RUN group showed a significant increase in the number of BrdU+ cells (F(1,22) = 7.13, P = 0.01; 1467± 158.9, n=6), compared to subjects in the stressed I-CON group (787.6 ± 100.7, n=6; Fig. 3.2B). In contrast, no difference was observed in the stressed socially housed subjects (stressed S-CON: 859.8 ± 111.7, n=8; stressed S-RUN: 778.7 ± 162.0, n=7). There was a main effect of social condition (F(1,22) = 7.56, P = 0.01) as well as an interaction between exercise and social condition (F(1,22) = 11.5, P = 0.003) reflected in the fact that stressed I-RUN mice continued to show a significant increase in cell proliferation, despite exposure to the stressor.  !  66  Figure 3.2 Acute Restraint Stress Selectively Modulates Proliferation in Exercising Mice. A, Schematic drawing of the experimental paradigm and BrdU injection protocol was similar to those used previously, except that a 15-minute restraint stress period was introduced immediately after BrdU administration. Mice were perfused two hours after BrdU administration. (d:day). B, BrdUlabeled cells in mice subjected to 15 minutes of restraint stress. Isolated runners subjected to acute stress still show a significant increase in cellular proliferation in comparison to their sedentary controls. This effect is not observed in social runners. C, Comparison of BrdUlabeled cells in non-stressed mice from Figure 3.1 (white bars) and those subjected to restraint stress (black bars). Decreases in cellular proliferation are observed in all restraint groups, however stressed social runners are more susceptible to restraint stress. D, Levels of corticosterone in mice following 15 minute restraint stress show no main effects of exercise (black bars = running; white bars = sedentary controls) or social condition. Error bars represent S.E.M. (*) denotes significance (P < 0.05) in comparison to controls (two-way ANOVA and Fisher post-hoc test).  !  !  67  It should be noted however, that in comparison to subjects not subjected to restraint stress, mice in all four stress conditions did show decreases in cell proliferation (Fig. 3.2C). Interestingly, the most pronounced reduction in the number of BrdU+ cells following acute stress was observed in the stressed S-RUN subjects. An analysis of variance on CORT levels, taken at the time of perfusion from the four groups, did not show any differences (P > 0.05; Fig. 3.2D).  3.3.3 Social Isolation has No Effect on the Production of New Neurons in Response to Voluntary Exercise. To investigate if the observed increase in cellular proliferation translates into an increase in the production of new neurons, we quantified the number of cells immunopositive for the immature neuronal marker DCX. DCX-immunopositive neurons were prevalent throughout the DG of mice from all groups (Fig. 3.3A-D). Both individually housed and socially housed subjects showed equitable neuronal numbers in control conditions (ICON: 33237 ± 1590.6, n=6; S-CON: 26893 ± 1177.8, n=3), however more DCX-labeled cells were apparent in isolated runners (I-RUN: 57525 ± 2821.1, n=6) and social runners (S-RUN: 54092 ± 2620.2, n=6; F(1,17) = 102, P < 0.000001). The social housing condition (isolated vs. group) did not produce an observable change in the number of DCX labeled cells (Fig. 3.3E).  !  68  ! Figure 3.3 Neurogenesis and Branching of New Neurons is Induced by Exercise, Irrespective of Social Condition. A-D, Representative photomicrographs of cells immunopositive for doublecortin (DCX) in the mouse dentate gyrus of isolated control (I-CON; A), isolated runner (I-RUN; B), social control (S-CON; C), and social runner (S-RUN; D) mice. Scale bar: 50 %m. Scale bar in high magnification insets: 10 %m. E, Both isolated and social running groups (black bars) show significantly more DCX-labeled cells than their sedentary counterparts (white bars). F, New neurons of exercising subjects (black bars) have more nodes per cell, indicating an increase in dendritic branching, in comparison to sedentary counterparts (white bars). Error bars represent S.E.M. (*) denotes significance (P < 0.05) in comparison to controls (two-way ANOVA and Fisher post-hoc test).  !  ! Additional analysis of the DCX-labeled cells revealed that the average numbers of nodes, or branch points for new neurons, was also increased in mice that engaged in  !  69  exercise. Immature neurons in individual runners had approximately twice the number of nodes as individual non-runners (I-RUN: 1.386 ± 0.066, n=6; I-CON: 0.7429 ± 0.059, n=6; Fig. 3.3F). Similarly, DCX-labeled neurons in social runners had approximately 50% more nodes than social non-runners (S-RUN: 1.494 ± 0.057, n=6; S-CON: 0.9111 ± 0.08889, n=3; F(1,19) = 1.91, P < 0.000001). There were no significant effects of social housing condition alone on the morphology of these new neurons, thus exercise seems to play a larger role in modifying the number and the morphology of new neurons in the mouse DG than social housing condition does.  3.4 Discussion This study provides evidence that voluntary exercise increases cell proliferation and neurogenesis in the mouse DG irrespective of social condition. This is in contrast to recent reports suggesting that voluntary exercise does not increase neurogenesis in isolated rats (Stranahan et al., 2006). The reason for these conflicting results is currently unclear. It is possible that variations in rodent species may account for some of these differences (as experiments performed by Stranahan and colleagues were conducted in rats and the present study was conducted in mice). Both environmental and physiological factors have been demonstrated to have differential effects on adult neurogenesis, depending on the type of rodent. Estrogen, for example, has been shown to stimulate adult neurogenesis in rats, but not in mice (Mazzucco et al., 2006; Lagace et al., 2007). It has been observed, however, that in some conditions voluntary exercise can reliably increase cell proliferation in rats housed in isolation (Christie et al., 2005; Eadie et al., 2005). Alternatively, as stress can be a regulatory factor for neurogenesis, differences in  !  70  experimental design or animal care management practices could account for the observed discrepancies. For example, in order to facilitate administration of BrdU, Stranahan and colleagues restrained rats for 60-90 seconds (Stranahan et al., 2006). Given that this group employed a multiple injection experiment protocol of 10 BrdU i.p. injections, this equates to 10 consecutive days of acute restraint. Thus, it may be possible that stress just prior to BrdU administration could modify proliferating cell numbers. We did not find any differences in CORT levels between mice with access to running wheels and the non-exercising control animals. This is in accordance with other research showing that corticosterone is only increased significantly immediately prior to the onset of physical activity (Droste et al., 2003; Stranahan et al., 2006). It was also recently shown that neither isolation nor variations in group size significantly affects corticosterone levels of male mice (Hunt and Hambly, 2006). Our study did not demonstrate statistically significant main effects of exercise or housing condition on CORT levels, however a trend for socially housed mice to have increased levels of CORT was apparent. Aggression among mice may lead to elevated CORT levels as male mice, like rats, commonly display aggressive behavior when housed together. Male mice, however, are least aggressive when housed in groups of three animals per cage, in comparison to groups of five and eight (Van Loo et al., 2001). Furthermore, in this study, socially housed males were monitored carefully for signs of aggression throughout the experiment. Thus, we believe that aggression-induced stress was not a major cause for the observed trend. This is consistent with our results showing that no effects of voluntary exercise or social conditions were observed in mice that were used for PCNA  !  71  immunohistochemistry rather than being subjected to the BrdU injection protocol (a potentially stressful procedure). Stressful situations can result in the near immediate downregulation of cell proliferation in the hippocampus by influencing levels of glucocorticoid (Falconer and Galea, 2003; Namestkova et al., 2005; Chen et al., 2006). Indeed, subjecting mice to 10 minute restraint stress following a BrdU injection can decrease the number of cell proliferation (Kim et al., 2005). Our results confirm that mice introduced to short-term restraint stress demonstrate an overall decrease in the amount of proliferating cells labeled with BrdU. Even under stressful conditions however, an increase in cell proliferation was observed in running mice housed in isolated conditions in comparison to non-running controls. Unexpectedly, under the same stressful conditions, exercise was unable to produce an increase in cellular proliferation in socially housed mice. While the mechanisms underlying this result are unclear, one possibility could be an enhancement of social stress immediately following restraint. Immediately after the 15 minute restraint paradigm, mice are returned to their cage where they remain until the time they are perfused. In isolated conditions, individual mice may be able to partially recover from the restraint stress during this period of time. In contrast, socially housed mice may not be able to recover from the induced stress as they are housed with their equally-stressed cage mates. Stressed animals housed together may produce a ‘social stress’ which could affect the levels of cellular proliferation in these social animals. Short-term restraint stress did not differentially alter CORT levels of mice in the different social and exercise conditions. Several studies have investigated how voluntary exercise and stress may interact. Some studies have reported that the levels of CORT in  !  72  running mice increase in response to restraint stress (Droste et al., 2003), while others have found that voluntary exercise was associated with increased adrenal sensitivity to restraint stress (Fediuc et al., 2006). One study showed an exaggerated increase in corticosterone immediately following a two hour restraint in exercising mice; this effect was not seen, however, within 10 hours of the restraint period (Adlard and Cotman, 2004). Our data supports the theory that exercise and social housing for short periods of time do not significantly influence CORT levels in stressed mice. We also found that voluntary exercise, but not social condition, also stimulates the production of new DCX-positive neurons, and these neurons show more branch points, or nodes. The effects of exercise were quite profound; after only 12 days with free access to a running wheel, a two-fold increase in the number of new neurons was observed. This estimate can be considered conservative as DCX quantification was only conducted on cells that had distinguishable membranes. The branching of neurons was also increased through exercise. This finding is particularly striking when one considers that after cell division, primary dendrites extend from neurons to molecular layer of the DG after approximately 1.5 weeks (Aimone et al., 2006), approximately the amount of time subjects have been exercising. One hypothesis is that exercise can independently upregulate factors that promote dendritic outgrowth and arbourization during neuronal development, in addition to increasing the number of new neurons produced. This conclusion is in accordance with recent Golgi-Cox analyses of neuronal morphology in exercising animals (Redila and Christie, 2006; Stranahan et al., 2007). Neurogenesis has often been correlated with performance on the Morris Water Maze, a task that is believed to test for hippocampal dependent spatial learning (van  !  73  Praag et al., 1999b; Cao et al., 2004; Snyder et al., 2005; Olson et al., 2006). Interestingly, male mice that have been housed in isolation for long periods of time showed no deficits in water maze learning when compared to aged-matched, socially housed animals (Voikar et al., 2005). Similarly, we were unable to observe differences in the structure of hippocampi of subjects housed in different social conditions in this study, indicating that social housing alone is not likely sufficient to induce long-term changes in the hippocampus that would benefit learning performance. Our study has several important implications. We have demonstrated that social condition does not regulate the exercise-induced promotion of cellular proliferation and neurogenesis in mice. We have also shown that the branching of new neurons is also upregulated by exercise, but not by social condition. Taken together, this suggests that voluntary exercise can promote cellular proliferation, neurogenesis and dendritic outgrowth in new neurons, irrespective of social condition. Finally, we have shown that restraint stress can produce differential effects on running-induced proliferation depending on social condition. This last point reveals a cautionary reminder that should be considered during the design of neurogenesis studies using mice, and that attention should be given to the level of stress that is experienced by these subjects.  !  74  4 The GluN2A Subunit of the NMDA Receptor is Critical for Bidirectional Synaptic Plasticity and Spatial Pattern Separation3 4.1 Introduction The N-methyl-D-aspartate (NMDA) receptor is an ionotropic glutamate receptor that is expressed throughout the central nervous system, and plays a key role in learning and memory (Morris et al., 1986; Staubli et al., 1989; Young et al., 1994; Tsien et al., 1996). The receptor is a heteromeric complex composed of two obligatory GluN1 (NR1) subunits and two regulatory GluN2 (NR2) or GluN3 (NR3) subunits. While variants of the GluN1 subunit impart subtle changes to NMDA receptor function (Traynelis et al., 1998), it is the regulatory subunits that confer dramatically different physiological properties on the receptor (Cull-Candy and Leszkiewicz, 2004). Indeed, GluN2A and GluN2B, the most highly expressed regulatory subunits in the hippocampus (Monyer et al., 1994; Wenzel et al., 1997), can distinctly alter an NMDA receptor’s open probability, decay rate and calcium transfer (Chen et al., 1999; Erreger et al., 2005). These differences have led to the proposal that the GluN2A and GluN2B subunits play distinct physiological roles (Liu et al., 2004; Liu et al., 2007; Yashiro and Philpot, 2008). The GluN2 subunits have been heavily investigated for their roles in hippocampal synaptic plasticity, specifically in the CA1 region. In the CA1, the threshold for longterm potentiation (LTP) is increased in GluN2A-/- mice (Sakimura et al., 1995; Kiyama et al., 1998), while GluN2B deletion inhibits long-term depression (LTD) (Kutsuwada et !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 3 A version of this chapter has been submitted for publication. Kannangara, T.S., Eadie, B.D., Bostrom, C.A., Brocardo, P.S., and Christie, B.R. The GluN2A subunit of the NMDA Receptor is Critical for Bidirectional Synaptic Plasticity and Spatial Pattern Separation !  75  al., 1996). Because of the poor subunit specificity of GluN2 antagonists, pharmacological studies experiments have thus far failed to elucidate a specific role for these subunits in LTP and LTD (Neyton and Paoletti, 2006; Berberich et al., 2007; Morishita et al., 2007) despite some promising early studies (Liu et al., 2004; Massey et al., 2004). Thus, the roles of GluN2 subunits in synaptic plasticity remain unknown. Closely neighboring the CA1 is the dentate gyrus (DG), a hippocampal region that offers a unique opportunity to explore the role of GluN2 subunits in synaptic plasticity and behaviour for several reasons. First, the DG prominently demonstrates NMDA receptor-dependent LTP and LTD (Morris et al., 1986; Fox et al., 2006; Vasuta et al., 2007). Second, GluN2 subunits are highly expressed in the DG (Wenzel et al., 1997; Nacher et al., 2007). Finally, the DG-specific process of spatial pattern separation, the enhancement of contrast between two spatial patterns (Kesner, 2007b), has been associated with NMDA receptor expression (McHugh et al., 2007), although the involvement of specific GluN2 subunits remains unclear. The necessity of spatial pattern separation, and by extension the DG, cannot be understated: loss of this ability would allow distinct patterns to converge into single, incorrect memories, rendering recall of a correct memory impossible (Deng et al., 2010). In this study, we examined the contribution of the GluN2A subunit to synaptic and behavioural plasticity in the adult DG of mice lacking GluN2A expression. Our results reveal for the first time an essential role for the GluN2A subunit in bidirectional synaptic plasticity and spatial pattern separation in the mouse DG.  !  76  4.2 Materials And Methods 4.2.1 Animals and Housing Conditions Male wild-type (WT) and GluN2A knockout (GluN2A-/-) mice with a C57BL/6 background, as characterized previously (Townsend et al., 2003), were housed in standard cages in a colony maintained at 21oC. Animals were maintained on a 12-hour light/dark cycle with access to food and water ad libitum. All procedures were approved by the University of Victoria Animal Care Committee and in accordance with the Canadian Council on Animal Care.  4.2.2 Electrophysiology 4.2.2.1 Electrophysiology Tissue Preparation Adult male animals (2-4 month) were anesthetized with isofluroane and rapidly decapitated. The brains were removed and immersed in ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 2.50 KCl, 1.25 NaH2PO4, 25.0 NaHCO3, 2.00 CaCl2, 1.30 MgCl2, and 10.0 dextrose, oxygenated with 95%O2/5%CO2. Transverse hippocampal slices (350 µm) were generated using a Vibratome Sectioning System 1500 (Ted Pella, Redding, CA). For whole cell patch clamp experiments, transcardial perfusions were performed prior to decapitation (Magee et al., 1995). Perfusions and tissue slicing were conducted using ice-cold choline ACSF containing (in mM): 2.50 KCl, 1.25 NaH2PO4, 25.0 NaHCO3, 0.500 CaCl2, 7.00 MgCl2, 20.0 dextrose, 1.30 ascorbate acid, 2.40 Na-pyruvate, 110 choline chloride. Slices were gently transferred to an incubation chamber filled with oxygenated ACSF and maintained at 30oC for a minimum of 1 hour post-dissection. Slices were transferred to a recording chamber and  !  77  superfused at a rate of 1-2 ml/min with 30oC, oxygenated ACSF. A concentric bipolar stimulating electrode (FHC, Bowdoin, ME) was placed under visual guidance into either the medial aspect of the DG molecular layer for DG experiments or the stratum radiatum of the CA1 for CA1 experiments, using an Olympus BX51W1 microscope (Center Valley, PA).  4.2.2.2 Field Electrophysiology Borosilicate recording electrodes (0.7-1.5 M#) filled with ACSF were placed in either the medial aspect of the DG molecular layer or the stratum radiatum of the CA1. The inputs from the CA3 to CA1 were severed prior to CA1 experiments. Field excitatory postsynaptic potentials (fEPSPs) were evoked using monophasic negative current pulses (120 µs, 10-80 µA) supplied via a digital stimulus isolation unit (Getting Instruments, San Diego, CA). Responses were acquired at 100 kHz using a MultiClamp 700B amplifier (Axon Instruments) and collected on a computer for offline analysis using Clampfit 10.2 (Axon Instruments). For each slice, stimulus intensity was adjusted to yield 50-55% of the maximal response slope (without population spikes). Baseline measurements were collected using individual fEPSPs evoked every 15 seconds. After a steady baseline of 20 minutes, a conditioning protocol was used to induce synaptic plasticity, followed by baseline measurements for one hour. In order to investigate the capacity for LTP in the DG, two high frequency stimulation (HFS) conditioning protocols were used: a weak HFS protocol (wHFS; 2 bursts of 50 pulses, delivered at 100 Hz; 2x100 Hz, 30 s inter-train interval), and a strong HFS protocol (sHFS; 4 bursts of 50 pulses, delivered at 100 Hz;  !  78  4x100 Hz, 30 s inter-train interval). Both HFS protocols were conducted in the presence of bicuculline methiodide (BMI, 5 %M, Sigma-Aldrich, MO), to block inhibition from !aminobutyric acid A receptors (GABAA) and facilitate LTP induction in the DG (Wigstrom and Gustafsson, 1983). LTP in the CA1 was evoked using similar protocols, but in the absence of BMI. LTD was induced using a low frequency stimulation (LFS) conditioning protocol (900 pulses delivered at 1 Hz), previously shown to be the most effective LFS protocol for inducing LTD in the DG (Trommer et al., 1996). To verify conditioning protocols induced NMDA receptor-dependent forms of synaptic plasticity, a series of experiments was conducted using the NMDA receptor antagonist 2-amino-5phosphovalerate (APV, 50 %M, Sigma-Aldrich, MO). Potentiation or depression was quantified by examining the average of the last 10 min of the post-conditioning baseline (50-60 min). Input-output curves and assays to examine presynaptic mechanisms were examined in separate, naive slices. Input-output curves were generated by measuring the slope of fEPSPs in response to increasing stimulus pulse widths (30-300 %s). Paired pulse plasticity was assayed by stimulating the medial perforant path with paired stimuli at 25, 50, 100, 200 and 500 ms interstimulus intervals. The paired pulse plasticity ratio was calculated by dividing the second fEPSP slope with the first fEPSP slope. The number of readily releasable pool vesicles was examined by measuring the decay rate of a series of fEPSPs evoked with a brief HFS protocol (50 pulses, delivered at 100 Hz) in the presence of APV (50 %M) and BMI (5 %M). To determine the rate at which the reserve pool of vesicles replenishes the readily releasable pool, a repetitive stimulation  !  79  protocol (300 pulses, delivered at 14 Hz) was administered in the presence of APV (50 %M) and BMI (5 %M).  4.2.2.3 Whole Cell Electrophysiology For whole-cell recordings, recording electrodes (4-8 M#) were backfilled with an internal recording solution consisting of (in mM): 120 Cs-Gluconate, 17.5 CsCl, 0.100 EGTA, 5.00 QX-314, 10.00 HEPES, 4.00 ATP, 0.300 TrisGTP, and 14.0 Phosphocreatine (pH 7.2). An Olympus BX51W1 microscope with oblique illumination was used to visually target dentate granule cells in the outer region of the dentate granular cell layer (Fig. 4.2A). Recordings were acquired using an Axopatch 200B (Molecular Devices, CA), filtered at 5 kHz using a four-pole Bessel filter and digitized at 20 kHz using a Digidata 1440A (Molecular Devices, CA). The resting membrane potential was acquired immediately following successful whole-cell break-in. The current response to a 5 mV, 300 ms step (from -90 mV to -85 mV) was fit to a double exponential function to calculate series resistance, cell membrane capacitance, and input resistance. Only cells with series resistance values less than 30 M# were used for analysis. Pipette and whole cell capacitances were compensated. In voltage-clamp, cells were maintained at -70 mV using a holding current to measure excitatory postsynaptic currents (EPSCs). AMPA ("-amino-3-hydroxyl-5-methylisoazole-propionate) receptor-mediated currents (AMPA-EPSCs) were determined by stimulating the medial aspect of the DG molecular layer while holding cells at -70 mV. NMDA receptor-mediated currents (NMDA-EPSCs) were isolated in the same cell by  !  80  exchanging ACSF with low (0.1 mM) Mg2+-ACSF in the presence of glycine (20 %M), AMPA receptor antagonist NBQX (5 %M), and BMI (5 %M). Amplitudes and decay time constants for evoked EPSCs were analyzed using Clampfit 10.2 (Molecular Devices, CA). Glutamate receptor amplitudes were measured as the difference between the pre-stimulation baseline and the evoked EPSC peak. Charge transfer was determined by integrating maximum amplitude NMDA-EPSC waveforms, from the beginning of the downward deflection to 300 ms after stimulus. Decay time constants for NMDA-EPSCs were estimated by using a double exponential fit between cursors placed just beyond the evoked EPSC peak and 300 ms after that point, and the weighted decay time constant was calculated using: &Weighted = &1 [A1/(A1+A2)] + &2 [A2/(A1+A2)], where & is the fitted time constant and A is the amplitude.  4.2.2.4 Electrophysiology Drugs We prepared BMI (Sigma-Aldrich, MO) and APV (Sigma-Aldrich, MO) as concentrated stock solutions in dH2O and diluted them with ACSF to the specified concentrations prior to each experiment. NBQX (Sigma-Aldrich, MO) was dissolved in 0.1% DMSO for stock solutions.  4.2.3 Golgi Staining 4.2.3.1 Golgi Impregnation and Slice Processing Male mice (2-4 month) were deeply anesthetized with urethane (10 mg/kg) and transcardially perfused with 0.9% saline. Brains were immediately removed and placed in vials containing 20 mL of modified Golgi-Cox solution (Gibb and Kolb, 1998). This  !  81  solution was replaced with fresh Golgi-Cox solution after 24 hours and stored in the dark for 14 days. Brains were then transferred to 30% sucrose (in dH2O) in and stored in the dark for a maximum of 21 days. Coronal sections (200 %m) were generated throughout the length of the hippocampus using a Leica vibratome V1400 (Leica, Nussloch, Germany) in 6% sucrose (in dH2O). Sections were immediately mounted onto 2% gelatin-coated slides and processed as previously described (Redila and Christie, 2006). Briefly, sections were placed in: dH2O (1 min), ammonium hydroxide (30 min), dH2O (1 min), Kodafix for film (30 min), dH2O (1 min), 50% ethanol (1 min), 70% ethanol (1 min), 95% ethanol (1 min), 100% ethanol (2 x 5 min), 100% ethanol/Citrisolv/chloroform (1:1:1 for 10 min) and then Citrisolv (2 x 15 min). After processing, all slides were coverslipped using Permount mounting medium (Fisher Scientific, Ottawa, Canada) and stored in the dark.  4.2.3.2 Dendritic Analysis All slides were coded prior to analysis, allowing the experimenter to conduct analyses blind to animal identity. Golgi-impregnated dentate granule cells were chosen from the outer 50% of the granular cell layer. Cells were selected if they fulfilled the following criteria: 1) cells had consistent impregnation throughout the extent of the cell body and dendrites, 2) cells were distinguishable from neighboring impregnated cells, and 3) cells had intact dendritic trees. Each selected cell was traced at 40x magnification using an Olympus BX51 microscope (Center Valley, PA) and Retiga-2000R camera (QImaging, Surrey, BC, Canada) connected to the Neurolucida and NeuroExplorer morphometry software (MicroBrightField, Williston, VT). Differences between WT and  !  82  GluN2A-/- cells were assessed by examining mean total dendritic branch length, branch order and the number of branch nodes. A Sholl analysis (Sholl, 1956) was performed by quantifying the number of dendritic processes crossing concentric circles located at 10 %m intervals.  4.2.4 Hippocampal-dependent Behavioural Tasks 4.2.4.1 Behavioural Apparatus The behavioural apparatus used for the metric and temporal ordering of visual objects consisted of a rectangular box measuring 60 cm in diameter. Eight objects (a set of small glass bottles, two different sets of LEGO pieces, and a set of plastic bottle water lids) measuring between 2.5–5 cm at the base and between 5 and 15 cm tall were used as stimuli in these tasks. These objects were chosen since they are texturally and visually unique and easily distinguishable for the mice. Between the habituation and test sessions, the mice were placed in a clean cage. The experimental box was wiped down with 70% ethanol after testing of each mouse to remove olfactory cues that could influence object exploration by subsequent mice. The metric spatial change and temporal ordering tasks have been used in transgenic mice previously (Hunsaker et al., 2009; Hunsaker et al., 2010).  4.2.4.2 Metric Spatial Change Task The metric spatial change task was comprised of one exploration session and one test session. Male mice (2-4 month) were placed in the experimental box with two objects placed 40 cm apart (Fig. 4.6A). The mouse was allowed 15 min to freely explore  !  83  the square and stimulus objects. After the 15 min exploration session, the mouse was removed to a clean cage for 5 min. During this intersession interval, the objects were moved closer to each other so that the objects were 10 cm apart. The mouse was then placed again in the experimental box and given 5 min to re-explore the objects during this test session. For the 15 min exploration session, total object exploration time (s) was calculated individually for the first, middle and last 5 min epochs to facilitate comparison between the last 5 min of the exploration session and the 5 min test session. An experimenter blind to the experimental conditions recorded active object exploration and general locomotor activity as dependent measures.  4.2.4.3 Temporal Ordering Task The temporal ordering task was comprised of three exploration sessions and one test session. Prior to exploration session 1, two identical copies of a first object (object 1) were placed in the extremities of the experimental box, 2.5 cm from the end walls and centered between the long walls (Fig. 4.7A). For exploration session 1, the mouse was placed in the center of the experimental box facing away from both objects. The mouse was given 5 min to freely explore the objects. After 5 min, the mouse was removed to a clean cage for 5 min. During this time, the first objects were replaced with two duplicates of a second object (object 2). The procedure for exploration session 1 was then repeated twice, with two duplicates of new objects each time (i.e. two copies of object 2 for exploration session 2 and two copies of object 3 for exploration session 3). After exploration session 3, the mouse was removed into a clean cage for 5 min and an  !  84  unused copy of the first object and an unused copy of the third object were placed into the box. The mouse was again placed into the box and allowed to explore the two objects (i.e., object 1 and object 3) during a 5 min test session. For the temporal ordering task, object exploration was defined as active physical contact with the object with the forepaws, whiskers, or nose. Data was collected across all three object exploration sessions as well as during the test session by an experimenter blind to the mouse genotype.  4.2.4.4 Behavioural Analysis An exploration ratio for the metric spatial change task was calculated to facilitate the comparison between the test session and the last 5 min of the exploration session, as previously described (Goodrich-Hunsaker et al., 2005). Briefly, the ratio was calculated as: [(exploration time during the 5 min test session)/ (exploration time during the 5 min test session + exploration time during the last 5 min of the exploration session)]. This constrained all the values between 0 and 1. With this ratio, increased exploration during the 5 min test session compared to the last 5 min of the habituation session is reflected as a ratio > 0.5, while decreased exploration (or continued habituation) is reflected as a ratio < 0.5. Exploration during the temporal ordering test sessions was also converted into a ratio score to constrain the values between -1 and 1. The ratio was calculated as follows: [(exploration of object 1- exploration of object 3)/(exploration of object 1 + exploration of object 3)].  !  85  4.2.5 Statistical Analysis Computed results were processed for statistical analysis using Excel 2007 (Microsoft) and Statistica 7.0 analytical software (Statsoft Inc., Tulsa, OK). For all studies, data was presented as mean ± standard error of the mean (S.E.M.). For all electrophysiology experiments, individual student’s t-tests were performed. For behavioural experiments, exploration data that was converted to ratio values was also analyzed using individual student’s t-tests. In addition, a two-way repeated measures ANOVA with genotype and session as factors was performed on the object exploration data in the temporal ordering task. For dendritic branch length and node number, individual unpaired, two-tail student’s t-tests were performed. A repeated measures ANOVA was performed for Sholl analysis and branch order, using genotype and either distance from soma, or branch order, as factors for Sholl and branch order analysis, respectively. The repeated measures ANOVA was followed by planned comparisons of least square means between genotypes. Differences were considered to be statistically significant when p < 0.05.  4.3 Results 4.3.1 Reduced NMDA:AMPA Ratio in Dentate Granule Cells of GluN2A-/- Mice Our initial experiments determined that deletion of the GluN2A subunit does not significantly alter excitatory synaptic transmission in the dentate gyrus (DG). Inputoutput curves for wild-type (WT) and GluN2A-/- mice were not significantly different (Fig. 4.1A). In addition, there was no effect of genotype observed in assays that investigated the probability of vesicular release (Fig. 4.1B), the number of vesicles in the readily releasable pool (Fig. 4.1C), the rate of reserve vesicle pool replenishment (Fig.  !  86  ! Figure 4.1 Synaptic transmission and presynaptic properties are normal in GluN2A-/- mice. A, Normal input-output curve in GluN2A-/- mice. Curves were generated by plotting fEPSP slopes against stimulus pulse width. No differences were found between GluN2A-/- and their WT counterparts. B, Normal paired-pulse activity in GluN2A-/- mice. Paired pulse plasticity was calculated from the ratio of the second fEPSP slope to the first, at different interstimulus intervals. No differences were seen between GluN2A-/- and controls. C, Normal responses to brief high frequency stimulation in GluN2A-/mice. Slices from GluN2A-/- and WT mice were supplied with 50 pulses, delivered at 100 Hz in the presence of APV (50 %M) and BMI (5 %M). No significant differences were observed between genotypes. Inset shows response at 40th stimuli. D, Normal responses to repetitive stimulation in GluN2A -/- mice. Slices from GluN2A-/- and WT mice were supplied with 300 pulses, delivered at 14 Hz in the presence of APV (50 %M) and BMI (5 %M). No significant differences were observed between genotypes. In all graphs, wild-type (WT): black; GluN2A-/-: white. Data is represented as mean ± SEM.  ! 4.1D), conducted as previously described (Cabin et al., 2002; Woo et al., 2005). Overall, these experiments indicate that presynaptic release properties are not significantly altered in the DG of GluN2A-/- mice. To examine if the deletion of the GluN2A subunit modifies the function of postsynaptic ionotropic glutamate receptors in the DG, whole cell recordings from mature  !  87  dentate granule cells were performed. Mature dentate granule cells were defined by their well characterized hyperpolarized resting membrane potential, low input resistance and soma location in the outer half of the dentate granular cell layer (Liu et al., 1996; Schmidt-Hieber et al., 2004; Redila and Christie, 2006; Mongiat et al., 2009)(Fig. 4.2A). There were no significant differences between AMPA-EPSCs recorded from WT (1096.8 ± 137.4 pA; n = 7 slices, 6 mice) and GluN2A-/- (-1313.4 ± 197.2 pA; n = 8 slices, 5 mice) mice (t(13) = 0.94, p > 0.1; Fig. 4.2C). NMDA-EPSCs isolated from these same cells were significantly attenuated in the GluN2A-/- mice (-253.8 ± 42.4 pA, t(13) = -5.93, p < 0.0001) when compared to WT (-1123.3 ± 173.8 pA). This reduction was also evidenced as a significant change in the NMDA:AMPA ratio (WT: 1.03 ± 0.08, GluN2A/-  : 0.20 ± 0.03, t(13) = 10.53, p < 0.000001) (Fig. 4.2D). In addition, NMDA-EPSC charge  transfer was significantly reduced in GluN2A-/- mice (WT: 95.06 ± 12.39, GluN2A-/-: 42.96 ± 8.57, t(13) = 3.80, p < 0.005) (Fig. 4.2E). Thus, loss of the GluN2A subunit significantly reduces the NMDA:AMPA ratio in the DG. We next examined the decay rate time constant of the NMDA-EPSC in dentate granule cells. The composition of GluN2 subunits in NMDA receptors modulates the decay rate of NMDA-EPSCs (Flint et al., 1997); specifically, NMDA receptors containing GluN2A demonstrate a small decay time constant, whereas receptors containing GluN2B demonstrate a significantly longer time constant. GluN2A-/- cells did indeed display a significantly longer NMDA-EPSC decay time constant than that observed in WT cells (WT: 56.6 ± 8.40 ms, GluN2A-/-: 128.5 ± 8.28 ms, t(13) = -6.53, p < 0.00005; Fig. 4.2G). Together, these results demonstrate that the loss of GluN2A in dentate granule cells significantly influences NMDA-EPSC kinetics in the DG.  !  88  !  ! ! Figure 4.2 Reduced NMDA:AMPA ratio and increased NMDA receptor decay rate in GluN2A-/mice. A, Image of a dentate granule cell, taken at 40x magnification using oblique illumination (ML: molecular layer, GCL: granule cell layer). B, Representative traces of EPSCs from WT and GluN2A-/synapses, recorded in control ACSF (1), low Mg2+ (0.1 mM) -ACSF with NBQX (5 %M) and glycine (20 %M) (NMDA-isolating ACSF), 2), and low-Mg2+ ACSF with NBQX, glycine and APV (50 %M) (NMDA-isolating ACSF + APV), 3). C, Maximum amplitudes of AMPA and NMDA-EPSCs in WT and GluN2A-/- cells revealed a significant reduction in NMDA-EPSCs at GluN2A -/- synapses. D, The NMDA:AMPA EPSC ratio was severely reduced at GluN2A-/- synapses. E, Charge transfer via NMDA receptors was significantly reduced at GluN2A-/- synapses. F, Representative traces of a GluN2A-/NMDA-EPSC (grey), scaled to a WT NMDA-EPSC (black). G, Weighted decay time constant of NMDA-EPSCs was significantly increased at GluN2A-/- synapses (constant generated through double exponential fit of decay phase). In all graphs, wild-type (WT): black; GluN2A-/-: white. Data is represented as mean ± SEM. * denotes statistical difference (p < 0.05). Scale bar for B = 200 pA, 20 ms, Scale bar for E = 50 ms.  ! !  89  4.3.2 Abolished LTP in the Dentate Gyrus and Reduced LTP in the CA1 of Adult GluN2A-/- Mice It had previously been reported that GluN2A-/- mice have reduced LTP in the CA1 region (Sakimura et al., 1995). This reduction appears to be the result of a higher threshold for induction, as LTP can be reinstated by increasing the intensity of the HFS (Kiyama et al., 1998). To test if there is a higher induction threshold for LTP in the DG, we tried inducing LTP using two HFS conditioning protocols: a weak HFS protocol (wHFS; 2x100 Hz), and a strong HFS protocol (sHFS; 4x100 Hz). The wHFS protocol produced LTP in WT mice (31.7 ± 10.9%; n = 8 slices, 7 mice), but was ineffective in GluN2A-/- mice (1.64 ± 9.47%, n = 7 slices, 7 mice, t(13) = 2.20, p < 0.05) (Fig. 4.3A). The sHFS protocol produced robust LTP in WT mice (43.6 ± 12.2%; n = 13 slices, 7 mice), but failed to induce LTP in the GluN2A-/- DG (0.67 ± 6.65%, n = 7 slices, 5 mice, t(18) = 2.56, p < 0.05) (Fig. 4.3B). LTP induced by the sHFS protocol was fully blocked by 50 %M APV, confirming that only NMDA receptor-dependent LTP was being induced (WT: -4.29 ± 5.38%, data not shown). In the CA1, wHFS induced robust LTP in WT animals (31.3 ± 10.6%; n = 8 slices, 6 mice). The GluN2A-/- mice showed modest LTP (10.30 ± 5.40%, n = 9 slices, 6 mice) that was significantly less than that observed in WT animals (t(14) = 2.03 (one-tail), p < 0.05; Fig. 4.3C). When the sHFS protocol was employed, WT and GluN2A-/- mice showed equivalent LTP in the CA1 subfield (WT: 27.3 ± 6.54%, n = 11 slices, 8 mice, GluN2A-/-: 29.0 ± 8.39%, n = 9 slices, 6 mice, t(18) = -0.17, p > 0.1; Fig. 4.3D). Together, these results demonstrate that  !  90  !  !  Figure 4.3 GluN2A-/- mice have abolished LTP in the DG, altered LTP in the CA1. A, B, Lack of LTP induced through a weak (2x100 Hz, A) and strong (4x100 Hz, B) HFS protocol at GluN2A-/synapses in the DG. Representative traces from WT and GluN2A-/- DG slices, before (1) and after (2) the high frequency conditioning protocol. C, Partially reduced LTP induced through a weak high frequency conditioning protocol (2x100 Hz) at GluN2A-/- synapses in the CA1. D, Normal LTP induced through a strong high frequency conditioning protocol (4x100 Hz) at GluN2A-/- synapses in the CA1. Representative traces from WT and GluN2A-/- CA1 slices, before (1) and after (2) the high frequency conditioning protocols. In all graphs, wild-type (WT): black; GluN2A -/-: white. Data is represented as mean ± SEM. * denotes statistical difference (p < 0.05). Scale bar = 0.2 mV, 5 ms.  !  91  the GluN2A subunit is essential for NMDA receptor-dependent LTP in the DG, while LTP in the CA1 can still be induced through the activation of other NMDA receptors.  4.3.3 Abolished LTD in the Dentate Gyrus and Intact LTD in the CA1 of Adult GluN2A-/- Mice A function for the GluN2B subunit in CA1 LTD has been postulated by some (Kutsuwada et al., 1996; Liu et al., 2004; Izumi et al., 2006), but this hypothesis remains controversial (Bartlett et al., 2007; Morishita et al., 2007). We have previously shown that the GluN2A antagonist NVP-AAM077, but not the GluN2B-specfic antagonist ifenprodil, blocks LTD in the DG of animals that exercise, but not in control animals (Vasuta et al., 2007), suggesting a complex role for GluN2A subunits in LTD in this region. To further examine this issue, we induced LTD in GluN2A-/- mice using a low frequency stimulation (LFS) conditioning protocol. This protocol produced strong LTD in WT animals (-22.6 ± 6.54%; n = 9 slices, 8 mice) but failed to induce LTD in GluN2A/-  mice (-5.03 ± 5.40%, n = 13 slices, 7 mice, t(18) = 2.21, p < 0.05) (Fig. 4.4A). In  contrast to the DG, LFS applied to the CA1 region produced equivalent LTD in both GluN2A-/- and WT mice (WT: -14.3 ± 7.43%, n = 8 slices, 5 mice; GluN2A-/-: -13.1 ± 6.23%, n = 13 slices, 6 mice, t(19) = -0.12, p >0.1) (Fig. 4.4B). These findings indicate that the GluN2A subunit also plays a significant role in LTD in the DG, but not in the CA1.  !  92  !  !  Figure 4.4 GluN2A-/- mice have abolished LTD in the DG, intact LTD in the CA1. A, Lack of LTD induced through a LFS conditioning protocol (1 Hz, 900 pulses) at GluN2A-/- synapses in the DG. Representative traces from WT and GluN2A-/- DG slices, before (1) and after (2) the low frequency conditioning protocol. B, Normal LTD induced through a low frequency conditioning protocol (1 Hz, 900 pulses) at GluN2A-/- synapses in the CA1. Representative traces from WT and GluN2A-/- DG slices, before (1) and after (2) the low frequency conditioning protocol. In all graphs, wild-type (WT): black; GluN2A-/-: white. Data is represented as means ± SEM. * denotes statistical difference (p < 0.05). Scale bar = 0.2 mV, 5 ms.  ! 4.3.4 Intact Dendritic Structure of Dentate Granule Cells in GluN2A-/- Mice Individual synaptic changes are integrated in dendrites, and are therefore greatly influenced by the morphology of the granule cell dendritic tree (Schmidt-Hieber et al., 2007). Our results show clear bidirectional synaptic plasticity impairments in the DG in the GluN2A-/- mice, and we next examined whether these impairments were due to reductions in dendritic morphology. Golgi impregnation was used to label the soma and dendritic tree of individual mature dentate granule cells in WT and GluN2A-/- mice.  !  93  ! Figure 4.5 Normal dendritic morphology of dentate granule cells in GluN2A-/- mice. A, Representative light micrographs, with Neurolucida-generated tracing overlay (white), of golgiimpregnated dentate granule cells, acquired at 40x magnification. B, GluN2A -/- cells did not show alterations in total dendritic length. C, A Sholl analysis revealed no main effect of genotype between WT and GluN2A-/- dentate granule cells. D, Branch order analysis revealed no main effect of genotype between WT and GluN2A -/- dentate granule cells. E, GluN2A-/- cells contained similar amounts of dendritic node points. In all graphs, wild-type (WT): black; GluN2A -/-: white. Data is represented as mean ± SEM. * denotes statistical difference (p < 0.05). Scale bar = 20 %m  !  !  Dendritic length did not differ between genotypes (WT: 1229 ± 63.25 %m, n = 18, GluN2A-/-: 1176 ± 76.92 %m, n = 26, t(42) = 0.51, p > 0.5) (Fig. 4.5B). Several analyses were then used to examine dendritic complexity. No main effects of genotype were observed when dendritic complexity was analyzed using a Sholl analysis (F(1,41) = 0.018, p > 0.5) (Fig. 4.5C). Similarly, no effect of genotype was observed in branch order (F(1,42) = 0.075, p > 0.5) (Fig. 4.5D) or number of dendritic nodes (WT: 8.06 ± 0.542, GluN2A-/-: 7.88 ± 0.631, t(42) = 0.20, p > 0.5) (Fig. 4.5E). Thus, the GluN2A does not impact dendritic morphology in the DG, indicating that morphological changes do not contribute to the bidirectional synaptic plasticity impairments observed in the DG of GluN2A-/- mice.  !  94  4.3.5 Compromised Spatial Pattern Processing in GluN2A-/- Mice GluN2A-/- mice have previously been shown to demonstrate impairments at the Morris water maze task (Sakimura et al., 1995). This well-established task, however, is dependent on the entire hippocampus (Morris et al., 1982), and does not provide the sensitivity needed to examine hippocampal region-specific deficits. Having established reduced bidirectional synaptic plasticity only in the DG of GluN2A-/- mice, we sought to investigate if these deficits were accompanied by impairments in behaviours dependent on the DG. We tested mice on a metric spatial change task previously shown to be DGspecific (Goodrich-Hunsaker et al., 2005, 2008; Hunsaker et al., 2008a). The metric spatial change task, which examines spatial pattern processing, allows mice to explore a pair of different objects spaced 40 cm apart (Fig 4.6A). Mice are then re-exposed to the environment, with the same objects moved closer together (10 cm apart). Normal rodents are able to recognize the pair of objects as being metrically displaced and re-explore the pair of objects for a significant amount of time, while rodents with impaired DG functioning fail to detect such differences and spend less time re-exploring the pair of objects (Gilbert et al., 2001; Goodrich-Hunsaker et al., 2008). The GluN2A-/- group showed significantly lower mean ratio scores, indicating that compared to controls, GluN2A-/- mice re-explored the pair of objects to a lesser extent after the metric change (mean ratio WT: 0.59 ± 0.03, n = 8, GluN2A-/-: 0.40 ± 0.02, n = 8, t(14) = 4.04, p < 0.001, Fig. 4.6B). Thus, loss of GluN2A in the DG was sufficient to compromise the DGspecific behaviour of spatial pattern processing.  !  95  !  !  Figure 4.6 Compromised spatial pattern separation in GluN2A-/- mice. A, Behavioural paradigm for the metric spatial change task. B, Decreased performance in metric spatial change task in GluN2A-/mice. Mean ratio score, reflecting the proportion of time re-exploring the metrically displaced objects, was significantly lower in GluN2A -/- mice in comparison to controls. In graphs, wild-type (WT): black; GluN2A -/-: white. Data is represented as mean ± SEM. * denotes statistical difference (p < 0.05)  !  4.3.6 Intact Temporal Pattern Processing in GluN2A-/- Mice To test whether there were also changes in CA1-dependent behaviour in GluN2A/-  mice, we used a temporal ordering task (Fig. 4.7A). The temporal ordering task allows  mice to explore three pairs of objects in sequence, followed by a test period, where they are exposed to the first and third object of the sequence. This task takes advantage of a rodent’s tendency to explore the earlier object in a sequence of objects, if given the choice between two, and performance of this task has been attributed to CA1 functioning (Gilbert et al., 2001; Hoge and Kesner, 2007). All mice similarly explored objects across sessions 1-3 and no significant differences in total object exploration during the test session were detected. Indeed, a two-way repeated measures ANOVA with genotype (WT, GluN2A-/-) and session (session 1, session 2, session 3, test session) as factors revealed no significant main effects of genotype (F(1,10) = 1.16, p > 0.1), session, (F(3, 10) = 1.40, p > 0.1), and no significant interaction between genotype and session (F(3, 30) = 0.57, p > 0.5). To determine the performance of GluN2A-/- mice in temporal ordering, a mean  !  96  ratio score was calculated for both groups. The mean ratio score for the temporal ordering task, which reflects the proportion of time spent exploring the first sequential object (object 1) during the test session, was similar between both genotypes (mean ratio WT: 0.22 ± 0.07, n = 6, GluN2A-/-:0.16 ± 0.05, n = 6, t(10) = 0.007, p > 0.5) (Fig. 4.7B). Thus, loss of GluN2A had no impact on the CA1-specific behaviour of temporal pattern processing.  !  !  Figure 4.7 Normal temporal pattern separation in GluN2A-/- mice. A, Behavioural paradigm for the temporal ordering task. B, Normal performance in temporal ordering task in GluN2A-/- mice. Mean ratio score, reflecting the proportion of time spent exploring object 1 during the test session, did not differ significantly between genotypes. In graphs, wild-type (WT): black; GluN2A -/-: white. Data is represented as mean ± SEM.  4.4 Discussion The present study provides strong evidence that the GluN2A subunit plays a critical role in the physiology and behavioural function of the adult DG. This role is also fundamentally different from its role in the CA1. Specifically, we found that: (1) bidirectional synaptic plasticity in the DG requires the presence of GluN2A subunits, whereas GluN2A plays a lesser role in the neighboring CA1 region; (2) the GluN2A subunit is necessary for optimal performance in the DG-specific task of spatial pattern separation. Conversely, GluN2A subunits were not involved in the CA1-specific task of  !  97  temporal pattern separation. Together, these results present a unique link between DG synaptic plasticity and behavioural function that is dependent on the GluN2A subunit.  4.4.1 Alterations in Synaptic NMDA Receptor Responses in the Dentate Gyrus Dentate granule cells from GluN2A-/- mice had normal AMPA-EPSCs, but reduced NMDA-EPSCs, corresponding to an ~80% reduction in NMDA:AMPA ratio. This is even greater than the change in NMDA:AMPA ratio previously shown in the CA1 (Sakimura et al., 1995). In addition, the NMDA:AMPA ratio in control cells was much higher in this study in comparison to those observed in the CA1 (~43%) (Sakimura et al., 1995). This discrepancy most likely reflects methodological differences in how NMDA receptor currents are isolated: factors such as holding potential, stimulation method, and extracellular Mg2+ concentration can alter the measured NMDA:AMPA ratio (Myme et al., 2003). Despite these methodological differences, reduced NMDA-EPSCs, and decreased NMDA:AMPA ratios in GluN2A-/- mice were observed in both studies. The decay rate of the NMDA-EPSCs in mutant mice was also more than twice that of controls. This is consistent with previous reports of the GluN2A subunit being associated with a reduced decay time constant for NMDA receptor currents (Flint et al., 1997; Bellone and Nicoll, 2007; Rauner and Kohr, 2011). The observed increase in decay time constant also confirms that the loss of GluN2A can alter synaptic NMDA receptor currents in the DG, a region not previously investigated in the GluN2A-/- mouse. In combination with the decreased maximum NMDA-EPSC amplitude, the increase in the decay time constant in mutant mice drastically changes the capacity for ion transfer. In mutant mice, calcium entry via NMDA receptor activation would produce an initially  !  98  small, but prolonged influx of ions. This may modify the activity of several calciumdependent enzymes including Calcium-calmodulin-dependent Kinase-II (CaMKII), and protein phosphatases, whose balance have been suggested to regulate synaptic plasticity (Lisman, 1994; Winder and Sweatt, 2001).  4.4.2 Role of GluN2 subunits in LTP Using pharmacological agents, several studies have associated the GluN2A subunit with LTP in the CA1 (Liu et al., 2004; Zhang et al., 2009), DG (Vasuta et al., 2007) and other non-hippocampal regions (Massey et al., 2004; Dalton et al., 2011). However, the poor subunit specificity for the antagonists used in these studies (NVPAAM077 and zinc) leaves these results open to question (Berberich et al., 2005; Kohr, 2006; Neyton and Paoletti, 2006; Paoletti and Neyton, 2007; Lorca et al., 2011). Due to the low specificity of currently available GluN2A antagonists, genetic manipulations offer a favorable alternative to examining GluN2 contributions to LTP. Here we show for the first time that NMDA receptor-dependent LTP is completely abolished in the DG of GluN2A-/- mice, even when strong levels of HFS are applied. In the neighboring CA1 region, GluN2A-/- mice only show partially reduced LTP, which is rescued using stronger stimulation. The results in the CA1 are in accordance with previous reports (Sakimura et al., 1995; Kiyama et al., 1998), and suggest that GluN2A deletion increases the threshold for LTP induction in the CA1. Furthermore, it indicates that GluN2B-containing receptors may be sufficient to induce LTP in the CA1. In contrast, the results in the DG suggest that GluN2B-containing receptors are not sufficient for LTP induction and that the GluN2A subunit is critical for LTP in the DG.  !  99  4.4.3 Role of GluN2 Subunits in LTD Surprisingly, NMDA receptor-dependent LTD was completely abolished in the DG of GluN2A-/- mice. However, LTD in the CA1 region remained intact in mutant mice, a finding consistent with previous experiments (Longordo et al., 2009). Previous pharmacological experiments have suggested that GluN2B-containing receptors play a primary role in LTD in several other hippocampal and non-hippocampal regions (Liu et al., 2004; Massey et al., 2004; but see Morishita et al., 2007). GluN2 antagonists specific to the GluN2B subunit have been available for electrophysiological studies, however their use is also not without problems. For example, the often-used GluN2B-specific antagonist ifenprodil has recently been shown to potentiate NMDA-EPSCs (Rauner and Kohr, 2011) and alter voltage-gated calcium channels (Delaney et al., 2011). Genetic investigations into the role of the GluN2B subunit have also been problematic, as global deletion of GluN2B produces mice that do not survive beyond postnatal day five (Kutsuwada et al., 1996). It was discovered that early post-natal mutant mice did not demonstrate CA1-LTD, despite the fact that there is a high concentration of GluN2B receptors at neonatal ages (Wenzel et al., 1997). Given that both NMDA subunit expression and synaptic plasticity may differ between early and adult ages (Dumas, 2010), it is difficult to extend any results obtained in very young mice with those obtained in adult mice. That said, recently Brigman and colleagues have generated a mouse with a GluN2B deletion isolated to CA1 and cortical pyramidal cells and observed a partial loss of CA1-LTP, and an abolishment of CA1-LTD (Brigman et al., 2010). It should be noted however that LTD was elicited with low frequency  !  100  stimulation in conjunction with tPDC, a glutamate transporter antagonist that was in itself sufficient to elicit LTD (Brigman et al., 2010).  4.4.4 Deletion of GluN2A Does Not Alter Dendritic Morphology Glutamate receptors, specifically AMPA and NMDA receptors, have been suggested to regulate the integration and modification of synaptic inputs (Myme et al., 2003), which might manifest through the alteration of dendritic morphology. As changes in dendritic morphology could alter synaptic plasticity and behaviour, it was prudent to examine the dendritic structure of dentate granule cells in the GluN2A-/- mice. The dendritic structure was found to be largely intact, as no differences were observed in dendritic length and dendritic arbourization in GluN2A-/- mice. To our knowledge, this is the first time that the contribution of the GluN2A subunit in dendritic morphology has been investigated in vivo, and clearly indicates that GluN2A does not play a role in the dendritic morphology of mature cells in the DG. Our results are in contrast to hippocampal culture work that showed increases in both dendritic length and arbourization after GluN2A RNA interference (Sepulveda et al., 2010). This difference could be attributed to many factors, including cell type examined (as we focused on dentate granule cells) or the differences between the cell culture system and the in vivo brain experiments presented here. Work in Xenopus tadpoles also suggests a complex role for the GluN2A subunit in morphology, as both overexpression and knock-down of GluN2A expression decreases branch clustering (Ewald et al., 2008). Overall, this study and others allude to a multifaceted role of GluN2 subunits in neuronal  !  101  structure, and further experiments will need to be completed before these mechanisms are fully understood.  4.4.5 GluN2 Subunit Contribution to Pattern Separation Although it has long been known that the hippocampus is important for the acquisition of memories (Morris et al., 1982), the contributions of the different hippocampal subfields have only been investigated more recently. Targeted lesion studies have associated the CA1 region with memory encoding and temporal pattern separation, the CA3 with spatial pattern completion, and the DG with spatial pattern separation (reviewed in Kesner et al., 2004). In this study, the deficits in spatial information associated with the DG were seen in GluN2A-/- mice, while processing associated with the CA1 was intact. This result is consistent with reports of compromised rapid spatial pattern separation in mutant mice lacking the obligatory GluN1 subunit in the DG (McHugh et al., 2007). Alterations in the production of newborn neurons in the adult DG, via a process referred to as adult neurogenesis, have also been associated with spatial pattern separation (Clelland et al., 2009). Notably, adult neurogenesis is unaltered in GluN2A-/- mice (Kitamura et al., 2003). Our findings therefore provide a novel link between GluN2A-containing receptors at DG synapses and the ability to discriminate similar spatial distance. Furthermore, these findings implicate a common mechanism between the GluN2A-/- deficits observed at both the behavioral and functional levels in the DG. In conclusion, our results demonstrate that NMDA receptor-dependent bidirectional synaptic plasticity is abolished in the adult DG of GluN2A-/- mice. These  !  102  deficits could not be attributed to alterations in presynaptic properties or synaptic transmission in the DG. Mature dentate granule cells exhibited significantly reduced NMDA receptor currents, however their dendritic structure remained unaltered. The behavioral experiments provide evidence for spatial processing deficits in GluN2A-/mice, specifically for processing metric relationships between objects, a process associated with DG function. We therefore conclude that both synaptic and behavioural plasticity in the adult DG are uniquely regulated by the GluN2A subunit.  !  103  5 NMDA Receptor GluN2A Subunits are Required for Normal Dendritic Development of Dentate Granule Neurons4 5.1 Introduction The generation of new neurons in the dentate gyrus (DG) of the hippocampus has been shown to persist throughout adulthood in many species (Altman and Das, 1965; Kaplan and Hinds, 1977; Kuhn et al., 1996), including humans (Eriksson et al., 1998). These cells are believed to originate from radial glial-like neural stem cells located within the subgranular zone, where they proliferate, adopt a neuronal phenotype, and migrate into the granule cell layer. Immature neurons then proceed to sprout multiple dendrites that extend into the molecular layer, before maturing into neurons that are functionally and structurally indistinguishable from older neurons (van Praag et al., 2002; Ge et al., 2008; Deng et al., 2010). This process of structural plasticity, formally referred to as adult neurogenesis, is tightly regulated, and can be modulated by synaptic activity (Cline, 2001). N-methyl-D-aspartate (NMDA) receptors, primarily known for their involvement in synaptic plasticity (Collingridge et al., 1983; Bliss and Collingridge, 1993) and learning and memory (Morris et al., 1986), have been also shown to modulate adult neurogenesis (Nacher and McEwen, 2006; Ewald and Cline, 2009). However, their exact role during this process is still under debate. For instance, under normal conditions, blocking NMDA receptor activity increases proliferation in the adult DG (Cameron et al., !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 4 A version of this chapter has been submitted for publication. Kannangara, T.S., Bostrom, C.A., Flamank, A., Thompson, L., Cater, R.M., Gil-Mohapel, J., and Christie, B.R. NMDA receptor GluN2A subunits are required for normal dendritic development in dentate granule neurons. !  104  1995; Petrus et al., 2009), while in models of stroke and epilepsy, blocking these receptors inhibits the well known stroke- and seizure-induced increase in cell proliferation (Arvidsson et al., 2001; Jiang et al., 2004). Furthermore, while immature dentate granule cells (DGCs) show drastic morphological changes after learning-induced activation of NMDA receptors, no such alterations are seen in mature cells (Tronel et al., 2010). The NMDA receptor itself is a heterodimer, comprised of two obligatory GluN1 (previously NR1) subunits, and two regulatory subunits from the GluN2 family (GluN2A-D; previously NR2A-D) or GluN3 family (GluN3A-B; previously NR3A-B) (Cull-Candy and Leszkiewicz, 2004). The regulatory subunits impart distinctive functional changes on the NMDA receptor, which include altering the receptor’s biophysical properties and affinity to intracellular binding proteins (Cull-Candy and Leszkiewicz, 2004). This certainly holds true for two of the most highly expressed regulatory subunits in the hippocampus, the GluN2A and GluN2B subunits. The distinct GluN2 subunits offer one possible explanation for the differential role of NMDA receptors during adult neurogenesis, as a transition of GluN2 expression occurs during development (Sheng et al., 1994; Wenzel et al., 1997). Early during neuronal development, the GluN2B subunit is predominantly expressed, while the GluN2A subunit has only minimal expression. Later in development, GluN2A expression increases to approximately equal levels to GluN2B (Wenzel et al., 1997). It is believed that this expression pattern holds true in DGCs during adult neurogenesis (Nacher et al., 2007), suggesting that the GluN2A subunit may not contribute to the early stages of neurogenesis. Indeed, in mice lacking the GluN2A subunit, cell proliferation in  !  105  the DG is intact (Kitamura et al., 2003). However, this temporal change in subunit expression also suggests that GluN2A-containing receptors may play a role in the later stages of neurogenesis, when dendritic arbourization and spine formation are being established. In the present study, we address how the GluN2A subunit may contribute to the different stages of neurogenesis by examining cell proliferation, neuronal differentiation, and neuronal morphology in a mutant mouse line lacking GluN2A expression (Sakimura et al., 1995). We found that the GluN2A subunit did not affect the earliest stages of adult neurogenesis, specifically levels of hippocampal cell proliferation and neuronal differentiation. However, GluN2A deletion produced significant alterations in dendritic morphology and spine density. Importantly, these deficits were restricted to immature neurons and disappear when cells become fully mature, suggesting that GluN2Acontaining NMDA receptors play an important role during neuronal maturation in the adult DG.  5.2 Experimental Procedures 5.2.1 Animals and Housing Conditions Adult (2-2.9 months) male wild-type (WT) and GluN2A knockout (GluN2A-/-) mice with a C57BL/6J background (Sakimura et al., 1995; Townsend et al., 2003) were housed in standard cages (2-3 mice per cage, minimal enrichment) in a colony maintained at 21oC. Animals were maintained on a 12-hour light/dark cycle with access to food and water ad libitum. All procedures were approved by the University of Victoria Animal Care Committee and in accordance with the Canada Council on Animal Care.  !  106  5.2.2 Genotyping 5.2.2.1 DNA Isolation DNA extraction was performed on ear or tail snip tissue stored at -20 °C using a PureLink Genomic DNA Purification Kit (Invitrogen Canada; Burlington, Ontario, Canada). Briefly, tissue was placed in a Dnase, Rnase-free 1.5-ml microtube with 180 µL genomic digestion buffer and 20 µL of Proteinase K, and incubated overnight at 55 °C, while mixing at 300 rpm with an Eppendorf Thermomixer (Model R, Brinkmann Instruments, Westbury, NY, USA). Samples were centrifuged for 3 min and the supernatant was transferred into new microtubes. Supernatant was incubated in 20 µL of RnaseA for 2 minutes. Following this step, 200 µL of Lysis Buffer/Binding Buffer and 200 µL of 100% ethanol was added sequentially to each microtube. Lysates were then transferred to Genomic spin columns and collection tubes and centrifuged at 10,000 g for 1 minute. Spin columns were placed into new collection tubes and samples were washed with 500 µL of Genomic Wash Buffer I, followed by centrifuging samples at 10,000 g for 1 minute. Collection tubes were replaced and samples were washed with 500 µL of Genomic Wash Buffer II, and centrifuged at 21,000 g for 3 minutes. Collection tubes were again replaced and 100 µL of Elution Buffer was added. Samples were incubated for 1 minute at room temperature, and then centrifuged at 21,000 g for 1 minute. Collection tubes containing DNA were stored at -20°C until being processed by polymerase chain reaction (PCR).  !  107  5.2.2.2 Polymerase Chain Reaction Each sample (2 µL DNA) was incubated in PCR master mix, consisting of: 14.85 µL Nuclease-free H2O, 2.5 µL 10$ PE Reaction Buffer, 1.4 µL (50 mM) MgCl2, 2.0 µL (2.5 mM) dNTP, 0.5 µL of NR2A1 Primer, 1.0 µL of NR2A3 Primer, 0.5 µL of Neo2A Primer, and 0.25 µL (5U/ µL) Taq DNA polymerase (Invitrogen Canada; Burlington, Ontario, Canada). The cycling parameters employed were as follows: first cycle of 4 minutes at 94 °C, then 29 cycles of: 30 s at 94 °C, 40 s at 60 °C and 60 s at 72 °C. Samples were left at 72 °C for 7 minutes and then stored at 4 °C until use. Primers used were NR2A1 (5’-TCT GGG GCC TGG TCT TCA ACA ATT CTG TGC-3’), NR2A3 (5’-CCC GTT AGC CCG TTG AGT CAC CCC T-3’) and Neo2A (5’-GCC TGC TTG CCG AAT ATC ATG GTG GAA AAT-3’) (Invitrogen Canada; Burlington, Ontario, Canada). PCR products were run on a 1.5% agarose gel with SYBR-safe and visualized using a trans-illuminator.  5.2.3 Immunohistochemistry 5.2.3.1 Tissue Preparation GluN2A-/- (n = 7) and WT littermate mice (n =5) were deeply anesthetized with an intraperitoneal (i.p.) injection of urethane (250 mg/ml in water; 10 mg/kg of body weight) and transcardially perfused with 0.9 % NaCl followed by 4 % paraformaldehyde (PFA). The brains were removed and left in 4 % PFA overnight at 4 ºC and then transferred to 30 % sucrose. Following saturation in sucrose, serial coronal sections were obtained on a vibratome (Leica VT1000S, Nussloch, Germany) at a 30 %m thickness. Sections were collected into a 1/6 section-sampling fraction and stored in an anti-freeze  !  108  cryoprotectant solution [0.04 M Tris-buffered saline (TBS), 30 % ethylene glycerol, 30 % glycerol] at 4 ºC.  5.2.3.2 Immunohistochemistry One in six sections were processed for detection of the following endogenous markers: 1) Ki-67, a nuclear protein expressed during all active phases of the cell cycle, but absent from cells at rest (Kee et al., 2002; for review, see Christie and Cameron, 2006), 2) proliferating cell nuclear antigen (PCNA), which is expressed during all active phases of the cell cycle and for a short period of time after cells become post-mitotic (for review, see Christie and Cameron, 2006), and 3) neurogenic differentiation protein (NeuroD), a basic helix-loop-helix transcription factor involved in neuronal differentiation (Brunet and Ghysen, 1999; Miyata et al., 1999). Briefly, after thorough rinsing, sections that were processed for Ki-67 and PCNA were incubated twice in 10 mM sodium citrate buffer (in 0.1 M TBS, pH = 6.0) at 95°C for 5 minutes. For all immunohistochemical procedures, sections were then quenched with 3% H202/10% methanol in 0.1 M TBS for 15 minutes and pre-incubated for 1 hour with either 5% normal goat serum (for Ki-67 and PCNA) or normal horse serum (for NeuroD). Sections were then incubated for 48 hours at 4°C with the respective primary antibody: 1) rabbit polyclonal anti-Ki67 (1:500; Vector Laboratories, Burlingame, CA, USA), 2) rabbit polyclonal anti-PCNA (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or 3) goat anti-NeuroD (1:200; Santa Cruz Biotechnology). After thorough rinsing, sections were incubated for 2 hours with a biotin-conjugated secondary antibody: goat anti-rabbit IgG (1:200; Vector Laboratories) for Ki67 and PCNA, or horse anti-goat  !  109  IgG (1:200; Vector Laboratories) for NeuroD. Bound antibodies were visualized using an avidin-biotin-peroxidase complex system (Vectastain ABC Elite Kit, Vector Laboratories) with diaminobenzidine (DAB; Vector Laboratories) as a chromogen. The sections were mounted onto 2% gelatin-coated microscope slides, dehydrated in a series of ethanol solutions of increasing concentrations followed by a 5 minutes incubation with a xylene substitute (CitriSolv, Fisher Scientific, Fair Lawn, NJ, USA), and coverslipped with Permount mounting medium (Fisher Scientific).  5.2.3.3 Cell Quantification All morphological analyses were performed on coded slides, with the experimenter blinded to animal identity, using an Olympus microscope (Olympus BX51, Center Valley, PA, USA) with 10x, 40x and 100x objectives. Image Pro-Plus software (version 5.0 for Windows TM, Media Cybermetic Inc., Silver Spring, MD, USA) and a Cool Snap HQ camera (Photometrics, Tucson, AZ, USA) were used for image capture. A modified stereological approach was used to estimate the total number of Ki-67-, PCNA-, and NeuroD-positive cells in the subgranular zone of the DG of the hippocampus following a procedure previously described by us (Kannangara et al., 2009; Kannangara et al., 2011) and others (Nixon and Crews, 2004; Kempermann, 2006). All sections containing the DG and spanning the entire dorsal/ventral axis of the hippocampus (from 1.34 mm posterior to Bregma to 3.52 mm posterior to Bregma (Paxinos et al., 2001)) were used for the analysis, resulting in 9-10 DG sections per animal. In each section, all cells positive for either Ki-67, PCNA, or NeuroD that were present within two to three nuclear diameters below the granule cell layer were counted. The results were expressed  !  110  as the total number of labeled cells in the DG sub-region of the hippocampus by multiplying the average number of labeled cells/DG section by the total number of 30 %m thick-sections that contain the DG (estimated as 73 sections). Images were processed with Adobe Photoshop 4.0 (Adobe Systems Mountain View, CA, USA). Only contrast enhancements and colour level adjustments were made; otherwise images were not digitally manipulated.  5.2.4 Golgi Staining 5.2.4.1 Golgi Impregnation GluN2A-/- (n = 6) and WT littermate mice (n = 6) were briefly anaesthetized with isoflurane (Abbott Laboratories, North Chicago, IL, USA) followed by an i.p. injection of urethane (250 mg/ml in water; 10 mg/kg of body weight). Mice were then transcardially perfused with 0.9% saline. Brains were immediately removed and placed in vials containing 20 mL of modified Golgi-Cox solution and stored at room temperature, in the dark (Gibb and Kolb, 1998). This solution was replaced with fresh Golgi-Cox solution after 24 hours and stored at room temperature in the dark for 14 days. The brains were then transferred to 30% sucrose solution and stored in the dark at room temperature for a maximum of 21 days.  5.2.4.2 Slice Preparation and Processing Coronal sections (200 %m) were generated throughout the length of the hippocampus using a vibratome (Leica, 1400, Nussloch, Germany). Sections were immediately mounted onto 2% gelatin-coated microscope slides and processed as  !  111  previously described (Redila and Christie, 2006). Briefly, sections were sequentially placed in: dH2O (1 min), ammonium hydroxide (30 min), dH2O (1 min), Kodafix for film (30 min), dH2O (1 min), 50% ethanol (1 min), 70% ethanol (1 min), 95% ethanol (1 min), 100% ethanol (2 x 5 min), 100% ethanol/HemoDe/chloroform (1:1:1 for 10 min) and finally HemoDe (2 x 15 min). After processing, all slides were coverslipped, using Permount mounting medium (Fisher Scientific) and stored in the dark.  5.2.4.3 Dendritic Analysis All morphological analyses were performed on coded slides, with the experimenter blinded to animal identity, using an Olympus CX21 light microscope with 10x and 40x objectives. Eight to eleven DGCs from each brain were classified into one of two groups based on the soma location in the granule cell layer: outer granular zone (OGZ) cells had somas located in the outer half of the granule cell layer (adjacent to the molecular layer), and inner granular zone (IGZ) cells had somas located in the inner half of the granule cell layer (adjacent to the SGZ and hilus), as previously described (Redila and Christie, 2006). Cells with somas located directly at the halfway point of the granule cell layer (between the molecular layer and the hilus) were excluded. For this study, 4-6 OGZ and 4-5 IGZ cells were analyzed per animal. Cells were uniformly distributed through the dorsal-ventral axis. Golgi-impregnated cells were selected if they fulfilled the following criteria: 1) had consistent impregnation throughout the extent of the cell body and dendrites, 2) were distinguishable from neighboring impregnated cells, and 3) had intact dendritic trees. Each selected cell was traced by hand using a camera lucida projection from the microscope at 40x magnification. Cells were then scanned at 300 dpi  !  112  and measured using the NeuroJ plugin (Meijering et al., 2004) for ImageJ (Rasband W.S., ImageJ, NIH, Bethesda, Maryland). Differences between WT and GluN2A-/- cells were assessed by examining mean total dendritic branch length and branch order. A Sholl analysis (Sholl, 1956) was performed by quantifying the number of dendritic processes crossing concentric circles located at 20 %m intervals. Digital images were obtained using a Cool Snap HQ CCD camera (Photometrics) and Image Pro-Plus software (version 5.0 for Windows TM, Media Cybermetic Inc.).  5.2.4.4 Spine analysis A subset of golgi-impregnated cells was randomly chosen for spine density analysis using an Olympus BX51 microscope with 40x and 100x objectives, and a Retiga-2000R camera (QImaging, Surrey, BC, Canada). The DG molecular layer was subdivided into three equal regions. For this study, the most outer region (outer molecular layer), and the middle region (middle molecular layer) were investigated, as they are the location of the lateral perforant path, and medial perforant path, respectively. For each cell, three 10 %m dendritic segments were randomly chosen, under x100 magnification, from each region. Z-stacks (stack depth varied depending on dendrite segment) were obtained for each segment, and the number of spines per 10 %m was quantified using Neurolucida and NeuroExplorer morphometry software (MBF Bioscience, Williston, VT, USA).  !  113  5.2.5 Statistical Analysis Computed results were processed for statistical analysis using Excel 2007 (Microsoft Office) and Statistica 7.0 (Statsoft Inc., Tulsa, OK, USA). For all studies, data were presented as mean ± standard error of the mean (S.E.M.). Individual unpaired, two-tail student’s t-tests were performed for Ki-67, PCNA, NeuroD and dendritic branch length analyses. A repeated measures analysis of variance (ANOVA) was performed for Sholl analysis and branch order, using genotype and either distance from soma, or branch order, as factors for Sholl and branch order analysis, respectively. The repeated measures ANOVA was followed by planned comparisons of least square means between genotypes. A 2-tailed 3x2 Fisher’s exact test was used to determine differences in the proportion of granule cells with different numbers of primary dendrites. Differences were considered to be statistically significant when p < 0.05.  5.3. Results 5.3.1 Cell Proliferation and Neurogenesis is Intact in GluN2A-/- Mice To assess whether the loss of the GluN2A subunit alters the levels of proliferation in the adult DG, we performed immunohistochemistry for the endogenous proliferation marker Ki-67, a nuclear protein expressed during all active phases of the cell cycle but absent in cells at rest (Kee et al., 2002; for review, see Christie and Cameron, 2006). GluN2A-/- mice showed a similar number of Ki-67-positive cells in comparison to their WT littermates (WT: 2263 ± 193, GluN2A-/-: 2464 ± 225, t(9) = -0.683, p > 0.5, Fig. 5.1A and B). Immunohistochemistry for proliferating cell nuclear antigen (PCNA), another endogenous marker of cell proliferation that is expressed during all phases of the cell  !  114  ! Figure 5.1 Intact cell proliferation in the adult dentate gyrus of GluN2A-/- mice. A, C, Representative images of dentate gyrus sections processed for Ki-67 (A) and PCNA (C) immunohistochemistry in both wild-type (WT, left panel) and GluN2A-/- (right panel) mice. B, D, No change in the number of Ki-67-positive cells (B) or PCNA-positive cells (D) in the adult dentate gyrus of GluN2A-/- mice in comparison to wild-type littermates. For both graphs, WT: black; GluN2A -/-: white. Data is represented as means ± SEM. Scale bar = 100 %m (A, C).  !  ! cycle, confirmed these results (WT: 2230 ± 114, GluN2A-/-: 2202 ± 115, t(9) = 0.169, p > 0.5, Fig. 5.1C and D). Together, these results strongly indicate that the absence of the GluN2A subunit does not alter cell proliferation in the adult DG. The levels of neuronal differentiation were determined by examining the number of cells that express the neurogenic differentiation protein, NeuroD (Brunet and Ghysen, 1999; Miyata et al., 1999). GluN2A-/- mice showed a similar number of NeuroD-positive cells when compared with their WT littermate controls (WT: 7369± 982, GluN2A-/-: 7450 ± 992, t(9)= -0.062 , p > 0.5, Fig. 5.2), indicating that neuronal differentiation is also unchanged by GluN2A expression.  !  115  ! ! Figure 5.2 Intact differentiation in the adult dentate gyrus of GluN2A-/- mice. A, Representative images of dentate gyrus sections processed for NeuroD immunohistochemistry in wild-type (WT, left panel) and GluN2A-/- (right panel) mice. B, No change in the number of NeuroD-positive cells in the adult dentate gyrus of GluN2A-/- mice, in comparison to wild-type littermates. For graph, WT: black; GluN2A -/-: white. Data is represented as means ± SEM. Scale bar = 100 %m (A).  !  ! 5.3.2 Impaired Dendritic Morphology in Late Immature Neurons of GluN2A-/- Mice The DG follows an outside-in gradient in regards to cell age, with mature cells located in the outer layer of the granular zone (i.e., in the OGZ), and immature cells located in the inner layer of the granular zone (i.e., in the IGZ) (for review, see Zhao et al., 2008). Another characteristic of immature cells is the presence of few primary dendrites, whereas mature cells often have multiple primary dendrites (Wang et al., 2000; Redila and Christie, 2006). To verify that cells present in the IGZ are primarily immature while cells within the OGZ are predominantly mature, we used the Golgi impregnation technique to examine the number of primary dendrites present in each cell (Fig. 5.3A and B). Consistent with previous findings, WT cells with somas located in the IGZ were significantly different when compared to those with somas located in the OGZ (2-tailed 3x2 Fisher’s exact test, p < 0.001, Fig. 5.3C and D): IGZ cells predominantly had one primary dendrite (1o: 85.7%, 2o: 14.3%. 3o+: 0.0%), while OGZ cells had multiple primary dendrites (1o: 33.3%, 2o: 37.5%, 3o+: 29.2%). This finding further confirms  !  116  previous studies suggesting that IGZ neurons are predominantly immature while OGZ neurons are mostly mature. The percentage of DGCs with multiple primary dendrites was unchanged in GluN2A-/- mice both in the IGZ (1o: 87.5%, 2o: 9.4%, 3o+: 3.1%; 2-tailed 3x2 Fisher’s exact test, p > 0.5, Fig. 5.3C) and the OGZ (1o: 25%, 2o: 50%, 3o+: 25%; 2-tailed 3x2 Fisher’s exact test, p > 0.5, Fig. 5.3D), indicating that the GluN2A subunit does not alter primary dendrite number.  !  !  Figure 5.3 GluN2A does not alter the number of primary dendrites in immature and mature cells in the adult dentate gyrus. A, B, Representative images of Golgi-impregnated cells from the inner granular zone (IGZ, A) and outer granular zone (OGZ, B). Light micrographs were acquired at 10x (left panels) and 40x (right panels) magnification. C, D, Percentage of dentate granule cells with different numbers of primary dendrites. GluN2A expression did not alter the number of cells with one (black), two (grey), and three or more (white) primary dendrites in IGZ (C) and OGZ (D) cells. Scale bar = 100 %m (left panels), 25 %m (right panels) (A, B).  ! To determine the contribution of GluN2A to dendritic morphology, we examined the length and complexity of dendritic arbours. Interestingly, a significant decrease in total dendritic length was observed in immature cells from GluN2A-/- mice (WT: 972.41 ± 57.39 %m, GluN2A-/-: 783.18 ± 77.11 %m, t(51) = 2.417, p < 0.05, Fig. 5.4A). A Sholl  !  117  analysis revealed main effects of both genotype (F(1, 51) = 5.26, p < 0.05, Fig. 5.4B;) and distance from soma (F(11, 561) = 109.8, p < 0.0001) in immature cells. Further analysis revealed significant reductions in dendritic complexity from 80 to 160 %m from the soma in immature GluN2A-/-cells (p < 0.05 for each 80-160 %m comparison). Branch order analysis also revealed a main effect of genotype in immature cells (F(1, 51) = 4.17, p < 0.05, Fig. 5.4C), with a significant reduction observed in third order branches in immature cells from GluN2A-/- mice (F(1, 51) = 7.25, p < 0.05). In contrast to immature cells, mature cells from GluN2A-/- mice showed no  !  !  Figure 5.4 Altered dendritic morphology in immature dentate granule cells from GluN2A-/- mice. A, D, Total dendritic length was significantly decreased in GluN2A-/- inner granular zone (IGZ) cells (A) and unaltered in GluN2A-/- outer granular zone (OGZ) cells (D). B, E, Sholl analysis of IGZ (B) and OGZ (E) cells. A repeated measures ANOVA revealed a main effect of genotype in IGZ cells (B), with GluN2A-/- IGZ cells having a lower number of dendrites per ring intersection from 80-160 %m from soma. No effect of genotype was seen in OGZ cells (E). C, F, Branch order analysis of IGZ (C) and OGZ (F) cells. A repeated measures ANOVA revealed a main effect of genotype in IGZ cells (C), showing a significantly less number of third-order branches in GluN2A-/- IGZ cells. OGZ cells did not show a main effect of genotype (F). For all graphs, WT: black; GluN2A-/-: white. Data is represented as means ± SEM. * denotes statistical significant difference (p < 0.05).  !  !  118  reduction in dendritic length (WT: 971.61 ± 50.56 %m, GluN2A-/-: 946.56 ± 42.56 %m, t(54) = 0.397, p > 0.5, Fig. 5.4D). A Sholl analysis only revealed a main effect of distance from the soma (F (10, 530) = 192.68, p < 0.0001, Fig. 5.4E), with no significant reductions in dendritic complexity between genotypes. Similarly, branch order analysis did not reveal a main effect of genotype, only branch order (F(6, 424) = 105.80, p < 0.0001, Fig. 5.4F).  5.3.3 Immature Neurons of GluN2A-/- Mice Show Localized Increases in Spine Density Spine density was analyzed in two locations, the middle molecular layer and the outer molecular layer, where DGCs receive afferent input from the medial perforant path and lateral perforant path, respectively. GluN2A-/- cells from the IGZ showed approximately a 21% increase in spine density in dendrites that were sampled from the middle molecular layer (WT: 15.8 ± 1.00, GluN2A-/-: 19.1 ± 1.22, t(21)= -2.16, p < 0. 05, Fig. 5.5C), but not from the outer molecular layer (WT: 15.8 ± 1.93, GluN2A-/-: 17.7 ± 0.88, t(20)= -1.25, p > 0.1). In comparison, GluN2A-/- cells from the OGZ, defined as mature cells, showed no differences in spine density in dendrites sampled from either the middle molecular layer (WT: 17.8 ± 0.59, GluN2A-/-: 17.0 ± 0.90, t(33) = 0.69, p > 0.5, Fig. 5.5F) or the outer molecular layer (WT: 15.6 ± 0.92, GluN2A-/-: 15.2 ± 0.80, t(32) = 0.33, p > 0.5).  !  119  !  ! Figure 5.5 Immature cells have localized increases in spine density in the adult dentate gyrus of GluN2A-/- mice. A, D, Representative tracings of inner granular zone (IGZ, A) and outer granular zone (OGZ, D) cells produced using Neurolucida. Three dendritic regions were chosen from both the middle molecular layer (MML) and outer molecular layer (OML) of the dentate gyrus. B, E, Representative images of dendritic regions from wild-type (WT, upper panel) and GluN2A -/- (lower panel) granule cells located in the IGZ (B), and OGZ (E). C, F, Spine density of IGZ (C) and OGZ (F) cells. An increase in spine density was observed in IGZ cells from GluN2A-/-, but only in the middle molecular layer (C). No changes in spine density were seen in OGZ cells, in either region of the molecular layer (F). GCL = Granular Cell Layer. Data is represented as means ± SEM. Scale bar =1 %m (B, E). * denotes statistical significant difference (p < 0.05).  5.4 Discussion The results presented here show for the first time that the GluN2A subunit plays an important role during a specific phase in the development of adult born DGCs (see Fig. 5.6 for summary). While the levels of cell proliferation and neuronal differentiation are unaltered by the absence of this NMDA receptor subunit, the maturation process of new neurons appears to be compromised. During this “late immature” stage, neurons show significant morphological alterations in GluN2A-/- mice when compared with their  !  120  WT littermate controls. This finding suggests that the GluN2A subunit plays a crucial role in dendritic morphology and spinogenesis of immature DGCs.  !  ! Figure 5.6 Summary schematic of the results. The GluN2A subunit alters the “late immature” stage of adult neurogenesis in the dentate gyrus, as GluN2A-/- neurons (white) show decreased dendritic arbourization and length, and increased spine density in the middle molecular layer, in comparison to wild-type neurons (black). Both earlier and later stages of adult neurogenesis in the dentate gyrus are intact in mutant mice. SGZ: Subgranular Zone.  !  5.4.1 Role of the GluN2A Subunit in Early Stages of Adult Neurogenesis The NMDA receptor and its major hippocampal subunits are not significantly expressed during the earliest stages of neurogenesis (i.e., in type-2a, type-2b and type 3 cells), although GluN1 and GluN2B subunits may be minimally present in type-1 radial glia-like neural stem cells (Nacher et al., 2007). Expectedly, we did not observe any changes in the number of proliferating cells and immature neurons in GluN2A-/- mice, as indicated by two independent, endogenous cell cycle markers (Ki-67 and PCNA) and the immature neuronal marker NeuroD, respectively. Our findings substantiate previous studies demonstrating intact proliferation in the same GluN2A-/- mouse model as assessed with the exogenous mitotic marker bromodeoxyuridine (BrdU) (Kitamura et al., 2003). While the number of proliferating cells in our study is significantly higher than those seen previously (Kitamura et al.,  !  121  2003), this difference can be attributed to the increased expression of endogenous markers during the cell cycle, while BrdU is only incorporated into proliferating cells during S-phase (Christie and Cameron, 2006). Importantly, no significant differences in the number of proliferating cells were observed between WT and GluN2A-/- groups, regardless of the proliferation marker used. While others have suggested that blocking GluN2A-containing receptors may produce subtle decreases in cell proliferation in the DG (Hu et al., 2009), that study used NVP-AAM077, a GluN2A antagonist that may lack NMDA receptor subunit specificity in rodent tissue (Neyton and Paoletti, 2006). A previous study examining NMDA receptor subunit expression during adult neurogenesis has indicated that NeuroD expression approximately coincides with a significant increase in GluN1 and GluN2B expression (Nacher et al., 2007). It is currently unknown when GluN2A expression begins in adult born DGCs, but it has been hypothesized that the adult neurogenesis timeline of GluN2A expression may mirror other systems during development, where the GluN2A subunit is only strongly expressed once both GluN1 and GluN2B subunits have been expressed (Monyer et al., 1994; Quinlan et al., 1999). Furthermore, GluN2A-containing receptors appear to be present only after synapse formation and not before (Tovar and Westbrook, 1999). This would indicate that strong GluN2A expression occurs after neuronal differentiation and our results, demonstrating intact levels of neuronal differentiation in the GluN2A-/- DG, are in agreement with this hypothesis.  !  122  5.4.2 Role of the GluN2A Subunit in the “Late Immature” Stage of Adult Neurogenesis Previous reports examining several neuronal populations (Cline and ConstantinePaton, 1989; Lee et al., 2005), including DGCs (Brewer and Cotman, 1989; Tronel et al., 2010), have suggested that NMDA receptor activity can modulate dendritic branching. Only recently have the roles of NMDA receptor subunits in neuronal development been investigated. Disrupting GluN2B expression alters dendritic arbourization in neurons from the trigeminal nucleus (Kutsuwada et al., 1996), and ventral spinal cord (Sepulveda et al., 2010), but produces only a modest change in hippocampal cells (Sepulveda et al., 2010). A mosaic-based mutant model that deleted GluN2B expression in individual DGCs demonstrated a reduction in the percentage of cells with multiple primary dendrites; notably, this effect was only observed in embryonic-born DGCs, and not adultborn cells (Espinosa et al., 2009). Work in Xenopus tectal neurons revealed that GluN2B-containing receptors may decrease branch clustering and stability (Ewald et al., 2008). Overall, these studies suggest a role for GluN2B-containing receptors in maintaining dendritic arbourization, but the role is still unclear. Currently, there is no consensus as to the role GluN2A-containing receptors play in dendritic morphology. Here we report that GluN2A deletion alters dendritic morphology at a specific time during DGC development in the adult mouse DG. GluN2A-/- DGCs located in the IGZ showed significant reductions in dendritic length and arbourization. Previous reports where GluN2A expression was silenced showed increases in dendritic length and complexity in hippocampal cultures, but not in ventral spinal cord cultures (Sepulveda et al., 2010). Furthermore, both overexpression and  !  123  knock-down of GluN2A in Xenopus tectal neurons were shown to decrease branch clustering without modifying dendritic length (Ewald et al., 2008). It is possible that the role of the GluN2A subunit in dendritic morphology may vary according to the cell type, and future experiments are warranted in order to elucidate the exact role of GluN2A in dendrites.  5.4.3 Role of the GluN2A Subunit in Spinogenesis NMDA receptor activity is capable of modifying spine formation, as mice lacking cortical NMDA receptors have decreased spine density and altered spine morphology (Ultanir et al., 2007). In regards to NMDA receptor subunits, it had been reported that targeted GluN2B deletion decreases spine density (Akashi et al., 2009; Brigman et al., 2010). A link between GluN2A and spine density has also been formed, although the role of GluN2A is still unclear. Knock-down of alpha 1-chimerin, a non-kinase phorbol ester receptor that binds to GluN2A, increases spine density in the CA1 region of the hippocampus (Van de Ven et al., 2005). A recent study in hippocampal cultures saw a significant decrease in spine addition due to early GluN2A expression and a trend for spine additions due to GluN2A knock-down, further suggesting that the GluN2A subunit has an inhibitory role on spine density (Gambrill and Barria, 2011). In contrast, deletion of IQGAP1, a synaptic scaffolding protein, lowers GluN2A expression and decreases spine density in the CA1, but not in the medial prefrontal cortex or basal amygdala (Gao et al., 2011). Here we provide direct in vivo evidence of a link between the GluN2A subunit and spine density. We detected a specific increase in spine density in immature GluN2A-  !  124  /-  neurons that was localized to the middle molecular layer. While it is unclear as to why  the middle molecular layer was affected by the loss of GluN2A subunit, this finding may be attributed to the type of afferent input located in this region. The DG receives major excitatory input from the perforant path, which can be subdivided into medial and lateral pathways based on anatomical and physiological properties. The medial perforant path innervates DGC dendrites in the middle molecular layer and demonstrates substantial NMDA receptor-dependent long-term potentiation, while the lateral perforant path innervates the outer molecular layer, and demonstrates less potentiation that can be regulated by opioid receptors (Bramham et al., 1991a; Colino and Malenka, 1993). Such studies suggest that the quantity of NMDA receptors at medial perforant path synapses, in the middle molecular layer, may be higher than at lateral perforant path synapses, in the outer molecular layer. Interestingly, autoradiography experiments indicate a higher density of NMDA receptors in the inner half of the molecular layer in comparison to the outer half (Monaghan and Cotman, 1985). Thus, the high concentration of NMDA receptors at spines in the middle molecular layer suggests that alterations in the expression of particular NMDA receptor subunits may have a greater impact on these spines. The present results reveal deficits in the dendritic morphology of DGCs in the GluN2A-/- mouse. Interestingly, these deficits were not observed in cells located in the OGZ, suggesting the possibility of compensatory mechanisms, perhaps utilizing GluN2B-containing NMDA receptors. An alternative possibility is that the loss of GluN2A subunit may delay maturation of DGC morphology, producing deficits in cells located in the IGZ, but intact morphology in cells located in the OGZ.  !  125  While the present results suggest an important role for the GluN2A subunit in the dendritic development of DGCs, two important factors should be considered. First, the Golgi impregnation technique used to label DGCs does not allow the co-labeling of cells with a second marker, preventing the assessment of a neuron’s exact age. However, an estimation of the age of a specific neuron can be made based on its morphological characteristics. Thus, the appearance of multiple dendrites that extend the full extent of the molecular layer to the hippocampal fissure (Esposito et al., 2005), the presence of spines in each dendrite (Zhao et al., 2006), and the location of the somas within the IGZ (Redila and Christie, 2006) suggest that these IGZ neurons are in a late phase of their immature stage. A second factor to be considered is that the global deletion of the GluN2A subunit may produce confounding effects on DGCs. For example, while gross brain anatomy is reported to be intact in GluN2A-/- mice (Sakimura et al., 1995), afferent input into the hippocampus may be altered. It is unlikely that presynaptic changes are involved though, as presynaptic NMDA receptors in the perforant path-DGC synapse appear to only contain GluN2B subunits (Jourdain et al., 2007). Future experiments using a mosaic model of GluN2A expression would aide in revealing whether the effects of the GluN2A subunit observed here are indeed cell autonomous.  5.5 Conclusions In conclusion, we have shown that the levels of adult hippocampal cell proliferation and neuronal differentiation are unaltered in GluN2A-/- mice, as is the morphology of mature DGCs. However, absence of the GluN2A subunit altered branch  !  126  arbourization and spine density of immature DGCs. Therefore, GluN2A-containing NMDA receptors play a role in the maturation of adult born DGCs.  !  127  6 General Discussion 6.1 Summary of Findings The purpose of this thesis was to resolve if the loss of GluN2A-containing NMDA receptors produces synaptic and structural plasticity deficits, and impairs information processing in the adult DG.  6.1.1 Establishment of Methodology We began by investigating two methodological aspects regarding plasticity in the DG. First, we determined the optimal amount of disinhibition required to induce LTP in the DG by using different concentrations of the GABAA receptor antagonist BMI. With our HFS protocol, 5 µM BMI provided the least amount of disinhibition required to maximally induce LTP. Due to this finding, LTP experiments in the DG were conducted in the presence of 5 µM BMI. Second, we examined how factors inherent to the design of adult neurogenesis experiments can alter the measured rates of cell proliferation and neuronal differentiation. We discovered that social housing conditions do not alter cell proliferation or differentiation in the adult mouse DG (Kannangara et al., 2009). We also confirmed that voluntary exercise has the capacity to increase both of these processes (Kannangara et al., 2009). In addition, we revealed that acute restraint stress lowers cell proliferation in both isolated and social housing conditions to a similar degree (Kannangara et al., 2009). Based on these findings, mice used in structural plasticity experiments were housed in social housing conditions.  !  128  6.1.2 Synaptic Plasticity To investigate the role of GluN2A in synaptic plasticity, we first examined NMDA receptor-mediated currents in the GluN2A-/- mouse. We hypothesized that mature DGCs from GluN2A-/- mice would have intact AMPA receptor-mediated currents but reduced NMDA receptor-mediated current. As hypothesized, we observed a significant decrease in the maximum evoked NMDA receptor-mediated current response, resulting in severe deficit in the NMDA:AMPA ratio. NMDA receptor-mediated currents from GluN2A-/- mice demonstrated a significantly prolonged decay rate, consistent with the idea that GluN2A incorporation into NMDA receptors shortens NMDA receptor decay rates. Previous reports examining synaptic plasticity in the CA1 of the GluN2A-/- mice had demonstrated that the threshold for LTP was impaired, while LTD and presynaptic properties were intact (Sakimura et al., 1995; Kiyama et al., 1998; Longordo et al., 2009). Therefore, we hypothesized that a similar deficit in LTP threshold would be the only observed deficit in the DG of the GluN2A-/- mouse. While we observed no presynaptic alterations, we revealed impaired NMDA receptor-dependent LTP and LTD in the DG of GluN2A-/- mice. Together, these results demonstrate impairments in bidirectional synaptic plasticity in the DG of the GluN2A-/- mouse.  6.1.3 Structural Plasticity Based on a previous report examining cell proliferation in the GluN2A-/- mouse (Kitamura et al., 2003), we hypothesized that the early stages of adult neurogenesis, termed in this thesis as the precursor and immature stages, would be unaffected in the  !  129  GluN2A-/- mouse. Consistently, we found that GluN2A-/- mice show no differences in cell proliferation or differentiation in the adult DG. While we hypothesized that early stages of adult neurogenesis would be unaffected, we also hypothesized that alterations in neuronal morphology may be observed in mature GluN2A-/- neurons, due to the late temporal expression pattern of GluN2A (discussed in 1.3.3, 5.1, 5.4). Interestingly, we found that mature DGCs in the GluN2A-/- mouse demonstrated no deficits in dendritic length and dendritic complexity, or spine density. Interestingly, DGCs with somas located in the IGZ, and presenting the morphological characteristics of late immature DGCs, demonstrated significant deficits in dendritic length and dendritic complexity. In addition, these late immature DGCs showed an increase in spine density, specific to the middle molecular layer. Thus, our work demonstrates morphological alterations in late immature DGCs from GluN2A-/- mice.  6.1.4 Behavioural Plasticity It had been previously demonstrated that GluN2A-/- mice have deficits in hippocampal-dependent behaviour (discussed in 1.4.5), and thus we hypothesized that GluN2A-/- mice would show impairments in both spatial pattern separation and temporal pattern separation, forms of information processing associated with the DG and CA1, respectively (Gilbert et al., 2001; Hoge and Kesner, 2007; Goodrich-Hunsaker et al., 2008). While GluN2A-/- mice demonstrated a significant deficit in the ability to discriminate objects that were spatially displaced, they demonstrated no impairment in the ability to discriminate objects that were presented in temporal sequence. These results reveal a specific deficit in DG (but not CA1) function in GluN2A-/- mice.  !  130  6.2 Hippocampal Plasticity in the GluN2A-/- Mouse 6.2.1 Synaptic Plasticity and GluN2 Subunits The neuronal mechanism most closely associated with the NMDA receptor is arguably hippocampal synaptic plasticity. Due to unique physiological properties of GluN2 subunits (discussed in 1.3.2.2), several theories involving GluN2 subunits and bidirectional synaptic plasticity have been proposed; these include the “subunit theory”, the “charge transfer theory”, the “ GluN2 structure theory”, and the “GluN2 ratio theory”. Our results (Chapter 4), demonstrating impaired bidirectional synaptic plasticity in the DG of the GluN2A-/- mouse, do not support any one theory alone, and suggest that GluN2 subunits may play a unique role in the DG in comparison to other hippocampal regions. Each theory will be discussed below, while the currently proposed model is discussed in section 6.3.  6.2.1.1 The Subunit Theory of Hippocampal Synaptic Plasticity The “subunit theory” of synaptic plasticity suggests that plasticity is governed by distinctive GluN2 subunits (Liu et al., 2004). Formulation of this theory was the result of elegant pharmacological studies in the CA1 and perirhinal cortex that proposed predominant roles for GluN2A in LTP and GluN2B in LTD (Liu et al., 2004; Massey et al., 2004). This theory was also supported by a study from our laboratory, where intraperitoneal injections of GluN2 antagonists revealed the selective contributions of the GluN2 subunits in synaptic plasticity in vivo (Fox et al., 2006). Additional support for the “subunit theory” includes pharmacological studies demonstrating that late forms of LTP are GluN2A-dependent (Zhang et al., 2009), and that blocking GluN2A and  !  131  GluN2B inhibits LTP and LTD, respectively, in the lateral amygdala (Dalton et al., 2011). Despite its initial popularity, the “subunit theory” has been under considerable debate, in part due to the specificity of the GluN2A antagonists used. Particularly, the aforementioned GluN2A antagonist NVP-AAM077 has been demonstrated to be only ~10-12 times more specific for GluN2A than GluN2B (Neyton and Paoletti, 2006), and has been shown to reduce NMDA receptor currents in GluN2A-/- mice, further suggesting non-subunit specific binding (Berberich et al., 2005). Another commonly used GluN2A antagonist, zinc, shows adequate specificity for GluN2A, but can only maximally inhibit GluN2A-containing NMDA receptors up to 70-80% (Kohr, 2006; Paoletti and Neyton, 2007). In addition, zinc may also modulate P2X receptors at similar concentrations to those used for in vitro synaptic plasticity experiments (Lorca et al., 2011). Notably, the role of GluN2B in LTD has also been questioned, with several independent groups, including our own, being unable to block LTD with GluN2B antagonists (Morishita et al., 2007; Vasuta et al., 2007). Genetic manipulations of GluN2 expression have also produced mixed results with respect to the “subunit theory.” One study demonstrated that GluN2B deletion reduces LTD in early post-natal mice (Kutsuwada et al., 1996). However, GluN2B deletion in CA1 pyramidal cells was shown to increase LTP threshold in addition to abolishing LTD (Brigman et al., 2010). In a different study, reduced synaptic GluN2B expression blocked LTP, but not LTD (Gardoni et al., 2009). Finally, transgenic rats overexpressing GluN2B showed increased LTP and normal LTD (Wang et al., 2009). As previously discussed (and replicated in this thesis, Chapter 4), the CA1 of GluN2A-/-  !  132  mice demonstrate alterations to LTP threshold (Sakimura et al., 1995; Kiyama et al., 1998), but shows intact LTD (Longordo et al., 2009). Our results in the CA1 replicate previous findings (Sakimura et al., 1995; Kiyama et al., 1998; Longordo et al., 2009) and partially support the “subunit theory,” in that GluN2A is exclusively involved in LTP (and not LTD) in the CA1. However, LTP can still be observed with stronger HFS in the GluN2A-/- mouse, thus the GluN2A subunit is not required for LTP in the CA1. Our laboratory has previously investigated the role of GluN2 subunits in synaptic plasticity in the DG. Previous work revealed that both GluN2A and GluN2B were necessary for LTP induction, while neither subunit exclusively regulated LTD (Vasuta et al., 2007). On the other hand, the results presented here (Chapter 4) show that GluN2A is critical for bidirectional plasticity in the DG. Perhaps the primary reason why the present results contrast those observed previously is that Vasuta and colleagues employed the aforementioned GluN2 antagonists (discussed in 6.2.11), making interpretation of these results difficult (Vasuta et al., 2007). In conclusion, while our results from the GluN2A-/- mouse appear to support the “subunit theory” for the CA1, this theory does not appear to extend to the DG.  6.2.1.2 The Charge Transfer Theory of Hippocampal Synaptic Plasticity Ionotropic receptors are characterized for their ability to flux charged ions. NMDA receptors are highly permeable for calcium in comparison to other ions (Sharma and Stevens, 1996), thus the transfer of charged ions (charge transfer) mediated by NMDA receptors is strongly correlated with calcium influx. How charge transfer  !  133  pertains to hippocampal synaptic plasticity was examined by Georg Kohr’s laboratory; they demonstrated a correlation between NMDA receptor-mediated charge transfer and LTP (Berberich et al., 2007). From these experiments, they proposed the “charge transfer theory”, arguing that if charge transfer (and by extension, calcium influx) was sufficiently blocked, the type of GluN2 antagonist used was of little consequence (Berberich et al., 2007). We observed a significant decrease in NMDA receptor-mediated charge transfer in GluN2A-/- mice - it is therefore possible that the lack of LTP in the GluN2A-/- DG may simply be credited to a lack of charge transfer. It should be noted however that the “charge transfer theory” has only been examined for LTP, and therefore it is unclear whether this theory is valid during LTD.  6.2.1.3 The GluN2 Structure Theory of Hippocampal Synaptic Plasticity The “GluN2 structure theory” proposes that the C-terminus domain of GluN2 subunits plays a large role in determining induction of LTP. In organotypic hippocampal cell cultures, removal of the GluN2B C-terminus domain inhibited LTP induction, while blocking GluN2B-containing receptor activity did not (Foster et al., 2010). This inhibition could be rescued if the GluN2B C-terminus domain was attached to the GluN2A subunit (Foster et al., 2010). In contrast, removal of the GluN2A C-terminus domain or removal of synaptic GluN2A-containing receptors enhanced LTP (Foster et al., 2010). These results argue for the importance of subunit structure, suggesting that the GluN2B structure plays a critical role for LTP, while the GluN2A structure is nonessential, and in fact, inhibitory. The results presented in this thesis are not consistent with the idea that GluN2  !  134  structure is the key determinant in LTP. In both hippocampal regions examined, we find that the loss of GluN2A alters LTP, whether it is LTP threshold in the CA1, or complete loss of LTP in the DG. If the proposed theory regarding the GluN2 C-terminus domains was valid, we would expect an enhancement of LTP in the GluN2A-/- mouse. It should be noted that Foster and colleagues used organotypic hippocampal cell cultures, and perhaps their theory cannot be extrapolated to the mouse model system. Indeed, mutant mice lacking the GluN2A C-terminus domain show a reduced LTP threshold in the CA1 (Kohr et al., 2003).  6.2.1.4 The GluN2 Ratio Theory of Hippocampal Synaptic Plasticity The “GluN2 ratio theory” suggests that the direction of synaptic plasticity (either potentiation or depression) is determined by the ratio of synaptic GluN2A-containing receptors to GluN2B-containing receptors. This theory was recently demonstrated in the CA1 by using priming stimulation prior to inducing LTP and LTD with HFS or LFS protocols, respectively (Xu et al., 2009). A low frequency priming protocol decreased the ratio of GluN2A:GluN2B, as determined by using the GluN2B antagonist ifenprodil (Xu et al., 2009). Low frequency priming also facilitated LTP induction, while hindering LTD induction (Xu et al., 2009). The opposite was demonstrated with a high frequency priming protocol, which increased the GluN2A:GluN2B ratio, hindered LTP and facilitated LTD (Xu et al., 2009). While other reports have demonstrated opposite effects to priming stimulation (e.g., low frequency priming simulation applied at the LPP-DGC synapse enhances LTD (Christie and Abraham, 1992b)), the “GluN2 ratio theory” has also been supported by non-priming experiments, such as the demonstration that GluN2A  !  135  transfection into organotypic hippocampal cell cultures inhibits LTP (Barria and Malinow, 2005). The results presented in this thesis also do not support the “GluN2 ratio theory”, as loss of the GluN2A subunit, which maximally lowers the GluN2A:GluN2B ratio, alters LTP threshold, without modifying LTD in the CA1. In the DG, bidirectional synaptic plasticity was impaired, also in contrast to this theory. In fact, LTP at the MPPDGC synapse investigated here is unaltered by priming protocols (Zhang et al., 2005), further suggesting that even if the “GluN2 ratio theory” was valid in the CA1, it may not be valid in the DG.  6.2.2 Structural Plasticity 6.2.2.1 The Role of the NMDA Receptor in Adult Neurogenesis Early evidence has demonstrated a potential role for NMDA receptors in adult neurogenesis in the DG. Single intraperitoneal injections of the NMDA receptor antagonist MK-801 increase cell proliferation in the adult DG (Cameron et al., 1995; Cameron et al., 1998). Lesions to the entorhinal cortex, the source of the perforant path, also increase proliferation (as well as differentiation and survival) (Cameron et al., 1995; Gama Sosa et al., 2004), suggesting that excitatory activity via NMDA receptor inhibits cell proliferation. In contrast to this theory, several lines of in vitro evidence suggest that the role of the NMDA receptors is more complex. Deisseroth and colleagues (2004) dissociated adult hippocampal neural precursor cells and found that glutamate application would cause cells to acquire a neuronal phenotype, an effect dependent on both L-type calcium  !  136  channels and NMDA receptors (Deisseroth et al., 2004). Consistently, direct application of MK-801 on cortical neuronal stem cells decreased proliferation (Hu et al., 2008). The two most prominent studies attempting to resolve the complex role of NMDA receptors in adult neurogenesis were conducted in the laboratories of Gerd Kempermann and Fred Gage. Using MK-801 and a transgenic mouse expressing green fluorescent protein driven by the Nestin gene, Petrus and colleagues painstakingly identified changes in each class of precursor cells in the adult DG (Petrus et al., 2009). Their results supported previous in vivo reports, and showed that NMDA receptor antagonists promote the differentiation of neuronal precursor cells into neurons (Petrus et al., 2009). An alternative approach was ingeniously performed by Tashiro and colleagues (2006), who created a retrovirus linked to Cre recombinase. This retrovirus was then injected into mutant mice whose GluN1 gene was flanked with a loxP site, ablating GluN1 expression in infected DGCs. They found that DGCs lacking GluN1 had a decreased survival rate two to three weeks post-injection, and proposed that NMDA receptors are thus important for survival during this period (Tashiro et al., 2006). These results suggest that NMDA receptor activity decreases cell proliferation and neurogenesis of DGCs. Briefly put, NMDA receptor activity appears to counteract adult neurogenesis in the DG.  6.2.2.2 The Role of GluN2 Subunits in Adult Neurogenesis The roles of the GluN2 subunits during the neurogenic process are still largely unknown. Two studies conducted by Hu and colleagues approached this problem by injecting GluN2 antagonists into the intraperitoneal cavity of rodents (Hu et al., 2008; Hu  !  137  et al., 2009). They found that injection of the GluN2B antagonist Ro 25-6981 increased cell proliferation and survival (Hu et al., 2008), while injection of the GluN2A antagonist NVP-AAM077 slightly decreased proliferation and survival (Hu et al., 2009). However, several methodological problems are present in these reports. Aside from the aforementioned complications of using NVP-AAM077 as a GluN2A antagonist (discussed in 6.2.1.1), no dose-response curves were provided for either drug. Thus, it is difficult to be certain that specific GluN2 subunits were even adequately blocked in these experiments. Surprisingly, questions regarding GluN2 subunits and adult neurogenesis have largely not been investigated in genetic mouse models. Apart from the results presented in Chapter 5, the only other study that has addressed this issue was conducted by Kitamura and colleagues (2003), who used BrdU to demonstrate that intact cell proliferation and neuron survival is present in the DG of GluN2A-/- mice (Kitamura et al., 2003). Our results are congruent with those presented by Kitamura and colleagues (2003), as we also observed intact cell proliferation in the GluN2A-/- mouse. This thesis also extends earlier results by demonstrating that neuronal differentiation is also intact in GluN2A-/- mice. Thus, the results presented in this thesis, in combination with those produced by Kitamura and colleagues (2003), suggest that GluN2A does not contribute to the ability of the NMDA receptor to negatively regulate adult neurogenesis in the DG.  6.2.2.3 Dendrite Formation and the NMDA Receptor A considerable body of evidence supports an essential role of NMDA receptors in dendrite formation and growth. First, NMDA receptor activity has been shown to  !  138  increase branch growth. For example, incubating hippocampal cultures in NMDA increased the total neurite length and number of branches (Brewer and Cotman, 1989), as well as the number of filopodia (Henle et al., 2012). Second, blocking NMDA receptor activity decreases arbourization, as NMDA receptor inhibition decreases the dendritic arbour of Xenopus tectal neurons (Rajan and Cline, 1998). Third, NMDA receptor activity is necessary for activity-dependent modifications of dendrites, as light-induced changes in dendritic branch growth of Xenopus tectal neurons require NMDA receptors (Sin et al., 2002). In short, NMDA receptor activity appears to play a major role in dendritic development. The prominent role of the NMDA receptor in dendritic development, as well as the unique temporal expression pattern of GluN2 subunits (discussed in 1.3.3), led to the proposal that the earlier expressed GluN2B subunit promotes dynamic synaptic changes; whereas the later expressed GluN2A subunit increases temporal resolution through refinement of the dendritic tree (Scheetz and Constantine-Paton, 1994). However, experimental evidence suggests the role of the GluN2 subunit may not be so simple. In Xenopus tectal neurons, both up- and down-regulation of GluN2A expression decreases branch clustering and causes formation of more branches closer to the soma, while GluN2B expression decreases dendritic branch clustering and stability (Ewald et al., 2008). However, in ventral spinal cord cultures, blocking GluN2A expression via interference RNA has no effect on dendritic morphology, while blocking GluN2B increases the number of branches and dendritic complexity (Sepulveda et al., 2010). These complex effects may suggest that the role of the GluN2 subunits in dendrite formation varies among different model systems.  !  139  In regards to the GluN2B subunit, a mosaic model abolishing GluN2B expression in individual adult-born DGCs showed no effect of GluN2B on dendritic complexity (Espinosa et al., 2009). Deletion of GluN2B in CA1 pyramidal neurons also produced no changes in dendritic morphology (Brigman et al., 2010). A study that silenced GluN2B expression in hippocampal cultures demonstrated only a small decrease in the number of secondary branches (Sepulveda et al., 2010), while GluN2B antagonists reduced the number of filopodia without influencing the number of secondary dendrites (Henle et al., 2012). Studies investigating the GluN2A subunit have shown that blocking GluN2A expression in hippocampal cultures increases dendritic length and complexity, (Sepulveda et al., 2010), while blocking GluN2A activity via NVP-AAM077 inhibits the formation of secondary dendrites, but not filopodia (Henle et al., 2012). Together, these previous reports indicate that GluN2B plays a minimal role in dendritic morphology in the hippocampus. The role of GluN2A, however, remains unclear. Our work (Chapters 4 and 5) is consistent with the proposal of Henle and colleagues, who suggested an inhibitory role of GluN2A on secondary dendrites (Henle et al., 2012). Our results, however, do not support the work conducted by Sepulveda and colleagues (2010). Importantly, our results provide the first mammalian in vivo evidence of a role for the GluN2A subunit in dendritic morphology. Both previous studies used hippocampal cultures, which lack the well-defined structure and organization of the intact hippocampus, and consequently, the clearly identified synaptic connections between neuronal cell types. This lack of intact cytoarchitecture may underlie the differences between previous studies and the results reported here.  !  140  We demonstrate (Chapter 4) that mature DGCs from GluN2A-/- mice have intact neuronal morphology, quantified using the Neurolucida quantification system. We later replicate this finding (Chapter 5) using the Camera Lucida quantification system, and extend our findings by revealing deficits in dendritic length and complexity in late immature DGCs from GluN2A-/- mice. The results from this study raise an important question: why does the deletion of GluN2A expression specifically alter immature neurons? It is possible that GluN2Acontaining receptors are incorporated into dendrites during this “late immature” stage of neuronal maturation, thus influencing the morphology of late immature neurons. It has been suggested that the type of synaptic NMDA receptors rapidly changes after synapse formation, from a largely GluN2B-containing NMDA receptor population to a mixed population of GluN2B-containing receptors and GluN2A-containing receptors (Tovar & Westbrook, 1999). This is consistent with reports in the DG, which show that synaptic NMDA receptors in late immature DGCs have a small but significant non-GluN2B NMDA receptor current (Ge et al., 2007). As such, it is possible that absence of the small number of synaptic GluN2A-containing receptors, as in the case of GluN2A-/- mice, may be able to produce significant alterations in morphology.  6.2.2.4 Spine Density and the NMDA Receptor Spines are small protrusions located on dendrites that act as key sites for excitatory transmission (Yuste, 2011). As such, considerable research has been conducted in investigating the role of NMDA receptors and spinogenesis. Incubating hippocampal neurons in NMDA produces a significant increase in the number of spines  !  141  (Tian et al., 2007). In addition, activating NMDA receptors during LTP induces an increase in spine size in small spines (Matsuzaki et al., 2004). While deletion of the GluN1 subunit in individual CA1 pyramidal cells does not alter spine density (Adesnik et al., 2008), uncaging glutamate onto dendritic segments triggers spine formation, in a process dependent on NMDA receptors (Kwon and Sabatini, 2011). Together, this suggests that NMDA receptor activity is sufficient, but not required for the modification of spine number and structure. The differential roles of GluN2 subunits in spinogenesis have been previously discussed in Chapter 5 (section 5.4.3). In brief, two studies have demonstrated that ablation of GluN2B decreases spine density (Espinosa et al., 2009; Brigman et al., 2010), suggesting that GluN2B-containing receptors positively regulate spine density. In contrast, there has been little consensus regarding the role of GluN2A in this process. Recently, it was demonstrated that endogenous GluN2A expression decreased spine density, while lowering GluN2A expression produced a non-significant trend in spine additions, suggesting that GluN2A-containing receptors negatively regulate spine density (Gambrill and Barria, 2011). Our results support the hypothesis that GluN2A-containing receptors are negative regulators of spine formation, as we observed increased spine density that was localized to the middle molecular layer of late immature GluN2A-/- DGCs.  6.2.3 Hippocampal-dependent Behaviour Recent studies indicate that the hippocampus has a role in discriminating between small changes in the spatial and temporal relationships of stimuli, suggesting that one  !  142  function of the hippocampus may be to encode and separate events in time and space by a process called pattern separation (Gilbert et al., 2001; Kesner et al., 2004; Kesner, 2007b). Spatial pattern separation involves determining the precise angles and exact distances that separate objects in the environment, without regard to the identity of the objects, and this process has been associated with DG function (Goodrich-Hunsaker et al., 2008). On the other hand, temporal pattern separation, involved in determining a temporal order for spatial location information, has been associated with CA1 function (Hoge and Kesner, 2007; Hunsaker et al., 2008b). As described in Chapter 4, GluN2A-/- mice were tested on two pattern separation tasks previously established in rodents: spatial pattern processing was tested using a spatial metric change task (Gilbert et al., 2001; Goodrich-Hunsaker et al., 2008), whereas temporal pattern processing was tested using a temporal ordering task (Hoge and Kesner, 2007). GluN2A-/- mice showed deficits in processing metric spatial patterns, but performed the temporal ordering task without difficulty. These results indicate that information processing in the DG is compromised in GluN2A-/- mice. Hippocampal-dependent spatial memory has been examined in GluN2A-/- mice using the standard, hidden platform version of the Morris water maze test. GluN2A-/mice demonstrated significant deficits in this test (Sakimura et al., 1995). While learning the standard version of the Morris water maze test has been classically associated with NMDA receptor function in the entire hippocampus (Morris et al., 1986), recent studies have suggested that the acquisition of this task may in fact be restricted to NMDA receptor function in the CA1. Indeed, mutant mice lacking the obligatory GluN1 subunit in the CA1 show deficits in Morris water maze performance (Tsien et al., 1996), while no  !  143  deficits are observed in mice lacking GluN1 expression in the DG (McHugh et al., 2007). It is therefore interesting that the GluN2A-/- mice, with their previously shown deficits in Morris water maze performance (Sakimura et al., 1995), only show information processing deficits in the DG. While it is unclear what may cause this discrepancy, it is possible that this deficit is associated with the morphological impairments observed in late immature neurons (Chapter 5). As previously discussed (section 1.2.2.2), changes in adult neurogenesis may impair performance in the Morris water maze test. Recently, a study from Fred Gage’s laboratory developed a unique way to examine the contribution of immature adult-born neurons to the performance on this task (Deng et al., 2009). These authors generated a transgenic mouse whose Nestin promoter was followed by an inducible lethal genetic marker (herpes simplex virus thymidine kinase gene; HSV-tk; (Mansour et al., 1988)). As Nestin expression is isolated to neural progenitor cells in the adult brain, activating the lethal marker via intraperitoneal injections of a specific nucleotide ablated age-specific transfected DGCs; thus, this technique provided tight temporal regulation of adult neurogenesis (Deng et al., 2009). This technique revealed that ablation of immature to late immature neurons produced significant deficits in Morris water maze performance (Deng et al., 2009). Therefore, the altered morphology observed in late immature GluN2A-/- DGCs may explain the fact that these mice show impaired Morris water maze performance (Sakimura et al., 1995) but intact information processing.  !  144  6.3 Current Model The present results indicate that DGCs in the GluN2A-/- mouse progress through the early stages of development normally, as indicated by intact progenitor cell proliferation and neuronal differentiation. As adult-born GluN2A-/- DGCs reach the “late immature” stage however, they demonstrate reductions in dendritic complexity, accompanied by localized increases in spine density. While neuronal morphology appears to be rescued as DGCs reach maturity, mature GluN2A-/- neurons have a significantly reduced NMDA:AMPA ratio, leading to the abolishment of bidirectional synaptic plasticity. The morphological deficits in late immature DGCs and/or bidirectional synaptic plasticity deficits in mature DGCs are manifested in the alterations in DG-specific behaviour (discussed in 6.3.2).  6.3.1 Neuronal Morphology of Late Immature Dentate Granule Cells Late immature DGCs from GluN2A-/- mice have reductions in dendritic morphology, but localized increases in spine density. How could GluN2A deletion produce this seemingly opposite changes in morphology? One theory is that GluN2A contributes to the developmental progression of DGCs. Dendritic arbours become more complex as a DGC matures (Redila & Christie, 2006; Zhao et al., 2006). In addition, spine density steadily increases during development, and is then reduced, suggesting that spinogenesis involves early overproduction and later pruning stages (Segal et al., 2000). The GluN2A subunit may allow late immature neurons to develop dendrites and prune spines at the appropriate time in development. Therefore, a loss of GluN2A expression would slow morphological maturation: DGCs would show decreases in dendritic  !  145  arbourization, but increases in spine density. This theory is supported by reports indicating that NMDA receptor antagonists may block spine pruning (Lau et al., 1992; Bock & Braun, 1999). In addition, the slowing, but not distorting of the developmental timeline would ultimately result in intact morphology in mature cells, a finding that is seen here. An alternative theory is that the spine increase observed in late immature DGCs of GluN2A-/- mice could be a compensatory mechanism for the decreased dendritic arbourization. A similar compensatory mechanism has been observed in cortical pyramidal cells depleted of norepinephrine (Kolb et al., 1997). Further experiments that can accurately estimate the age of adult-born DGCs may shed light on this issue (discussed in 6.4.4).  6.3.2 Role of the GluN2A Subunit in Specific Hippocampal Regions Our findings strongly indicate that the GluN2A subunit plays a significant role in plasticity specific to the DG, however the mechanisms underlying this regional specificity are unclear. Interestingly, the DG expresses approximately equal amounts of GluN1 and GluN2A, but less GluN2B than the CA1 (Coultrap et al., 2005). Thus, GluN2B-containing receptors may be less numerous, and perhaps less integral than GluN2A-containing receptors in the DG. Our results, which demonstrate that GluN2Bcontaining receptors are insufficient for both synaptic plasticity and behavioural function in the DG, support this hypothesis.  !  146  6.3.3 Impairments in the Dentate Gyrus that Underlies Behavioural Deficits In this thesis, we present three major impairments in the DG of the GluN2A-/mouse: loss of synaptic GluN2A-containing receptors in DGCs, an impairment in bidirectional synaptic plasticity in mature DGCs, and an impairment in the morphology of late immature DGCs. An argument could be made that any or all of these impairments account for the DG-specific behavioural deficit reported here. In the simplest scenario, the loss of synaptic GluN2A-containing NMDA receptors may account for the observed deficits in pattern separation. DGCs, in comparison with CA1 pyramidal cells, have a short temporal window for EPSP summation at distal dendritic sites (approximately 10 ms) (Schmidt-Hieber et al., 2007). In essence, if a synaptic input is activated in succession, within a small time window, the resulting depolarizations will sum and effectively depolarize the dendritic region. It is immediately apparent that the requirement of DGCs to respond to only the most similar of patterns would be optimal for pattern separation. Notably, the need for a short temporal time window may be reflected in an abundance of NMDA receptors with fast overall kinetic properties, such as GluN2A-containing receptors (discussed in 1.3.2.2). Without GluN2A-containing receptors, as seen in the GluN2A-/- mouse, synaptic activity would produce a slow, prolonged rise in intracellular calcium that would allow for similar patterns to be encoded in an overlapping manner, negatively influencing the capacity to enhance contrast between spatial patterns. In a second scenario, the deficits in bidirectional synaptic plasticity may underlie the deficits in spatial pattern separation. Hippocampal synaptic plasticity has classically been linked to hippocampal function (discussed in 1.2.1.1.2, 1.2.1.2.2). For example, in  !  147  the DG, NMDA receptor antagonists blocks LTP and impairs performance on the Morris water maze (Morris et al., 1986). It has been proposed that pattern separation may be achieved in the DG by individual mature DGCs “learning” to responds to patterns of entorhinal cortex activity (Rolls, 2010). This “learning” process for individual mature DGCs may occur at a synaptic level, where different spatial patterns could differentially induce LTP or LTD in different sets of synapses. Impairments in bidirectional synaptic plasticity would therefore disable the “learning” processes of individual mature DGCs, producing a DG-specific behavioural deficit. In a third scenario, alterations in morphology of late immature neurons may primarily contribute to the observed behavioural deficit. The “late immature” stage of neuronal development appears to be critical for DGCs, both morphologically and functionally. Morphologically, late immature DGCs undergo a dynamic process of dendritic arbourization and spine formation, which allows them to receive afferent synaptic input and integrate into the hippocampal network (Zhao et al., 2006). Functionally, late immature DGCs can more easily fire action potentials and demonstrate more long-term potentiation than mature DGCs (Schmidt-Hieber et al., 2004; Ge et al., 2007), which highlights their potential to undergo activity-dependent changes. Indeed, recent studies suggest that late immature DGCs may be extremely important for hippocampal-dependent tasks (Kee et al., 2007; Tashiro et al., 2007; Deng et al., 2009). However, it is still unknown how these late immature DGCs contribute to spatial pattern separation. Recently, Aimone and colleagues (2011) proposed a unified model for late immature and mature DGCs in pattern separation. They hypothesized that mature DGCs, which are only sparsely activated in response to activity, may be finely tuned to encode  !  148  only very specific content in a spatial environment (Aimone et al., 2011). Late immature DGCs, which show a facilitated ability for action potential firing and LTP, have been hypothesized to respond to a broad range of spatial patterns (Aimone et al., 2011), which may allow for broad encoding of all objects in the environment. In the GluN2A-/- mouse, alterations to the late immature DGCs would therefore inhibit the ability to broadly encode the environment, and due to their sparse activity profile, mature DGCs alone may not be able to project sufficient amounts of information for pattern separation. As such, the disruptions in neuronal morphology observed in late immature DGCs of GluN2A-/mice might produce the behavioural deficit in the DG.  !  149  6.4 Limitations and Pitfalls 6.4.1 Using a Global Mutant Mouse Model The GluN2A-/- mouse used in this thesis has a complete deficiency in GluN2A expression throughout the entire central nervous system (Sakimura et al., 1995). As deletion of GluN2A is not spatially restricted to the hippocampus, or temporally restricted to post-natal mice, it is possible that the observed effects in synaptic, structural and behavioural plasticity may be at least partially attributed to confounding effects resulting from the absence of this subunit in other regions of the brain. For example, deletion of GluN2A during development may alter the development and wiring of the DG. The DG receives a considerable amount of cortical input from regions other than the entorhinal cortex, including (but not limited to) the pre- and parasubiculum, medial septum, hypothalamus and the ventral tegmental area (reviewed in Amaral et al., 2007), and modifications to these inputs could impact plasticity in the DG. To resolve this issue, a mosaic-based mouse model approach could be used, similar to those previously employed by the laboratories of Fred Gage and Liqun Luo (Tashiro et al., 2006; Espinosa et al., 2009). Both laboratories used retroviruses injected into the DG to ablate NMDA receptor subunit expression. As retrovirus infection is often sporadic, only subsets of DGCs demonstrate subunit ablation. Use of this technique for GluN2A deletion would help determine if the results presented in this thesis are indeed cell autonomous.  6.4.2 Synaptic Plasticity Deficits due to Loss of GluN2A or the NMDA Receptor? DGCs from GluN2A-/- mice demonstrate a major reduction in the observed NMDA:AMPA ratio, largely due to a severely reduced NMDA-EPSC. Given the major  !  150  reduction in NMDA receptor response and charge transfer, it is possible that the lack of bidirectional plasticity observed in the GluN2A-/- DG is not due to a lack of GluN2Acontaining receptors, but rather a lack of overall NMDA receptors in the DG. This possibility would undercut the idea of GluN2A-containing receptors being critical for synaptic plasticity in the DG. Rather, the finding would suggest that NMDA receptors, which are predominantly GluN2A-containing in the DG, are critical for synaptic plasticity. This in itself is a novel finding, as it suggests that the majority of functional synaptic NMDA receptors in the DG contain the GluN2A subunit. Future experiments may help elucidate this specific hypothesis. For example, determining the levels of the obligatory subunit GluN1 through Western blots would demonstrate whether overall NMDA receptors are indeed decreased in the DG of GluN2A-/- mice.  6.4.3 Performance in the Temporal Ordering Task in Mice The temporal pattern task (Chapter 4) used a paradigm designed by the laboratory of Raymond Kesner. While the task has previous been used to assay CA1 function in mice (Hunsaker et al., 2010), it was first designed as a task for rats (Hoge and Kesner, 2007). This is not uncommon, as most behavioural tasks are initially designed for rats, and then adapted for use in mice (Crawley, 1999). Modifications of complex tasks originally designed for rats can be problematic, as mice often perform worse on complex tasks, including the Morris water maze (Gerlai and Clayton, 1999). Whether differences in performance between rats and mice are also observed with the temporal pattern task employed in this study is a question that still needs further clarification.  !  151  The present results from the temporal patterning task show no differences in GluN2A-/- mice in comparison to wild-type littermates, although our results show a significant amount of variation. Even though the temporal patterning task may be the best test currently available to assess CA1-dependent behaviours (R. Kesner, personal communication), it may not be sensitive enough to properly probe this type of behaviour in mice. As such, the development of novel behavioural paradigms specifically designed for mice is a recognized priority. Alternatively, performing the temporal patterning task again with a larger cohort of animals may also minimize the observed variation.  6.4.4 Limitations of the Golgi Impregnation Technique In Chapter 5, we demonstrated deficits in dendritic morphology specific to DGCs located in the IGZ, and proposed that late immature DGCs are altered in the GluN2A-/mouse. In order to confirm that altered DGCs are indeed in the late immature stage, the specific age of the adult-born DGCs analyzed here needs to be determined. One technique well suited for this task is retroviral-labeling (van Praag et al., 2002). The retrovirus system used by van Praag and colleagues only infects mitotic cells (van Praag et al., 2002), so the exact age of adult-born DGCs can be conducted. In addition, retroviral labeling has several other advantages over Golgi impregnation. Retroviral infection is sporadic so that only a randomized subset of DGCs is labeled; this is advantageous over Golgi impregnation as Golgi-impregnated cells often overlap in clusters, which limits the number of cells that can be individually analyzed. In addition, tissue from retroviral-injected mice can be co-labeled for other markers; this cannot be achieved with Golgi-treated tissue.  !  152  6.5 Future Directions 6.5.1 Contribution of the GluN2A Subunit to Late Immature Dentate Granule Cell Physiology The present results show impaired bidirectional synaptic plasticity in the adult DG of GluN2A-/- mice. However, it is currently unknown whether subsets of DGCs retain the capacity for synaptic plasticity in these mice. Late immature DGCs demonstrate increased LTP that is dependent on the GluN2B subunit (Wang et al., 2000; Snyder et al., 2001; Schmidt-Hieber et al., 2004; Ge et al., 2007). As the results presented here indicate that the structure of late immature DGCs is impaired in GluN2A-/- mice, investigations into the synaptic plasticity of these cells would be insightful. In addition, the appearance of GluN2B-dependent LTP in the late immature GluN2A-/- DGCs would demonstrate that the synaptic plasticity deficits observed in GluN2A-/- mice is due to the specific loss of the GluN2A subunit, and not NMDA receptors as a whole (discussed in 6.4.2). Two separate approaches can be used to investigate synaptic plasticity in immature to late immature DGCs. The first approach, introduced by the laboratory of J.M. Wojtowicz, consists of inducing LTP in field EPSPs without using GABAA receptor antagonists (termed ACSF-LTP) (Snyder et al., 2001). As presented in Chapter 2, we were unable to show significant LTP in the DG of wild-type mice without using the GABAA receptor antagonist BMI. The reasons underlying this result are currently unknown. One possibility is the induction protocol used: our HFS protocol (four trains of 50 pulses, applied at 100 Hz, with a 30 s inter-train interval), which has been used to reliably elicit LTP in the DG (Vasuta et al., 2007; Eadie et al., 2012), is slightly weaker than that used by Snyder and colleagues (2001) (20 s inter-train interval), and  !  153  significantly weaker than the protocol used by most other laboratories that show ACSFLTP (four trains of 100 pulses, applied at 100 Hz, with 15 s inter-train interval (Saxe et al., 2006; Wang et al., 2008) or 10 s inter-train interval (Garthe et al., 2009)). Further investigations of the differences among the various types of LTP induction protocols are needed in order to demonstrate how ACSF-LTP should be elicited. A second approach to investigate the contribution of late immature DGCs would be to conduct whole-cell experiments, targeting late immature and mature neurons. Several laboratories have performed this technique, revealing various physiological characteristics that can be used to identify the age of DGCs, including resting membrane potential, input resistance, cell capacitance and firing properties (Wang et al., 2000; Ambrogini et al., 2004; Overstreet et al., 2004; Schmidt-Hieber et al., 2004; Esposito et al., 2005).  6.5.2 Role of GluN2A Subunit in Exercise-Induced Changes in Synaptic Plasticity in the Dentate Gyrus Voluntary exercise has been shown to uniquely alter the adult DG via several mechanisms, including through modifications to synaptic plasticity (van Praag et al., 1999b; Vasuta et al., 2007). Several lines of evidence indicate that the GluN2A subunit contributes to these exercise-induced changes in synaptic plasticity. Using GluN2 antagonists, our laboratory has previously shown that voluntary exercise increases the contribution of GluN2A in LTD (Vasuta et al., 2007). Preliminary evidence, examining the ultrastructure of DGC synapses, suggest that GluN2A-containing receptors may move to more central locations in the postsynaptic density in response to exercise (Janssen et  !  154  al., 2007). How exercise influences synaptic plasticity in the GluN2A-/- mouse remains to be determined.  6.5.3 Role of the GluN2A Subunit in Activity-dependent Increases in Cell Proliferation in the Dentate Gyrus Here, we showed that cell proliferation and neuronal differentiation is unaltered in the GluN2A-/- mouse. Interestingly, while GluN2A-/- mice show intact cell proliferation, they fail to show increases in proliferation in response to voluntary exercise (Kitamura et al., 2003), a potent stimulator of hippocampal cell proliferation (van Praag et al., 1999b; Eadie et al., 2005; Kannangara et al., 2009). This inability of GluN2A-/- mice to demonstrate activity-dependent changes in cell proliferation may be due to an inability to traffic GluN2A-containing receptors, as NMDA receptors containing GluN2A (but not GluN2B) are added to synaptic regions in an activity-dependent manner (Barria and Malinow, 2002; Tang et al., 2010). The question regarding the role of GluN2A in activity-dependent changes in cell proliferation can be assayed using the NMDA receptor antagonist MK-801. Previous reports have shown that blocking NMDA receptor activity increases cell proliferation in the adult DG (Cameron et al., 1995; Gould and Cameron, 1997; Gould et al., 1997)(discussed in 6.2.2.1). Notably, several lines of evidence suggest that MK-801 may also alter GluN2A expression. Prolonged exposure of hippocampal cultures to MK-801 produced a selective increase in synaptic GluN2A, but not GluN2B subunits (von Engelhardt et al., 2009). MK-801 has been demonstrated to increase GluN2A mRNA in all hippocampal regions (Wilson et al., 1998). An effect of MK-801 on GluN2A has also  !  155  been shown in other systems, such as the spinal cord (Alilain and Goshgarian, 2007). Taken together, these results suggest that NMDA receptor activity may modulate cell proliferation and neurogenesis through the regulation of synaptic GluN2A-containing receptors, and as such, warrants future experiments.  !  156  6.6 Conclusions The experiments presented in this thesis reveal the unique contribution of the GluN2A subunit of the NMDA receptor to plasticity in the adult DG. DGCs in the GluN2A-/- mice have severely reduced NMDA receptor-mediated currents, and the lack of GluN2A expression produces two distinct impairments in neuronal plasticity: altered dendritic morphology during the “late immature” stage of development of adult-born neurons, and deficits in bidirectional synaptic plasticity in mature DGCs. 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