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N-methyl-D-aspartate receptors of the central nervous system : network connectivity, trafficking, and… She, Kevin 2012

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N-methyl-D-aspartate RECEPTORS OF THE CENTRAL NERVOUS SYSTEM: NETWORK CONNECTIVITY, TRAFFICKING, AND PLASTICITY  by  KEVIN SHE  B.A., Cornell College, 2000 M.Sc., 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)  September 2012 © Kevin She, 2012  Abstract  Activity through NMDA type glutamate receptors sculpts connectivity in the developing nervous system and is typically studied in the visual system in vivo where individual synapses are difficult to visualize. Here, we developed a model of NMDA-receptor dependent synaptic competition in dissociated cultured hippocampal neurons. GluN1 -/- (KO) mouse hippocampal neurons were cultured alone or in defined ratios with wild type (WT) neurons. Synapse development was assessed by immunofluorescence for PSD-95 apposed to VGlut1. Synapse density was specifically enhanced only onto minority WT neurons co-cultured with majority KO neighbour neurons and this increased synapse density was dependent on activity through NMDA receptors. This enhanced synaptic density onto NMDA receptor-competent neurons in minority co-culture represents a cell culture paradigm for studying synaptic competition. Trafficking of NMDA receptors to the cell surface is critical for proper brain function. Recent evidence suggest that surface trafficking of other ionotropic glutamate receptors requires ligand binding for exit from the endoplasmic reticulum. We show that glutamate binding is required for trafficking of NMDA receptors to the cell surface by expressing a panel of GluN2B ligand binding mutants in heterologous cells and primary rodent neurons and found that glutamate efficacy correlates with surface expression. Such a correlation was found even with inhibition of endocytosis indicating differences in forward trafficking. These results indicate that ligand binding is critical for receptor trafficking to the cell surface. NMDA receptors mediate many forms of synaptic plasticity. GluN2B is proposed to bind and recruit CaMKII to synapses to mediate multiple forms of synaptic plasticity. We find that accumulation of CFP-CaMKIIα at synapses is induced in wild-type but not in KO neurons by bath stimulation of NMDA receptors or by a chemical long-term potentiation protocol. Stimulated synaptic accumulation of CFP-CaMKIIα was rescued in KO neurons by YFPGluN2B or chimeric GluN2A/2B tail but not by GluN2A, chimeric GluN2B/2A tail, or GluN2B with point mutations in the CaMKII binding site. Thus, activity-regulated synaptic aggregation of CaMKII is dependent on the cytoplasmic CaMKII binding site of GluN2B and not on differential permeation properties between GluN2B and GluN2A.  ii  Preface  For Chapter 2, a version of this chapter has been published (She, K. and Craig, A.M. 2011. NMDA receptors mediate synaptic competition in culture. PLoS One, 6(9): e24423. Epub 2011 Sep 15). Published under Creative Commons Attribution License. Conceived and designed the experiments: Kevin She (KS), Ann Marie Craig (AMC). Performed the experiments: KS. Analyzed the data: KS, AMC. Wrote the paper: KS, AMC. Excellent technical assistance was provided by Xiling Zhou, Nazarine Fernandes, and Amanda Rooyakkers. For Chapter 3, a version of this chapter has been submitted for peer review (She, K., Ferriera, S.J., Carvalho, A.L., and Craig, A.M. 2012. Glutamate binding to GluN2B controls surface trafficking of NMDA receptors). Conceived and designed the experiments: KS, AMC. Performed the experiments: KS. Analyzed the data: KS, AMC. Wrote the paper: KS, AMC. Additional biochemical experiments not included in this dissertation conducted by Joana S. Ferreira and Ana Luisa Carvalho. For Chapter 4, Figure 35 was generated by Jacqueline Rose Dr. Xiling Zhou provided expert support for all primary neuron cultures and Nazarine Fernandes and Shuqiang Sun provided animal care support for all experiments involving transgenic mice. All animal experiments were approved by the University of British Columbia Animal Care Committee according to Protocol A09-0278.  iii  Table of Contents Abstract ........................................................................................................................................... ii Preface............................................................................................................................................ iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................ vii List of Figures .............................................................................................................................. viii List of Abbreviations ...................................................................................................................... x Acknowledgements ....................................................................................................................... xii Chapter 1. Introduction ................................................................................................................... 1 1.1 Excitatory Synapses of the Central Nervous System ............................................................ 2 1.1.1 The Pre-synaptic Terminal ............................................................................................. 4 1.1.2 The Post-synaptic Specialization ................................................................................. 10 1.1.3 The Synaptic Cleft, Cell Adhesion Molecules, and Synaptogenesis ........................... 15 1.2 Structure of N-methyl-D-aspartate (NMDA) Receptors ..................................................... 18 1.2.1 Subunits of the NMDA Receptor ................................................................................. 22 1.2.2 GluN1 Splice Variants ................................................................................................. 25 1.2.3 GluN2 Structure ........................................................................................................... 27 1.2.4 Switching of GluN2B and GluN2A ............................................................................. 31 1.3 NMDA Receptors and Brain Development ........................................................................ 34 1.3.1 Signalling Through NMDA Receptors at Glutamatergic Synapses ............................ 36 1.3.2 NMDA Receptor Activity and Neuronal Network Development................................ 38 1.3.3 Molecular Genetic Studies of NMDA Receptor Function ........................................... 40 1.4 Trafficking of NMDA Receptors ........................................................................................ 45 1.4.1 Forward Trafficking ..................................................................................................... 46 1.4.2 Surface Mobility of NMDA Receptors ........................................................................ 51 1.4.3 Internalization .............................................................................................................. 53 1.4.4 GluN2 Selective Synapse Accumulation ..................................................................... 54 1.5 NMDA Receptors and Synaptic Plasticity.......................................................................... 57 1.5.1 Long Term Potentiation ............................................................................................... 58 1.5.2 Role of NMDA Receptor Subtype in Plasticity ........................................................... 61 1.6.3 Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII) .................................... 64 1.7 Primary Rodent Neural Tissue Culture ............................................................................... 69 1.8 Thesis Hypotheses and Objectives...................................................................................... 71 1.8.1 A Cell Culture Model of Synaptic Competition .......................................................... 71 iv  1.8.2 ER NMDA Receptor Ligand Binding as a Quality Control Checkpoint ..................... 72 1.8.3 Structure of the GluN2B Tail Mediates CaMKII Translocation ................................. 72 Chapter 2: NMDA Receptors Mediate Synaptic Competition in Culture .................................... 74 2.1 Introduction ......................................................................................................................... 75 2.2 Materials and Methods ........................................................................................................ 77 2.2.1 Ethics Statement........................................................................................................... 77 2.2.2 Cell Culture .................................................................................................................. 77 2.2.3 Immunocytochemistry ................................................................................................. 78 2.2.4 Imaging and Quantitative Fluorescence Analysis........................................................ 79 2.3 Results ................................................................................................................................. 80 2.3.1 Hippocampal GluN1 -/- Neurons Exhibit Normal Survival and Synapse Density in Culture.......................................................................................................................... 80 2.3.2 Synaptic Competition Occurs in Mixed Wild Type and GluN1 -/- Co-culture of Defined Ratio ............................................................................................................... 81 2.3.3 Synaptic Competition in GluN1 -/- and Wild Type Co-culture Requires NMDA Receptor Activity ......................................................................................................... 86 2.4 Discussion ........................................................................................................................... 87 Chapter 3: Glutamate Binding to GluN2B Controls Surface Trafficking of NMDA Receptors . 94 3.1 Introduction ......................................................................................................................... 95 3.2 Materials and Methods ........................................................................................................ 96 3.2.1 DNA Constructs ........................................................................................................... 96 3.2.2 Cell Culture .................................................................................................................. 97 3.2.3 Live Antibody Labelling and Immunocytochemistry .................................................. 99 3.2.4 Transferrin Uptake Assay and Induction of Surface Receptor Endocytosis.............. 100 3.2.5 Imaging and Data Analysis ........................................................................................ 101 3.3 Results ............................................................................................................................... 102 3.3.1 Mutagenesis of the GluN2B Glutamate Binding Pocket Protects Against Toxicity of Expressed NMDA Receptors ..................................................................................... 102 3.3.2 Glutamate Binding Regulates Cell Surface Delivery of NMDA Receptors in Heterologous Cells ..................................................................................................... 105 3.3.3 Co-expression of Wild Type GluN2B Cannot Rescue Surface Trafficking Deficits of GluN2B Glutamate Binding Mutants ........................................................................ 110 3.3.4 Glutamate Binding Regulates Surface Expression of NMDA Receptors in Cultured Neurons ...................................................................................................................... 110 3.3.5 A Constitutively Closed Cleft GluN2B Mutant is Similar to Wild Type in Surface Expression .................................................................................................................. 114 v  3.3.6 Glutamate Binding of GluN2B Regulates Synaptic Expression Upon Rescue in ..... 115 GluN2B -/- Hippocampal Neurons ..................................................................................... 115 3.4 Discussion ......................................................................................................................... 120 Chapter 4: Intracellular Determinants of GluN2 Control Synaptic Recruitment of CaMKIIα .. 125 4.1 Introduction ....................................................................................................................... 126 4.2 Materials and Methods ...................................................................................................... 128 4.2.1 DNA Constructs ......................................................................................................... 128 4.2.2 Cell Culture ................................................................................................................ 130 4.2.3 Stimulation Protocols ................................................................................................. 131 4.2.4 Immunocytochemistry ............................................................................................... 132 4.2.5 Imaging and Data Analysis ........................................................................................ 132 4.3 Results ............................................................................................................................... 135 4.3.1 GluN2B is Required for Globally but not Locally Induced CaMKII Translocation . 135 4.3.2 Exogenous Rescue of YFP-GluN2B Restores CaMKIIα Translocation ................... 137 4.4.3 GluN2B Cytoplasmic Tail and CaMKII Binding Site is Essential for Globally Induced CaMKII Translocation ............................................................................................... 139 4.4.4 GluN2 Cytoplasmic Tail is Essential for CaMKII Translocation Induced by Chemical Long Term Potentiation ............................................................................................. 144 4.5 Discussion ......................................................................................................................... 147 Chapter 5: Conclusion................................................................................................................. 152 5.1 Summary of Findings and Future Directions .................................................................... 152 5.1.1 NMDA Receptors Mediate Synaptic Competition in Culture ................................... 152 5.1.2 Glutamate Binding to GluN2B Controls Surface Trafficking of NMDA Receptors. 154 5.1.3 Intracellular Determinants of GluN2 Control Synaptic Recruitment of CaMKIIα ... 155 Bibliography ............................................................................................................................... 157  vi  List of Tables  Table 1: NMDA receptor nomenclature ....................................................................................... 18 Table 2: GluN2 subunit specific permeation and gating properties ............................................. 31  vii  List of Figures  Figure 1: A young (DIV3) rat hippocampal neuron in culture ....................................................... 2 Figure 2: Schematic of an excitatory synapse................................................................................. 3 Figure 3: Schematic diagram of CAZ proteins ............................................................................... 5 Figure 4: Molecular model of an average brain synaptic vesicle ................................................... 7 Figure 5: The synaptic vesicle cycle. .............................................................................................. 9 Figure 6: Variability in spine shape and size ................................................................................ 12 Figure 7: The post-synaptic density .............................................................................................. 13 Figure 8: Extracellular record of a spinal motoneurone in vivo ................................................... 20 Figure 9: Glycine as a co-agonist for NMDA receptor activation ................................................ 22 Figure 10: Tetrameric NMDA receptor ........................................................................................ 23 Figure 11: Schematic structures of the 7 isoforms of the NMDA receptor .................................. 26 Figure 12: Molecular architecture of NMDA receptors ............................................................... 28 Figure 13: GluN agonist binding sites .......................................................................................... 29 Figure 14: GluN/GluN2 receptor subtypes differ in their glutamate deactivation kinetics .......... 30 Figure 15: Structure of CaMKII ................................................................................................... 65 Figure 16: Ca2+ frequency decoding mechanism by CaMKII autophosphorylation ................... 66 Figure 17: Hippocampal GluN1 -/- neurons develop normal synapse density in an equal coculture with wild-type neurons. ................................................................................... 82 Figure 18: Paradigm for generating synaptic competition with unequal co-culture of GluN1 -/and wild-type neurons. ................................................................................................ 84 Figure 19: Synaptic competition: wild-type neurons develop increased synapse density only when in a minority with predominantly GluN1 -/- neighbours ................................... 85 Figure 20: Synaptic competition in wild-type and GluN1 -/- neuron co-culture is dependent on NMDA receptor activity. ............................................................................................. 87 Figure 21: Potential mechanisms for the observed NMDA receptor-dependent synaptic competition. ................................................................................................................. 89 Figure 22: Design of glutamate binding deficient GluN2B mutants and protection against NMDA receptor mediated toxicity in heterologous cells ....................................................... 103 Figure 23: Glutamate binding to GluN2B regulated surface levels of NMDA receptors in heterologous cells ...................................................................................................... 106 Figure 24: Reduced surface levels of glutamate binding deficient GluN2B mutants are due to reduced forward trafficking ....................................................................................... 108 Figure 25: Reduced surface levels of glutamate binding deficient GluN2B mutants are not rescued by co-expression of wild-type GluN2B ....................................................... 111 Figure 26: Glutamate binding of GluN2B regulated surface levels in neurons .......................... 113 Figure 27: A constitutively closed cleft GluN2B mutant is similar to wild-type in surface expression .................................................................................................................. 116 Figure 28: Glutamate binding of GluN2B regulates rescue of synaptic surface GluN2B in GluN2B -/- neurons ................................................................................................... 118 Figure 29: Quantitative thresholding of CFP-CaMKIIα ............................................................. 134 Figure 30: Bath induced CaMKII translocation was abolished in GluN2B -/- cells, but locally induced translocation was maintained ....................................................................... 136 viii  Figure 31: Exogenous rescue of GluN2B -/- with YFP-GluN2B restores bath induced CaMKIIα translocation .............................................................................................................. 138 Figure 32: GluN2 expression of PSD-95 immunoreactivity is comparable across all GluN2 rescued cells .............................................................................................................. 140 Figure 33: Bath induced CFP-CaMKIIα translocation is impaired in the presence of GluN2B CaMKII binding mutant, GluN2A, and GluN2B 2AT chimera but rescued by GluN2A 2BT chimera .............................................................................................................. 142 Figure 34: cLTP induced CaMKII translocation is impaired in the presence of GluN2B CaMKIIα binding mutant, GluN2A, and GluN2B 2AT chimera but rescued by GluN2A 2BT chimera ...................................................................................................................... 146  ix  List of Abbreviations  ABD, agonist binding site AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors AP5, D-2-amino-5-phosphono-pentanoate, qv APV APV, D-2-amino-5-phosphono-pentanoate, qv AP5 BDNF, brain-terived neurotrophic factor c-tail, intracellular C-terminal cytoplasmic tail CaMKII, calcium/calmodulin-dependent protein kinase II CAZ, cytomatrix at the active zone chapsyn-110, channel-associated protein of synapse-110 CNS, central nervous system CREB, cAMP response element-binding Dlg1, Drosophila disc large tumor suppressor DLG2, disks large homolog 2 DIV, days in vitro EAAT, excitatory amino acid transporter EPSP, excitatory post-synaptic potential EPSC, excitatory post-synaptic current ER, endoplasmic reticulum ERK, extracellular signal-regulated kinase FGF, fibroblast growth factor GABA, gamma-Aminobutyric acid GKAP, guanlyate kinase-associated protein GTPase, guanosine triphosphate hydrolysing enzyme GUK, guanylate kinase KO, knock-out LRRTM, leucine-rich repeat transmembrane neuronal protein LTD, long term depression LTP, long term potentiation MAGUK, membrane-associated guanylate kinase MAPK, mitogen activated protein kinase MEF2, myocyte enhancer factor-2 MK-801, (+)-5-methyl-10,11,-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate NC-IUPHAR, nomenclature committee-International Union of Basic and Clinical Pharmacology NMDA, N-methyl-D-aspartate NMDAR, N-methyl-D-aspartate receptor NO, nitric oxide NSF, N-ethyl-maleimide-sensitive factor NTD, N-terminal domain PAGE, poly-acrylamide gel electrophoresis PDZ, post-synaptic density protein (PSD-95), Drosophila disc large tumor suppressor (Dlg1), zonula occludens-1 protein (zo-1) x  PI3 kinase, phosphatidylinosital 3-kinase PIP2, phosphatidylinositol 4,5-bisphophate PKA, protein kinase A, cAMP-dependent protein kinase PKC, protein kinase C PSD, post-synaptic density PSD-93, post-synaptic density protein 93 PSD-95, post-synaptic density protein 95 RRP, readily releasable pool SAP-90, synapse-associated protein 90 SAP-97, synapse-associated protein 97 SAP-102, synapse-associated protein 102 Ser, serine SH3, SRC homology 3 domain SNAP25, synaptosomal-associated protein 25 SNARE (SNAP (Soluble NSF Attachment Protein) REceptor) Src, (sarcoma), proto-oncogene tyrosine-protein kinase Src SV, synaptic vesicle Thr, threonine VAMP, vesicle-associated membrane protein VGAT, vesicular GABA transporter VGCC, voltage gated calcium channel VGlut, vesicular glutamate transporter VIAAT, vesicular inhibitory amino acid transporter WT, wild-type zo-1, zonula occludens-1 protein  xi  Acknowledgements  I am very grateful to my family for their patience, to dad for teaching me self reliance, and to Granduncle Ralph and Auntie Helen for their support of my family and my academic pursuits. Many thanks go to Ann Marie Craig for superlative mentorship and encouragement. It has been an unbelievably amazing opportunity. I am indebted to my supervisory committee Drs. Shernaz Bamji, Lynn Raymond, and Yu Tian Wang for their generosity of time and advice.  xii  Chapter 1. Introduction  1  1.1 Excitatory Synapses of the Central Nervous System  The central nervous system (CNS) comprising primarily the brain and spinal cord receives and processes information received from all parts of the body and in turn coordinates the behaviour of the organism. While the brains of various species contain varying proportions of important supportive glia, 25% in the fruit fly, to 65% in the mouse, to 90% in humans (Allen and Barres, 2009), it is the neuron that makes up the core of the CNS. Neurons generally consist of a soma, the central portion of the neuron, a network of dendrite branches that sample the cell’s local area for other neurons, and an axon, which when mature can extend tens of thousands of times the diameter of the soma, with which to communicate with other cells (Fig 1). These highly specialized electrically excitable cells integrate and transmit information via electrical and chemical signalling primarily through synapses which are dedicated junctions between the axon of one cell and the dendrite or soma of another. Synapses consist of a pre-synaptic compartment, a synaptic cleft, and a post-synaptic terminal (Fig 2). The adult human brain contains on average 86 x1012 neurons (Azevedo et al., 2009) and each neuron in the neocortex possesses approximately 7x103 synapses (Tang et al., 2001). Figure 1: A young (DIV3) rat hippocampal neuron in culture Phase contrast micrograph of a growing axon, soma, and dendrites of an immature rat hippocampal neuron in culture. Scale bar 20 µm. (KS)  Synapses are typically classified by the composition of neurotransmitter receptors that accumulate within them; signalling through synapses containing excitatory neurotransmitter 2  receptors allow the temporary flow of positively charged ions into the cell contributing to membrane depolarization. Conversely, inhibitory synapses contain neurotransmitter receptors that allow the temporary flow of negatively charged ions that contribute to membrane hyperpolarization. The most common excitatory neurotransmitter is glutamate whereas glycine and gamma-aminobutyric acid (GABA) are the typical inhibitory neurotransmitters. If the temporal and spatial summation of these events depolarizes the cell past a threshold an action potential is generated. This dissertation focuses primarily on excitatory synapses containing glutamatergic N-methyl-D-aspartate (NMDA) receptors.  Figure 2: Schematic of an excitatory synapse Synaptic vesicles containing neurotransmitter in the pre-synaptic terminal are separated from the receptors present at the post-synaptic terminal by the synaptic cleft. A constellation of trans-synaptic adhesion molecules span this cleft and provide a physical molecular connection between the pre- and postsynaptic membranes. (KS)  In contrast to morphologically distinct type II inhibitory synapses that form at axodendritic and at axo-somatic junctions (Gray, 1959), type I excitatory synapses in vivo form predominantly on dendritic spines (Hendry et al., 1983; Harris and Kater, 1994). Dendritic spines are small protrusions from the dendrite of neurons that have highly complex actin dynamics and undergo continuous remodelling (Ethell and Pasquale, 2005). Although originally observed by 3  Santiago Ramon y Cajal in 1891 by staining brain tissue with methylene blue and subsequently iconically imaged by silver impregnation methods developed by Camilo Goli, the ultrastructural and molecular organization of spines have only recently begun to be explored.  1.1.1 The Pre-synaptic Terminal  Axons can project over long distances from the soma to synapse with their targets. Typically the sites of synaptic junctions are marked by axonal varicosities or boutons, physical swellings along the axon, which can form en passant along the length of the axon or at terminal bulbs at the end of an axon. These pre-synaptic axonal boutons are host to an active zone, numerous proteins densely concentrated where the pre-synaptic plasma membrane comes into close contact with the post-synaptic plasma membrane, and a pool of neurotransmitter containing synaptic vesicles (SV) with an entourage of proteins responsible for loading vesicles with neurotransmitter in addition to facilitating exocytosis. The active zone, first coined in 1970 (Couteaux and Pecot-Dechavassine, 1970), is the site of neurotransmitter release and its superstructure is now referred to as the cytomatrix at the active zone (CAZ). When viewed with an electron microscope this appears as a highly electron dense web-like structure extending approximately 50nm into the cytoplasm, reflecting the presence of a tightly packed protein scaffold (Pfenninger et al., 1969). This pre-synaptic scaffold not only recruits SVs (Landis, 1988) and regulates their release both temporally and spatially but also contains numerous trans-synaptic adhesion proteins that reach into the synaptic cleft (Phillips et al., 2001).  4  In part due to the density and number of interactions between proteins of the active zone structure it is highly insoluble and requires very harsh treatments to disrupt in order to biochemically investigate its composition (Dresbach et al., 2001). However, a number of proteins and protein families have been identified including trans-synaptic adhesion molecules, cytoskeletal proteins actin and tubulin (Morales et al., 2000), scaffolding proteins containing post-synaptic density-95 (PSD)/Drosophila disc large tumor suppressor (Dlg1)/zonula occludens1 protein (zo-1) (PDZ) binding domain such as piccolo (Shibasaki et al., 2004) and rab3interacting molecule RIM (Wang et al., 1999), SV fusion proteins N-ethyl-maleimide-sensitive factor (NSF) (Rothman, 1994) and Munc-18 (Aravamudan et al., 1999), and voltage-gated calcium channels (VGCC) (Coppola et al., 2001). Together, these and other proteins interact to form a large complex that recruits, primes, releases, and recycles SV (Fig 3).  Figure 3: Schematic diagram of CAZ proteins Schematic of CAZ proteins and the resulting network of interactions at the active zone. Adapted with permission from (Schoch and Gundelfinger, 2006).  Synaptic vesicles ranging in diameter between 35nm and 55nm are recruited to the nerve terminal and are assorted into three general pools; a small fraction (~1-2%) of readily releasable  5  pool (RRP) of SVs docked at the active zone, a reserve pool, and a recycling pool (Cingolani and Goda, 2008). In cultured rat hippocampal neurons at steady state, there are approximately 200 SVs present in each pre-synaptic terminal of which only four to eight are docked at the active zone in the RRP at any time (Sudhof, 2004). The glutamate concentration in each SV is estimated to be 50-100 mM resulting in 250-5000 molecules of glutamate (Schikorski and Stevens, 1997). At most synapses the distribution of SVs appears constrained suggestive of a protein cage tethering the reserve pool of vesicles to individual synapses (Landis et al., 1988). While initial hypotheses on the nature of the scaffold focused on actin, phalloidin staining for Factin revealed that actin does not co-localize with SVs at all but instead was concentrated between clusters of vesicles (Dunaevsky and Connor, 2000). While F-actin may not be directly involved in tethering vesicles its strategic position suggests a role in restricting vesicle dispersion. Indeed, electrical activity at pre-synaptic terminals enhances actin polymerization and while dispersal of actin networks with latrunculin-A fails to disrupt SV localization, this manipulation impairs synapsin localization suggesting that actin is important as a chemical scaffold to increase the local concentration of SV regulatory molecules (Sankaranarayanan et al., 2003). Piccolo, a long and flexible 420kDa protein in contrast does co-localize with, but is not a component of, SVs (Cases-Langhoff et al., 1996). Another candidate for SV tethering is the synapsin family of proteins which when genetically ablated selectively disrupts the reserve pool of SVs in pre-synaptic terminals (Rosahl et al., 1995). The surface of a typical SV is studded with a surprising amount and diversity of proteins (Fig 4) and these account for almost 60% of the vesicle’s dry weight (Takamori et al., 2006). These proteins can be broadly categorized as being involved in the transport of molecules into the vesicle or the trafficking of the vesicle itself including exocytosis at the active zone. The first 6  category of proteins include vacuolar H+ ATPase which generates the proton gradient that facilitate neurotransmitter uptake although neurotransmitter specific transporters are also required (Edwards, 2007) such as excitatory amino-acid transporters (EAAT) VGlut1, 2, and 3 (vesicular glutamate transporters ) or vesicular inhibitory amino acid transporter (VIAAT) VGAT (vesicular GABA transporter). While the localizations of VGlut1 and 2, and VGAT are conventionally considered to be strictly segregated at individual pre-synaptic terminals giving rise to uni-functional excitatory or inhibitory synapses (Ottersen and Landsend, 1997), there is emerging evidence for a limited amount of coexistence of EAAT and VIAATs. Indeed a small subset of SVs bear both VGlut2 and VGAT where the loading of negatively charged glutamate alters the electrochemical gradient to improve GABA uptake (Zander et al., 2010). Figure 4: Molecular model of an average brain synaptic vesicle Outside view of a vesicle based on space filling models of all macromolecules at near atomic resolution. Adapted with permission from (Takamori et al., 2006).  The second category of SV surface proteins, those involved in vesicular trafficking, prominently include synaptobrevin, also referred to as VAMP1, 2 (vesicle-associated membrane protein) (Sollner et al., 1993), which binds to plasma membrane bound syntaxin and SNAP25 7  and progressively zippers together the vesicle and plasma membrane (Sutton et al., 1998). While it is likely that there are many other proteins involved in SV exocytosis, this synaptobrevinsyntaxin-SNAP25 vesicular SNARE (SNAP (Soluble NSF Attachment protein) REceptor) complex represents the minimal apparatus for SV fusion allowing the exocytosis of the vesicle’s contents into the synaptic cleft (Weber et al., 1998). Another notable SV surface protein important for vesicle fusion and exocytosis is synaptotagmin, a rapid response calcium sensor that interacts with the SNARE complex and the plasma membrane (Martens et al., 2007). Synaptic vesicles docked at the active zone spontaneously fuse with the plasma membrane at a constant, albeit low, rate. However, when the pre-synaptic terminal senses threshold-exceeding depolarization, the influx of Ca2+ via VGCC rapidly and dramatically increases the probability that the SV docked in the active zone undergo fusion and release its neurotransmitter payload into the synaptic cleft. The rapid kinetics of SV fusion, within 100 µs of cytosolic Ca2+ elevation (Rizo and Rosenmund, 2008), suggest a direct conformational change in the pre-fusion complex at the active zone via a complex including at least synaptotagmin rather than an indirect pathway involving Ca2+ responsive enzymes such as protein kinases (Chapman, 2002; Fernandez-Chacon et al., 2002). Once the SV has discharged its contents, it is broadly accepted that the surface components of the SV is recycled (Holtzman et al., 1971; Ceccarelli et al., 1973; Heuser and Reese, 1973) to ensure a constant supply of releasable vesicle at the nerve terminal. However, the details of this process continue to be investigated (Cremona and De Camilli, 1997; Brodin et al., 2000; Leitz and Kavalali, 2011) (Fig 5). One model of SV recycling is referred to as “kissand-run” where after a transient fusion event the vesicle may be retrieved rapidly back into the synaptic terminal without losing its identity and is available to be reloaded with neurotransmitter. 8  While this model of exocytic vesicle recycling has been directly demonstrated in non-neuronal cells (Breckenridge and Almers, 1987), there is only indirect evidence for this phenomenon in neurons. Using miniature capacitance measurements directly at a synaptic release face (He et al., 2006) to resolve the fusion and retrieval of SV, the measured rate of endocytosis decreases as the rate of exocytosis increases and the most rapid endocytic event had a time constant of 56 ms (Sun et al., 2002) suggesting that the kiss-and-run model is at least possible at neuronal synapses. Further evidence of kiss-and-run relies on the use of styryl dyes that assort into SVs and de-stain upon vesicle exocytosis (Betz et al., 1992). Under certain stimulation conditions, exocytosis of neurotransmitter can occur while a significant fraction of the dye is retained in the exocytosed vesicle consistent with a transient fusion event predicted by the kiss-and-run model (Klingauf et al., 1998).  Figure 5: The synaptic vesicle cycle. Synaptic vesicles fuse with the pre-synaptic membrane at the active zone releasing neurotransmitter into the synaptic cleft. The membrane of the fused vesicle diffuses laterally to areas outside of the active zone and retrieved by clathrin-mediated endocytosis. The reconstituted vesicle is subsequently sorted and transported to the reserve pool of SV or to endosomal compartments for recycling. Adapted with permission from (Shupliakov and Brodin, 2010).  9  A more established mechanism of SV retrieval after complete fusion with the presynaptic plasma membrane is the clathrin-mediated endocytosis process that occurs in the periactive zone away from the site of exocytosis (Dittman and Ryan, 2009). Clathrin-mediated endocytosis in non-neuronal cells is a constitutive process and in neuronal cells participates in internalizing receptors and their ligands (Man et al., 2000; Lee et al., 2002). Clathrin-coated elements are rarely seen in resting nerve terminals but are conspicuously present shortly after stimulation inducing vesicle release (Heuser and Reese, 1973). Only after a number of exocytosis events are AP-2 adaptor proteins observed to be recruited to bind to synaptotagmin to serve as docking sites for clathrin (Li et al., 1995). Clathrin associates as triskelion motifs composed of three heavy and three light chains (Pearse, 1976) which form into planar hexagonal lattices that subsequently fold into icosahedral structures that physically warps the plasma membrane into an invagination (Heuser, 1989). Dynamin is a GTPase that oligomerizes into rings and these have been observed to develop at the neck of deeply invaginated endocytic buds (Takei et al., 1995) which are cleaved from the plasma membrane upon GTP hydrolysis as the dynamin rings constrict. Clathrin rapidly dissociates from the vesicle after fusion, likely with the aid of polyphosphoinositide phosphatase synaptojanin mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) (Cremona et al., 1999), and the vesicle is free for neurotransmitter refilling.  1.1.2 The Post-synaptic Specialization  In contrast to inhibitory synapses, in vivo and under certain culture conditions, the majority of excitatory synapses form between a pre-synaptic varicosity and a post-synaptic 10  dendritic spine (Bourne and Harris, 2008). While the development of spines from filopodia has not been shown directly, live imaging of developing hippocampal cells in culture show a concomitant decrease in the number of transient filopodia with an increased number of stable dendritic spines apposed to functioning pre-synaptic varicosities (Ziv and Smith, 1996). Dendritic spines vary in size from 0.5 to 2µm in length (Svitkina et al., 2010), spine head volumes range from 0.001µm3 to 1µm3 (Nimchinsky et al., 2002), and most spines have constricted necks (Fig 6); their size and morphology strongly suggest a role in providing a spatially separate compartment allowing each spine to function as a partially independent unit isolating biochemical cascades in response to pre-synaptic input. Although spines are less motile than filopodia, spines are still highly dynamic and the spine volume is correlated with synaptic strength (Popov et al., 2004). Interestingly, in the dendrite, mitochondria not only carry out aerobic respiration but also actively buffer intracellular Ca2+. Changing the abundance of dendritic mitochondria by genetic manipulation of mitochondrial fission and fusion proteins results in the concomitant increase or decrease in spine density (Li et al., 2004). When viewed by electron microscopy there is a highly electron dense structure about 3040nm thick and several hundred nm wide at the head of the spine that is termed the post-synaptic density (PSD). The PSD is located across the synaptic cleft and is tightly aligned with the analogous CAZ of the pre-synaptic active zone. Like the CAZ the PSD is a tightly packed protein scaffold that contains an even more vast network of interacting proteins and is highly insoluble in non-ionic detergents and thus can be purified to a substantial degree by differential centrifugation (Carlin et al., 1980). Indeed, one study using a number of purification strategies and analysis by liquid chromatography tandem mass spectrometry and large scale immunoblotting identified over 1100 unique proteins associated with the PSD and of these 11  Figure 6: Variability in spine shape and size A three-dimensional reconstruction of a hippocampal dendrite (gray) from EM illustrating different spine shapes including mushroom (blue), thin (red), stubby (green), and branched (yellow). The PSD (red) also vary in size and shape. Upper Left: A graph plotting the ratio of head diameters to neck diameters for the spines on the reconstructed dendrite. Adapted with permission from (Bourne and Harris, 2008).  over 400 have been validated by their previous detection in two or more studies (Collins et al., 2006) (Fig 7). These proteins can be categorized into several general groups including cytoskeletal proteins such as actin, scaffolding and adaptor proteins including PSD-95 and other MAGUK proteins, membrane-bound channels and neurotransmitter receptors including α-amino3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors and N-methyl-D-aspartate (NMDA) receptors, kinase and phosphatase signalling molecules including calcium/calmodulindependent protein kinase II (CaMKII), and trans-synaptic cell adhesion proteins (Boeckers, 2006). 12  Figure 7: The post-synaptic density The PSD is comprised of cytoskeletal proteins, scaffolding and adaptor proteins, membranebound channels and neurotransmitter receptors, kinase and phosphatase signalling molecules, and cell adhesion proteins. Adapted with permission from (Feng and Zhang, 2009).  An abundant constellation of scaffolding proteins localizes to, and constitutes the core of the PSD architecture. These scaffolds have diverse but crucial roles in anchoring and clustering glutamate receptors, linking these receptors with their downstream signalling molecules, and interfacing with and regulating the cytoskeletal structure (Scannevin and Huganir, 2000; Kim and Sheng, 2004). These functions are dynamic and directly affect the morphology of the PSD and dendritic spine and therefore crucial in facilitating synaptic plasticity.  13  The most common protein-protein interaction module among PSD scaffold proteins is the post-synaptic density-95 (PSD)/Drosophila disc large tumor suppressor (Dlg1)/zonula occludens1 protein (zo-1) PDZ domain (Feng and Zhang, 2009). These PDZ domains generally bind to short motifs present in the distal region of the C-terminus of other proteins (Cowburn, 1997) with relatively weak binding affinities (Zhang and Wang, 2003). Combined with the high number of PDZ domains present at the PSD relative to the number of glutamate receptors and other transmembrane targets, it is tempting to speculate that anchoring of these targets is more of a consensus effort than in a direct 1:1 scaffold:target manner. For example, receptors residing at the central core of the PSD membrane are less mobile than receptors at the periphery where the density of PDZ domains is lower (Chen et al., 2008b). In addition to specific binding to scaffolding proteins, the presence of obstacles and the geometry of the synapse are also involved in regulating the lateral mobility of cell surface proteins (Rusakov et al., 2011). Interestingly, the abundance of scaffolding proteins containing this PDZ binding domain raises questions regarding the functional specificity and redundancy of these scaffolds. For example, the membrane-associated guanylate kinase (MAGUK) family of PDZ domaincontaining scaffolding proteins including PSD95, PSD93, SAP102, and SAP97 are all expressed in excitatory synapses but have different roles in trafficking glutamate receptors. While the relative proportion of these scaffolds change depending on the developmental stage and experience of the synapse (Sans et al., 2000), which explains in part their specificity in particular roles, studies on genetically ablated knockout mice and acute knock-down experiments with short hairpin RNA (shRNA) reveal a remarkable degree of functional compensation for the missing family member (Migaud et al., 1998; Elias et al., 2006).  14  1.1.3 The Synaptic Cleft, Cell Adhesion Molecules, and Synaptogenesis  Alluded to in previous sub-chapters but not discussed in detail are the trans-synaptic adhesion molecule partners that originate from the pre- and post- synaptic protein densities and meet in the synaptic cleft. Synaptic specializations on both sides of the synaptic cleft are aligned with exquisite precision marrying chemically matched pre-synaptic vesicles to post-synaptic neurotransmitter receptors and their attendant scaffolding and signalling molecules. Dendritic spines and filopodia are highly dynamic and undergo morphological rearrangements over very short (< 1 minute) time courses although this motility declines with neuronal maturation and is not affected by activity blockade or induction (Dunaevsky et al., 1999). Both inhibitory (Dobie and Craig, 2011) and excitatory (Bresler et al., 2001) synapses show varying degrees of translocation, up to 6 µm in total path length within 20 hours, but strikingly the inter-puncta distance between the centres of mass of the pre- and post-synaptic components remain remarkably stable with an average separation of 0.4 µm. Additional evidence for a very high degree of mechanical stability between the pre- and post- synaptic specializations is apparent from the ability to recover intact synaptosomes, pinched off sections of pre- and postsynaptic membrane that remain tightly adherent, from mechanically homogenized brain tissue (Gray and Whittaker, 1962). The precise alignment of the pre- and post- synaptic machinery, their highly coordinated mobility, and their mechanical stability owe in large part to transsynaptic adhesion molecules spanning the synaptic cleft that provide the physical molecular connection – a connection that begins during synaptogenesis, the birth of a synapse. Synaptogenesis, the formation of new synapses, occurs extensively in the CNS during embryonic development and continues throughout the lifetime of the organism. In addition to 15  plastic changes in existing synapses adult synaptogenesis is implicated in learning and memory. There is evidence that the founding of a nascent excitatory synapse typically begins with an initial contact of the axon with a dendritic filopodium, the latter which exhibit transient extension and retraction events reminiscent of exploratory behaviour suggesting that these dendritic extensions actively sample the immediate milieu for nearby axons (Ziv and Smith, 1996). Electron microscopic studies on the developing rat hippocampus reveal numerous nascent asymmetric synapses being formed at the point of contact between axons and dendritic filopodia (Fiala et al., 1998) and time-lapse fluorescent imaging of cultured neurons show that synaptic proteins accumulate rapidly on both the pre- and post-synaptic sides upon contact between the axon and filopodium (Ahmari et al., 2000). While the formation of synapses between an axon and the vertebrate neuromuscular junction requires the secretion of extracellular-matrix proteins (Sanes and Lichtman, 2001) direct contact between the axonal and dendritic membranes appear to be the initiating event for synaptogenesis in the CNS (Li and Sheng, 2003). The initiation and the specificity, ensuring chemically matched pre- and post-synaptic specializations, of emerging synapses rely on trans-synaptic adhesion molecules. Of these molecules some are required for synapse formation but are insufficient to induce the formation of synapses by themselves and others are capable of and sufficient by themselves to induce the formation of pre- or postsynaptic specializations when presented to axons or dendrites, respectively. Synaptogenic adhesion complexes consist of pre- and post-synaptic partners that bind in trans across the synaptic cleft. While many interacting partners exhibit bidirectional activity, the ability of both pre- and post- synaptic partner to induce synaptic differentiation on the opposite membrane, other binding pairs only possess unidirectional activity. One of the best characterized examples of synaptogenic complexes are the neurexin-neuroligin and neurexin-LRRTM 16  interactions (Ichtchenko et al., 1996; Craig and Kang, 2007; Sudhof, 2008; Linhoff et al., 2009). The neurexin family, itself structurally diverse in having a β and an α isoform and possessing a number of alternative splicing sites, astonishingly bind to multiple structurally diverse partners. The first characterized neurexin binding partners were the neuroligins. The major glutamatergic partner for β-neurexin is neuroligin-1 with an insert at its B splice site while neuroligin-2 is specifically involved at GABAergic synapses and binds all neurexins. Additionally leucine-rich repeat transmembrane neuronal proteins (LRRTMs), initially discovered in an unbiased expression screen (Linhoff et al., 2009), are also trans-synaptic binding partners for neurexins. Both glutamatergic LRRTM1 and 2 bind to both β and α neurexins lacking an insert at splice site 4 (-S4). In neuronal cultures LRRTM2 is even more potent at inducing synaptogenesis than neuroligin-1. Remarkably a significant portion of the binding face of neurexin-1β (-S4) is shared between the structurally distinct neuroligins and LRRTMs yet the two families of neurexinbinding partners cooperate in an additive manner (Siddiqui et al., 2010). The differential binding code of neurexin isoforms and their diverse partners is intriguing and suggests a further role in synaptic plasticity beyond merely facilitating synaptogenesis although the physiological significance is not yet fully understood.  17  1.2 Structure of N-methyl-D-aspartate (NMDA) Receptors  The discovery of ionotropic glutamate receptors, the delineation between the NMDAquisiqualate/AMPA-kainate subtypes, indirect evidence for multiple NMDA-class receptor subtypes, through the development of a constellation of pharmacological agonists and antagonists, and the concurrent cloning of the NMDA receptor subunits from different species by different labs has followed a byzantine path and begat a baffling collection of inconsistent nomenclature, excellently reviewed by (Lodge, 2009). Fortunately, the nomenclature committee of The International Union of Basic and Clinical Pharmacology (NC-IUPHAR) chaired by Graham L. Collingridge have recommended a unified nomenclature for ligand-gated on channels that participate in fast synaptic transmission (Collingridge et al., 2009) (Table 1).  Table 1: NMDA receptor nomenclature Human Gene Name  NC-IUPHAR Subunit Nomenclature  GRIN1  Human Previous Nomenclature Chromosomal Location 9q34.3 NMDA-R1, NR1, GluRζ1, GLUN1  GRIN2A  16p13.2  GluN2A  GRIN2B  12p12  GRIN2C  17q25  GRIN2D  19q13.1  GRIN3A  9q31.1  NMDA-R2A, NR2A, GluRε1, GLUN1 NMDA-R2B, NR2B, GluRε2, GLUN2B NMDA-R2C, NR2C, GluRε3, GLUN2C NMDA-R2D, NR2D, GluRε4, GLUN2D NMDA-R3A, NMDAR-L, χ-1  GRIN3B  19p13.3  NMDA-R3B, GLUN3B  GluN3B  GluN1  GluN2B GluN2C GluN2D GluN3A  18  Similar to initial work investigating synaptogenesis, inquiries into the modes of synaptic transmission began with the study of the neuromuscular junction which led to the realization that fast synaptic transmission was likely to be chemical rather than electrically evoked (Feldberg and Gaddum, 1934). Whether synaptic transmission in the CNS could also be chemically (humorally) mediated was a matter of some debate as mainstream electrophysiologists of the time had derisively referred to proponents of chemical synaptic transmission as “soup physiologist.” (Krnjevic, 2005) The first solid evidence for the chemical hypothesis in the CNS was presented by John Eccles who succeeded in recording synaptic potentials inside spinal motoneurons (Brock et al., 1951). In addition to observing excitatory post-synaptic potentials (EPSP), which however were clearly not cholinergic as in the neuromuscular junction, inhibitory post-synaptic potentials were also observed which could not be explained by electrical transmission. The identity of substances that mediate chemical synaptic transmission would remain elusive, although acidic amino acids L-glutamate and L-aspartate were suspected as they are found in high concentrations throughout the brain (Berl and Waelsch, 1958), induce convulsions and spreading depression when applied to the cerebro-cortical surface (Van Harreveld, 1959), and was able to elicit depolarization of the crayfish muscle (Robbins, 1959). It was not until David R. Curtis and Rosamond Eccles developed a microelectrophoresis technique to apply exogenous compounds to individual neurons (Curtis and Eccles, 1958) that further progress would be made to provide evidence that acidic amino acids were the mediators of chemical synaptic transmission in the CNS (Curtis et al., 1960) (Fig 8) although doubts continued to linger. Curtis along with John W. Phillis, using L-glutamic acid analogues synthesized by Jeffrey C. Watkins, would proceed to pioneer the delineation of the different subtypes of glutamate receptors with the discovery that N-methyl-D-aspartate (NMDA) was 10 19  times more potent than L-glutamate which had similar efficacy as D-glutamate and N-methyl-Laspartate (Curtis and Watkins, 1963) in stimulating a particular population of receptors. Naturally, the potency of the D-isomer was at odds with the predominant chirality (L-) of terrestrial biological amino acids (Breslow and Cheng, 2009) although some species are able to synthesize peptides from D-isomer amino acids, such as by non-ribosomal peptide synthetases found in some Streptomyces bacteria (Miao et al., 2006). It was only a decade later that the differences in efficacy between the D- and L- isomers of N-methyl-aspartate were explained by the observation that the L- isomers were much more efficiently removed from the extracellular environment resulting in a higher apparent potency of the D- isomer (Balcar and Johnston, 1972). Figure 8: Extracellular record of a spinal motoneurone in vivo A) A synaptic response with action potential discharges coincident with the field potential, B,C) the firing of this neurone in response to the electrophoretic ejection of Lglutamate (20 and 120 nA respectively). Modified with permission from (Curtis et al., 1960). These are some of the earliest records showing the effects of acidic amino acids on single neurons.  Although early investigations tentatively suggested the existence of different pools of receptors that preferred either aspartate or glutamate (Duggan, 1974) and the synthesis and response to kainate (Shinozaki and Konishi, 1970) preferentially activated the presumed glutamate preferring receptors (McCulloch et al., 1974), it was the synthesis of a highly selective competitive NMDA receptor antagonist D-2-amino-5-phosphono-pentanoate (AP5 or APV)  20  (Davies et al., 1981) that would show that L-glutamate is most likely the endogenous ligand for NMDA receptors as L-glutamate displaced APV binding much more potently than L-aspartate (Olverman et al., 1984). Additionally, around this era, glycine was discovered to be a co-agonist of this subset of glutamate receptors but not for quisiqualate (AMPA) or kainate responsive glutamate receptors (Johnson and Ascher, 1987) (Fig 9). It would be another decade after the synthesis of APV before the cloning of the NMDA receptor subunits from multiple species in rapid succession by multiple groups that bestowed upon them a variety of designations (Table 1). The first subunit cloned was named NMDAR1 or NR1 from rat (Moriyoshi et al., 1991). Of historical interest, however, while it was immediately apparent that the GluN1 subunit bound glycine and not glutamate, (Moriyoshi et al., 1991) were able to show glutamate-activated currents after expressing GluN1 alone in Xeonpus oocytes. Subsequently the homologous human clone maintained this designation (Karp et al., 1993) while the mouse clone was called NMDA receptor channel subunit ζ1 (Yamazaki et al., 1992). Shortly after four further subunits from rat, NR2A-D, were successfully cloned (Monyer et al., 1992; Monyer and Seeburg, 1993) concurrent with the cloning of mouse ε1 (Meguro et al., 1992), mouse ε1-3 (Kutsuwada et al., 1992) and mouse ε4 (Ikeda et al., 1992). The four identified nonGluN1 NMDA subunits, termed NMDAR2A-D, were also independently cloned and characterized (Ishii et al., 1993). The naming convention for NMDA receptors have now been unified and modern designation for individual receptor subunits lack an “R” given that many of these subunits do not form functional receptors when expressed alone (Collingridge et al., 2009). Although the NR (NMDA Receptor) designation (NR1, NR2A, NR2B, &c.) has been historically popular, the subunits of the NMDA receptor will be subsequently referred to in this manuscript using NC- IUPHAR recommended nomenclature (Table 1) 21  Figure 9: Glycine as a co-agonist for NMDA receptor activation A) Whole cell currents from a patch clamp recording from a neurone in culture, showing minimal current induced by NMDA or glycine alone, but a robust response when applied together. B) Current induced by glutamate alone or glutamate and glycine C,D) Glycine does not potentiate quisiqualate (AMPA) or kainate induced current. Adapted with permission from (Johnson and Ascher, 1987).  1.2.1 Subunits of the NMDA Receptor  Ion channels and receptors involved in transmembrane signalling are often multimeric complexes. NMDA receptors in the CNS are excitatory receptors that are fundamental in neuronal development, synaptic transmission, and synaptic plasticity consist of three subunit families: GluN1, GluN2, and GluN3 (Nakanishi, 1992; Sun et al., 1998). While there is only one member of the GluN1 family, it has three alternative splice sites that can give rise to eight distinct GluN1 isoforms (Moriyoshi et al., 1991). The GluN2 subfamily consists of four subunits (A-D) and GluN3 consists of two subunits (A, B). GluN1 shares only a 25% homology with GluN2 but has a very low homology with the AMPA and kainate receptor subtypes. Within the GluN2 family, GluN2A and GluN2B are the most closely related to each other (57% homology) in contrast to GluN2C or GluN2D (43-47% homology), but which are themselves closely related to each other (54% homology) (Monaghan and Jane, 2009). Although the overall sequence identity seems low, the sequence encoding the pore region is quite highly conserved and the majority of the differences lie in the sequence of the C-terminal cytoplasmic tail region. The  22  GluN3A and GluN3B subunits share only a 17-21% sequence homology with GluN1 and GluN2 but are closely related to each other (47% homology) (Chatterton et al., 2002). Canonically, NMDA receptors are heteromeric tetramers containing two obligatory glycine-binding GluN1 subunits and two other regulatory subunits, either from the glutamate binding GluN2 or the glycine binding GluN3 subfamily (Laube et al., 1998) (Fig 10). Although tri-heteromeric receptors containing different GluN2 subunits is possible as well as triheteromeric receptors containing both GluN3A and GluN3B, receptors containing a GluN2 and GluN3 either do not traffic to the cell surface or the inclusion of one subunit excludes the incorporation of the other (Ulbrich and Isacoff, 2008).  Figure 10: Tetrameric NMDA receptor Transmembrane architecture of an NMDA Receptor. A) Schematic side view. GluN2 (NR2) and GluN1 (NR1) subunits are composed of several conserved regions. The extracellular region includes the N-terminal domain that can contain modulatory sites that bind Zn2+ or ifenprodil. The S1 and S2 domains form the binding site for glutamate in NR2 and for glycine in NR1. The channel-lining region (M2) is a re-entrant “pore loop” that enters the membrane from the intracellular surface. The ion channel is permeable to Na+, K+, and Ca2+. Extracellular Mg2+ causes voltage-sensitive block by binding deep within the pore. The Cterminal tail of GluN2 and GluN1 binds to synaptic kinases and structural proteins. B) Schematic isometric view. The type of GluN2 subunit involved is critical in determining the receptorchannel properties. NMDA receptors form as tetramers composed of two copies of GluN1 and two copies of GluN2. Adapted with permission from (Cull-Candy and Leszkiewicz, 2004). Right) Top-down perspective. Presumed gating movements of the M3 transmembrane segment leading to pore opening. Adapted under Creative Commons Attribution- Noncommercial-Share Alike 3.0 (Talukder and Wollmuth, 2011). 23  The composition of the non-GluN1 subunit confers upon the receptor distinct physiological properties. While all GluN2 subunits possess a glutamate binding site, the different subunits have different kinetics, have subtly different channel lining structures and thus have different ionic permeation properties (Monyer et al., 1992), and small differences in the glutamate binding site which can be exploited pharmacologically with antagonists showing partial specificity for particular subunits (Feng et al., 2005). The four GluN2 subunits are differentially distributed in the brain with patterns of expression that change during development (Monyer et al., 1994). In the rat brain, most neurons at the early developmental stage strongly express GluN2B, and to a lesser extent GluN2D, whereas the expression of GluN2A and GluN2C subunits increase over time. In the adult brain, GluN2A is ubiquitously expressed while GluN2B becomes partially restricted to the forebrain and GluN2C to the cerebellum. Interestingly, while tri-heteromeric GluN2 containing NMDA receptors are possible in heterologous systems, GluN2A and GluN2B can be coimmunoprecipitated from primary neurons (Chazot and Stephenson, 1997), and while receptors with intermediate electrophysiological properties consistent with tri-heteromers have been observed (Brickley et al., 2003; Jones and Gibb, 2005), they have not been directly detected by electrophysiological means in synapses possibly due to limitations in the resolution of singlechannel recordings at those locations or that one of the two GluN2 subunits may act in a dominant manner (Gotz et al., 1997; Lozovaya et al., 2004). Additionally, NMDA receptors containing GluN2B predominates early in the life of a neuron while GluN2A containing receptors increases over time suggesting that there is a developmental switch transitioning synapses from a more plastic to a less plastic state (Wenthold et al., 2003). 24  Interestingly however, the expression of more than one GluN2 subunit within the same neuron can lead to the apparent distribution of nearly homogenous populations of di-heteromeric receptors to discrete parts of the cell. For example, in neocortical layer 5 neurons, excitatory post-synaptic currents (EPSC) from intra-cortical synaptic inputs appear to be mediated primarily by GluN2B containing NMDA receptors while inputs from the collosum in the same neuron are mediated primarily by GluN2A containing NMDA receptors (Kumar and Huguenard, 2003). Likewise, in CA3 hippocampal pyramidal neurons NMDA EPSCs evoked from commissural/associational inputs appear to be mediated primarily by GluN2B containing NMDA receptors while EPSCs from fimbrial inputs by GluN2A (Ito et al., 1997). These observations of input-specific regional segregation of different di-heteromeric receptor populations has been proposed as a method for cells to differentially integrate and process signals from different sources. However, the authors do not present a mechanism for cells to actively target specific receptor subtypes to specific inputs. A possible mechanism could be that the histories of those synapses were different and experienced different kinds of signals. Instead of active assortment to different compartments, activity-dependent plasticity could have moulded the receptor subtype present in those synapses over the lifetime of the cell.  1.2.2 GluN1 Splice Variants  After the initial cloning of GluN1, a number of reports described the insertion of a splice cassette near the amino terminal and deletions of two independent consecutive splice cassettes from the carboxy terminus. GluN1, in rat, is encoded by a single 22 exon gene that can generate eight distinct GluN1 isoforms through alternative splicing of exons 5, 21, and 22 (Moriyoshi et  25  al., 1991) (Fig 11). The alternative splice at exon 5 of GluN1, a 21-amino acid sequence that maps to the N-terminal extracellular domain, alters responses to pH and polyamines such as spermine (Traynelis et al., 1995). Alternative splicing of exons 21 and 22, mapping to the intracellular C-terminal, generates four splice variants varying in the C1, C2, or C2’ cassettes and differentially regulates protein-protein interactions, early receptor trafficking, and GluN1 phosphorylation. The C1 cassette is encoded by exon 21, the C2 cassette is encoded by exon 22, and the C2’ arises in the absence of exon 22 (Fig 11). The C1 cassette is involved in interactions with calmodulin (Ehlers et al., 1996), neurofilament-L (Ehlers et al., 1998), importin α (Jeffrey et al., 2009), and yotiao, a scaffolding protein that physically links PP1 and PKA to NMDA receptors and regulates channel activity (Westphal et al., 1999). The naming convention for these splice variants was proposed by Hollmann et al. (Hollmann et al., 1993) where 1 = no deletion; 2, 3, and 4 = deletions 1, 2, 1+2, and ‘a’ and ‘b’ indicate the absence or presence of the 21-amino acid insertion. Figure 11: Schematic structures of the 7 isoforms of the NMDA receptor The protein-coding region is depicted by boxes while the non-coding region is displayed by solid lines. 4 putative transmembrane segments are shown in filled boxes. The inserted sequence and the alternatively created Cterminal sequences are indicated by differently hatched boxes. The modern nomenclature and the presence of N1, C1, C2, and C2’ cassettes are indicated. Adapted with permission from (Sugihara et al., 1992).  26  The expression patterns and levels of these GluN1 splice variants changes over development (Laurie and Seeburg, 1994) and can be regulated by neuronal activity (Durand et al., 1992; Xie and Black, 2001). In the rat brain, total GluN1 mRNA begins to be detectable globally in the embryonic day 14 brain and increases gradually during development and reaches adult levels by the third postnatal week.  1.2.3 GluN2 Structure  NMDA receptors are transmembrane proteins arranged as a tetramer with two obligate glycine binding GluN1 subunits and two other GluN subunits. These relatively large subunits form a central ion channel pore that is selective for cations Na+, K+, and Ca2+. Additionally, NMDA receptors exhibit a signature voltage dependent Mg2+ blockade (Dingledine et al., 1999). When assembled, the receptor consists of four discrete domains; extracellular N-terminal domains (NTD) and agonist-binding domains (ABD), a membrane channel pore domain made of the M2 membrane re-entrant loop and three transmembrane segments M1, M3, and M4, and an intracellular C-terminal tail (c-tail) (Mayer, 2006) (Fig 12). The NTD encompasses approximately the first 380 amino acids of the protein forming a bi-lobate or clamshell-like structure and is involved in subunit assembly and affects receptor properties. Strikingly, the NTD domains between the different GluN2 subunits are highly divergent and share only approximately a 19% sequence homology. The NTD forms a highaffinity binding site for Zn2+, concentrated in SV at many glutamatergic synapses (Paoletti et al., 2009), which acts as a potent allosteric inhibitor (Williams et al., 1994) particularly of the GluN2A receptor subtype (Paoletti et al., 1997). The NTD is also important in conferring further 27  subunit specific properties; like the ABD, in particular the S2 segment, it can alter the glycine sensitivity of GluN1 (Chen et al., 2008a). Experiments using chimeric receptor subunits with truncated or swapped NTDs reveals that it also influences the deactivation kinetics and channel open probabilities (Yuan et al., 2009).  Figure 12: Molecular architecture of NMDA receptors A) Domain organization of n individual subunit. B) X-ray crystal structure of a full GluA2 homotetramer. Red – NTD (Nterminal domain). Blue – ABD (agonist binding domain). Gray – Pore region. Green – Linkers. C) Three possible subunit quaternary arrangements. Interactions between subunits via ABDs are depicted as black ovals. Adapted with permission from (Paoletti, 2011).  The ABD is formed by approximately the next 300 amino acids that is split into two discontinuous S1 and S2 segments by transmembrane segments M1, M3, and the M2 re-entrant loop. The ABD, like the NTD, also forms as a typical bi-lobate structure with the ligand binding in the central inter-lobe cleft (Furukawa et al., 2005). As opposed to the NTD, the ABD shares a 63% sequence homology among the GluN2 subunits especially in the protein sequences that 28  directly interact with the ligand (Fig 13). Likewise, the membrane domains are highly conserved sharing a 73% sequence homology. Mutagenesis experiments have identified asparagine residues in the M2 region of both GluN1 and GluN2, near the point of maximal constriction of the pore, that are critical for both Ca2+ permeability as well as the signature voltage-dependent channel block by external Mg2+ (Sakurada et al., 1993). Despite the high degree of conservation between the GluN2 subunits, there is heterogeneity in the amino acid sequence of the pore forming M1M4 regions that are likely to contribute to the diversity of permeation properties. However, the precise determinants that are responsible for these differences between the GluN2 isoforms are still not well described. Figure 13: GluN agonist binding sites Sequence alignment for the S1 and S2 domains of NR2 subunits. Amino acids lining the ligand-binding cleft in the GluR2-based homology models are shown in boxes. The amino acid residues that are nonidentical among the four NR2 subunits and lining the ligandbinding cleft are shown in bold. The single ligand-binding pocket amino acid differing between rat and human sequences (NR2A R711) is shown with an arrow.. Adapted with permission from (Kinarsky et al., 2005).  Like the NTD, the amino acid sequences of the intracellular C-terminal tail (c-tail) are highly divergent and share at most only a 2% sequence homology. The four GluN2 subunits possess unusually long c-tails; from 401 amino acids for GluN2C to >620 amino acids for GluNs 2A and 2B, especially when compared to their invertebrate orthologues. Genetic analyses suggest that this extension was specific to GluN2 and occurred before the duplication into the 29  four isoforms and the subsequent explosion of the c-tail divergence in the vertebrate lineage (Ryan et al., 2008). In contrast with the extracellular domains, the c-tails do not form welldefined structures, but instead consist of an unstructured but flexible series of short motifs that interact with intracellular factors involved in the trafficking and clustering, focusing of ionic and conformational signals to downstream effectors, and for direct modulation of the receptor properties by various post-transcriptional modification factors. While a number of motifs and post-translation modification sites have been identified, there are likely a wealth of as yet unidentified auxiliary proteins and post-translational modification sites that can influence essential receptor properties as have been well established in other glutamate receptors (Traynelis et al., 2010). The diversity in the physical structure of the GluN2 subunits gives rise to the biophysical and pharmacological attributes of the different subtypes of NMDA receptors depending on which non-GluN1 subunit is incorporated (Fig 14a, Table 2). Indeed, even the identity of the GluN1 subunit that is incorporated affects the channel kinetics (Rumbaugh et al., 2000) (Fig 14b).  Figure 14: GluN/GluN2 receptor subtypes differ in their glutamate deactivation kinetics A) NMDA receptor-mediated currents recorded from transfected HEK cells were mediated by a brief application of saturating glutamate. B) Influence of the GluN1 exon 5 splice variant (N1 cassette). Adapted with permission from (Paoletti, 2011).  30  Table 2: GluN2 subunit specific permeation and gating properties GluN1-1a+ GluN2A GluN2B GluN2C GluN2D Conductance (pS) Main Sub Mean open time (ms) EC50 (Glycine), µm EC50 (Glutamate), µm τoff (Glycine), ms τoff (Glutamate), ms Ρo, peak Ρf (Ca2+), % IC50 (Mg2+), µM (νm = -100mV)  50 38 3-5 1.7 4 140 40 0.4-0.5 18 2  50 38 3-5 0.4 2 300 0.1-0.2 18 2  37 18 0.5-1 0.3 1 680 300 ~0.01 8 12  37  0.5-1 0.1 0.4 2000 ~0.01 n.d. 12  1.2.4 Switching of GluN2B and GluN2A  GluN1 mRNAs can be detected as early as embryonic day 14 and continues to be ubiquitously expressed throughout the lifespan of an organism. While GluN2B mRNA is detected coincident with the expression of GluN1, GluN2A mRNA is undetectable during the entire embryonic period and only begins to be expressed around post-natal day 7 (Monyer et al., 1994). During post-natal development, NMDA receptor currents become increasingly resistant to the partial GluN2B NMDA receptor specific antagonist ifenprodil and develop a progressive shortening of NMDA EPSC consistent with the faster decay of GluN2A NMDA receptors (Sheng et al., 1994). These correlations indicate the increasing incorporation of GluN2A NMDA receptors into excitatory synapses and could contribute to the observed loss of cortical plasticity that accompanies the maturation of the brain and this shift is thought to alter the threshold for inducing NMDA receptor-mediated synaptic plasticity (Yashiro and Philpot, 2008). Additionally, the incorporation of GluN2A NMDA receptors is likely dependent on synaptic  31  activity. Synapses in the visual cortex normally follow this developmental incorporation of GluN2A NMDA receptors but this is impaired in animals deprived of vision and thus synaptic activity in this region of the brain (Carmignoto and Vicini, 1992). Surprisingly, in dark-reared post-natal day 21 – 23 rats, even brief exposure to light induces the incorporation of GluN2A NMDA receptors in the visual cortex within one hour of exposure (Quinlan et al., 1999). However, the precise molecular mechanisms of how GluN2A NMDA receptors are preferentially incorporated into maturing synapses, beyond differences in the temporal expression of the GluN2 subunit genes, remain largely unknown although increasing evidence implicates the differential roles of the different scaffolding molecules of the post-synaptic density. Although commonly referred to as a “switch,” GluN2B subunits persist in excitatory synapses and it is largely the ratio of GluN2B:GluN2A that changes. The PSD-95-like membrane-associated guanylate kinases (PSD-MAGUK) family of proteins form the primary multimeric scaffolding at excitatory post-synaptic sites, and include PSD-95 (SAP-90), PSD-93 (chapsyn-110), SAP97, and SAP102. This family of proteins are characterized by the inclusion of three N-terminal PDZ domains, a Src-homology 3 (SH3) domain, and a C-terminal catalytically inactive guanylate kinase (GUK) domain. Although the gross domain structure is fairly conserved, the N-terminal amino acid sequences are quite distinct, and each PSD-MAGUK exist as several isoforms with further variance in N-terminal amino acid sequence and domain structure organization (Elias and Nicoll, 2007). However, there are remarkable similarities between the PSD-MAGUKS in terms of protein-protein interactions and a high degree of functional compensation. For example, there are no apparent structural or functional abnormalities in the PSD-93 knockout mouse (McGee et al., 2001). Likewise, synaptic NMDA receptor currents, subunit expression, localization, and 32  synaptic morphology is surprisingly unaffected in the PSD-95 transgenic knockout mouse, although there are differences in experimentally induced plasticity which is consistent with the idea that different PSD-MAGUKs may mediate distinct downstream signalling pathways (Migaud et al., 1998). SAP102 knockout mice are also viable and show no alterations in GluN1, GluN2B, or GluN2A localization but have impaired synaptic plasticity and show learning deficits (Cuthbert et al., 2007). On the other hand, SAP97 germline knockout mice have neonatal lethal feeding deficits although cultured embryonic cortical neurons appear to have normal synaptic clustering of glutamate receptors (Klocker et al., 2002). Nonetheless, there is a temporal coincidence of the postnatal developmental switch from GluN2B to increasing GluN2A NMDA receptors and the switch from SAP102 to increasing PSD-95 expression that suggest the switches may be related (van Zundert et al., 2004). Indeed, this GluN2B to GluN2A switch is abolished in PSD-93/PSD-95 double knockout mice (Elias et al., 2006). In a subsequent study using over-expression and shRNA knockdown, SAP102 and PSD-95 was shown to have distinct roles between synaptogenesis and synapse maturation. SAP102 is expressed early and is required for the trafficking of NMDA receptors during synaptogenesis but is not required for synapse maturation. Recently, there is evidence that SAP102 can be alternatively spliced at its N-terminal to contain a PDZ-independent domain that specifically binds GluN2B (Chen et al., 2011). PSD-95, on the other hand, despite being able to interact with all GluN2 subunits and the lack of effect on synaptic transmission in PSD-95 germline knockout animals (Elias et al., 2006), appears to be required for synapse maturation and the incorporation of GluN2A NMDA receptors at least via in utero shRNA knockdown and overexpression from E16 to 17 (Elias et al., 2008).  33  The functional significance of this shift from GluN2B to GluN2A has been demonstrated by overexpression of GluN2B or GluN2A by biolistic transfection of organotypic hippocampal slices during the beginning of expression and synaptic incorporation of GluN2A (post-natal day 6). Cells overexpressing GluN2B at this time does not affect the number nor growth rates of synapses although there is an increase in spine motility. Overexpression of GluN2A in this system however, reduces the number of synapses, the volume of spines, and spine motility (Gambrill and Barria, 2011). Despite the shift from predominantly GluN2B containing NMDA receptors to increasing ratios of GluN2A containing NMDA receptors, GluN2B appear to activate unique cellular processes that cannot be replaced by GluN2A as demonstrated in mice where the GluN2B allele is disrupted and replaced with GluN2A. Indeed, the genetic replacement of GluN2B with GluN2A rescues GluN1 immunoreactivity at synapses that is lost in both the global GluN1 and GluN2B knockout animal however, these genetically replaced animals exhibit high rates of lethality, suppressed feeding, and depressed social exploratory behaviour (Wang et al., 2011).  1.3 NMDA Receptors and Brain Development  Ionotropic glutamate receptors are ligand-gated ion channels that mediate the vast majority of excitatory neurotransmission in the vertebrate CNS. NMDA receptors are the most complex of these receptors and play pivotal roles in the tightly regulated control of neuronal communication required for high cognitive functions such as memory formation and learning (Rao and Finkbeiner, 2007). While mild perturbations may impair learning, memory, and behaviour, highly abnormal signalling through NMDA receptors can lead to catastrophic results. 34  Overstimulation through NMDA receptors have been implicated in Huntington’s Disease (Reilmann et al., 1994), epilepsy (Mody and Heinemann, 1987), spongiform encephalopathy (Scallet and Ye, 1997), and ischemia-related glutamate excitotoxicity (Lipton and Rosenberg, 1994). Conversely, improperly reduced NMDA receptor activity can also lead to pathologies such as neuronal apoptosis associated with foetal alcohol syndrome (Olney et al., 2001) and schizophrenia (Goff and Coyle, 2001). However, NMDA receptor-mediated signalling is also important in the development of neuronal connectivity and brain development. The importance of NMDA receptor mediated synaptic activity in the development of neuronal networks was first established when it was shown that chronic intra-cortical infusion of NMDA receptor antagonist APV rendered kitten striate cortices resistant to the effects of monocular deprivation (Kleinschmidt et al., 1987). Additional evidence for the importance of NMDA receptors comes from the development of transgenic mice. GluN1 knockout mice suffer from perinatal death despite the absence of gross morphological brain abnormalities (Forrest et al., 1994). Similarly, GluN2B transgenic knockout mice also suffer from perinatal death primarily owing to defects in respiration and suckling response leading to the lack of nutrition (Kutsuwada et al., 1996). In both of these lines there were no differences in non-NMDA receptor mediated evoked excitatory post-synaptic currents compared to wild-type littermates but NMDA components of synaptic potentials were completely abolished despite the normal expression of other NMDA receptor subunit mRNAs.  35  1.3.1 Signalling Through NMDA Receptors at Glutamatergic Synapses  A typical glutamatergic excitatory post-synaptic specialization is host to both AMPA and NMDA receptors, although some lack AMPA receptors and are functionally silent (Malenka and Nicoll, 1997). Electron-microscopic evidence suggest that even in synapses containing both receptor types they occupy slightly different regions within the synaptic membrane (Kharazia and Weinberg, 1997). In a characteristic synapse in the hippocampal CA1 region there are an estimated 0-150 AMPA receptors and only 5-30 NMDA receptors (Spruston et al., 1995). AMPA receptors have a relatively low affinity for glutamate (EC50 ~3-30 mM) and open quickly (<1 ms), only briefly, and most AMPA receptor subtypes are selectively permeable to Na+ (Hollmann and Heinemann, 1994). However, if this brief signal through AMPA receptors sufficiently depolarizes the post-synaptic terminal, the Mg2+ ion that normally resides in, and blocks, the NMDA receptor channel can be released (Kupper et al., 1998). This requirement for simultaneous binding of glutamate to NMDA receptors coincident with the membrane depolarization that allows the channel to open makes them exquisite coincidence detectors of pre- and post-synaptic activity and could be considered a biological logic AND gate in neuronal processing. NMDA receptors have a higher affinity for glutamate (EC50 ~0.4-1.7 mM) than AMPA receptors but require micromolar concentrations of glycine for full activity, at least in experimental conditions. Interestingly, while free glycine is actively loaded into inhibitory SVs and is co-released with GABA (Russier et al., 2002) this is not the case for excitatory SVs. However, D-serine is up to three times more potent than glycine at the glycine binding site (Matsui et al., 1995). Moreover, D-serine and serine racemase, an enzyme that generates D-  36  serine from L-serine, are found in the mouse brain in a pattern consistent with the distribution of NMDA receptors. In contrast with the loading of glycine with GABA in inhibitory SVs, D-serine is released through a cytosolic pathway and exogenous application of D-serine deaminase diminishes NMDA elicited excitotoxicity suggesting that D-serine is the endogenous co-agonist for NMDA receptors (Schell et al., 1997; Kartvelishvily et al., 2006). In addition, other allosteric modulators such as spermine, Zn2+, and H+, and opioid peptides can all affect NMDA receptor response. Although NMDA receptors take longer to open (> 2ms) than AMPA receptors, NMDA receptors remain open longer and are much more permeable to Ca2+ than even homomeric AMPA receptors. Most AMPA receptor channels have a Ca2+/Na+ conductance ratio of ~0.1, AMPA channels lacking an edited GluR2 subunit is ~2, and NMDA receptors have a ratio in excess of 5 (Dingledine et al., 1999). A single typical AMPA receptor channel may admit ~10 Ca2+ ions during a single opening, a GluR1 homomeric channel may admit an order of magnitude more, ~100 Ca2+ ions, but a single NMDA receptor channel may admit yet another order of magnitude more, ~1000 Ca2+ ions. There is general agreement that an elevation of Ca2+ ions is the single most important factor for inducing changes in synaptic plasticity (Bliss and Collingridge, 1993; Berridge, 1998); not only does Ca2+ induce short term changes such as the activation of Ca2+/calmodulin-dependent protein kinase (CaMKII) (Miyamoto, 2006) but it is also involved in long term changes via neuronal gene expression when combined with other signal transduction cascades (Morgan and Curran, 1988). Additionally, it has been shown that single synapses in the hippocampal CA1 region can be switched rapidly between long term potentiation (LTP) and long term depression (LTD) (Heynen et al., 1996) and that both LTP and LTD are mediated by Ca2+ (Bear and Malenka, 1994; Linden, 1994). 37  1.3.2 NMDA Receptor Activity and Neuronal Network Development  While the gross morphology of brains of all transgenic mice lacking individual NMDA receptor subunits appear normal suggesting that NMDA receptors are not essential for brain development, analysis of the fine structures of axonal and dendritic branches belie this supposition as both axonal and dendritic arborization is impaired in the absence of NMDA receptor signalling. Chronic and specific blockade of Rana pipiens tadpole optic tectal NMDA receptors with APV or MK-801 ((+)-5-methyl-10,11,-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate), an irreversible open-channel blocker (Wong et al., 1986; Huettner and Bean, 1988), disrupts the development of eye-specific stripes of retinal ganglion cell axon arbors (Cline et al., 1987). This requirement for NMDA receptor signalling in activity-dependent sensory map formation is evolutionarily conserved and generalizable to rodents; rats treated chronically with NMDA receptor antagonists during the first two postnatal weeks develop aberrant topographical ordering of retinal connections (Simon et al., 1992). Furthermore, axonal arbor establishment and refinement in the whisker systems of GluN1 and GluN2B transgenic knockout mice are impaired (Li et al., 1994; Kutsuwada et al., 1996). In wild-type mice, peripheral and central trigeminal ganglion cells innervate the whisker pads on the snouts of rodents and the precise arrangement of the whiskers are mapped back onto the brainstem trigeminal nuclei which is subsequently mapped onto the thalamus and barrel cortex creating a distinct pattern with each barrel receiving input from individual whiskers. These barrel structures fail to form in the GluN1 and GluN2B knockout mice as the axon arbors show exuberant elaboration in the absence of activitydependent synapse stabilization preventing the development of segregated patches. Since  38  canonical NMDA receptors reside in the post-synaptic membrane while retinal arbors are presynaptic, these effects are likely to be mediated by a retrograde signal generated downstream of Ca2+ entry through NMDA receptors that direct the behaviour of the pre-synaptic retinal terminals. However, the identity and indeed more likely the multiple identities of these retrograde signals remains elusive (Schmidt, 2004) although a number of candidate diffusible and non-diffusible factors have been proposed. Nitric oxide, brain-derived neurotrophic factor, and arachidonic acid are diffusible factors that may modulate retrograde signalling while the upregulation of synapse-specific cell adhesion molecules, ephrins, and neurexin/neuroligin/LRRTMs may mediate activity-driven synapse stabilization or increase the likelihood of local synaptogenesis. Recently, studies in Xenopus tadpole retinotectal synapses demonstrated that LTP and LTD induced in vivo undergo rapid long-range retrograde spread from the optic tectum to the retina, resulting in potentiation or depression of bipolar cell synapses on the dendrites of retinal ganglion cells. Interestingly, the retrograde spread of LTP is mediated through BDNF and its receptor TrkB whereas the spread of LTD requires nitric oxide (NO) signalling through protein kinase G (Du et al., 2009). Dendritic arbor growth is also regulated by NMDA receptor signalling although the results are less consistent. One of the experimental systems that permit the direct observation of neuronal development in intact animals is the Xenopus laevis model where the transparency of the tadpole allows for direct in vivo time lapse imaging of developing optic tectal neurons (Witte et al., 1996). Normally, after the extension of the axon, the dendritic arbor undergoes a highly dynamic and rapid phase of growth and elaboration that gradually subsides as the arbor is stabilized. Blockade of NMDA receptor activity reduces dendritic dynamism and growth rates (Rajan et al., 1999). In the same system, visual stimulation induced NMDA receptor-mediated 39  synaptic activity promotes dendritic arbor growth (Sin et al., 2002). However the opposite was observed in the mammalian optical system. When chronically treated with APV in vivo, the dendrites of neurons in the ferret kit lateral geniculate nucleus developed exuberant branching suggesting that NMDA receptor blockade removes the stabilization signals provided by productive synaptic transmission and interferes with dendritic arbor refinement (Rocha and Sur, 1995).  1.3.3 Molecular Genetic Studies of NMDA Receptor Function  One method of investigating the normal role of NMDA receptors is to observe the system in their absence. However, elucidation of the roles that NMDA receptors play in the formation and structure of synapses are complicated by the perinatal lethality of global transgenic GluN1 knockout mice (Forrest et al., 1994) as are investigations on the role of GluN2B on synapse function as the global transgenic GluN2B knockout mice also suffer from a similar perinatal lethality (Kutsuwada et al., 1996). Interestingly, the large cytoplasmic tails of NMDA receptors appear to be extraordinarily important for their proper localization as mutant knock in mice expressing NMDA receptors without the cytoplasmic tail of any one of GluN2B, GluN2A, or GluN2C phenotypically resemble mice lacking the entire subunit (Sprengel et al., 1998). While genetic tools such as overexpression and short hairpin RNA (shRNA) knockdown have been useful, the development of conditional and spatially restricted knockout animals and the development of genetic replacement strategies have provided additional evidence for how NMDA receptors- and the specific roles of individual subunits- shape synaptic structures and neuronal connectivity.  40  One spatially restricted transgenic knockout mouse, in an effort to bypass the perinatal lethality associated with GluN1 global knockout, made use of the Cre-LoxP system (Tsien et al., 1996) and inserted the Cre recombinase gene into the Emx1 locus, a homeobox gene expressed exclusively in the dorsal telencephalon from embryonic stages into adulthood (Gulisano et al., 1996). As the GluN1 subunit it obligatory for the formation of functional NMDA receptors, ablation of GluN1 is sufficient to eliminate all NMDA receptors (McIlhinney et al., 2003). In this mouse (CxNR1KO), GluN1 protein was reduced to 5% in the cortex while GluN1 protein in the thalamus and brainstem were unaffected and homozygous knockout mice were viable into adulthood (Iwasato et al., 2000). Like the global GluN1 KO mouse (Forrest et al., 1994), these mice also failed to develop proper patterning in the barrel cortex up to post-natal day 41 despite normal patterning in the brainstem and thalamic somatosensory relay stations. Interestingly, these mice show increased spine density, a marker for excitatory synapses in vivo, in layer IV spiny stellate cells. A similar spatially restricted transgenic KO mouse has also been generated (Ultanir et al., 2007) from GluN1-floxed mice crossed with Nex-Cre mice which express Cre recombinase under the NEX promoter, a neuron specific basic helix-loop-helix transcription factor expressed exclusively and abundantly in pyramidal neurons of the hippocampus, cerebellum, and several neocortical areas (Bartholoma and Nave, 1994). In contrast with the CxNR1KO mouse, spine density is decreased in cortical layer II/III pyramidal neurons which is accompanied by increases in the pre-synaptic axon bouton volume, post-synaptic density area, and an increase in miniature excitatory post-synaptic current amplitude and frequency (Ultanir et al., 2007). However, the barrel cortex of these mice also fail to pattern correctly, suggesting that NMDA receptors play different roles in synaptogenesis or synaptic stability in different neurons  41  types and areas of the brain although the timing of genetic ablation of GluN1 may also contribute to the observed differences. Alternative strategies to study the contribution of NMDA receptors on the formation and long-term stabilization of glutamatergic synapses are through pharmacological blockade and acute knockdown of gene transcript by shRNA interference. In organotypic slice preparations, chronic pharmacological blockade of NMDA receptors with APV slightly increases the number of transient spines but does not affect spine numbers, which increased over time at a rate indistinguishable from control organotypic slices. Sparse ablation of GluN1 by shRNA from individual pyramidal neurons results in a severe reduction in NMDA receptor-mediated synaptic currents whereas AMPA receptor-mediated currents were still observed. In these transfected neurons however, there is an increase in the number of transient spines similar to cells chronically treated with APV but this increase is accompanied by a concomitant decrease in the formation of new spines and the loss of existing spines without affecting dendritic branch complexity suggesting that the physical presence of NMDA receptors may have a mechanistic role distinct from ion flux (Alvarez et al., 2007). In a similar set of experiments to address the requirement of NMDA receptors in the development of neuronal circuitry, sparse GluN1 knockout was achieved by biolistically transfecting P5-P9 organotypic slice cultures from GluN1 floxed (GluN1fl/fl) mice with CRE recombinase and cultured for an additional 12-17 days. Since the transfection efficiency was low, few cells were ablated of GluN1 and NMDA-mediated currents while the vast majority of neighbouring cells were unaffected (Adesnik et al., 2008). While the prevailing model predicts that the ablation of NMDA receptors would strongly impair the maturation of excitatory circuits, cells depleted of GluN1 paradoxically developed pronounced enhancement of synaptic 42  transmission mediated by AMPA receptors. Investigations on the role of NMDA receptors in excitatory glutamatergic synapses are complicated by the presence of both AMPA and NMDA receptors at a typical synapse. Some excitatory synapses, however, contain only NMDA receptors but no AMPA receptors rendering the synapse silent as normal AMPA receptormediated membrane depolarization is absent and NMDA receptors, due to their voltage dependent Mg2+ block, remain inactive under typical conditions (Montgomery et al., 2001). In the converse experiment in order to determine any effects of synaptic competition between NMDA receptor competent and NMDA receptor lacking cells, organotypic slice cultures were prepared from Nex-Cre mice and sparse rescue of GluN1 was achieved via biolistic transfection with GFP-tagged GluN1. These GluN1 rescued neurons exhibited significantly reduced synaptic AMPA currents compared with neighbouring untransfected knockout neurons suggesting that normal NMDA receptor function during network activity limits the number of functional synapses made onto pyramidal neurons. Further, GluN1fl/fl embryos were electroporated in utero to produce the sparse knockout phenotype before the development of synapses. These sparse NMDA receptor deficient neurons also showed an increase in AMPA receptor mediated synaptic currents but surprisingly no increase in either spine density or disruption of dendritic morphology indicating that a greater proportion of spines have functional AMPA receptors than in NMDA receptor competent neurons. Additionally, in this system, these changes were also seen when GluN1 was locally ablated in a majority of neurons via high-titre adenovirus expressing Cre recombinase suggesting that synaptic competition was not a major mechanism (Adesnik et al., 2008) unlike the formation of the neuromuscular junction (Buffelli et al., 2003). At most early forebrain excitatory synapses, NMDA receptors predominantly contain only the GluN2B subunit in addition to the obligate GluN1, which are gradually replaced or 43  supplemented by GluN2A containing receptors. This switch is bi-directionally regulated by experience and activity (Bellone and Nicoll, 2007) and is thought to alter the threshold for inducing NMDA receptor-mediated synaptic plasticity (Yashiro and Philpot, 2008). Using single-cell deletion strategies by transcranial stereotactic injection of P0-P1 GluN2Bfl/fl and GluN2Afl/fl mice with adeno-associated virus expressing Cre-recombinase, viable P18 mice were generated with sparse knockout of either GluN2B or GluN2A. Examining these mice, it was found that GluN2B and GluN2A regulate the insertion of AMPA receptors to synapses by distinct mechanisms (Gray et al., 2011). While post-natal GluN1 deletion increases AMPA receptor mediated currents, a similar increase is also seen when both GluN2B and GluN2A are ablated from CA1 pyramidal neurons. Surprisingly, deletion of either GluN2B or GluN2A alone also resulted in similar increases in AMPA receptor mediated currents. The deletion of GluN2B was similar to the ablation of GluN1 given its prominent expression in early post-natal development while the ablation of GluN2A increases AMPA receptor insertion by a distinct mechanism likely to involve the increase in miniature excitatory post-synaptic currents seen when GluN2A alone is ablated (Gray et al., 2011). However, in the genetic replacement transgenic mice where the GluN2B allele is disrupted and replaced with GluN2A, there is a similar increase in AMPA receptor mediated miniature excitatory post-synaptic current (mEPSC) amplitudes (Wang et al., 2011). Evidence suggesting distinct mechanisms on the regulation of AMPA receptor insertion into synapses comes from knockdown and genetic replacement experiments. The acute knockdown by shRNA of GluN2B is sufficient to induce increases in AMPA mEPSC. Co-expression of GluN2B shRNA and a shRNA resistant GluN2B silent mutant with R1300Q/S1303D calcium/calmodulin-dependent protein kinase II (CaMKII) binding mutant (Bayer et al., 2006) 44  fails to prevent the increase in AMPA mEPSC suggesting that suppression of synaptic AMPA receptor incorporation requires interactions between GluN2B and CaMKII. In addition to regulating the synaptic insertion of AMPA receptors, the cytoplasmic tails of GluN2B and GluN2A differentially regulate synapse organisation. A transgenic mouse was derived from crossing floxed GluN2B and Cre-recombinase driven by the GluK4 gene that only begins to ablate GluN2B from the CA3 region of the hippocampus at P0 leading to the complete loss of GluN2B by 2 months of age. The loss of GluN2B after the normal embryonic development of synapses surprisingly results in the virtual loss of evoked synaptic NMDA receptor currents and LTP induction in the CA3 region, but not extra-synaptic currents, despite having the normal amount of GluN2A protein. Although there were normal levels pre-synaptic synaptophysin and PSD-95 at the post-synapse, the detergent solubility of PSD-95 and GluN2A was increased concomitant with a decreased amount of synaptic F-actin and the number of spines suggesting that the physical presence of GluN2B, but not GluN2A, selectively affects the equilibrium of the actin cytoskeleton (Akashi et al., 2009).  1.4 Trafficking of NMDA Receptors  The current understanding of the cell biology of the NMDA receptors is still in the early stages and questions regarding the assembly of the receptor complex, their trafficking in the neuron, and the regulated insertion and removal from synapses have only recently been addressed. In contrast with AMPA receptors, which are readily added and removed from synapses based on activity, NMDA receptors had been viewed as a more stable component of synapses (Ehlers, 2000). However, there is increasing evidence for the rapid internalization 45  (Roche et al., 2001) and regulated insertion of NMDA receptors into synapses in response to activity (Quinlan et al., 1999; Grosshans et al., 2002). While NMDA receptors are concentrated at excitatory synapses, functional NMDA receptors are present on the plasma membrane at extrasynaptic sites and exhibit a fairly high degree of mobility between these sites (Tovar and Westbrook, 2002). The differences in spatial localization and the co-localization of accessory proteins can influence the outcome of signalling between these two populations, for example NMDA receptor signalling at synaptic sites promotes neuronal survival while activation of extrasynaptic NMDA receptors are implicated in ischemia-related excitotoxicity (Hardingham and Bading, 2003).  1.4.1 Forward Trafficking  NMDA receptors are tetramers consisting of two obligate GluN1 and two other regulatory subunits. Unique amongst ligand-gated ion channels, this tetrameric receptor requires two agonists, glycine and glutamate where the GluN1 subunit contains the glycine binding site and GluN2 the glutamate. In neurons, there is a large excess of GluN1 subunits relative to GluN2 and this pool of GluN1 remain as unassembled monomers that are rapidly degraded, with a half life of two hours, if not assembled, stabilized, and transported to the cell surface (Huh and Wenthold, 1999) suggesting that the availability of GluN2 subunits is the rate limiting factor in generating new functional receptors. Assembled and stabilized receptor complexes on the cell surface have a half life of 34 hours. Newly synthesized proteins in the endoplasmic reticulum (ER) undergo a variety quality control processes to ensure that cytoplasmic and transmembrane proteins conform to their proper 46  states before being exported. Since NMDA receptors are large compound complexes it is to be expected that it undergoes quality control at multiple levels. Many transmembrane proteins contain short amino acid sequences in their cytoplasmic or ER luminal domains that when left exposed serve as ER retention signals (Teasdale and Jackson, 1996). Two ER retention motif families have been identified for transmembrane proteins. However, a third motif has been discovered specific to multi-transmembrane domain proteins, the RXR (arginine-X-arginine) motif (Zerangue et al., 1999). GluN1 subunits when expressed alone in heterologous systems do not form functional receptors with the exception of the anomalous findings by (Moriyoshi et al., 1991) although it has been proposed that Xenopus laevis oocytes express low levels of endogenous NMDA receptor subuntis (XenGluN1and XenGluN2) that under some circumstances may assemble with exogenous GluN1 (Green, 2002{Schmidt, 2006 #773; Schmidt and Hollmann, 2009){Schmidt, 2006 #773}. Interestingly, the RXR retention motif of GluN1 resides in the C1 cassette present in splice variants GluN1-1 (C1C2) and -3 (C1C2’) but missing from GluN1-2 (C2) and -4 (C2’) (Fig 11) (Scott et al., 2001). Indeed, GluN1-2 and -4 can traffic to the cell surface in heterologous systems however GluN1-3 alone can as well, due to the PDZ binding domain (STVV) at the distal end of the C2’ cassette. Possible mechanisms promoting this exit from the ER may be by the interaction with PDZ proteins that then occlude the RXR retention signal in the C1 cassette. However, homomeric GluN1 is not detected on the surface of neurons likely due to additional ER retention signal in the third transmembrane domain (M3) present in all GluN subunits (Horak et al., 2008). Additional studies have recently suggested that some ionotropic glutamate receptors undergo further quality control inspection for ligand binding prior to release from the ER. Hints 47  that ligand binding is important for forward trafficking arose from early work investigating the structural homology between AMPA and kainate receptor S1 and S2 segments and their contributions to channel gating and desensitization properties. Interestingly, single amino acid point mutants of GluR6 (kainate receptor GluK2) near the putative ligand binding site when expressed in heterologous cells had highly impaired ligand response and defects in surface expression (Fleck et al., 2003). Some of these mutant receptors were investigated in more detail; mutants with undetectable [3H]kainate binding and electrophysiological response to both glutamate or kainate were retained intracellularly and fail to traffic to the cell surface. However, these mutant receptors were shown to successfully multimerize through co-immunoprecipitation and native PAGE. Moreover, these intracellularly retained pools of receptors were sensitive to Endo-β-N-acetylglucosaminidase H (Endoglycosidase H; EndoH) that specifically cleaves asparagine-linked mannose-rich oligosaccharides that are attached to many proteins destined for the cell surface while in the endoplasmic reticulum (ER) prior to subsequent modifications once the protein has passed into the compartments of the Golgi apparatus. Together, these are some of the first evidence that non-functional ionotropic glutamate receptors are retained in the ER through some functional quality control checkpoint (Mah et al., 2005). The finding that GluK2 receptors with abolished affinity to its ligands were unable to traffic to the surface of heterologous cells was subsequently shown to apply also to GluK1 receptors. Although able to successfully assemble into tetramers, ligand binding mutants of GluK1 and GluK2 were unable to traffic to the surface of both heterologous cells and cultured hippocampal neurons (Valluru et al., 2005) AMPA receptor mRNA editing gives rise to distinct pools of receptors with differential conductance and permeation properties. In the course of investigating the assembly properties of 48  these variants GluA2 subunits with mutations at R385K or E705A, amino acid residues at the ligand binding pocket that interact with L-glutamate, are retained in the ER and fail to traffic to the surface of heterologous cells (Greger et al., 2006). However, a pore-loop mutation (Q608R) that eliminates channel activity has no effect on receptor trafficking suggesting that while ligand binding affects ER export, ion flux is not strictly required (Coleman et al., 2006). Interestingly, surface expression of the ligand binding mutants is fully rescued by overexpression of stargazin,(calcium channel, voltage-dependent, gamma subunit 2), an integral membrane protein that is involved in the transport of AMPA receptors to the synaptic membrane (Coleman et al., 2009). Mechanistically, it has been proposed that reversible gating motions of AMPA receptors in response to glutamate binding promotes secretion from the ER. AMPA receptor undergo distinct conformations upon ligand binding; state 1 involves ligand binding to the upper lobe followed by state 2 where hydrogen bonds form between the ligand and the lower lobe. Subsequently state 3 is achieved when additional hydrogen bonds stabilize the closed-cleft conformation. In an elegant study, AMPA receptor mutants were created where the receptor was locked into various states. Mutations that shift the receptor into either irreversibly open or irreversibly closed states result in ER retention while mutants that reversibly stabilized the closed- cleft state, as compared to wild-type, promotes optimal ER export (Penn et al., 2008). Similarly, a kainate receptor (GluK2) mutant with its ligand binding domain locked in a closed conformation through cysteine mutagenesis decreased surface trafficking levels of full length receptor although the same mutation in soluble ligand binding domain protein promotes its secretion. However, the surface expression of this disulfide-bond closed cleft mutant is rescued in heterologous cells by incubation with dithiothreitol (DTT), a small-molecule redox reagent that is an unusually strong reducing agent that is effective at breaking disulfide bonds, 49  which when applied to cells expressing the wild-type receptor did not further potentiate its expression (Gill et al., 2009). Compared with AMPA and kainate receptors, less is known about the role of glutamate binding in surface delivery of NMDA receptors. However, a recent study of a co-agonist binding mutant suggested that glycine binding to GluN1 is essential for cell surface delivery of NMDA receptors (Kenny et al., 2009). In this system, a GluN1-1a D732A mutant in the S2 domain was assessed for glycine efficacy in Xenopus oocytes by co-transfection with GluN2A was and found to have a 35,000 fold decrease in apparent glycine affinity compared to wild-type GluN1-1a. This mutant receptor subunit when co-expressed with GluN2A in heterologous cells fails to traffic to the cell surface. Intriguingly, treatment with cell-permeable 5,7-di-chlorokynurenic acid, a competitive antagonist that acts at the glycine binding site of GluN1 was able to partially rescue surface expression of the mutant di-heterodimer but did not potentiate increased surface expression of the wild-type receptor. Once assembled and upon passing the different layers of quality control, membrane proteins including NMDA receptors are further processed in the Golgi apparatus and then distributed to the trans Golgi network. Although first discovered in neurons (Golgi, 1989) (originally published in 1898), much of the experimental cell biology examining the structure, distribution, and functions of this organelle were performed in relatively simpler cells such as in fibroblasts. Neurons are geometrically complicated cells with axons that can extend great distances from the cell body and possess morphologically heterogeneous dendritic branching patterns. Additionally, neurons have surface areas up to 10,000 times greater than most other cell types and the proper function of neurons depend on the precise delivery of proteins to very specific parts of the cell (Horton and Ehlers, 2004). In response to these challenges, the Golgi 50  apparatus in neurons are distributed throughout the dendrites and even in dendritic spines and provide a mechanism for rapid responses at localized sites.  1.4.2 Surface Mobility of NMDA Receptors  Evidence that NMDA receptors are tightly associated within synapses includes their extremely high resistance to detergent extraction from the post synaptic density (Kennedy, 2000) and their high degree of enrichment at synaptic versus extra-synaptic sites (Petralia et al., 1994; Rao and Craig, 1997). However, evidence that other ionotropic receptors move laterally into and out of synaptic sites (Akaaboune et al., 1999; Meier et al., 2001; Borgdorff and Choquet, 2002) suggested that NMDA receptors may also share this feature. Indeed, NMDA receptors show a surprising degree of lateral mobility. Making use of the irreversible open-channel blocker MK-801 which permanently displaces the Mg2+ ion once it is released upon membrane depolarization and channel opening, synaptic signalling was induced in autaptic hippocampal neurons in culture in the presence of MK-801. This initially completely blocked synaptic NMDA receptor response to synaptically released glutamate, but the NMDA receptor mediated synaptic response recovered to 40% within 20 minutes of MK-801 washout from the media (Tovar and Westbrook, 2002). Conversely, when both synaptic and extrasynaptic ion flux were blocked by the co-application of NMDA and MK-801, synaptically evoked NMDA receptor responses failed to recover, strongly suggesting that functional NMDA receptor can diffuse laterally from extra-synaptic to synaptic sites. Subsequent studies using single-molecule tracking have also identified differences in the lateral mobility between NMDA receptor subtypes. Specifically, GluN2A containing receptors laterally diffuse more slowly (~2 51  x10-4µm2sec-1) than GluN2B containing receptors (~50 x10-4µm2sec-1) and a decreasing mobility of NMDA receptor clusters parallels the progressive inclusion of GluN2A containing receptors as synapses mature (Groc et al., 2004; Groc et al., 2006). Furthermore, some mechanisms of NMDA receptor mobility have been identified as being mediated by the GluN1 subunit. The synaptic accumulation of NMDA receptors is regulated by activity in a homeostatic manner. In general, long-term NMDA receptor blockade enhances the synaptic accumulation of receptors in a PKA dependent manner whereas increased synaptic activity promotes receptor dispersal (Rao and Craig, 1997). Investigating more closely however, chronic activity blockade increases the expression of C2’-containing GluN1 while increased activity results in the enhanced expression of C2-containing GluN1 subunits (Mu et al., 2003). Further the rapid dispersal of NMDA receptors from synaptic to extra-synaptic sites is mediated by PKC (Fong et al., 2002) and indeed phosphorylation of Ser-890 induces dispersal of GluN1 clusters in non-neuronal cells (Ehlers et al., 1995) but phosphorylation of Ser-896 and 897 increases surface expression of GluN1 (Scott et al., 2001). In sum, it is attractive to hypothesize that GluN1 splice variants are differentially expressed and is a mechanism for longterm activity-regulated synaptic targeting or activity-induced dispersal of NMDA receptors. The lateral mobility of NMDA receptors may also play a role in the formation of synapses as they cycle in and out of nascent synapses; as the synapse matures, they capture an increasing number of receptors (Washbourne et al., 2004). A recent report studying the synaptic expression of NMDA receptors, their homeostatic regulation of synaptic clustering, and their rapid dispersal elegantly made use of GluN1 transgenic knockout hippocampal and cortical tissue in culture whereby individual splice variants were reintroduced by high titre Lentiviral transduction. While all splice variants were 52  able to rescue the synaptic clustering of GluN1, surprisingly all GluN1 splice variants showed significant enhanced synaptic accumulation upon prolonged activity blockade and all showed rapid dispersal upon activation of PKC. Given this evidence, major mechanisms mediating homeostatic activity and PKC regulation of synaptic NMDA receptor accumulation appear to occur independently of GluN1 splice isoform (Ferreira et al., 2011).  1.4.3 Internalization  It follows that since the forward trafficking and insertion of NMDA receptors to synapses is regulated, that the internalization of these receptors be also. GluN2B containing NMDA receptors are abundant early in development and at immature synapses. Under basal conditions at immature synapses, these receptors are rapidly internalized (Carroll and Zukin, 2002). Coexpression of the MAGUK family scaffolding molecule PSD-95 in heterologous cells stabilizes NMDA receptors at the cell surface and reduces the rate of internalization. Conversely, disrupting the interaction between PSD-95 and NMDA receptors by truncating a distal PDZbinding site increased the rate of internalization (Roche et al., 2001). Consistent with the instability of NMDA receptors at immature synapses, the expression of PSD-95 is initially low and increases over time (Sans et al., 2000). Additionally, regulated disruption of the interaction between NMDA receptors and PSD-95 can be mediated by PKC activity. Once released from this stabilizing scaffold, the receptor is free to move into extra-synaptic sites where they can be targeted for internalization. Indeed endocytic proteins including clathrin, AP-2, and dynamin are concentrated in domains tangential to the synapse and away from the post-synaptic density (Racz et al., 2004).  53  The specific molecular mechanisms of active internalization of NMDA receptors were investigated upon the identification of an internalization motif, YEKL (tyrosine-glutamic acidlysine-leucine), in the distal cytoplasmic tail of GluN2B (Prybylowski et al., 2005) and is a member of the well-characterized family of tyrosine-based internalization signals present in a number of other membrane proteins (Roche et al., 2001). This motif is recognized by the adaptor protein AP-2 that recruits clathrin to form invaginations that are then excised from the plasma membrane by dynamin, much like the recycling of synaptic vesicles. The internalization of GluN2B containing NMDA receptors is attenuated by the co-expression of a dominant-negative form of dynamin. Likewise, although GluN2A containing NMDA receptors are relatively stable at mature synapses of the hippocampus and cortex (Ehlers, 2000), they too are subject to the regulated removal from the membrane surface. GluN2A containing NMDA receptors expressed in heterologous cells can be gradually removed from the cell surface upon repeated long-term application of agonist independent of ion flux. This use-dependent internalization was dependent on tyrosine dephosphorylation suggesting the presence of a tyrosine-based motif that is recognized by AP-2 (Vissel et al., 2001).  1.4.4 GluN2 Selective Synapse Accumulation  Early experiments overexpressing GluN2 subunits in hippocampal slice cultures suggested that while GluN2B containing NMDA receptors traffic to synapses at a basal rate and is unaffected by APV or 5,7-dichlorokynurenic acid, competitive inhibitors at the glutamate and glycine binding site respectively, the incorporation of GluN2A containing NMDA receptors 54  require activation of synaptic NMDA receptors (Barria and Malinow, 2002). However, simulated ligand binding by treatment with the glutamate binding site competitive antagonist APV can also drive GluN2A into dendritic spines whereas chronic treatment with MK-801, a NMDA receptor pore blocker, is insufficient suggesting that the insertion of GluN2A may rely on the use dependent internalization of GluN2B (Vissel et al., 2001). In this system, however, the exogenous GluN subunits do not associate with endogenous GluN subunits to create hybrid NMDA receptors (Barria and Malinow, 2002). Interestingly, while GluN2B incorporate into synapses in a constitutive manner whereas GluN2A required activity or sensory experience, the determinants of these behaviours do not lie in the cytoplasmic tail, which carries elements crucial for proper receptor localization and function. The expression of GluN2 chimeras in hippocampal slices cultures, GluN2B with a 2A tail or GluN2A with a 2B tail, incorporate into synapses identically to their wild-type counterparts. Instead, the determinants for basal- and activity dependent- synaptic incorporation lie in the extracellular loop between M3 and M4 and differential N-glycosylation at these sites may segregate different NMDA receptor subtypes into separate trafficking pathways with different requirements to reach the synaptic membrane (Storey et al., 2011). A different group wished to examine the relationship between the availability of GluN2 subunits and whether overexpression could increase synaptic incorporation. Using cerebellar granule cell cultures, tagged GluN1-1a, GluN2B, or GluN2A were expressed in these cultures by calcium phosphate transfection. In contrast with the overexpression experiments done in hippocampal slice cultures, exogenous GluN-1-1a, GluN2B, and GluN2A can all co-assemble with endogenous subunits and form surface puncta at synaptic sites (Prybylowski et al., 2002). Interestingly, while exogenous GluN1-1a was able to incorporate into synapses, overexpression 55  of GluN1 did not alter NMDA mediated currents nor alter the number of synaptic puncta likely due to the fact that under normal conditions, there is a large excess of endogenous GluN1 that remain as unassembled monomers that if not assembled, stabilized, and transported to the cell surface is rapidly degraded (Huh and Wenthold, 1999).The overexpression of either GuN2B or GluN2A, though, significantly increased both current- and cluster- density although many of these supernumerary puncta do not co-localize with synaptophysin (Prybylowski et al., 2002). Despite differences in the incorporation of exogenous GluN subunits, both of these sets of experiments showed that the distal amino acids in the cytoplasmic tails, including the PDZ domain in GluN2B, is important for NMDA receptor incorporation into synapses since both GluN2B and GluN2A distal tail truncation mutants incorporated less efficiently than the exogenous wild-type subunit (Barria and Malinow, 2002) and expression of ΔPDZ GluN2B was unable to alter synaptic excitatory post-synaptic potentials like the overexpression of the wildtype subunit (Prybylowski et al., 2002). However, in a different set of experiments, an S1462A mutant of GluN2A, analogous to the PDZ binding domain mutant of GluN2B, was able to incorporate into synapses and alter the NMDA-mEPSCs comparable to the expression of wildtype GluN2A (Prybylowski et al., 2005). In contrast with the PDZ binding motifs in the very distal amino acids residues of the GluN2 cytoplasmic tails, a clathrin adaptor protein (AP-2) binding motif, YEKL, has been identified in GluN2B.Again, using the cerebellar granule cell culture system, expression of exogenous wild-type GluN2B increases the current density and slows the deactivation kinetics in response to NMDA/D-serine application. Expression of the AP-2 binding mutant GluN2B in these cells increased the current density and fluorescence intensity of immunolabeled synaptic puncta, defined as co-localization with bassoon, even further presumably by decreasing the rates 56  of GluN2B receptor endocytosis. Interestingly, expression of a double AP-2- and PDZ- binding site mutant increased current density and slowed deactivation kinetics indistinguishably from the overexpression of wild-type GluN2B (Prybylowski et al., 2005). While AP-2 mediated receptor internalization is likely a major mechanism for the removal of GluN2B containing NMDA receptors from synapses, phosphorylation of the PDZ binding domain by protein (casein) kinase 2 (CK-2), a highly conserved serine/threonine kinase (Chung et al., 2004), provides an alternative means of removing GluN2B containing NMDA receptors from synapses. CK2 preferentially phosphorylates GluN2B over GluN2A and is abolished in the S1480A PDZ binding domain GluN2B mutant. Treatment of cortical neurons in culture with 4,5,6,7tetrabromobenzotriazole (TBB), a specific ATP site-directed inhibitor of CK2 (Sarno et al., 2001), increased surface synaptic accumulation of GluN2B and reduced the ratio of surface synaptic GluN2A to total GluN2A. Additionally, inhibition of CK2 strongly reduced the endocytosis rates of GluN2B containing NMDA receptors from synapses (Sanz-Clemente et al., 2010).  1.5 NMDA Receptors and Synaptic Plasticity  The pioneering work of Ramon y Cajal in 1891 in identifying specialized junctions between neurons initiated the idea that information is stored in the brain by changing the efficiency of signalling between neurons through what Sir Charles Scott Sherrington would coin as synapses in 1897. This idea was further refined by Donald Olding Hebb and Jerzy Konorski in the 1940’s who proposed that synapses between two cells strengthen if both cells are active at the same time. Long lasting enhancement of synaptic effectiveness following electrical stimulation  57  of excitatory synapses was first demonstrated by Timothy Vivian Pehlham Bliss and Terje Lømo in 1973 and was termed long term potentiation (LTP) (Bliss and Lomo, 1973). Although LTP could be disrupted and reversed by synaptic activity, a phenomenon referred to as depotentiation (Barrionuevo et al., 1980), homosynaptic long term depression (LTD) can also be reliably elicited by low frequency electrical stimulation of excitatory synapses (Dudek and Bear, 1992). Since their original discovery, a huge variety of different stimulation protocols for different regions of the brain and different synapse types have been developed for the induction of LTP and LTD (Hunt and Castillo, 2012), sometimes resulting in conflicting findings. It is now generally accepted that long-term alterations in the strength of synaptic transmission are likely cellular correlates of learning and memory (Linden, 1994). Thus far, however, the majority of work investigating neuronal plasticity has been performed in vitro under fairly unphysiological experimental conditions and the current challenge is to determine the contributions of LTP and LTD to in vivo experience-driven changes in memory and behaviour.  1.5.1 Long Term Potentiation  NMDA receptors are classically considered as coincidence detectors and are essential for many, but not all, forms of long term potentiation (LTP). The integrative qualities of NMDA receptors owe to their Mg2+ block at resting potentials, high permeability to Ca2+, and the slow kinetics of NMDA-EPSPs allowing for robust temporal summation. Experimental in vitro NMDA receptor-dependent LTP can be achieved through a very wide variety of induction protocols (Hunt and Castillo, 2012). Central to the induction of LTP though is the influx of Ca2+ through the NMDA receptor channels leading to an elevation in 58  spine Ca2+ concentration (Lynch et al., 1983). Although a transient 2-3second rise in spine Ca2+ is sufficient to induce LTP, it is unknown whether Ca2+ entry from NMDA receptors is sufficient or whether additional Ca2+ influx, either from voltage-gated Ca2+ channels or release from intracellular stores, is required (Bliss and Collingridge, 1993). However, regardless of induction method calcium/calmodulin-dependent protein kinase II (CaMKII) is required for NMDA receptor-dependent LTP (Lisman et al., 2002). An exception to the requirement for CaMKII is in the juvenile system using slice preparations of the visual cortex from neonatal rats, where the expression of CaMKII is very low (Kirkwood et al., 1999). Although additional protein kinases including PKA (Yasuda et al., 2003), PKC and its isozyme PKMζ (Hrabetova and Sacktor, 2001), the mitogen activated protein kinase (MAPK) cascade leading to extracellular signalregulated kinase (ERK) activation (Sweatt, 2004), and phosphatidylinosital 3-kinase (PI3 kinase) and the tyrosine kinase Src (Man et al., 2003) have all been implicated in being important for LTP, although it is unclear whether they directly mediate LTP or act as modulators (Malenka and Bear, 2004) and there may be different manifestations of LTP depending on which pathway has been triggered. The end result of these signalling cascades is the long lasting increase in synaptic strength. While there has been ongoing debate on whether this is due primarily to post-synaptic modification or a pre-synaptic changes in neurotransmitter release, it is clear that LTP involves increasing the number of post-synaptic AMPA receptors (Malinow and Malenka, 2002; Bredt and Nicoll, 2003) in addition to post-translational modifications to these receptors (Benke et al., 1998; Lee et al., 2003). Since experimentally induced NMDA receptor-dependent LTP is triggered post-synaptically, any relatively rapid pre-synaptic modifications would require a retrograde messenger. Although a number of diffusible candidates, such as nitric oxide and 59  arachidonic acid, have been ruled out (Malenka and Bear, 2004), the current focus for retrograde messaging is on the trans-synaptic adhesion molecules which span the synaptic cleft and interact with ligands on the pre-synaptic plasma membrane. Once LTP is induced, the question arises as to how it is maintained. Like many longlasting biological phenomena, the maintenance of LTP requires gene transcription and protein synthesis (Abraham and Williams, 2003). One striking feature of LTP is that the spines of newly potentiated synapses enlarge rapidly (Matsuzaki et al., 2004) and accumulate both F-actin and AMPA receptors (Lisman and Zhabotinsky, 2001; Fukazawa et al., 2003). Additionally, the presynaptic active zone closely matches the post-synaptic spine in size (Lisman and Harris, 1993), further suggesting the possibility that following LTP there is an insertion of trans-synaptic adhesion molecules in the post-synaptic density which interacts with and instructs the presynaptic specialization. While many forms of experimental LTP can be induced in cultured neurons using electrical stimulation, specific activation of synaptic NMDA receptors can also induce a form of LTP in contrast with the specific activation of extra-synaptic NMDA receptors, which results in long-term depression (LTD) (Lu et al., 2001).One method of specifically stimulating synaptic NMDA receptors takes advantage of the spontaneous release of one or two quanta of neurotransmitter glutamate from pre-synaptic terminals resulting in miniature excitatory postsynaptic currents (mEPSCs) which would only be accessible to receptors at synapses. While LTP is not routinely induced by spontaneous activity, brief application of supersaturating levels of NMDA receptor co-agonist glycine and the removal of Mg2+ that mediates the voltage sensitive blockade from the bathing solution is sufficient to rapidly increase the frequency of receptor stimulation and enhances the NMDA component of the mEPSC. This brief stimulation is 60  sufficient to induce AMPA receptor accumulation to synaptic cell surfaces (Lu et al., 2001) as well as induce long lasting changes in the synaptic accumulation of GluR1 AMPA receptors and calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) .  1.5.2 Role of NMDA Receptor Subtype in Plasticity  Interestingly, the NMDA receptor subtype that is activated may be important in preferentially mediating LTP or long term depression (LTD), a weakening of synaptic strength primarily due to the dephosphorylation of AMPA receptors at Ser-845 resulting in decreased channel open probability (Banke et al., 2000) accompanied by rapid removal of AMPA receptors from the synapse (Carroll et al., 1999; Beattie et al., 2000), that also requires Ca2+ flux albeit typically at lower frequencies (Dudek and Bear, 1992; Hunt and Castillo, 2012). NMDA receptors containing different GluN2 subunits have different ionic permeation and gating properties, cytoplasmic tails with potentially different protein interaction sites, and are differentially trafficked depending on synaptic activity, and thus it is attractive to consider that the subunit composition at a given synapse may optimize the threshold for inducing potentiation or depression (Yashiro and Philpot, 2008). However, investigations regarding differences in the involvement of GluN2B- and GluN2A- containing NMDA receptors have delivered conflicting results. Given that GluN2B containing NMDA receptors have much slower decay kinetics than GluN2A containing NMDA receptors and thus carry more Ca2+ per channel opening event (Monyer et al., 1994; Sobczyk et al., 2005) it is tempting to speculate that a high GluN2B/GluN2A ratio favours the induction of LTP. Indeed, several lines of evidence suggest 61  that the induction of LTP is more easily facilitated through GluN2B than GluN2A. Firstly, LTP is enhanced in transgenic mice overexpressing GluN2B (Tang et al., 1999). In a transgenic mouse lacking GluN2B in pyramidal neurons of cortex and CA1 sub-region of the hippocampus, a sub-saturating LTP induction protocol was impaired whereas a saturating LTP induction protocol was intact (Brigman et al., 2010). Pharmacological experiments using ifenprodil, a GluN2B specific agonist (Reynolds and Miller, 1989; Korinek et al., 2011), LTP induction is abolished in immature hippocampal slice cultures where GluN2B is the predominant NMDA receptor subunit (Barria and Malinow, 2005). In this same system, overexpression of GluN2A also attenuates the induction of LTP (Barria and Malinow, 2005). Conversely, transgenic ablation of GluN2A does not block the induction of LTP in the dorsolateral bed nucleus of the stria terminalis (Weitlauf et al., 2005) and pharmacological blockade of GluN2A with NVPAAM077 also fails to block LTP in transverse hippocampal slices from P28 mice. However, in the same sets of experiments, partial blockade of all NMDA receptors subtypes with APV to levels similar as that achieved with NVP-AAM077 also fails to block LTP induction (Berberich et al., 2005). It has also been reported that GluN2A containing NMDA receptors mediate LTP in that NVP-AAM077 blockade of GluN2A function occludes LTP induction in hippocampal slices. (Liu et al., 2004) Similarly, LTP induction is also disrupted by NVP-AAM077 blockade in perirhinal cortical slices (Massey et al., 2004). However, the specificity of NVP-AAM077 has been questioned. While NVP-AAM077 was reported to be 100 times more selective for human GluN1/GluN2A over GluN1/GluN2B NMDA receptors expressed in heterologous cells and that 0.4 µM NVP-AAM077 blocks 52% of NMDAR receptor-mediated EPSCs, with the residual EPSC being GluN2B specific ifenprodil sensitive (Liu et al., 2004), NVP-AAM077 shows much 62  lower, only 6-12 fold, specificity for rodent GluN1/GluN2A over GluN1/GluN2B NMDA receptors expressed in heterologous cells. (Berberich et al., 2005; Weitlauf et al., 2005). Pharmacological blockade experiments have been performed to investigate differential subunit dependencies for the induction of long term depression (LTD) has generated conflicting results. In one study, GluN2B specific antagonist ifenprodil was shown to completely block LTD. However, subsequent investigations again using ifendprodil suggest that the activation of GluN2B containing NMDA receptors is not required for LTD induction (Morishita et al., 2007). In response to this controversy, it was subsequently shown that slice orientation affects the requirement of GluN2B for the induction of LTD. While LTD can be induced in brain slices prepared in the sagittal orientation independent of GluN2B, LTD induction in coronal slice preparations were GluN2B sensitive likely due to differences in downstream signalling effectors such as Ras-GRF1 (Bartlett et al., 2011). However, experiments have been carried out in a transgenic mouse lacking GluN2B in pyramidal neurons of the cortex and CA1 sub-region of the hippocampus showing that low-frequency stimulation induced LTD is abolished in these animals suggesting that GluN2B can mediate both LTP and LTD (Brigman et al., 2010). Attempts have been made to directly address the role of GluN2A for the induction of LTD. Normally in the visual cortex the standard 1 Hz stimulation of 900 pulses induces LTD in wild-type mice. However, in the GluN2A knockout mouse, this protocol gives rise to LTP instead. Moreover, a lower frequency (0.5 Hz) stimulation of 900 pulses is able to induce LTD in both the GluN2A knockout- and wild-type- mouse suggesting that GluN2A may normally raise the threshold for the induction of LTP and the loss of GluN2A lowers the threshold for LTP induction and restricts the window for LTD induction (Philpot et al., 2007).  63  Taken together, it appears that the ratio of GluN2B and GluN2A at a particular synapse may control the threshold for the induction of LTP/LTD due to differences in open probability and Ca2+ charge transfer. However, indirect downstream effectors such as SynGAP (Ge et al., 2010) or direct effectors such as Ras-GRF1 (Bartlett et al., 2011) and calcineurin (Morishita et al., 2001), may modulate the response to NMDA receptor activity independent of subunit composition. Indeed calcium/calmodulin-dependent protein kinase II (CaMKII), a vital effector of many forms of LTP (Malinow et al., 1989), has preferential affinity for GluN2B over GluN2A (Barria and Malinow, 2005) and may be a major mechanism of how the GluN2B/GluN2A ratio modulates the threshold for inducing LTP.  1.6.3 Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII)  CaMKII is a Ca2+ activated enzyme expressed ubiquitously in the brain in diverse neuronal compartments and is highly abundant constituting 1-2% of total protein in the hippocampus (Ouimet et al., 1984). The CaMKII family is encoded by four genes, α, β, γ, and δ and can be alternatively spliced. While the γ and δ isoforms are expressed in most tissues, the α and β isoforms are primarily restricted to neural tissue. CaMKII is comprised of an N-terminal catalytic domain, a central regulatory domain within which an autoinhibitory domain resides, a Ca2+/calmodulin (CaM) binding motif, a variable sequence, and the C-terminal subunit association domain (Soderling, 1996). This protein exists as a holoenzyme oligomeric protein structure consisting of twelve 50-60 kDa subunits arranged as two stacked hexameric rings. The C-terminal association domains form the central core of each ring with the N-terminal catalytic domains pointing outwards (Kolodziej et al., 64  2000) (Fig 15). In the absence of bound Ca2+/CaM, CaMKII is inactive due to interactions between the autoinhibitory and the catalytic domain. Upon Ca2+/CaM binding there is immediate autophosphorylation on Thr-286 within the oligomeric complex between adjacent subunits that have bound the Ca2+/CaM, restricting intramolecular autophosphorylation within each of the two hexameric rings. This rapid autophosphorylation slows the release of Ca2+/CaM from less than a second to several hundred seconds and the holoenzyme remains active even after the dissociation of the substrate. Thus, a transient elevation of intracellular Ca2+ can give rise to a prolonged response through this constitutive activity of autophosphorylated CaMKII and this activity appears to be crucial for the induction of LTP by specific high frequency stimulation (Meyer et al., 1992).  Figure 15: Structure of CaMKII A) CaMKII subunit domain organization consisting of an N-terminal catalytic domain, a central regulatory region with overlapping autoinhibitory and calmodulin binding regions, a variable region, and a C-terminal subunit association domain. The colour coding corresponds with the organization of the holoenzyme in B. B) The holoenzyme structure of CaMKII consists of a gear-shaped body ~140 Å in diameter with a height of 100 Å composed of 12 subunits arrange in two sets of six subunits that form a stack of hexagonally shaped rings. This top view is based on three-dimensional electron microscopy. Used with permission from (Soderling et al., 2001).  These properties of CaMKII has been hypothesized to be a decoding mechanism for integrating transient Ca2+ spikes in response to the high frequency electrical stimulation that induces LTP. Indeed, the kinetics of autophosphorylation and Ca2+/CaM dissociation allows for 65  the summation of specific magnitude, frequency, and duration of Ca2+ spikes for maximal constitutive activity of the CaMKII holoenzyme (De Koninck and Schulman, 1998) (Fig 16). Interestingly, while T286 phosphorylation can induce the binding of CaMKII to GluN2B (Strack and Colbran, 1998; Strack et al., 2000a), stimulation of CaMKII by Ca2+/CaM is sufficient to mediate this interaction upon which CaMKII further phosphorylates GluN2B (Bayer et al., 2001). Binding to GluN2B locks CaMKII into a persistent active Ca2+/calmodulin trapping state that cannot be reversed by phosphatases and suppresses inhibitory autophosphorylation at T305/306 that would otherwise promote dissociation of the kinase from synaptic sites (Bayer et al., 2001). Global cell stimulation of NMDA receptors induces translocation of CaMKII from the  Figure 16: Ca2+ frequency decoding mechanism by CaMKII autophosphorylation This figure illustrates conditions where CaM is limiting relative to CaMKII (depicted as a decamer) three Ca2+ spikes at a frequency and amplitude such that no all Ca2+/CaM has dissociated from CaMKII after the first spike. The magnitude and duration of each spike allows three subunits of CaMKII to bind Ca2+/CaM (blue). During the first spike, by chance, no adjacent subunits bind CaM; therefor, no Thr-286 autophosphorylation occurs, and at the end of the spike the Ca2+/CaM rapidly dissociates. The second spike occurs at a frequency when one nonphosphorylated subunit still has bound Ca2+/CaM, increasing the probability that a proximal subunit would bind one of the three additional Ca2+/CaM and undergo intersubunit autophosphorylation (P). Between the second and third Ca2+ spikes, Ca2+/CaM dissociates more slowly from autophosphorylated subunits (solid purple line) than from nonphosphorylated subints (dashed orange line). During the third Ca2+ spike additional adjacent subunits autophosphorylate. The constitutive activity of the CaMKII holoenzyme is the summation of the autophosphorylated subunits (blue), which in turn depends on the magnitude, frequency, and duration of the Ca2+ spikes. Used with permission from (Soderling et al., 2001).  66  dendritic shaft to spines (Shen and Meyer, 1999). Synaptically localized CaMKII is optimally activated by Ca2+ entry through NMDA receptors and voltage-gated calcium channels and this localization allows CaMKII to phosphorylate synaptic targets including AMPA receptors, NMDA receptors, SynGap, and nNOS (Soderling et al., 2001). The source of Ca2+ entry appears to be important for inducing the translocation of CaMKII. In a mutant mouse exclusively expressing GluN1(N598R) which is Ca2+ impermeable (Burnashev et al., 1992; Rudhard et al., 2003), stimulation with NMDA and glycine was still able to evoke a large APV sensitive Ca2+ signal through the cell soma and dendrites comparable with wild-type neurons, possibly through voltage gated calcium channels, Na+/Ca2+ exchangers, or intracellular stores (Thalhammer et al., 2006). While CaMKII translocates primarily to synaptic sites upon Ca2+ entry through NMDA receptors, the NMDA/glycine evoked Ca2+ rise in GluN1(N598R) cells is insufficient to induce CaMKII translocation although the expression of the constitutively active T286D CaMKII mutant in these cells was able to overcome the absence of Ca2+ flux through NMDA receptors suggesting that CaMKII normally requires local NMDA receptor-mediated Ca2+ signalling (Thalhammer et al., 2006). CaMKII preferentially binds to GluN2B, as opposed to GluN2A, as a holoenzyme through the catalytic domain. The binding site is in GluN2B’s cytosolic tail residues 1290-1309 (Strack et al., 2000a). Phosphorylation of GluN2B at Ser-1303 by CaMKII inhibits binding and promotes slow dissociation of pre-formed CaMKII/GluN2B complexes (Strack et al., 2000a). Activation of CaMKII following productive Ca2+/CaM binding increases its affinity for GluN2B (Strack et al., 1997). However, this translocation to GluN2B containing synapses can occur reversibly as transient translocation to synapses occurs after brief stimulation while longer stimulation increases the resident time of CaMKII at synapses (Bayer et al., 2006). Paired 67  stimulation induced LTP in young hippocampal slices can be blocked (Barria and Malinow, 2005) by either GluN2B specific antagonist ifenprodil or CaMKII specific inhibitor KN93(Mamiya et al., 1993). In this system, over-expression of GluN1/GluN2A drives GluN2A into synapses and occludes LTP, possibly by the removal of enough GluN2B containing NMDA receptors from synapses. Interestingly, over-expression of GluN1 with a GluN2AΔIN mutant with increased affinity for CaMKII (Mayadevi et al., 2002) was sufficient to rescue LTP induction suggesting the importance of synaptically localized binding sites for CaMKII (Barria and Malinow, 2005). Complementarily, and consistent with the idea that CaMKII activation is optimally achieved through synaptic Ca2+ flux, disruption of CaMKII binding to synaptically localized GluN2B highly impairs CaMKII function. In a transgenic mouse line where the cytoplasmic tail of GluN2B was fused to a tamoxifen-dependent mutant of the estrogen receptor ligand-binding domain, inducing the expression of the chimera disrupts synaptic GluN2B/CaMKII interactions, reduces Thr286 phosphorylation, attenuates phosphorylation of CaMKII substrates such as GluR1, and produces deficits in hippocampal LTP and spatial learning (Zhou et al., 2007). Similarly, in hippocampal slice cultures, shRNA knockdown of GluN2B impairs, nominally CaMKII dependent, LTP (Foster et al., 2010) as does [R-(R,S)-α-(4-hydroxyphenyl)-β-methyl-4(phenylmethyl)-1-piperidine propranol] Ro25-6981, a selective GluN2B antagonist (Nikam and Meltzer, 2002).Rescue of GluN2B knockdown with wild-type GluN2A nor a chimeric GluN2B with a GluN2A tail fails to rescue LTP induction whereas the reverse chimera, a GluN2A with a GluN2B tail, restores LTP induction (Foster et al., 2010). Taken together, these data suggest that CaMKII function, specifically CaMKII translocation to synapses where they can be optimally activated by NMDA receptor mediated 68  Ca2+ influx and subsequently phosphorylate synaptic targets, relies on synaptically localized binding partners including GluN2B.  1.7 Primary Rodent Neural Tissue Culture  One of the earliest experiments in culturing nervous tissue was performed by Ross Granville Harrison in 1907, while investigating competing theories of axon growth. In this study, a portion of tadpole neural tube, containing neuroblasts, was isolated and cultured in frog lymph on an inverted coverslip demonstrating that axons were a continuous extension of the neural cell. Gary A. Banker and W. Maxwell Cowan pioneered the culture of dissociated pyramidal cells from the hippocampi of foetal rats and were able to maintain them for up to a week, although after 72 hours there was a rapid loss of neurons (Banker and Cowan, 1977). Advances since then have allowed the culture of glia and post-mitotic neurons from both vertebrates and invertebrates by the late 1980’s and these advances have been paralleled by greater understanding of the biology of developing neurons (Banker and Goslin, 1988). Neural cell culture remains a popular and widely used tool for the study of neurobiology and there exist techniques for culturing lowdensity dissociated hippocampal tissue that routinely survive up to and beyond four weeks (Kaech and Banker, 2006). Primary hippocampal cultures are a well accepted preparation in the study of synapse formation and remodelling. Typically, these dissociated cultures are prepared from the hippocampus of embryonic day 17-18 (E17-18) rats or mice. These cultures are very accessible for observation and manipulation; low-density cultures are less complex than neural tissue but still form physiologically relevant neuronal connections. The spatial simplicity of these systems 69  allows for high resolution imaging by multiple microscopic techniques for investigations on the subcellular localization and trafficking of neuronal proteins. Additionally, neurons in these cultures become appropriately polarized, develop extensive axonal and dendritic arbors, and form numerous functional synaptic connections with one another. An additional benefit of using cultured neural tissue is the reduced use of animals. The hippocampi from a single embryo are sufficient for a few to a few dozen experimental assays. In the original work that identified Nmethyl-D-aspartate as an agonist for a specific population of glutamate receptors, the eponymous NMDA receptors, it was reported that a statistical minimum of 100 kittens were consumed in the course of the experiment (Curtis and Watkins, 1963) whereas the entire experiment could be replicated with one or two pregnant rat dames using modern cell culture techniques. Primary rat and mouse hippocampal and cortical culture techniques for imaging used in the subsequent chapters of this dissertation were based on updated methods by Kaech and Banker (Kaech and Banker, 2006). In brief, hippocampi or cortices were freshly dissociated and 3 x105 cells were plated on poly-L-lysine treated glass coverslips in a 60 mm dish. In addition to this treatment, each coverslip also has three paraffin dots attached to its surface. When the cell bodies become attached to the treated glass substrate, after approximately four hours, the coverslips are reversed over a confluent monolayer of rat glia in serum-free media (Neurobasal) supplemented with L-glutamine, B-27 supplement, and in the case of mouse cultures, insulin. The paraffin dots physically separate the low density neuronal monolayer from the feeder glia layer. Cytosine arabinoside is added on 2 DIV to suppress the proliferation of glia on the glass coverslip. These culture preparations allows for chemical manipulation of discrete networks of neurons and importantly allows the imaging of single neurons including the cell body and large areas of the proximal dendritic arbor with little interference from neighbouring cells. 70  1.8 Thesis Hypotheses and Objectives  1.8.1 A Cell Culture Model of Synaptic Competition  Activity through NMDA type glutamate receptors sculpts connectivity in the developing nervous system. This topic is typically studied in the visual system in vivo, where activity of inputs can be differentially regulated, but in which individual synapses are difficult to visualize and mechanisms governing synaptic competition can be difficult to ascertain (Huberman et al., 2008). Here, we make use of a GluN1 -/- (KO) transgenic mouse line (Forrest et al., 1994) where the loss of the obligate GluN1 subunit abolishes NMDA inducible increases in intracellular calcium and membrane currents. In conjunction with an Amaxa nucleofection system, a relatively high proportion of hippocampal neurons of different genotypes can be labelled with different coloured fluorescent proteins prior to being cultured alone or in defined ratios with cells of the opposite genotype. Due to the importance of NMDA receptors signalling in activitydependent sensory map formation, refinement of barrel cortex development, and dendrite arbor refinement (Cline et al., 1987; Li et al., 1994; Rocha and Sur, 1995), we hypothesize that NMDA receptors are important in refining the balance of synaptogenesis and synapse maintenance in culture were NMDA receptor competent cells are expected to be a preferred post-synaptic partner for synapse development.  71  1.8.2 ER NMDA Receptor Ligand Binding as a Quality Control Checkpoint  Trafficking of NMDA receptors to the surface of neurons and to synapses is critical for proper brain function and activity dependent plasticity. Recent evidence suggests that surface trafficking of other ionotropic glutamate receptors requires ligand binding for exit from the endoplasmic reticulum (ER), presumably as a form of quality control. However, most of these studies used a limited number of ligand binding null or otherwise highly deficient mutants resulting in the appearance of a binary dichotomy between normal or complete abolishment of receptor trafficking (Mah et al., 2005; Coleman et al., 2009; Gill et al., 2009). We hypothesise that the glutamate binding by the GluN2 subunit of NMDA receptors is also important for ER export and forward trafficking for this class of receptor and additionally that the efficiency of receptor trafficking is correlated to the affinity of the receptor to its ligand.  1.8.3 Structure of the GluN2B Tail Mediates CaMKII Translocation  Alterations in the protein-protein interactions and activity states of synaptic molecules have been proposed as a mechanism whereby transient stimuli can induce persistent changes in the excitability of synapses. NMDA receptors and calcium-calmodulin dependent protein kinase II (CaMKII) are strongly implicated in mediating long-term potentiation (LTP), an experimental model correlate of learning and memory (Lynch et al., 1983; Lisman et al., 2002). CaMKII activity is dependent not only on the activation of the holoenzyme but also determined by its subcellular localization. Synaptically localized CaMKII is maximally activated by Ca2+ entry through NMDA receptors and is in a most expedient position to phosphorylate synaptic targets including AMPA receptors, NMDA receptors, SynGAP, and nNOS (Soderling et al., 2001). 72  Indeed, global cell stimulation of NMDA receptors induces translocation from dendritic shaft to spines, increasing its synaptic levels (Shen and Meyer, 1999). Here, we show that GluN2B -/(KO) neurons fail to translocate CaMKII with similar stimulation and we use a rescue approach to delineate the contributions of specific domains and interaction sites of GluN2B to assess the synaptic accumulation of CaMKII.  73  Chapter 2: NMDA Receptors Mediate Synaptic Competition in Culture  74  2.1 Introduction  Synaptic activity mediated by NMDA type glutamate receptors sculpts the wiring of the nervous system, regulating functional and structural connectivity (Constantine-Paton et al., 1990; Katz and Shatz, 1996). As studied most intensely in the developing visual system (Huberman et al., 2008), activity in part via NMDA receptors controls axon and dendrite arbor development and refines connectivity. For example, in the developing tadpole retinotectal system, NMDA receptor antagonists enhance rates of axon branch addition and retraction, and reduce retinotopic precision (Rajan et al., 1999; Ruthazer and Cline, 2004). NMDA receptors mediate selective connectivity by acting as calcium permeable molecular coincidence detectors gated by both ligand and voltage (MacDonald and Nowak, 1990; Mori and Mishina, 1995). NMDA receptors open to allow calcium entry only hen glutamate release from pre-synaptic inputs coincides with sufficient post-synaptic depolarization to relieve the magnesium block. NMDA receptordependent signalling cascades (Saneyoshi et al., 2010) and gene transcription (Greer and Greenberg, 2008) regulate synapse assembly, maturation, and refinement. The contribution of NMDA receptors to detect differences in patterns of activity among inputs is thought to mediate selective elimination of some inputs and retention and strengthening of other inputs Although NMDA receptors sculpt much of the developing circuitry in the nervous system, the precise rules of competition and the underlying molecular mechanisms are as yet not well understood. Targeted deletion of the essential GluN1 subunit results in ablation of all NMDA receptor function in mice (Forrest et al., 1994) and can be used to study NMDA receptor mediated aspects of development. Whisker-related somatosensory patterning fails to develop in mice lacking GluN1 (Li et al., 1994), or with insufficient levels of GluN1 (Iwasato et al., 1997) or bearing  75  GluN1 N598R deficient in calcium permeability and in magnesium block and thus coincidence detection (Rudhard et al., 2003). The altered synaptic connectivity in GluN1 deficient mice is associated with unsegregated exuberant terminal axon arbors and unoriented longer dendrite arbors in the trigeminal principle nucleus (Lee et al., 2005a). Cortex-restricted knockout of GluN1 results in a similar lack of refinement of both primary somatosensory cortical dendrite arbors and thalamocortical axon arbors in addition to a general loss of patterning (Iwasato et al., 2000; Lee et al., 2005b). Single cell deletion of GluN2B in the barrel cortex revealed cell autonomous roles of NMDA receptors in dendrite patterning (Espinosa et al., 2009). There may be altered synapse density on cortical GluN1 -/- neurons, with increased spine density reported on layer IV spiny stellate cells (Datwani et al., 2002) and reduced spine density reported on layer II/III pyramidal neuron basal dendrites (Ultanir et al., 2007). Cell culture confers advantages of clarity in visualizing all synapses onto a given neuro and ease of manipulating neurons for assessing mechanisms. GluN1 -/- cortical or hippocampal neurons differentiate in dissociated culture, forming structural and functional synapses with apparently normal AMPA receptor mediated miniature excitatory post-synaptic currents bu with reduced dendritic spine density (Tokita et al., 1996; Okabe et al., 1998; Ultanir et al., 2007). Here, we test whether NMDA receptor mediated synaptic competition can be modeled in primary neuron culture. We find that GluN1 -/- and wild-type hippocampal neurons develop activity-dependent differences in synapse density only when forced to compete in co-cultures of defined ratio. Based on these results, we discuss mechanisms underlying NMDA receptor mediated synaptic competition involving a retrograde ‘reward’ signal.  76  2.2 Materials and Methods  2.2.1 Ethics Statement  This study was conducted with approval of the University of British Columbia Animal Care Committee according to Protocol A09-0278  2.2.2 Cell Culture  GluN1 +/- mice were kindly provided by Drs. Michisuke Yuzaki and Tom Curran (Forrest et al., 1994) and genotyping was performed according to the previously described protocol. Heterozygous mice were mated to obtain GluN1 -/- and littermate GluN1 +/+ wild-type embryos. Hippocampi were dissected from individual 17-18 day embryos and stored overnight at 4oC in Hibernate E (Brain Bits) supplemented with B-27 (Invitrogen or Stemcell Technologies) pending genotyping of brainstem and tail tissue. Hippocampi were dissociated with papain (20 units/mL, 15 min, 37oC). Prior to plating, 5 x105 cells per dish were electronucleoporated using an Amaxa Nucleofector II (program O-005) with plasmids to express eCFP or eYFP from the CAG promoter consisting of the CMV immediate early enhancer and the chicken β-actin promoter (Niwa et al., 1991; Kaech and Banker, 2006) (a kind gift of S. Kaech and G. Banker with permission of J. Miyazaki for the CAG promoter). Nucleofected cells were plated onto poly-L-lysine treated glass coverslips in 60 mm petri dishes, for an effective estimated plating density of 3 x105 cells per dish. Neurons were maintained in Neurobasal medium (Invitrogen) supplemented with B-27 and 25 µg/mL of bovine pancreatic insulin (Sigma-Aldrich) on glass coverslips inverted over a glial feeder layer. In the activity blockade experiments, 7.5 µM MK77  801 (Enzo Life Sciences) NMDA receptor channel blocker was added chronically to culture media starting from DIV 0. MK-801 was reapplied during culture feeding on DIV6 and DIV 12.  2.2.3 Immunocytochemistry  For most experiments, neurons cultured on glass coverslips were fixed for 15 min in prewarmed PBS with 4% paraformaldehyde and 4% sucrose followed by permeabilization with 0.25% Triton X-100 in PBS. For GluN1 staining, coverslips were fixed in 4% paraformaldehyde/sucrose for 2 min followed by -20oC MeOH for 10 min followed by 0.04% Triton X-100 in PBS for 1 min. Fixed neuron cultures were blocked with 10% bovine serum albumin (BSA) in PBS (30 min, 37oC). Coverslips were washed six times for 2 min with PBS following each antibody incubation. The following antibodies were used: ant-synapsin I (rabbit; polyclonal; 1:1000; Millipore, AB1543P), anti-PSD-95 family (IgG2a; clone 6G6-1C9; 1:1000; Thermo Fisher Scientific, MA1-045; recognizes PSD-95, PSD-93, SAP102, and SAP97) anti-VGlut1 (guinea pig; polyclonal; 1:4000; Millipore; AB5905), anti-MAP2 (chicken; polyclonal; 1:10,000; AbCam, ab5392) and anti-GluN1 (mouse IgG; 1:1000; Millipore, 05-432 and IgG2a; clone 54.1; 1:1000; Invitrogen, 32-0500). Secondary antibodies were high cross-adsorbed antibodies mainly generated in goat: Alex-568 anti-rabbit, Alexa 568 anti-IgG2a, Alexa-647 anti-guinea pig, Alexa-568 anti-pan-mouse (1:500; Invitrogen), and AMCA conjugated anti-chicken IgY (donkey IgG; 1:200; Invitrogen).  78  2.2.4 Imaging and Quantitative Fluorescence Analysis  All imaging and analysis was done blind to cell genotype and culture composition. Images were acquired on a Zeiss Axioplan2 microscope with a 63X 1.4 numerical aperture oil objective and Photometrics Sensys cooled CCD camera using MetaVue imaging software (Molecular Devices) and customized filter sets. Individual antibodies were tested with single colour secondary staining to confirm no detectable bleed through between channels AMCA, CFP, YFP. Alexa-568, and Alexa-647. Images in each channel were acquired in grey scale from individual channels using the same exposure time across all cells, and pseudo-colour overlays for presentation were prepared using Adobe Photoshop. Healthy CFP- and YFP- positive cells were randomly selected for imaging and 2-3 dendrites per cell were chosen by fluorescent fill for quantitative analysis. Synaptic marker channels were thresholded by intensity and puncta per dendrite length counted. To define co-localized objects, thresholded puncta from the VGlut1 channel were dilated by 2 pixels and compared for pixel overlap with thresholded puncta from the PSD-95 channel. Statistical analyses were performed with Prism (GraphPad Software).  79  2.3 Results  2.3.1 Hippocampal GluN1 -/- Neurons Exhibit Normal Survival and Synapse Density in Culture To test whether neurons lacking functional NMDA receptors compete less effectively for synapses in a cell culture model system, we cultured hippocampal neurons from GluN1 -/- mice (KO neurons) together with hippocampal neurons from littermate wild-type (WT neurons). To identify neurons in mixed co-culture, WT neurons were nucleofected before plating with an expression vector for YFP and KO neurons with an expression vector for CFP, or vice versa (Fig 17A). Transfection efficiency was on average 23.9. Only neurons expressing YFP or CFP and thus of identified genotype were studied further in the resultant cultures. Cultures of pure WT, pure KO, and co-cultures of 50% WT and 50% KO were generated such that potential effects of YFP or CFP expression or differential nucleofection would not bias results (Fig 17A). Cultures were analyzed at 14 days in vitro (DIV). Cell survival was assessed by counting YFP- and CFP- positive neurons of identified genotype and total number of neurons per field. There was no significant difference according to genotype or culture composition in numbers of surviving YFP and CFP expressing neurons, nor in the fraction of surviving neurons expressing YFP or CFP (Fig 17B; ANOVA p > 0.05). There was also no significant difference in total neuron survival among cultures of different genotype composition (ANOVA p > 0.05). Thus, in these optimized low density serum-free cultures with a glial feeder layer, the absence of GluN1 does not affect hippocampal neuron survival, unlike high density cultures grown with serum (Okabe et al., 1998; Bradley et al., 2006) but like in vivo conditions (Tsien et al., 1996; Iwasato et al., 2000).  80  Immunofluorescence for the pre-synaptic marker synapsin showed a similar density of input synapses onto KO neurons and WT neurons both in pure cultures and in the 50:50 coculture (Fig 17C). Indeed, quantitation performed blind to genotype and culture composition revealed no significant differences in density of synapsin puncta indicating pre-synaptic terminals onto KO neurons or WT neurons in pure cultures or in the 50:50 co-culture (Fig 17D; ANOVA p > 0.1). We next assessed excitatory synapses by immunolabeling for the glutamatergic post-synaptic scaffold PSD-95 family and the excitatory pre-synaptic vesicular glutamate transporter VGlut1. There was no significant difference according to genotype or culture composition in the density of PSD-95 puncta (ANOVA p > 0.1) or of PSD-95 puncta colocalized with VGlut1 marking excitatory synapses (Fig 17D; ANOVA p > 0.1). Thus, hippocampal GluN1 -/- neurons develop a steady-state synapse density equivalent to wild-type neurons when cultured alone or when forced to compete in a 50:50 mixed co-culture with wildtype neurons.  2.3.2 Synaptic Competition Occurs in Mixed Wild Type and GluN1 -/- Co-culture of Defined Ratio We next increased potential competitive pressure for synaptogenesis, reasoning that KO neurons grown in a minority with WT, or WT neurons grown in a minority with KO, may reveal differential abilities to develop or maintain synapses. We generated mixed co-cultures of 90% WT with 10% KO and 90% KO with 10% WT, in comparison with pure WT and pure KO cultures. In one experiment, WT neurons were labelled with YFP and KO neurons were labelled with CFP (Fig 18A). In a complementary experiment performed at the same time, labels were reversed (WT CFP and KO YFP) to ensure no bias due to YFP or CFP expression or differential  81  Figure 17: Hippocampal GluN1 -/neurons develop normal synapse density in an equal co-culture with wild-type neurons. A) Hippocampal neurons from littermate wild-type or GluN1 -/- mice were labelled by nucleofection and grown in co-cultures as indicated. B) There was no significant difference in cell survival at 14 DIV according to genotype or culture composition. Values indicate number and percent of cells per microscope field labelled with YFP or CFP representing the indicated genotype; the 50% WT and 50% KO data come from the same coverslips of YFP WT mixed with CFP KO and of CFP WT mixed with YFP KO; ANOVA p > 0.05; n = 60 from two independent experiments. C) At 14 DIV, neurons were fixed and immunolabeled for synaptic markers. Neurons of defined genotype were identified by the YFP or CFP dendrite fill. Synapse density appeared similar regardless of genotype and culture composition. D) There was no significant difference according to genotype or culture composition in synapsin puncta marking total synapse density or in co-localized PSD-95 and VGlut1 puncta marking excitatory synapse density; the 50% WT and 50% KO data come from the same coverslips of YFP WT mixed with CFP KO and of CFP WT mixed with YFP KO; ANOVA p > 0.1; n ≥ 50 from 3 independent experiments. All data are presented as mean ± SEM.  82  nucleofection. GluN1 immunofluorescence was used to verify the genotype of YFP and CFP expressing neurons (Fig 18B). Mixed genotype cultures of unequal composition were immunolabeled at 14 DIV for the glutamatergic post-synaptic scaffold PSD-95 family and the excitatory pre-synaptic vesicular glutamate transporter VGlut1 (Fig 19A). Quantitation performed blind to genotype and culture composition revealed a significant difference among genotypes in the co-cultures in density of PSD-95 puncta, VGlut1 puncta, and PSD-95 co-localized with VGlut1 marking excitatory synapses (Fig 19B; ANOVA p = 0.0004 for PSD-95, p < 0.0001 for VGlut1, and p < 0.0001 for PSD-95 co-localized with VGlut1; n ≥ 45 cells for each condition from 3 independent experiments). WT neurons in the 90% KO and 10% WT co-culture showed a 25% increase in PSD-95 puncta density, a 26% increase in VGlut1 puncta density, and a 22% increase in PSD95/VGlut1 co-localized puncta density compared with KO neurons on the same coverslips (p < 0.01 for PSD-95, p < 0.001 for VGlut1, and p < 0.01 for co-localized PSD-95/VGlut1 by 0.01 for PSD-95, p < 0.001 for VGlut1, and p < 0.01 for co-localized PSD-95/VGlut1 by Bonferroni’s post hoc multiple comparison test). WT neurons in the 90% KO and 10% WT co-culture showed a 28% increase in PSD-95 puncta density, a 41% increase in VGlut1 co-localized puncta density, and a 36% increase in PSD-95/VGlut1 co-localized puncta density compared with sister pure WT cultures (p < 0.001 for PSD-95, p < 0.001 for VGlut1, and p < 0.01 for co-localized PSD95/VGlut1 by Bonferroni’s post hoc multiple comparison test). Furthermore, in each of the 3 culture condition always showed the highest value among the 6 conditions for density of PSD95, VGlut1, and co-localized PSD-95/VGlut1 puncta (ANOVA p = 0.016, p = 0.055, and p = 0.013 for individual experiments for co-localized PSD-95/VGlut1). In the combined data analyzed by full pairwise Bonferroni’s multiple comparison test, the WT neurons in the 90% KO 83  Figure 18: Paradigm for generating synaptic competition with unequal co-culture of GluN1 -/- and wild-type neurons. A) Hippocampal neurons from littermate wild-type or GluN1 -/- mice were labeled by nucleofection and grown in co-culture. This diagram represents one form of the experiment; in the other form, the wild-type neurons were labeled with CFP and GluN1 -/- neurons labeled with YFP and co-culture in similar proportions to ensure no bias due to the nucleofection label. B) Sample co-culture fields were immunoabeled for GluN1 and the dendrite marker MAP2. In co-cultures generated as in panel A), all YFP neurons were confirmed immunopositive for GluN1, and CFP neurons confirmed immunonegative for GluN1, with the genotype of untransfected neurons overall as expected for the culture composition.  84  Figure 19: Synaptic competition: wild-type neurons develop increased synapse density only when in a minority with predominantly GluN1 -/- neighbours A) Hippocampal neurons from littermate wild-type or GluN1 -/- mice were labelled by nucleofection and grown in co-culture as in Figure 24. At 14 DIV, neurons were fixed and immunolabeled for glutamatergic post-synaptic marker PSD-95 and pre-synaptic marker VGlut1. Neurons of defined genotype were identified by the YFP or CFP dendrite fill. B) There was a significant difference among co-cultures in density of PSD-95 puncta (ANOVA p = 0.0004), VGlut1 puncta (ANOVA p < 0.0001), and co-localized PSD-95 and VGLut1 puncta marking excitatory synapses A(ANOVA p < 0.0001); n ≥ 45 from 3 independent experiments. Post hoc Bonferroni’s test comparing the 10% WT condition to neighbour 90% KO neurons and sister 100% WT neurons showed significant differences for PSD-95, VGlut1, and PSD-95/VGlut1 colocalized puncta (*p < 0.01, ** p < 0.001). Complete pairwise post hoc Bonferroni’s multiple comparison test revealed additional significant differences of the 10% WT condition to other conditions but no other significant differences. Data are presented as mean ± SEM (top) and cumulative frequency histograms (bottom). 85  and 10% WT mix differed significantly from multiple other conditions, and no condition other than the WT neurons in the 90% KO and 10% WT mix showed any significant difference from any other condition. More specifically, the density of PSD-95, VGlut1, and PSD-95/VGLut1 colocalized puncta on KO neurons in the 90% WT and 10% KO co-culture did not differ significantly from WT neurons on the same coverslips or from sister pure KO cultures. This, in all 90:10 mixed genotype co-culture combinations, only the minority WT neurons exhibited a consistent difference in steady state excitatory synapse density, out-competing their majority GluN1 -/- neighbours.  2.3.3 Synaptic Competition in GluN1 -/- and Wild Type Co-culture Requires NMDA Receptor Activity The enhanced innervations onto WT neurons in the 90% KO and 10% WT co-culture could result from two general mechanisms. One possibility is that activity and ion flux through functional NMDA receptor channels confers and advantage to the WT neurons compared with KO neighbours. The other possibility is that the physical presence of GluN1 and associated GluN2 (Fukaya et al., 2003) at synapses is sufficient to confer an advantage to WT neurons, for example, by stabilizing post-synaptic adhesion molecules through molecular interactions (Dalva et al., 2000). To differentiate between these possibilities, we repeated the 90% KO and 10% WT co-culture in the chronic presence of the NMDA receptor channel blocker MK-801. Chronic blockade of NMDA receptor channel activity abolished the effect of genotype of synapse density in 90:10 mixed genotype co-cultures (compare Fig 21 with Fig 19B). In the 90:10 mixed genotype co-cultures treated chronically with MK-801, there was no significant difference among genotypes or culture composition in the density of PSD-95 puncta, VGlut1 puncta, or  86  PSD-95/VGlut1 co-localized puncta (Fig 20; ANOVA p > 0.1). Thus NMDA receptor channel activity is required for enhanced synapse density onto minority WT neurons compared to their majority GluN1 -/- neighbours.  Figure 20: Synaptic competition in wild-type and GluN1 -/- neuron co-culture is dependent on NMDA receptor activity. Hippocampal neurons from littermate wild-type or GluN1 -/- mice were labelled by nucleofection and grown in co-culture as in Figure 2, except that the NMDA receptor antagonist was added chronically from 0 DIV. At 14 DIV, neurons were fixed and analyzed as in figure 25. There was no significant difference among co-culture conditions in density of PSD-95 puncta, VGlut1 puncta, and co-localized PSD-95 and VGlut1 puncta marking excitatory synapses; ANOVA p > 0.1; n = 30 from 2 independent experiments (except n = 15 for 100% WT). Data are presented as mean ± SEM.  2.4 Discussion  We present here a cell culture paradigm of NMDA receptor-dependent synaptic competition. Wild-type hippocampal neurons when cultured together with predominantly GluN1 -/- neighbours developed increased synapse density compared with their GluN1 -/- neighbours and compared with sister wild-type neurons cultured alone. The physical presence of GluN1 was  87  not sufficient to mediate this form of synaptic competition, NMDA receptor activity was required. The development of genotype-specific differences in synapse density was dependent on a defined ratio of genotypes in the circuit, occurring in a 10:90 WT:KO co-culture but not in a 50:50 WT:KO co-culture or in a 90:10 WT:KO co-culture. This simple model system may prove useful for defining rules and understanding mechanisms of NMDA receptor mediated synaptic competition. Multiple mechanisms may contribute to the NMDA receptor-dependent synaptic competition observed here. One contributing factor may be altered network activity in cultures with predominantly NMDA receptor-deficient neurons compared with predominantly wild-type neurons (Fig 21A). These hippocampal neuron cultures grown in serum-free media with a separated glial feeder layer show irregular patterns of spontaneous activity (Verderio et al., 1999), unlike the highly synchronized spontaneous oscillatory activity of neurons grown in serum on glia (Murphy et al., 1992; Verderio et al., 1999) but perhaps more like hippocampal CA1 neurons in vivo e.g. (Hirase et al., 2001; Epsztein et al., 2010). However, in multiple culture systems and in vivo, genetic or pharmacological ablation of NMDA receptor activity alters network activity. Loss of NMDA receptor function typically reduces the amplitude or transforms the synchronous oscillatory activity into more irregular activity in culture (Murphy et al., 1992; Bacci et al., 1999), perhaps also increasing AMPA receptor-mediated transmission (Ultanir et al., 2007), and reduces co-ordinated place-related firing in CA1 (McHugh et al., 1996) and gamma frequency activity in the striatum (Ohtsuka et al., 2008). Thus, it is likely that the 90% KO and 100% KO cultures here may exhibit different patterns of network activity than the 90% WT and 100% WT cultures. It is possible that the WT neurons respond in an NMDA receptordependent way to the altered network activity in the 90% KO culture, through calcium 88  Figure 21: Potential mechanisms for the observed NMDA receptor-dependent synaptic competition. A) Cultures with a majority of NMDA receptor-deficient neurons (100% KO, and 90% KO with 10% WT) may exhibit altered network firing patterns compared with cultures with a majority of NMDA receptorcompetent neurons (100% WT, or 10% KO with 90% WT). The altered network firing may be specifically transduced in a manner dependent on NMDA receptor function into a retrograde signal that enhances synapse development. Thus, among all the culture conditions, only WT neurons in the 90% KO with 10% WT co-culture are subjected to the altered network firing and capable of transducing it to a retrograde signal to alter synapse development. B) Synapses bearing functional NMDA receptors are proposed to generate a retrograde signal. In pure WT culture and cocultures with a majority of WT neurons (10% KO with 90% WT), if each axon forms numerous synapses with WT neurons, total retrograde signal will be above a proposed threshold. In co-cultures with only a minority of WT neurons (0% KO with 10% WT), the total retrograde signal generated from initial random axon contact with WT neurons may not reach threshold. Additional synapses are selectively formed and/or stabilized into WT neurons until the total retrograde signal to the pre-synaptic neuron reaches threshold. Since GluN1 -/- KO neurons in this mixed culture generate no retrograde signal, synapse development onto them is not changed. In pure GluN1 -/KO cultures, no synapses are ale to generate retrograde signal and thus there is no basis for altering synapse density. Note: Mechanisms A) and B) are not mutually exclusive, nor exhaustive; for example, more complicated scenarios may occur, such as selective synapse development according to both pre-synaptic and post-synaptic genotype. 89  signalling and perhaps differential gene expression, generating a cell surface or secreted retrograde signal that results in enhanced synapse density. Other potential mechanisms independent of changes in network activity but also involving a retrograde signal could contribute to the observed NMDA receptor-dependent synaptic competition (Fig 21B). A retrograde signal generated only by NMDA receptorcompetent synapses could be sensed by each input neuron, and total retrograde signal compared with a target range. Only if the total retrograde signal does not reach a threshold following initial synapse development, then further selective synapse development may occur. In the simplest scenario, axons from each input neuron may randomly develop synapses onto neighbour neurons to a typical density (although there is also a possibility that pre-synaptic genotype may influence partner selection). If all, 90%, or even 50% of the synapses made by the input neuron were onto wild-type neurons, the total retrograde signal would be sufficient to reach threshold. However, in the 10:90 WT:KO co-culture, only 10% of synapses would randomly occur onto wild-type neurons, insufficient for the total retrograde signal to the input neuron to reach threshold. Such neurons lacking sufficient total retrograde signal may then develop additional synapses onto NMDA receptor competent neurons to increase the total retrograde signal to reach threshold. In the pure GluN1 -/- culture, or in the 10:90 WT:KO co-culture grown chronically in NMDA receptor channel blocker, threshold is not reach, but since no synapses are capable of generating this signal there is no drive for increasing synapse density on a specific subset of neurons. There are many candidates for potential secreted or cell surface retrograde signals that could function in such a mechanism. Local NMDA receptor activation might increase production or local secretion of classic messengers such as nitric oxide, endocannabinoids, or retinoic acid (Aoto et al., 2008; Regehr et al., 2009), or of growth factor type signals such as BDNF and FGFs 90  (Snider and Lichtman, 1996; Vicario-Abejon et al., 2002; Terauchi et al., 2010), or glial secretion of TNFα (Stellwagen and Malenka, 2006). Alternatively, local NMDA receptor activation might increase post-synaptic insertion or reduced endocytosis or altered conformation of surface proteins with trans-synaptic signalling capability. Neuroligins, LRRTMs, TrkC, NGLs, EphBs, ephrins, SynCAMS, NCAM, and cadherins are all candidates to convey such a signal (Dalva et al., 2000; Shen and Scheiffele, 2010; Siddiqui and Craig, 2011; Takahashi et al., 2011). If a threshold-type sensor is involved (Fig 21B), two key features – global summation and comparison with a threshold or target range – share commonalities with sensors for forms of homeostatic synaptic plasticity (Turrigiano and Nelson, 2004; Turrigiano, 2008). BDNF and TNFα have been implicated as signals mediating both homeostatic synaptic plasticity (Stellwagen and Malenka, 2006; Jakawich et al., 2010) and experience-dependent synaptic competition in vivo (Cabelli et al., 1995; Kaneko et al., 2008). The cadherin-β-catenin complex is also implicated in homeostatic synaptic plasticity (Okuda et al., 2007), and NMDA receptor activity increased N-cadherin and associated β-catenin levels at the synapse (Bozdagi et al., 2000; Tai et al., 2007). The more recently discovered cell surface synaptic organizing complexes have not been intensively studied in the context of activity-dependent synaptic modification. However, the potent ability of neuroligin or LRRTM binding to neurexins (Siddiqui and Craig, 2011), or of TrkC or NGL-3 binding to PTPRs (Woo et al., 2009; Takahashi et al., 2011), to trigger complex pre-synaptic differentiation suggests they may also generate far reaching signals for integration. Previous hippocampal culture experiments using mixed genotypes or transfection revealed activity-dependent effects on synapse density according to post-synaptic hyperpolarization (by expression of Kir2.1) (Burrone et al., 2002), post-synaptic levels of BDNF (Singh et al., 2006) or CaMKII (Thiagarajan et al., 2002; Pratt et al., 2003), and pre-synaptic  91  levels of synaptophysin (Tarsa and Goda, 2002). At least some of these components may operate in the pathway triggered by the differential presence of functional NMDA receptors. With respect to cell biological mechanisms of the observed synaptic competition, input neurons may selectively increase their rate of synapse formation and/or reduce their rate of synapse elimination, only onto WT targets in the 10:90 WT:KO co-culture. In wild type rat hippocampal cultures, NMDA receptor antagonists reduce rates of both synapse formation and synapse elimination (Okabe et al., 1999). In our competitive paradigm, perhaps reduced synapse elimination, i.e. enhanced stabilization rather than enhanced de novo formation, seems most likely, as in the classic model for studying synaptic competition, the neuromuscular junction (Kasthuri and Lichtman, 2003). However, differential synapse densities of silenced (by tetanus toxin light chain) versus active glutamatergic inputs onto retinal ganglion cells occurred by selective synapse formation rather than selective stabilization (Kerschensteiner et al., 2009). Perhaps it is surprising that GluN1 -/- neurons in 50:50 or 0:90 co-culture with wild-type neurons exhibited normal synapse density with no reduction. These results are consistent with the finding of normal spine density following single cell in utero deletion of GluN1 in cortical culture (Ultanir et al., 2007) or of GluN2B in vivo (Espinosa et al., 2009), and activityindependent reduction in spine and synapse density with GluN1 shRNA in hippocampal slice culture (Alvarez et al., 2007). The effects of NMDA receptor transmission are also complex (Hall et al., 2007; Ultanir et al., 2007; Adesnik et al., 2008), perhaps depending on synapse type, developmental stage, and environment. More generally, NMDA receptor dependent synaptic competition in sensory system is thought to involve selective stabilization of preferred inputs (inputs with greater activity or  92  specific patterns of activity) and destabilization of non-preferred inputs. Our results suggest that the Hebbian ‘reward’ signal mediating selective formation or stabilization of preferred inputs may be distinct from the ‘punishment’ signal mediating selective reduction of non-preferred inputs. In our cell culture model of synaptic competition, wild-type neurons were rewarded but GluN1 -/- neurons were not punished. Reduction of non-preferred synapses may require more demanding in vivo conditions such as limiting glial derived factors or higher neuron density altering the economics of synaptic competition. Cell culture models testing rules and mechanisms of synaptic competition in defined circuits may contribute to a better understanding of the role of activity in sculpting nervous system connectivity during development. Targeted genetic modifications could be further combined with microfluidic chambers in which subsets of neurons can be pharmacologically manipulated (Taylor et al., 2010) to aid in defining signalling pathways and temporal parameters mediating activity-regulated neuronal connectivity.  93  Chapter 3: Glutamate Binding to GluN2B Controls Surface Trafficking of NMDA Receptors  94  3.1 Introduction  N-methyl-D-aspartate (NMDA) receptors are a subtype of ionotropic glutamate receptor present at excitatory synapses in the central nervous system. Comprised of two obligate GluN1 subunits and two GluN2 or more rarely GluN3 subunits, channel opening requires L-glutamate binding to GluN2 and co-agonist glycine or D-serine binding to GluN1 (Mori and Mishina, 1995; Traynelis et al., 2010). In neurons, membrane depolarization is also required to clear Mg2+ from the pore to allow ion flux. Thus, NMDA receptors are exquisite coincidence detectors or pre- and post- synaptic activity. The residues in the receptor responsible for the Mg2+ block are also responsible for high permeability to Ca2+ (Burnashev et al., 1992) which triggers signal transduction cascades that mediate many forms of synaptic plasticity. NMDA receptors play key roles in brain development and in learning, memory, and cognitive functions (Wang et al., 2006; Lee and Silva, 2009). NMDA receptors are also key pharmacological targets for therapeutic protection during stroke, epilepsy, and traumatic brain injury and for relief of neuropathic pain and multiple psychiatric disorders (Traynelis et al., 2010). Ionotropic glutamate receptors are multimeric protein complexes that require quality control mechanisms to ensure correct assembly before exit from the endoplasmic reticulum (ER). For example, ER retention signals in the cytoplasmic region C1 cassette of GluN1-1a and in the membrane domain M3 of both GluN1 and GluN2 are masked upon correct assembly into tetramers (Hawkins et al., 2004; Yang et al., 2007; Horak et al., 2008). Additional studies suggest that some ionotropic glutamate receptors undergo further quality control inspection for ligand binding prior to release from the ER. Mutations in GluK2 and GluK5 kainate receptors and GluA2 and Glua4 AMPA receptors that abolished agonist binding resulted in retention in the  95  ER despite multimeric assembly (Mah et al., 2005; Valluru et al., 2005; Greger et al., 2006; Coleman et al., 2009). Studies of differentially spliced and edited isoforms of GluA2 and of mutant ligand binding domains of GluA4 and GLuK2 suggest that ligand-induced clamshell closure is required for forward trafficking (Penn et al., 2008; Coleman et al., 2009; Gill et al., 2009). Altogether, much evidence supports the idea that intracellular glutamate binding is required for surface delivery of AMPA and kainate receptors (Fleck, 2006; Coleman et al., 2010). Compared with AMPA and kainate receptors, little is known about the role of glutamate binding in surface delivery of NMDA receptors. A recent study of a co-agonist-binding mutant suggested that glycine binding to GluN1 is essential for cell surface delivery of NMDA receptors (Kenny et al., 2009). Here, we test the role of glutamate binding to GluN2 in cell surface delivery of NMDA receptors in heterologous cells, in cultured rat neurons, and upon rescue in GluN2B -/- mouse neurons. Further, since it can be difficult to differentiate between a specific role for loss of ligand binding or more non-specific conformational effects of mutants, we assessed a panel of GluN2B ligand binding mutants differing in glutamate efficacy. Our results support a critical role for glutamate binding in forward trafficking of NMDA receptors.  3.2 Materials and Methods  3.2.1 DNA Constructs  The dual promoter expression vector was derived from pVIVO2 (InvivoGen); the SV40 enhancer region upstream of the heavy chain human ferritin composite promoter was replaced 96  with a CMV enhancer from ECFP-N1 (Clontech). Enhanced CFP (Clontech) with 3x nuclear localization signals (DPKKKRKV) generated by PCR was inserted into the downstream multiple cloning site (MCS) 1. The control vector is this modified backbone with an empty MCS 2 (pV2.5-). Wild-type YFP-GluN2B (YFP-GluN2B) was developed from rat GluN2B ((Kim et al., 2005); a gift of M. Sheng, Genentech) by inserting enhanced YFP (Clontech) followed by the PCR generated linker LVPRGSRSR between amino acids 2 and 4 of the mature N-terminus. This coding region was inserted into MCS 2 driven by the light chain human ferritin promoter and the sequence of the coding region was confirmed. Single amino acid glutamate binding deficient mutants R493K, E387A, S664G, F390S, and V660A as well as K462C/N662C (KNCC) were generated by site-directed mutagenesis or overlap PCR and the sequence of the coding region and mutations were confirmed. Wild-type myc-GluN2B (myc-GluN2B-WT) was generated by replacing te YFP region of YFP-GluN2B by PCR. GW-GluN1-1a expresses rat GluN1-1a cDNA from the CMV promoter in vector GW1 (a gift from M. Sheng). Dominant negative (DN) HA-dynamin-K44A and wild-type (WT) HA-dynamin (Ochoa et al., 2000) expression vectors were gifts from Pietro De Camilli (Yale University).  3.2.2 Cell Culture  Dissociated primary rat hippocampal neurons cultures were prepared from embryonic day 18 rats as described (Kaech and Banker, 2006). GluN2B +/- mice (Kutsuwada et al., 1996) were maintained and genotypes as described (Liu et al., 2007); these heterozygous mice were timedmated to obtain GluN2B -/- embryos. Hippocampi or cortices were dissected from individual 1718 day embryos and stored overnight at 4oC in Hibernate E (Brain Bits) supplemented with B-27  97  (Invitrogen) pending parallel genotyping of brainstem and tail tissue. Mouse hippocampi were dissociated with papain (20 units/mL0, 15 min, 37oC) and the dissociation solution for cortices was supplemented with 85 units/mL deoxyribonuclease I (Invitrogen). 5 x105 freshly dissociated neurons were pelleted at 80 x g for 4 min for nucleofection using an Amaxa Nucleofector II (Lonza) (program O-003 and program O-005 for rat and mouse neurons, respectively) with 6 µg of plasmid DNA. Nucleofected cells were plated onto poly-L-lysine treated glass coverslips in 60 mm dishes for an effective estimated plating density of 3 x105 cells per dish and inverted over a feeder layer of rat glia. Rat neurons were maintained in Neurobasal medium (Invitrogen) supplemented with B-27 and L-glutamine (Gibco) and mouse neurons were additionally supplemented with 25 µg/mL bovine pancreatic insulin (Sigma-Aldrich). After 2 days in vitro (DIV), proliferation of glia was suppressed by addition of 5 µM cytosine arabinoside (Calbiochem). Neurons were analyzed at DIV6 and DIV14. COS-7 cells (ATCC CRL-1651) were maintained in DMEM (Sigma-Aldrich) with 10% foetal bovine serum (Gibco). 2.2 x104 or 3.5 x104 trypsin-dissociated cells were plated on 18 mm glass coverslips and transfected immediately with TransIT-LT1 transfection reagent (Mirus) according to manufacturer’s protocol. Cells were co-transfected with 0.5 µg each of GW-GluN11a and the YFP-GluN2B construct with 3 µL LT1; 0.5 µg each of GW-GluN1-1a, YFP-GluN2B construct, and WT or DN HA-dynamin with 4.5 µL LT1; and 0.5 µg each of GW-GluN1-1a, YFP-GluN2B construct, and vector control or myc-GluN2B with 4.5 µL LT1. All plasmids were pre-mixed prior to addition to the transfection reagent. Cells were grown without (control) or with 11.25 µM MK-801 ((5R,10S)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten5,10-imine hydrogen maleate) (Enzo Life Science) and 150 µM APV ((2R)-amino-5phosphovaleric acid) as indicated. Cells were analyzed at 48 hours after transfection. 98  3.2.3 Live Antibody Labelling and Immunocytochemistry  Coverslips were incubated with anti-GFP antibody (rabbit polyclonal; 1:500; Invitrogen A11122) in conditioned media for 30 min at 37oC (neurons) or in extracellular solution (ECS) containing 168 mM NaCl, 2.6 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM D-Glucose, and 10 mM HEPES, pH 7.2 for 30 min at 4oC (COS-7 cells). Coverslips were washed 6x 2 min in 37oC culture media (neurons) or room temperature ECS (COS-7 cells) prior to fixation for 15 min in pre-warmed PBS with 4% paraformaldehyde and 4% sucrose pH 7.4, followed by permeabilization with 0.25% Triton X-100 in PBS. Neuronal cultures for immune-detection of GluN1 were fixed and permeabilized by 2 min incubation in pre-warmed PBS with 4% paraformaldehyde and 4% sucrose pH 7.4, then 10 min in -20oC 100% methanol, and rehydrated in PBS for 30 sec. Fixed and permeabilized cultures were blocked in PBS with 8% bovine serum albumin (BSA) and 1.2% normal goat serum (NGS) for 30 min at 37oC prior to incubation with primary antibodies in PBS with 3% BSA and 0.3% NGS for 2 hours at 37oC and secondary antibodies for 45 min at 37oC. Coverslips were washed 6x 2 min with PBS following each antibody incubation. For experiments with DAPI (4’,6-diamido-2-phnylindole) nuclear staining, coverslips were incubated with 300 nM DAPI in PBS for 10 min at room temperature and washed in parallel with the secondary antibody wash. Coverslips were mounted in Elvanol (TrisHCl, glycerol, polyvinyl alcohol, and 2% 1,4-diazabicyclo [2,2,2] octane). The following antibodies were used: anti-GluN2B (IgG2b; 1:200; UC David/NIH NeuroMab Facility N59/36), anti-GluN1 (IgG2a; 1:500 (neurons) 1:10,000 (COS-7 cells); Thermo Fisher Scientific clone 6G6-1C9; recognizes PSD-95, PSD-93, SAP102, and SAP97), anti-VGlut1 (guinea pig polyclonal; 1:4000; Millipore AB5905), anti-MAP2 (chicken IgY  99  polyclonal; 1:10,000; AbCam ab5392), anti-myc (IgG1; 1:500; Millipore clone 9E10), and antiHA (rat IgG1; 1:1000; Roche clone 3F10). Secondary antibodies were mainly generated in goat and were high cross-adsorbed: Alexa-647 or Alexa-568 conjugated anti-rabbit, anti-mouse-IgG, IgG1, -IgG2a, -IgG2b (1:500; Invitrogren), AMCA conjugated anti-guinea pig (1:200; Invitrogen), AMCA conjugated anti-chicken IgY (1:200; Invitrogen), and AMCA conjugated anti-rat IgG (1:200; Leinco).  3.2.4 Transferrin Uptake Assay and Induction of Surface Receptor Endocytosis  COS-7 cells on glass coverslips transfected with TransIT-LT1 with 0.5 µg of DNA HAdynamin-K44A or WT HA-dynamin for 48 hours were serum starved for 1 hour at 37oC and incubated with 0.3 µM Alexa-568 conjugated human transferrin (Invitrogen) in conditioned media for 20 min at 37oC. Coverslips were washed 3x with cold PBS and acid stripped in 0.5M NaCl and 0.2M acetic acid for 4 min on ice and re-washed with cold PBS prior to fixation, permeabilization, and antibody staining for HA. Induction of surface receptor endocytosis was performed on COS-7 cells 48 hours after transfection with 0.5 µg each of GW-GluN1-1a, YFP-GluN2B-WT or YFP-GluN2B E387A, and DN- or WT- dynamin. Coverslips were first incubated with anti-GFP antibody (rabbit polyclonal; 1:500; Invitrogen A11122) in conditioned media for 30 min at 37oC and returned to their original growing media. The concentration of APV was increased to 300 µM and kynurenic acid was added to 5 µM for 1 hour. Coverslips were washed 3x with cold PBS and acid stripped in 0.5M NaCl and 0.2M acetic acid for 4 min on ice and re-washed with cold PBS prior to  100  fixation, permeabilization, blocking, and antibody staining for HA and GluN1 and detection of anti-GFP antibody.  3.2.5 Imaging and Data Analysis  All imaging and analysis was done blind to culture conditions and transfected GluN2B and/or dynamin construct. When possible, cells were confirmed to be co-expressing GluN1, HAdynamin or myc-GluN2B. Images were acquired on a Zeiss Axioplan2 microscope with a 63X 1.4 numerical aperture (Figs. 29-35) or 25X 0.8 numerical aperture (Fig 22) oil objective and Photometrics Sensys cooled CCD camera using MetaVue imaging software (Molecular Devices) and customized filter sets. Images in each individual channel were acquired in grey scale using the same exposure time across all cells, and pseudo-colour overlays for presentation were prepared using Adobe Photoshop. The frequency of live transfected cells was performed by counting random 25X optical fields for DAPI stained and CFP positive nuclei. For surface/total YFp-GluN2B ratio, the surface anti-YFP immunofluorescence integrated intensity and YFP integrated intensity were measured for the entire COS-7 cell area and the ratio calculated after subtracting the average integrated intensity from non-transfected cells for each channel. In DIV6 neurons, a mask of the entire somatodendritic domain within the image was generated by dilating the MAP2 fluorescence signal by 4 pixels prior to measuring the integrated intensity of the YFP and surface YFP fluorescent signals. Quantitation of the synaptic puncta of surface receptor, detected by live surface labelling, was determined by applying an intensity threshold to the surface YFP channel ad counting the number of puncta that exhibited any pixel overlap with the  101  thresholded PSD-95 channel. Statistical analyses were performed with Prism (GraphPad software).  3.3 Results  3.3.1 Mutagenesis of the GluN2B Glutamate Binding Pocket Protects Against Toxicity of Expressed NMDA Receptors To assess the role of glutamate binding to GluN2 in surface trafficking of NMDA receptors, we studied a series of point mutants in rat GluN2B with reduced glutamate efficacy. The choice of mutants was based on previous work from (Laube et al., 1997) reporting the concentration of glutamate for half-maximal activation (EC50) of a series of GluN2B mutants coexpressed with GluN1 in oocytes. From these potential mutants, we chose a series exhibiting a range of glutamate efficacies that mapped well (Fig 22A) to the more recent crystallographic data for the glutamate binding pocket of GluN2A (Furukawa et al., 2005) and thus likely reflect differences in glutamate affinity. Additionally, the selected point mutant amino acid residues are all conserved across GluN2A, GluN2B, GluN2C, and GluN2D (Fig 22B). GluN2B mutants V660A, F390S, S664G, and E387A show reductions in apparent glutamate affinity of 18-fold to 237-fold compared with wild-type GluN2B, while mutant R493K was the most severe giving no detectable glutamate response (Laube et al., 1997) (numbers correspond to the mature polypeptide after signal sequence cleavage. We generated the series of GluN2B mutants with an N-terminal YFP tag to facilitate measures of surface trafficking in a dual promoter mammalian expression vector co-expressing nuclear-targeted CFP (nCFP) to visualize transfected cells.  102  Figure 22: Design of glutamate binding deficient GluN2B mutants and protection against NMDA receptor mediated toxicity in heterologous cells A) Amino acid backbone structure (left) and the ligand interacting amino acid side chains (right) of GluN2A S1S2 dimer with bound glutamate. PDB ID: 2A5T (Furukawa et al., 2005). The positions of the mutants used in this study are indicated by circles (left) and elements interacting directly with the ligand are indicated in red (right). B) Amino acid alignment of rat GluN2A, GluN2B, GluN2C, and GluN2D in regions that comprise part of the glutamate binding pocket. Amino acids conserved between all four subunits are highlighted in yellow and the amino acids which were mutated for this study are highlighted in blue. Amino acid number is based on GluN2B, in bold. C) COS-7 cells transfected with GluN1-1a and a dual promoter vector coexpressing nuclear CFP (nCFP) with either YFP-GluN2B-wildtype or YFP-GluN2B-R493K. Cells were grown without (no inhibitor) or with inhibitors MK-801 and APV for 48 hours then fixed and stained with DAPI. D) In COS-7 cells transfected with the dual promoter vector, nCFP and YFP-GluN2B were always co-detected and nCFP was generally brighter. Thus, survival was assessed as the frequency of DAPI positive COS-7 cells expressing nCFP when grown without (white bar) or with inhibitors (black bar). All YFP-GluN2B mutants results in significantly less toxicity compared with WT under control (no inhibitor) conditions but there was no difference in cultures grown in MK-801/APV (control conditions ANOVA p < 0.0001 and post-hoc Bonferroni multiple comparison test compared with WT all p < 0.001; MK-801/APV conditions ANOVA p > 0.05; n = 20-30 fields for each condition from two independent experiments). In direct comparison of control with MK-801/APV conditions for each construct, only the WT and V660A mutant showed a significant different (* t-test p < 0.0001). Scale bars, 100 µm.  103  Activation of expressed NMDA receptors by free L-glutamate and glycine in normal cell culture media has been shown to be toxic to transfected heterologous cell lines (Cik et al., 1993) primarily through the unregulated influx of calcium. We used this characteristic to functionally test the series of glutamate binding GluN2B mutants upon co-expression with GluN1-1a in COS7 cells. Cells were transfected with GluN1-1a and nCFP vector bearing each of the YFP-GluN2B variants and cell survival 48 hours later was assessed as the frequency of DAPI positive cells expressing nCFP. In the absence of NMDA receptor inhibitors, COS-7 cell cultures cotransfected with GluN1-1a and nCFP vector bearing wild-type GluN2B (YFP-GluN2B-WT) had a dramatically decreased number of surviving transfected cells compared to control cultures cotransfected for GluN1-1a and nCFP vector alone (Fig 22C). Survival of YFP-GluN2B-WT transfected cells was rescued to levels comparable to vector alone by chronic administration of NMDA receptor inhibitors MK-801 and APV (MK-801/APV). In contrast to YFP-GluN2B-WT, even in the absence of NMDA receptor inhibitors, survival of cells transfected with GluN1-1a and nCFP vector bearing YFP-GluN2B R493K, E387A, S664G, and F390S mutants was not significantly different than that of control cells (Fig 22D). Survival of cells expressing the YFPGluN2B V660A mutant with the highest glutamate efficacy was reduced by 21% relative to control, and this toxicity was rescued by MK-801/APV. Comparing all GluN2B constructs under control conditions, cell survival was significantly different (ANOVA p < 0.0001), and all YFPGluN2B mutants resulted in significantly enhanced survival compared to WT (post-hoc Bonferroni’s multiple comparison test p < 0.001). In contrast, when cells were grown with MK801/APV, cell survival did not differ significantly among GluN2B constructs. Cell survival comparing control and MK-801APV conditions was significantly different only for the YFPGluN2B-WT and YFP-GluN2B-V660A constructs (t-test p < 0.0001). These protective effects  104  against excitotoxicity in a heterologous cell line support the functional glutamate binding defects in this series of GluN2B mutants, indicating glutamate affinities below or at the threshold for spontaneous excitotoxicity in culture media.  3.3.2 Glutamate Binding Regulates Cell Surface Delivery of NMDA Receptors in Heterologous Cells To determine the effect of reduced glutamate binding on the surface expression, wildtype or mutant YFP-GluN2B constructs were co-expressed with GluN1-1a in COS-7 cells and grown chronically with NMDA receptor inhibitors to prevent glutamate-induced excitotoxicity. After 48 hours, transfected COS-7 cells were incubated live with membrane impermeant antiGFP antibody cross-reactive with the N-terminal YFP tag. After washing, cells were fixed, permeabilized, and stained for GluN1 (Fig 23A) to confirm co-expression, and the ratio of labelled surface receptor integrated immunofluorescence over the entire cell area was calculated to estimate what fraction of total GluN2B is present on the cell surface. All of the mutants showed significantly decreased levels of surface expression compared to the wild-type GluN2B (Fig 23B; ANOVA p < 0.0001 and Bonferroni’s multiple comparison test p < 0.001). Furthermore, the relative level of surface expression of the mutant GluN2B constructs correlated well with the reported glutamate efficacy (Laube et al., 1997) (Fig 23C; Pearson’s coefficient of determination r2 = 0.95 p = 0.0044 for WT, V660A, F390S, S664G, and E87A; R493K was excluded because it had no measurable glutamate response). There was no difference in total YFP intensity per COS-7 cell area among the YFP-GluN2B constructs, indicating no difference in overall expression (ANOVA p > 0.1, n = 27 per construct from two independent experiments). This data suggest that ligand binding is necessary for NMDA receptors to accumulate at steady-  105  Figure 23: Glutamate binding to GluN2B regulated surface levels of NMDA receptors in heterologous cells A) COS-7 cells co-transfected with untagged GluN1-1a and YFP-GluN2B-WT or the indicated glutamate binding mutant were surface immunolabeled live for YFP-GluN2B then fixed, permeabilized, and immunolabeled to confirm co-expression of GluN1. B) Quantitation of surface YFP-GluN2B tot total YFP-GluN2B ratio normalized to WT revealed surface levels of the mutants (ANOVA p < 0.0001 and Bonferroni’s multiple comparison test compared to WT * P < 0.001; n = 27-60 per construct from at least two independent experiments). C) Correlation of relative surface levels from B with the log of the reported half maximal effective concentration of each GluN2B construct for glutamate (Laube et al., 1997) shows a correlation between glutamate efficacy of GluN2B and surface levels of the expressed NMDA receptor (Pearson’s coefficient of determination r2 = 0.95 p = 0.0044 excluding R493K which has no detectable efficacy). Scale bar, 20 µm.  106  state on the cell surface of heterologous cells, with apparent glutamate affinity directly regulating surface expression. Surface expression of NMDA receptors is subject to a number of regulatory mechanisms including ER retention limiting forward trafficking and dynamin-dependent endocytosis via the AP-2 clathrin adaptor protein binding site present in the cytoplasmic tail of GluN2B (Prybylowski et al., 2005; Lau and Zukin, 2007). To separate the relative contributions of forward trafficking from endocytosis, COS-7 cells were co-transfected with either dominant negative (DN) HA-dynamin K44A to inhibit clathrin-dependent endocytosis (Herskovits et al., 1993) or WT HA-dynamin along with the GluN2B and GluN1-1a constructs (Fig 24A). Surprisingly, there were no differences in the ratio of cell surface to total NMDA receptors between cells co-expressing DN dynamin or WT dynamin (Fig 24B), and results were similar to cells without dynamin co-expression (Fig 23B). Relative surface levels were not significantly different for any of the YFP-GluN2B mutants with DN dynamin co-expression compared with WT dynamin co-expression (Fig 24B; t-test p > 0.1). Even in the presence of DN dynamin to inhibit endocytosis, all of the mutant GluN2B constructs exhibited reduced surface levels compared to WT (Fig 24B; ANOVA p < 0.0001 and Bonferroni’s multiple comparison test p < 0.001 for R493K, E387A, and S664G, p < 0.01 for F390S, and p < 0.05 for V660A). Inhibition of transferrin uptake after serum starvation was used to confirm the efficacy of the DN dynamin in inhibiting endocytosis (Fig 24C) (Herskovits et al., 1993). To directly confirm the efficacy of DN dynamin in impairing receptor endocytosis COS-7 cells were co-transfected with GluN1-1a, YFP-GluN2B-WT, and either WT- or DN- dynamin. 48 hours after transfection, cells were surface labelled live with anti-YFP antibody for 30 min and returned to their original culture media whereupon ion flux independent receptor stimulation was used to induce AP-2 dependent 107  Figure 24: Reduced surface levels of glutamate binding deficient GluN2B mutants are due to reduced forward trafficking A) COS-7 cells were co-transfected with dominant negative (DN) HA-dynamin-K44A to inhibit endocytosis and with GluN1-1a and YFP-GluN2B-WT or the indicated glutamate binding mutants. Cells were immunolabeled live for surface YFP-GluN2B then fixed, permeabilized, and immunolabeled to confirm co-expression of DN HA-dynamin-K44A and GluN1. B) Quantitation of surface YFP-GluN2B to total YFP-GluN2B ratio normalized to WT revealed reduced surface 108  levels of the mutants, with similar results upon co-expression of WT HA-dynamin or DN HAdynamin-K44A. Comparison of WT dynamin with DN dynamin conditions for each GluN2B construct revealed no significant differences (t-test p > 0.1; n = 24 per construct from two independent experiments). With co-expression of DN dynamin, all GluN2B mutant surface levels were reduced compared to WT (ANOVA P < 0.0001 and Bonferroni’s multiple comparison test compared with WT p < 0.001 for R493K, E387A, and S664G, p < 0.01 for F390S and p < 0.05 for V660A). C) Efficacy of DN HA-dynamin-K44A but not WT HAdynamin to inhibit endocytosis of Alexa-568 conjugated transferrin by serum-starved cells. Cells were immunolabeled with HA to confirm the expression of DN or WT dynamin. D) Efficacy of DN dynamin but not WT dynamin or HA-dynamin negative to inhibit AP-2 dependent competitive antagonist induced receptor endocytosis. Cells were immunolabeled to confirm the co-expression of DN- or WT- HA-dynamin and GluN1. Scale bars, 20 µm.  receptor endocytosis (Vissel et al., 2001). For COS-7 cells co-transfected with GluN1-1a and YFP-GluN2B-WT, receptor endocytosis can be induced in the presence of MK-801 by the dual presence of competitive glutamate binding site antagonist APV and competitive glycine binding site antagonist kynurenic acid (KyA). Indeed, one hour after raising APV concentration to 300 µM and adding 5 µM of KyA in the presence of MK-801, after acid-washing antibody from the cell surface, COS-7 cells co-transfected with GluN1-1a, YFP-GluN2B-WT, and WT dynamin showed significant amounts of internalized receptor as were YFP- and GluN1-1a positive cells not expressing either WT- or DN- dynamin. However, in COS-7 cells co-expressing GluN1-1a, YFP-GluN2B-WT, and DN dynamin, receptor endocytosis was successfully blocked (Fig 24D). These data suggest that a difference in forward trafficking to the cell surface is the primary contributor to differences in cell surface levels of the GluN2B mutants, implying that impaired glutamate binding reduces forward trafficking.  109  3.3.3 Co-expression of Wild Type GluN2B Cannot Rescue Surface Trafficking Deficits of GluN2B Glutamate Binding Mutants Since NMDA receptors are tetramers comprised of two GluN1 and two GluN2 subunits, we co-expressed WT myc-GluN2B with each of the YFP-GluN2B mutants and GluN1-1a to attempt to rescue surface trafficking. In this situation, some of the YFP-GluN2B mutant subunits may be in GluN1/YFP-GluN2B-mutant di-heteromers while some may be in GluN1/YFPGluN2B-mutant/myc-GluN2B-WT tri-heteromers. We wondered whether glutamate binding with normal affinity to one WT subunit in such a tri-heteromer might increase surface levels of associated mutant YFP-GluN2B. Thus, we co-transfected COS-7 cells with GluN1, YFPGluN2B-mutant, and either myc-GluN2B-WT or empty vector for comparison and assayed for surface FP staining relative to total YFP signal in cells immunopositive for myc-GluN2B-WT (Fig 25A). However, co-expression of myc-GluN2B-WT did not rescue the surface expression of the GluN2B mutants; they still exhibited poor surface expression compared to WT (Fig 25A; ANOVA p < 0.0001). Comparison of myc-GluN2B-WT co-expression with the empty vector condition for each YFP-GluN2B construct revealed no significant difference in surface levels (Fig 41B; t-test p > 0.1). These results suggest that occupancy of the glutamate binding sites on both GluN2B subunits is required for proper surface delivery and that the presence of one glutamate binding-deficient subunit is dominant in reducing surface expression.  3.3.4 Glutamate Binding Regulates Surface Expression of NMDA Receptors in Cultured Neurons To assess the role of glutamate binding in surface expression of NMDA receptors in their native cell type, we transfected YFP-GluN2B and the series of mutants with reduced glutamate efficacy into cultured rat hippocampal neurons. Neurons were transfected at plating, grown under 110  Figure 25: Reduced surface levels of glutamate binding deficient GluN2B mutants are not rescued by coexpression of wild-type GluN2B A) COS-7 cells were co-transfected with myc-GluN2B-WT, GluN1-1a, and the indicated YFP-GluN2B constructs. Cells were surface immunolabeled live for YFP then permeabilized and immunolabeled to confirm coexpression of myc-GluN2B. B) Quantitation of surface YFP-GluN2B to total YFP-GluN2B ratio normalized to WT revealed reduced surface levels of the mutants, with similar results upon co-expression of myc-GluN2BWT or the empty vector control (ANOVA p < 0.0001). Comparison of myc-GluN2B-WT co-expression with the empty vector condition for each YFP-GluN2B construct revealed no significant difference (t-test p > 0.1; n = 24 per construct from two independent experiments). Scale bar, 20 µm.  111  normal conditions in the absence of inhibitors, and analyzed at 6 DIV, allowing time for the YFP-GluN2B to associate with endogenous GluN1 and traffic to the cell surface. We chose 6 DIV for initial analysis as a developmental stage when receptors are present on the neuron surface but not yet well clustered at synapses in cultured hippocampal neurons, to assess general mechanisms of surface expression relatively independent of synapse-specific mechanisms regulating trafficking. Transfected neurons were incubated live with membrane impermeant antiGFP antibody to detect surface YFP-GluN2B and then fixed, permeabilized, and immunolabeled for the somatodendritic marker MAP2. In DIV 6 rat hippocampal neurons, YFP-fluorescence corresponding to YFP-GluN2BWT was detected in the soma and dendrites and surface labelled YFP-GluN2B receptors were also detected through the somatodendritic domain (Fig 26A). We observed similar somatodendritic expression patterns of total YFP fluorescence for all YFP-GluN2B glutamatebinding deficient mutants. However, the mutants exhibited varying degrees of diminished surface immunofluorescence relative to the wild-type (Fig 26A shows examples for R493K, S664G, and V660A).Some non-uniformity was seen in the surface label for all receptors, presumably due to surface patching of the diffusible receptors during live cell antibody incubation, as previously reported for GluR1 in immature neurons (Mammen et al., 1997). Thus, for quantitation we measured the ratio of surface YFP-GluN2B to total YFP-GluN2B over the entire MAP2-positive somatodendritic domain per field for transfected cells chosen by the CFP and YFP channels. All mutants analyzed showed significant surface expression deficits compared to the YFP-GluN2B-WT (Fig 26B; ANOVA p < 0.0001 and Bonferroni’s multiple comparison test p < 0.001 for each mutant compared to wild-type). Furthermore, the relative surface expression of these mutants in neurons correlated well with their reported glutamate 112  Figure 26: Glutamate binding of GluN2B regulated surface levels in neurons A) Rat hippocampal neurons were transfected at plating with the indicated YFP-GluN2B construct and labelled live at 6 DIV for surface YFP-GluN2B then fixed, permeabilized, and immunolabeled for the somatodendritic marker MAP2. B) Quantitation of somatodendritic surface YFP-GluN2B to total YFP-GluN2B ratio normalized to WT revealed reduced surface levels of all mutants (ANOVA p < 0.0001 and Bonferroni’s Multiple Comparison Test compared to WT * p < 0.001; n = 45 per construct from three independent experiments). C) The relative surface levels correlated well with the log of the reported EC50 for glutamate (Laube et al., 1997) for the series of YFP-GluN2B constructs (Pearson’s coefficient of determination r2 = 0.93 p = 0.0088 excluding R493K which had no measured response). D, E) Similar experiments in GluN2B -/- mouse cortical neurons revealed similarly reduced surface levels of all mutant YFPGluN2B receptors (ANOVA p < 0.0001 and Bonferroni’s multiple comparison test compared to WT * p < 0.001; n = 30 per construct from two independent experiments) and correlation between glutamate efficacy of GluN2B and surface levels (Pearson’s coefficient of determination r2 = 0.95 p = 0.0056 excluding R493K). Scale bar, 10 µm.  113  efficacies (Fig 26C; Pearson’s coefficient of determination r2 = 0.93 p = 0.0088 for WT, V660A, F390S, S664G, and E387A). Since the presence of endogenous GluN2B in the rat hippocampal neurons may affect the surface expression of exogenously expressed mutant GluN2B either negatively through a competitive mechanism or positively through an associative mechanism, we also investigated the surface expression of our panel of YFP-GluN2B constructs in GluN2B -/- mouse cortical neurons at DIV 6. Similar to the results in rat hippocampal neurons, in cultured mouse cortical neurons lacking native GluN2B, the YFP-GluN2B glutamate binding deficient mutants exhibited reduced surface expression relative to wild-type. The fraction of YFP-GluN2B found on the cell surface was significantly reduced for all the mutants compared to YFP-GluN2B-WT (Fig 26D; ANOVA p < 0.0001 and Bonferroni’s multiple comparison test of each mutant compared to wild-type p < 0.001). Furthermore, consistent with results in rat hippocampal cells and heterologous COS-7 cells, relative surface expression in GluN2B -/- neurons correlated well with the reported glutamate efficacies (Fig 26E; Pearson’s coefficient of determination r2 = 0.95 p = 0.0056 for WT, V660A, F390S, S664G, and E387A).  3.3.5 A Constitutively Closed Cleft GluN2B Mutant is Similar to Wild Type in Surface Expression It has been suggested that reversible gating motions induced by ligand binding are required for GluA2 surface trafficking, that transition through multiple states is essential (Penn et al., 2008). This idea was based in part on the finding that a constitutively closed cleft GluA2 mutant was retained in the ER (Penn et al., 2008). On the other hand, if a simple closed cleft conformation promotes surface trafficking, then one might expect a mutant with a constitutively 114  closed cleft to traffic to the surface to the same extent or more efficiently than wild-type. To test these ideas, we generated YFP-GluN2B K462C/N662C (KNCC). These two residues are close enough to form a disulfide bond between the S1 and S2 lobes, stabilizing the closed cleft conformation, and indeed the homologous GluN2A KNCC mutant when expressed with GluN1 generates a constitutively active receptor with 90% maximal current in the absence of glutamate (Blanke and VanDongen, 2008). Glutamate could still activate the GuN2A KNCC mutant receptor to 100% with an EC50 of 0.76 µM (Blanke and VanDongen, 2008). In the cell surface expression assays in COS-7 cells, the GluN2B KNCC mutant behaved indistinguishably from wild-type GluN2B. Surface expression of YFP-GluN2B KNCC was essentially identical to that of wild-type GluN2B and was not affected by co-expression of either dominant negative dynamin or wild-type myc-GluN2B (Fig 27A, B; ANOVA p > 0.1). Thus, forward trafficking of GluN2B was neither enhanced nor reduced by the KNCC mutation generating a constitutive closed cleft conformation. Furthermore, surface expression of YFP-GluN2B KNCC was also indistinguishable from that of wild-type YFP-GluN2B upon expression in mouse GluN2B -/cortical neurons at 6 DIV (Fig 27C, D; t-test p > 0.1).  3.3.6 Glutamate Binding of GluN2B Regulates Synaptic Expression Upon Rescue in GluN2B -/- Hippocampal Neurons The pool of NMDA receptors in neurons that mediate most forms of synaptic plasticity are those that are localized to synapses. Thus we assessed the role of glutamate binding to GluN2B in trafficking to synapses in mature cultured neurons. We first established conditions to essentially replace the endogenous synaptic GluN2B with recombinant YFP-GluN2B-WT. Hippocampal neurons from GluN2B -/- mice were transfected at plating with YFP-GluN2B  115  Figure 27: A constitutively closed cleft GluN2B mutant is similar to wild-type in surface expression A) COS-7 cells were transfected with GluN1-1a and YFP-GluN2BWT or –KNCC plus either wild-type dynamin or dominant negative (DN) HA-dynamin-K44A and processed as in Fig 24. Quantitation of surface YFP-GluN2B to total YFP-GluN2B ratio normalized to WT revealed no effect of the KNCC mutation on surface expression, with similar results upon co-expression of WT HA-dynamin or DN HA-dynaminK44A (ANOVA p > 0.1; n = 24 per construct from two indepedent experiments). B) COS-7 cells were co-transfected with myc-GluN2B WT, GluN1-1a, and YFP-GluN2BWT or –KNCC and processed as in Fig 25. Quantitation of surface YFPGluN2B to total YFP-GuN2B ratio normalized to WT revealed no effect of the KNCC mutant on surface expression, with similar results upon co-expression of mycGluN2B-WT or the empty vector control (ANOVA p > 0.1; n = 24 per construct two two independent experiments. C) GluN2B -/- mouse cortical neurons were transfected at plating with YFP-GluN2B-WT or –KNCC and processed as in Fig 26. Quantitation of somatodendritic surface YFP-GluN2B to total YFP-GluN2B ratio ormalized to WT revealed no effect of the KNCC mutant on surface expression (t-test p < 0.1; n = 30 per construct from two independent experiments). D) Representative images of GluN2B -/- mouse cortical neurons expressing YFPGluN2B-WT or –KNCC and labeled live at 6 DIV for surface YFP-GluN2B then fixed, permeabilized, and immunolabeled for the somatodendritic marker MAP2. Scale bar, 10 µm.  116  under control of the human ferritin promoter and analyzed at 14 DIV. In this rescue system, the recombinant YFP-GluN2B localized appropriately to synapses apposed to synapsin and colocalized with GluN1 (Fig 28A). Furthermore, we were fortunate to obtain just the right expression level of YFP-GluN2B to rescue anti-GluN2B immunoreactive puncta number and intensity in the GluN2B -/- neurons to levels indistinguishable from that of wild-type sister neurons (Fig 28B; ANOVA p < 0.0001 and Bonferroni’s multiple comparison test p < 0.001 for GluN2B -/- compared to WT, p > 0.05 for GluN2B -/- plus YFP-GluN2B compared to WT). We next assessed synaptic surface accumulation of the glutamate binding deficient GluN2B mutants compared with wild-type in this rescue system. Mouse GluN2B -/hippocampal neurons were transfected with YFP-GluN2B constructs at plating and analyzed at DIV 14. Neurons were immunostained live for surface YFP-GluN2B and then fixed, permeabilized, and immunostained for excitatory post-synaptic scaffolding PSD-95 protein family and excitatory pre-synaptic vesicular glutamate transporter VGlut1. The glutamate binding deficient YFP-GluN2B mutants exhibited variable and reduced surface immunoreactivity compared with YFP-GluN2B-WT, although the surface punctuate immunoreactivity that was detected was generally co-localized with PSD-95 apposed to VGlut1 as for wild-type (Fig 28C). A difference in pattern of total YFP-GluN2B was also noted. Whereas total YFP-GluN2B-WT was mainly punctuate at synapses, total YFP-GluN2B for the mutants was more diffuse along dendrites despite a more punctuate surface signal. This pattern suggests that much of the mutant YFP-GluN2B in dendrites may be intracellular in diffusely distributed organelles such as the ER which in neurons extends throughout dendrites (Huh and Wenthold, 1999).  117  Figure 28: Glutamate binding of GluN2B regulates rescue of synaptic surface GluN2B in GluN2B -/- neurons A) Mouse hippocampal GluN2B -/- neurons were transfected at plating with YFP-GluN2B-WT and immunolabeled at 14 DIV for GluN2B and either synapsin (left) or GluN1 (right). The recombinant YFP-GluN2B localized appropriately opposite synapsin-labeled terminals (arrows) and co-localized with endogenous GluN1. Arrowheads indicate dendrites from a neighbour nontransfected neuron lacking GluN2B. B) Quantitation of GluN2B immunofluorescent puncta confirmed a complete absence in GluN2B -/- cultures and showed rescue to levels indistinguishable from WT neurons in GluN -/- neurons transfected with YFP-GluN2B 118  (ANOVA p < 0.0001 and Bonferroni’s multiple comparison test * p < 0.001 for GluN2B -/compared to WT neurons, p > 0.05 for GluN2B -/- plugs YFP-GluN2B-WT compared to WT neurons n = 12-15). C) Mouse GluN2B -/- hippocampal neurons were transfected at plating with the indicated YFP-GluN2B constructs and analyzed at 14 DIV. Neurons were immunolabeled live for surface YFP-GluN2B then fixed, permeabilized, and immunolabeled for PSD-95 and VGlut1. D) Quantitation of the number of surface YFP-GluN2B puncta co-localized with PSD95 per dendrite length showed reduced synaptic levels for all GluN2B mutants (ANOVA p < 0.0001 and Bonferroni’s multiple comparison test compared to WT * p < 0.001). E) The density of surface synaptic puncta correlated well with the log of the reported EC50 for glutamate for the series of YFP-GluN2B constructs (Pearson’s coefficient of determination r2 = 0.99 p = 0.0003 excluding R493K). F) There was no difference in the density of PSD-95 puncta among groups (ANOVA p > 0.05). Scale bars 5 µm.  Quantitatively, surface synaptic accumulation of all GluN2B glutamate binding mutants was reduced relative to wild-type (Fig 28A; ANOVA p < 0.0001 and Bonferroni’s multiple comparison test compared to WT p < 0.001). Furthermore, the density of surface synaptically localized puncta of YFP-GluN2B correlated with the log of their reported EC50 for glutamate (Fig 28E; Pearson’s coefficient of determination r2 = 0.99 p = 0.0003 excluding R493K). The integrated intensity of surface synaptically localized YFP-GluN2B per dendrite length was similarly reduced for all glutamate binding mutants (ANOVA p < 0.0001) and the reduction correlated well with the log of their reported EC50 (Pearson’s coefficient of determination r2 = 0.82 p = 0.01). As a control for culture conditions, there were no differences in the density of puncta for pSD-95 (Fig 28F) or VGlut1 or in their integrated intensity (data not shown) among rescue groups (ANOVA p > 0.05). These data indicate that accumulation of NMDA receptors at their site of primary function, the post-synaptic surface, requires glutamate binding and further suggests that glutamate affinity can regulate synaptic accumulation, at least over a low affinity range.  119  3.4 Discussion  Here we used a range of GluN2B ligand binding point mutants with reduced glutamate efficacy to assess the role of glutamate binding in surface trafficking of NMDA receptors. In both heterologous cells co-expressing GluN1 and in primary neuron cultures, surface expression of the GluN2B mutants correlated with apparent affinity for glutamate. Similar results were found in the presence of dominant negative dynamin to inhibit endocytosis, suggesting that glutamate binding is required for forward trafficking of NMDA receptors to the cell surface. Coexpressed wild-type GluN2B did not enhance surface expression of mutant GluN2B, suggesting that the ligand binding sites of both GluN2 subunits in a tetramer need to be occupied for surface expression. Further, in a molecular replacement strategy, we expressed YFP-GluN2B and the mutants in cultured neurons from GluN2B -/- mice and found that apparent glutamate affinity controlled surface expression in young neurons and surface synaptic levels in mature neurons. Altogether, these data strengthen the accumulating evidence that glutamate is required for forward trafficking of multiple ionotropic glutamate receptors (Fleck, 2006; Coleman et al., 2010) and indicate the NMDA receptors share this requirement. Prior studies on AMPA and kainate receptors were reported in a more binary fashion, in the sense that severe ligand binding mutants were studied and found to be strongly and similarly impaired in surface expression (e.g. (Mah et al., 2005; Valluru et al., 2005; Greger et al., 2006; Coleman et al., 2009). For example, compared to GluK2 wild-type glutamate EC50 191 µM or GluA4 wild-type EC50 2 mM, mutants impaired in surface trafficking had undetectable response (Mah et al., 2005; Coleman et al., 2009). Here, using a panel of GluN2B ligand binding mutants covering a spectrum of glutamate efficacies, we observed a correlation between glutamate  120  efficacy and surface expression, both in heterologous cells and in neurons. Compared with glutamate EC50 1.5 µM for wild-type GluN2B, mutants V660A, F390S, S664G, and E387A with EC50 in the range of 27 µM to 355 µM and R493K with undetectable response (Laube et al., 1997) showed a corresponding range of surface expression in heterologous cells (Fig 24) and in neurons (Fig 27). Combined with the evidence of a role for glutamate binding at the stage of forward trafficking (Fig 25), these results suggest that the rate of receptor release for forward trafficking is dependent on the rate of ligand binding. ER retention of mutant AMPA and kainate receptors has been demonstrated by sensitivity to endoglycosidase H (EndoH) (Mah et al., 2005; Greger et al., 2006; Coleman et al., 2009). EndoH cleaves N-linked high mannose-containing sugars on immature receptors found in the ER, while maturation through the Golgi renders receptors EndoH-resistant. We were not able to detect pools of GluN2B with differential EndoH sensitivity (data not shown), and previous studies could not detect EndoH-resistant GluN1, all GuN1 was EndoH-sensitive despite evidence for a large cytoplasmic pool (Huh and Wenthold, 1999), suggesting that ER localized NMDA receptors may be largely EndoH-sensitive. Nonetheless, the reduced forward trafficking of GluN2B mutants compared to wild-type even during inhibition of endocytosis (Fig 25) is consistent with the idea that the mutant receptors accumulate in the ER. While accurate estimates of glutamate concentration in the ER lumen are lacking, glutamate is readily detected in the ER and average intracellular concentrations are thought to be in the millimolar range (e.g. 4-16 mM in various brain regions (Berger et al., 1977; Meeker et al., 1989; Fleck, 2006)). The finding here that the constitutively closed cleft GluN2B KNCC traffics to the surface like wild-type GluN2B and does not exhibit enhanced surface expression (Fig 28) suggests that glutamate concentrations are not normally limiting for forward trafficking of GluN2B. Furthermore, the conserved 121  glutamate binding pocket and similar affinities of GluN2A, GluN2B, GluN2C, and GluN2D subunits for glutamate, ranging from EC50 0.51 µM to 3.3 µM (Erreger et al., 2007; Traynelis et al., 2010), suggest that glutamate is not typically limiting for forward trafficking of native NMDA receptors. The range of glutamate concentration that mediates maximal activation of wild-type GluN2B while detecting differences in activation of the GluN2B mutants is roughly from 50 µM to 1 mM (Laube et al., 1997). Assuming that the glutamate pool that controls forward trafficking is the same for AMPA, kainate, and NMDA receptors, thus also taking into account surface trafficking of GluA2 and GluK2 and one mutant of each with reduced glutamate efficacy (Mah et al., 2005; Coleman et al., 2009), would put the relevant glutamate concentration at the high end of our range. Interestingly, co-expression of WT GluK2(Q) partially rescued surface expression of a glutamate binding deficient mutant GluK2(R) E738G, assayed by rectification ratio in patchclamp recordings (Mah et al., 2005). In contrast, surface expression of GluN2B glutamate binding deficient mutants was not enhanced by co-expression of WT GluN2B (Fig 26). The apparent difference between these results may lie partly in the nature of the assay, as electrophysiological tagging can be very sensitive to detect low surface levels, or may reflect a difference between ionotropic receptor subtypes. Our results suggest that ligand must interact at both GluN2B subunits for proper trafficking, consistent with the requirement for two molecules of glutamate (and tow of glycine) for activation of NMDA receptors (Benveniste and Mayer, 1991; Clements and Westbrook, 1991) and with recent structural studies (Furukawa et al., 2005). Surface expression of NMDA receptors appears to be limited by the least avid ligand binding site on the two GluN2B subunits. Partial rescue of surface trafficking was also achieved for ligand binding deficient mutants of GluA4 by co-expression of stargazin (Coleman et al., 2009), and of 122  GluN1-D732A by treatment with a cell-permeable competitive glycine site antagonist (Kenny et al., 2009). However, stargazin does not interact with NMDA receptors, and while the idea that binding of a competitive antagonist might mimic ligand binding to rescue surface trafficking is attractive, the low cell permeability of competitive antagonists for GluN2B precluded the ability to test this idea. Previous studies, as discussed above, assayed the role of ligand binding to ionotropic glutamate receptors in surface trafficking in heterologous cells and by overexpression in neurons. Here, we extend these assays to include rescue of synaptic GluN2B at an appropriate expression level in GluN2B -/- neurons (Fig 28). These results indicate that differences in ligand binding correlate with differences in surface synaptic accumulation in mature neurons. Thus, ligand binding controls delivery of this functionally important pool of NMDA receptors. NMDA receptor accumulation at synapses is also controlled by other features of GluN1 and GluN2 subunits (Ferreira et al., 2011; Storey et al., 2011) and multiple interacting proteins regulated by activity and kinases (Lau and Zukin, 2007; Sanz-Clemente et al., 2010; Nolt et al., 2011). The precise mechanism by which ligand binding promotes forward trafficking of ionotropic glutamate receptors is not well understood. The mechanism is not likely to require ion flux, since mutations in the isolated S1-S2 ligand binding domain of GluA4 or GluK2 affected their secretion in a parallel manner to effects on surface expression of the full length receptors (Coleman et al., 2009; Gill et al., 2009). One idea is that reversible gating motions associated with reversible cleft closure induced by ligand binding are sensed in the ER to promote export (Penn et al., 2008). In support of this idea, a constitutively closed cleft GluK2 ENCC mutant showed reduced surface expression compared with WT GluK2 (Gill et al., 2009). However, in our study (Fig 27), the constitutively closed cleft GluN2B KNCC mutant trafficked to the surface 123  indistinguishably from WT GluN2B. These results suggest that GluN2B does not have to undergo reversible gating motions or reversible cleft closure, and that other aspects of the ligandinduced conformation promote forward trafficking to the cell surface. Other recent studies on AMPA receptors also support a mechanism of ligand-induced conformation changes promoting forward trafficking independent of activity, and suggest that the ligand binding domain is the primary site for ER quality control of AMPA receptors (Coleman et al., 2010). Ligand binding appears to control forward trafficking for all ionotropic glutamate classes, glutamate binding to AMPA and kainate receptors (Fleck, 2006), glycine binding to GluN1 (Kenny et al., 2009), and glutamate binding to GluN2B (here). A primary reason may be for quality control, so that only functional assembled ligand-gated ion channels traffic to the surface. An intriguing corollary is that NMDA receptors may be functional in the ER. Indeed, the proposed requirement for glutamate and glycine binding for ER exit suggests that NMDA receptors would be at least transiently open at intracellular sites. Interestingly, activation of the metabotropic receptor mGluR5 on intracellular membranes triggers different signalling cascades than activation of cell surface mGluR5, resulting in differential gene transcription (Jong et al., 2009). Thus, it is possible that ligand binding to intracellular receptors may have a function in addition to quality control, that sodium and calcium efflux from the ER or transport intermediates to the cytosol mediated by NMDA receptors may trigger local signalling in neurons.  124  Chapter 4: Intracellular Determinants of GluN2 Control Synaptic Recruitment of CaMKIIα  125  4.1 Introduction  Long term potentiation (LTP), as a consequence of heightened temporally synchronized synaptic activity, has been a major model for the study of the cellular and molecular basis of memory (Bliss and Collingridge, 1993; Lee and Silva, 2009). While a very wide variety of induction protocols has been developed to reliably elicit LTP in a variety of preparations (Hunt and Castillo, 2012), central to the induction of LTP is the influx of Ca2+ through the NMDA receptor channel leading to elevations in spine Ca2+ concentrations that activates calcium/calmodulin-dependent protein kinase II (CaMKII) (Martin et al., 2000; Lisman et al., 2002; Malenka and Bear, 2004). Not only does this Ca2+ influx activate the autophosphorylating holoenzyme CaMKII which acts as a decoding mechanism that integrates transient Ca2+ influxes and becomes constitutively active (De Koninck and Schulman, 1998; Soderling et al., 2001), this Ca2+ influx also promotes binding of CaMKII to NMDA receptors (Strack and Colbran, 1998; Leonard et al., 1999) and subsequent accumulation to synaptic sites (Strack et al., 1997; Shen and Meyer, 1999). Once at synaptic sites, CaMKII is in position to be optimally activated by further Ca2+ entry through NMDA receptors in addition to having direct access to its phosphorylation targets that include AMPA receptors, NMDA receptors, SynGap, and nNOS (Soderling et al., 2001). Global cell stimulation through NMDA receptors enhances synaptic levels of CaMKII by inducing translocation from the dendritic shaft to spines. This translocation requires calcium/calmodulin binding to CaMKII and is facilitated by autophosphorylation at Thr-286 (Meyer et al., 1992). Although CaMKII preferentially binds GluN2B (Bayer et al., 2006) and this interaction is important for LTP induction (Barria and Malinow, 2005) and memory  126  consolidation (Halt et al., 2012), CaMKII self association (Hudmon et al., 2005) and binding to other synaptic targets such as GluN1, GluN2A, densin-180, and α-actin (Colbran, 2004) may also be important. Globally induced accumulation of CaMKII at synapses was previously reported to be mediated at least in part by binding to the NMDA receptor subunit GluN2B. CaMKII mutants F98K, E139R, I205K, and W237K inhibit binding to GluN2B and inhibit globally induced synaptic accumulation (Bayer et al., 2006). However, these mutants could also affect binding to other CaMKII partners. Here, we test whether a double point mutation in GluN2B that blocks CaMKII binding also blocks CaMKII translocation to synapses. Multiple paradigms other than global cell stimulation of NMDA receptors can induce synaptic accumulation of CaMKII. Selective activation of synaptic NMDA receptors using a chemical LTP protocol aggregates CaMKII and induces their persistent translocation to synapses (Sharma et al., 2006). Additionally, local dendritic stimulation by application of glutamate puffs not only induces translocation of CaMKII to synapses at the site of stimulation, but leads to subsequent spreading translocation of CaMKII within dendrites to the distal dendrite arbor resulting in global accumulation of CaMKII to synapses. This locally induced propagating synaptic accumulation of CaMKII requires activation of NMDA receptors and L-type Ca2+ channels (Rose et al., 2009). Here, we made use of GluN2B -/- mouse hippocampal culture system to first examine the role of GluN2B in mediating local or global stimulation induced CaMKII translocation. Surprisingly, while the loss of GluN2B abrogates global cell stimulation induction of CaMKII translocation, the propagating accumulation of CaMKII at synapses in response to local stimulation is unaffected. Like the loss of CaMKII translocation in response to global cell stimulation through NMDA receptors, chemical LTP also fails to elicit CaMKII translocation in 127  KO neurons. Additionally, we south to investigate differential roles of GluN2B and GluN2A in mediating global cell stimulation induced CaMKII translocation through a rescue approach by expressing YFP-tagged GluN2 constructs introduced into GluN2B -/- (KO) hippocampal neurons in culture. However, stimulated synaptic accumulation of CaMKII in KO neurons by both global cell stimulation and chemical LTP were rescued by the expression of YFP-GluN2B or chimeric GluN2A/2B tail but not by GluN2A, chimeric GluN2B/2A tail, or GluN2B with point mutations in the CaMKII binding site. These results suggest that the activity-regulated synaptic aggregation of CaMKII is dependent on the cytoplasmic CaMKII binding site of GluN2B and not on differential permeation properties between GluN2B and GluN2A.  4.2 Materials and Methods  4.2.1 DNA Constructs  GluN2 constructs were inserted into the multiple cloning site 2, driven by the light chain human ferritin promoter, of a modified expression vector derived from pVIVO2 (InvivoGen). Wild type YFP-GluN2B (GluN2B) was developed from rat GluN2B ((Kim et al., 2005), a gift of M. Sheng, Genentech) by inserting enhanced YFP (Clontech) followed by the PCR generated linker LVPRGSRSR between amino acids 2 and 4 of the mature N-terminus. Wild type YFPGluN2A (GluN2A) was developed from rat GluN2A (a gift of M. Sheng, Genentech) by inserting the enhanced YFP from GluN2B, including the signal sequence and linker, in frame upstream of GluN2A alanine 24 and subcloned into the modified pVIVO2 expression plasmid. The GluN2B tail swap chimera (GluN2B 2AT) has the GluN2A tail inserted after leucine 841  128  (NCBI ref seq: NM_012574.1), the third amino acid after the last amino acid of the M4 domain. The GluN2A tail insert starts at valine 840 (NCBI ref seq: NM_012573.3). The GluN2A tail swap chimera (GluN2A 2BT) has the GluN2B tail inserted after tryptophan 837, the last amino acid of the M4 domain, and the GluN2B tail begins at tryptophan 844. These chimeras were generated with artificial hybrid primers. The CaMKII binding mutant (GluN2B RS-QD) was generated by site-directed mutagenesis changing both arginine 1300 to serine and glutamine 1303 to aspartic acid using the QuickChange II XL kit (Agilent) (Strack et al., 2000a). CFP-CaMKIIα was derived from GFP-CaMKIIα ((Shen and Meyer, 1999), a gift of T. Meyer, Stanford) by replacing the GFP with enhanced CFP (Clontech) and subcloned into a modified pLentiLox3.7 vector with the CMV promoter replaced with a synapsin promoter (a gift of A. El-Husseini, UBC). For packaging into lentivirus, eGFP-CaMKIIα was also subcloned into this modified pLentilox3.7 expression vector (pLL-GFP-CaMKIIα). Plasmids for third generation lentiviral packaging pMD2.G, pRSV-Rev, and the pLL-GFP-CaMKIIα construct were transfected with Lipofectamine 2000 into HEK 293FT cells. Media was harvested 48 hours later, centrifuged, and filtered supernatant was used for viral transduction of neurons at DIV 3-5. Soluble EYFP was expressed from a plasmid bearing the CAG promoter consisting of the CMV immediate early enhancer and the chicken β-actin promoter (Niwa et al., 1991)(kind gifts of S. Kaech and G. Banker, with permission of J. Miyazaki for the CAG promoter) (pBA-YFP). All coding sequences were confirmed by sequencing.  129  4.2.2 Cell Culture  Dissociated primary transgenic mouse hippocampal neuron cultures were prepared from embryonic day 18 mouse embryos as described (Kaech and Banker, 2006). GluN2B +/- mice (Kutsuwada et al., 1996) were maintained and genotyped as described (Liu et al., 2007); these heterozygous mice were timed-mated to obtain GluN2B -/- embryos. Hippocampi were dissected from individual 17-18 day embryos and stored overnight at 4oC in Hibernate E (Brain Bits) supplemented with B-27 (Invitrogen) pending parallel genotyping of brainstem and tail tissue. Mouse hippocampi were dissociated with papain (20 units/mL, 15 min, 370C). In some experiments, 3 x 105 freshly dissociated hippocampal neurons were plated on poly-L-lysine treated glass coverslips in 60 mm petri dishes for viral transduction. In the remaining experiments, 5 x105 freshly dissociated neurons were pelleted at 80 x g for 4 minutes for nucleofection using an Amaxa Nucleofector II (Lonza) (program O-005) with 6 µg of YFPGluN2 plasmid or 3 µg pBa-YFP and 4 µg of CFP-CaMKIIα. Nucleofected cells were plated onto poly-L-lysine treated glass coverslips in 60 mm petri dishes for an effective estimated plating density of 3 x105 live cells per dish and inverted over a feeder layer of glia. Neurons were maintained in Neurobasal medium (Invitrogen) supplemented with B-27, L-glutamine (Gibco), and 25 µg/mL bovine pancreatic insulin (Sigma-Aldrich). After 2 d in vitro (DIV), proliferation of glia was suppressed by the addition of 5 µM cytosine arabinoside (Calbiochem). Cells were maintained with 100 µM APV ((2R)-amino-5-phosphonovaleric acid) (Sigma-Aldrich) which was washed out prior to manipulation and analysis on DIV17.  130  4.2.3 Stimulation Protocols  Local Stimulation was performed on coverslips placed in a custom-built chamber and continuously perfused by gravity flow with extracellular solution (ECS in mM: 168 NaCl, 2.6 KCl, 2 CaCl2, 1.3 MgCl2, 10 D-Glucose, and 10 HEPES, pH 7.2) by positioning a 3-10 MΩ pipette filled with 100 µM glutamate and 10 µM glycine very close to the dendrite and generating a single pressure ejection of 10-20 psi and 10-25 ms via a Picospritzer (General Valve). The pipette was immediately removed from the perfusion bath following stimulation. Bath stimulation was performed on hippocampal neuron culture coverslips washed free of APV. Coverslips were incubated in 37oC ECS with 100 µM glutamate and 10 µM glycine (stimulated) or without amino acids (mock stimulated control) for 3 min and transferred to ECS without amino acids for 7 min followed by fixation. For chemical LTP (cLTP) stimulation, coverslips were washed free of APV and incubated in 37oC ECS without Mg2+ with 200 µM glycine (stimulated) or without glycine (mock stimulated control) for 10 min and transferred to normal ECS for a 20 min recovery period followed by fixation. For the nifedipine block of calcium channels, coverslips were washed free of APV, pre-incubated in normal ECS with 10 µM nifedipine for 1 min, then stimulated or mock stimulated normally in the continued presence of 10 µM nifedipine. All ECS incubations were conducted at 37oC with 5% CO2.  131  4.2.4 Immunocytochemistry  Coverslips were fixed for 15 minutes in pre-warmed PBS with 4% paraformaldehyde and 4% sucrose pH 7.4, washed in PBS, permeabilized with 0.25% Triton X-100 in PBS, and washed again. Fixed and permeabilized neuron cultures on coverslips were blocked in 8% bovine serum albumin (BSA) and 1.2% normal goat serum (NGS) for 30 min at 37oC prior to incubation in primary antibodies diluted in PBS with 3% BSA and 0.3% NGS for 2 hours at 37oC and secondary antibodies for 45 min at 37oC. Coverslips were washed 6x 2 min with PBS following each antibody incubation. Coverslips were rinsed in ddH2O and mounted in Elvanol (Tris-HCl, glyverol, polyvinyl alcohol, and 2% 1,4-diazabicyclo[2,2,2]octane). The following antibodies were used: anti-PSD-95 family (IgG2a; 1:1000; Thermo Fisher Scientific clone 6G6-1C9; recognizes PSSD-95, PSD-93, SAP102, and Sap97) and anti-MAP2 (chicken IgY polyclonal; 1:10,000; AbCam ab5392). Secondary antibodies were Alexa-568 conjugated anti-IgG2a, generated in goat and was highly cross-adsorbed (1:1000; Invitrogen) and AMCA conjugated anti-chicken IgY, generated in donkey (1:400; Invitrogen).  4.2.5 Imaging and Data Analysis  All imaging and analysis was done blind to genotype, nucleofected GluN2 construct, and stimulation history. For experiments involving GluN2 constructs and CaMKIIα translocation, cells were chosen by visual examination of the YFP signal and cells that resembled the wild type expression pattern were selected for imaging. Cells without co-transfection with CFP-CaMKII were discarded as were cells where the CFP signal was too highly overexpressed based on channel intensity saturation when acquired at a fixed 500ms exposure. Images were acquired on 132  a Zeiss Axioplan2 microscope with a 63X 1.4 numerical aperture oil objective and Photometrics Sensys cooled CCD camera using MetaVue imaging software (Molecular Devices) and customized filter sets. Images in each channel were acquired in grey scale using the same exposure time across all cells, and pseudo-colour overlays for presentation were prepared using Adobe Photoshop. Quantification of image linescans for Figure 30 was conducted with Metamorph by tracing the stimulated dendrite with a 8-20 pixel wide line and measuring the maximum intensities of the peaks (synaptic clusters) both before and after stimulation. For each cell, the average of the maximum post-stimulation peak intensities was divided by the average maximum intensity for the identical pre-stimulation region, with background subtraction, in Microsoft Excel. For figures 31-34, the thresholding of CFP-CaMKIIα for each cell was determined by 1) masking the CFP channel with a binarized dendrite marker MAP2 image, dilated by 2 pixels, from the same cell, 2) drawing a region of interest over a length of dendrite (bath stimulation 58.2 ± 14.0 µm per cell; cLTP stimulation 65.3 ± 14.8 µm per cell) and measuring the total area and total CFP intensity, which was 3) used to generate a value for the average total shaft CFP intensity which was multiplied by 1.75, that was 4) used to threshold the CFP channel for that cell for analysis of punctuate CFP-CaMKIIα (Fig 29). Quantification of PSD-95 was manually thresholded. YFP-GluN2 average shaft intensity was determined by applying the same dilated MAP2 mask over the YFP channel and synaptic YFP intensity was determined by masking the YFP channel with a 1 pixel dilated binarized image of the thresholded PSD-95 channel. YFP puncta density was thresholded manually.  133  For all experimental conditions including stimulated or mock-stimulated, a minimum of 24 cells from at least two independent cultures were acquired and analyzed. Statistical analyses were performed with Prism (GraphPad Software).  Figure 29: thresholding CaMKIIα  Quantitative of CFP-  To threshold the CFPCaMKIIα signal along a dendritic region of interest, the corresponding region from the MAP2 image channel for the same cell was binarized and dilated by two pixels to create a MAP2 mask. This mask was applied to the CFP channel to measure total integrated CFP intensity as well as area. The average shaft CFP intensity was calculated from total CFP intensity divided by the area. This number was multiplied by 1.75 to create thresholding levels for each dendritic region of interest for each cell.  134  4.3 Results  4.3.1 GluN2B is Required for Globally but not Locally Induced CaMKII Translocation  Here we generated neuron cultures from transgenic mice lacking GluN2B expressing exogenous GFP-CaMKIIα to investigate the effects of global and local stimulation. As predicted, GluN2B -/- neurons were unable to accumulate CaMKIIα at synapses upon bath stimulation with glutamate and glycine (Fig 30B). As a control, neuron cultures from littermate wild-type mice were also stimulated and accumulated CaMKIIα at spines in response to global stimulation, similar to rat neurons, showing a 206 ± 6% increase in GFP-CaMKII intensity at puncta (Fig 30A, C; p < 0.0001). Although quantitative differences from wild-type neurons have not been assessed, GluN2B -/- neurons still possess GluN2A mediated NMDA currents (Tovar et al., 2000) ruling out a trivial explanation for the lack of CaMKIIα accumulation upon global stimulation. Surprisingly, GluN2B -/- neurons exhibit unimpaired synaptic accumulation of CaMKIIα following local stimulation with glutamate and glycine (Fig 30D). GluN2B -/- neurons showed a 214 ± 24% increase in GFP-CaMKIIα intensity at non-stimulated puncta in response to local stimulation indistinguishably from wild-type neurons (Fig 30C, F; p > 0.1). Thus, global NMDA receptor induced synaptic accumulation of CaMKIIα requires Ca2+ flux specifically through GluN2B containing receptors and/or binding to GluN2B whereas locally induced propagation of CaMKIIα accumulation to synapses requires neither. This differential dependence on GluN2B clearly indicates disparate underlying mechanisms for globally versus locally induced CaMKIIα accumulation.  135  Figure 30: Bath induced CaMKII translocation was abolished in GluN2B -/- cells, but locally induced translocation was maintained Hippocampal neurons were cultured from littermate GluN2B -/- or +/+ mice, transduced to express GFP-CaMKIIα, and imaged live before (left) and >3 min after (right) stimulation as indicated. A) Upon bath stimulation with glutamate and glycine, wild-type mouse neurons accumulated CaMKIIα at spines, similar to rat neurons. B) In contrast, GluN2B -/- neurons showed no change in CaMKII distribution upon bath stimulation with glutamate and glycine. C) For quantitation, post-stimulation peak intensities of GFP-CaMKIIα were divided by prestimulation GFP-CaMKIIα intensities for the same regions, after background subtraction, and multiplied by 100. Horizontal line marks no change in CaMKIIα distribution. Statistical analysis revealed a significant difference between genotypes in ability to cluster CaMKIIα following bath stimulation (t(23) = 15.23, ** p < 0.0001). D-F) Upon local stimulation with glutamate and glycine applied from a pipet to a small region of primary dendrite (not shown), both GluN2B-/and wild-type neurons showed propagating accumulation of CaMKIIα at spines, indistinguishably. Quantitation was performed as for the bath stimulation in panel C and showed no difference between genotypes in ability to cluster CaMKIIα following local stimulation (t(16) = .18, p = 0.86). Scale bar 10 µm.  136  4.3.2 Exogenous Rescue of YFP-GluN2B Restores CaMKIIα Translocation To assess the differential roles of GluN2B and GluN2A and their C-terminus cytosolic tails in mediating global CaMKIIα translocation, we first established conditions to essentially rescue GluN2B in GluN2B -/- neurons. Knockout hippocampal neurons were nucleofected at plating with YFP-GluN2B under control of the human ferritin promoter and analyzed at 14 DIV. In this rescue system, the recombinant YFP-GluN2B localized appropriately at PSD-95 positive synapses (Fig 31A). Furthermore, we were fortunate that the combination of nucleofection protocol, amount of plasmid, and the expression system was able to rescue anti-GluN2B immunoreactive puncta number and intensity level in the GluN2B -/- neurons to levels indistinguishable from that of littermate wild-type sister neurons (Fig 31A, B; ANOVA p < 0.0001 and Bonferroni’s multiple comparison test p < 0.001 for GluN2B -/- compared to WT, p > 0.05 for GluN2B -/- plus YFP-GluN2B compared to WT). In this rescue system, concomitant with the re-introduction of YFP-GluN2B to knockout neurons, YFP-GluN2B expressing neurons regain the global translocation of co-nucleofected CFP-CaMKIIα to YFP-GluN2B positive and PSD-95 positive synapses in response to bath stimulation with glutamate and glycine similar to soluble EYFP transfected wild-type neurons. The CFP-CaMKIIα signal remains diffuse throughout the dendrite in both the soluble EYFP transfected GluN2B -/- stimulated neuron and in all mock stimulated cells (Fig 31C).  137  Figure 31: Exogenous rescue of GluN2B -/- with YFP-GluN2B restores bath induced CaMKIIα translocation A) GluN2B -/- hippocampal neurons in culture were nucleofected at plating with YFP-GluN2B and stained for GluN2B and PSD-95 immunoreactivity at DIV 14. The GluN2B antibody recognizes clusters of YFP-GluN2B with 100% fidelity and both antibody and YFP signal colocalizes to PSD-95 positive excitatory synaptic puncta. A dendrite from a neighbouring untransfected neuron shows normal PSD-95 immunoreactivity but not YFP or GluN2B antibody signal. B) The number of GluN2B puncta and integrated intensity of the GluN2B antibody signal were indistinguishable between YFP-GluN2B transfected GluN2B -/- neurons and sister wild138  type neurons (ANOVA p < 0.0001 and Bonferroni’s multiple comparison test * p < 0.001 for GluN2B -/- compared to WT, p > 0.05 for GluN2B -/- transfected with YFP-GluN2B compared to WT. C) Bath glutamate and glycine stimulation induced CFP-CaMKIIα translocation in soluble EYFP transfected WT neurons is lost in soluble EYFP transfected GluN2B -/- neurons. In mock stimulated GluN2B -/- cells co-transfected with YFP-GluN2B and CFP-CaMKIIα, the CFP signal remains diffusely localized along the dendritic shaft. Bath stimulation induced CFPCaMKIIα translocation is rescued in YFP-GluN2B expressing cells. Scale bar 10 µm.  4.4.3 GluN2B Cytoplasmic Tail and CaMKII Binding Site is Essential for Globally Induced CaMKII Translocation Given that the YFP-GluN2B rescue restored bath stimulation induced translocation of CFP-CaMKIIα to synapses, we investigated some of the characteristics of NMDA receptors required for the translocation of CaMKIIα. To assess specific binding of GluN2B to CaMKII, a CaMKII binding deficient GluN2B with R1300 mutated into glutamine and S1303 mutated into aspartic acid was constructed (GluN2B RQ-SD) (Strack et al., 2000a). GluN2B and GluN2A were also compared as were chimeras of GluN2B with the GluN2A cytosolic tail (GluN2B 2AT) and GluN2A with the cytosolic tail from GluN2B (GluN2A 2BT). Each of these GluN2 constructs was expressed in the YFP-tagged form from the human ferritin light chain promoter and co-expressed with CFP-CaMKIIα. During the acquisition of images, cells were selected based on the qualitative YFP-GluN2 expression pattern and levels that were within the similar range as wild-type. Therefore, although no claims can be made regarding the potential differences in targeting among the YFP-GluN2 constructs, post hoc analyses were performed to ensure that any differences in CaMKIIα response were not due to large differences in the number of NMDA receptors at synapses.  139  First, the number of thresholded PSD-95 puncta and the average size of these puncta were comparable across all GluN2 constructs and stimulation history (Fig 32A, B; ANOVA p > 0.05). The total integrated YFP-GluN2 intensity for the analysed dendritic region and the  Figure 32: GluN2 expression of PSD-95 immunoreactivity is comparable across all GluN2 rescued cells A, B) There were no differences between the density or average size of PSD-95 puncta. The number of PSD-95 puncta was the raw number of thresholded PS-95 puncta and the average PSD-95 puncta size was calculated for each cell. C-D) There was no differences between total shaft YFP-GluN2, synaptic YFP-GluN2 intensity, number of YFPGluN2 puncta, or the number of synaptic YFPGluN2 puncta. Total shaft YFP-GluN2 was calculated from a two pixel dilated binary MAP2 mask and synaptic YFP-GluN2 intensity was measured through a one pixel dilated binary PSD-95 mask. The number of YFP-GluN2 puncta was the raw number of thresholded YFP-GluN2 puncta while the number of synaptic YFP-GluN2 puncta were YFP-GluN2 puncta that co-localized by at least three pixels with PSD95. (ANOVA p > .05, n = 24).  140  synaptic integrated YFP-GluN2 intensity were similarly equal regardless of YFP-GluN2 construct or stimulation history (Fig 32C, D; ANOVA p > 0.05). Likewise, the total number of thresholded YFP-GluN2 puncta and the number of synaptic YFP puncta were comparable across all conditions (Fig 32E, F; ANOVA p > 0.05). As expected, rescue with the CaMKII binding mutant GluN2B RQ-SD resulted in impaired aggregation of CFP-CaMKIIα in response to bath glutamate and glycine stimulation compared to rescue with GluN2B although there was still a significant degree of CFP-CaMKIIα aggregation compared to mock-stimulated controls (Fig 33A,B; ANOVA p < 0.0001 and Bonferroni’s multiple comparison test * p < 0.05 compared to mock-stimulated but ††† p < 0.001 compared to GluN2B stimulated). The numbers of synaptically localized CFP-CaMKIIα aggregates were not significantly different compared to mock-stimulated controls (Fig 33A, C; ANOVA p < 0.0001 and Bonferroni’s multiple comparison test p > 0.05 compared to mock stimulated control). GluN2A also directly binds CaMKII, perhaps with a lower affinity than GluN2B, but binding is potentiated by CaMKII autophosphorylation at T286 and thus should be enhanced by stimulation (Gardoni et al., 1999; Colbran, 2004). Since GluN2A abundance is lower than that of GluN2B, at least in the hippocampal CA3 region (Akashi et al., 2009), levels of total GluN2 in the GluN2B -/- neurons may not be sufficient to mediate translocation of CaMKII. Thus we tested whether expression of YFP-GluN2A in GluN2B -/- neurons at similar levels as YFPGluN2B could rescue CaMKII translocation. This experiment will reveal directly whether there are features unique to GluN2B not present in GluN2A that mediate CaMKII translocation. We found that bath stimulation with glutamate and glycine in the presence of exogenous GluN2A, but in the absence of GluN2B receptors, is insufficient for inducing  141  Figure 33: Bath induced CFP-CaMKIIα translocation is impaired in the presence of GluN2B CaMKII binding mutant, GluN2A, and GluN2B 2AT chimera but rescued by GluN2A 2BT chimera GluN2B -/- neurons were nucleofected at plating with YFP-tagged GluN2B, GluN2B RQ-SD CaMKII binding mutant, GluN2A, GluN2B with a GluN2A tail chimera (GluN2B 2AT), or GluN2A with a GluN2B tail chimera (GluN2A 2BT). In the GluN2B rescued cells, in response to bath stimulation with glutamate and glycine, CFP-CaMKIIα translocates from a diffuse dendritic distribution to discrete puncta. The vast majority of CFP-CaMKIIα puncta are colocalized with PSD-95 (arrowheads) but a small number appear extra-synaptic (arrows). CFPCaMKIIα translocation is impaired in the cells rescued with GluN2B RQ-SD CaMKII binding mutant, GluN2A, and GluN2B 2AT with reduced overall thresholded CFP-CaMKIIα. While there was a significant degree of CFP-CaMKIIα translocated in the GluN2B RQ-SD rescued neurons as individual puncta after bath glutamate and glycine stimulation, many of these puncta were extra-synaptic (ANOVA p < 0.0001, Bonferroni’s multiple comparison test * p < 0.05, *** p < 0.001 asterisks comparing paired control mock stimulated and bath glutamate and glycine stimulated, ††† p < 0.001 daggers comparing stimulated with GluN2B stimulated). Synaptic CFP-CaMKIIα puncta were defined as those with a minimum three pixel overlap with PSD-95. Scale bar 10 µm.  142  aggregation of CFP-CaMKIIα to synapses (Fig 33; ANOVA p < 0.0001 and Bonferroni’s multiple comparison test ns p > 0.05 compared to mock-stimulated but ††† p < 0.001 compared to GluN2B stimulated). Next, to investigate the effects of differences in the cytoplasmic tails between GluN2A and GluN2B in mediating CaMKII aggregation and translocation, we tested a chimera with an intact wild-type GluN2B N-terminal domain (NTD), ABD (agonist binding domain), and intracellular pore region but with its C-terminal cytoplasmic tail replaced with that from GluN2A (GluN2B 2AT). In P7 organotypic hippocampal slices, rescue with similar GluN2B and GluN2A tail chimeras after RNAi knockdown of GluN2B both rescue the basal NMDA EPSC amplitude to similar levels and EPSC half width of the GluN2B 2AT chimera was indistinguishable from that of GluN2B whereas the GluN2A 2BT showed approximately 50% decrease in the EPSC half width (Foster et al., 2010). These data suggest that current flow is primarily controlled by the portions of the chimeras’ N-terminal to the cytoplasmic tails, and thus that differences associated with the tails may reflect molecular interactions. In our experiments, in neurons rescued with the GluN2B 2AT, CFP-CaMKIIα showed little aggregation in response to bath glutamate and glycine stimulation (Fig 33A, B; ANOVA p < 0.0001 and Bonferroni’s multiple comparison test ns p > 0.05 compared to mock-stimulated but ††† p < 0.001 compared to GluN2B stimulated). There was a slight but significant increase in synaptically localized CFP-CaMKIIα clusters although synaptic aggregation was much reduced compared to the stimulated rescued GluN2B (Fig 33A, C; ANOVA p < 0.0001 and Bonferroni’s multiple comparison test * p < 0.05 compared to mock-stimulated but ††† p < 0.001 compared to GluN2B stimulated). These data strongly suggest the importance of the C-terminal cytoplasmic tail of GluN2B in mediating CaMKII translocation. Next, we tested a chimera consisting of a wild-type GluN2A  143  NTD, ABD, and pore but with a GluN2B C-terminal cytoplasmic tail. This GluN2A 2BT chimera mediated a complete rescue of CFP-CaMKIIα aggregation (Fig 33A, B; ANOVA p < 0.0001 and Bonferroni multiple comparison test *** p < 0.001 compared to mock stimulated and ns p > 0.05 compared to GluN2B stimulated) and these aggregates localized properly to synapses (Fig 33A, C; ANOVA p < 0.0001 and Bonferroni multiple comparison test *** p < 0.001 compared to mock stimulated and ns p > 0.05 compared to GluN2B stimulated). These results suggest that the permeation properties of GluN2A are not limiting to CaMKII translocation whereas interactions between the cytoplasmic tail and CFP-CaMKIIα, possibly through intermediaries, are more crucial.  4.4.4 GluN2 Cytoplasmic Tail is Essential for CaMKII Translocation Induced by Chemical Long Term Potentiation As the bath application of glutamate and glycine indiscriminately activates both synaptic and extra-synaptic receptors, we wished to determine the effects of stimulation solely through synaptically localized receptors. In low density cortical and hippocampal cultures the removal of Mg2+ from the microenvironment and the addition of glycine induces spontaneous synaptic responses through NMDA receptors that mobilize CaMKII and AMPA receptors to synapses and mediate the induction of LTP (Lu et al., 2001; Sharma et al., 2006). Using this chemical LTP (cLTP) induction protocol we once again tested our panel of GluN2 constructs. The response from YFP-GluN2B nucleofected GluN2B -/- neurons (4.61 fold increase in CFP-CaMKIIα clusters and 5.78 fold increase in synaptic clusters) were similar to GluN2B +/+ neurons while cLTP failed to induce CFP-CaMKIIα translocation in non-rescued GluN2B -/- neurons. Unexpectedly, in GluN2B RQ-SD rescued cells there was a significant degree of CFPCaMKIIα aggregation in response to cLTP induction when compared to mock-stimulated control 144  although this is still impaired compared to GluN2B stimulated (Fig 34A, B; ANOVA p < 0.0001 and Bonferroni’s multiple comparison test *** p < 0.001 compared to mock stimulated, ††† p < 0.001 compared to GluN2B stimulated). However, many of these clusters failed to localize properly at synapses (Fig 34A, C; ANOVA p < 0.0001 and Bonferroni’s multiple comparison test ns p > 0.05 compared to mock-stimulated, ††† p < 0.001 compared to GluN2B stimulated). Again, this cLTP protocol induced a significant but impaired amount of CFP-CaMKIIα aggregation through GluN2A and GluN2B 2AT (Fig 34A, B; ANOVA p < 0.0001 and Bonferroni multiple comparison test * p < 0.05, ** p < 0.01 compared to mock stimulated) but only a significant number of the clusters were localized at synaptic sites through GluN2B 2AT (Fig 34A, C; ANOVA p < 0.0001 and Bonferroni’s multiple comparison test ** p < 0.01 compared to mock stimulated, ††† p < 0.001 compared to GluN2B stimulated). Identically with bath stimulation, the GluN2A 2BT tail swap chimera fully rescued synaptic CFP-CaMKII aggregation (Fig 34A, B, C; ANOVA p < 0.0001 and Bonferroni’s multiple comparison test *** p < 0.001 compared to mock-stimulated, ns p > 0.05 compared to GluN2B stimulated).  145  Figure 34: cLTP induced CaMKII translocation is impaired in the presence of GluN2B CaMKIIα binding mutant, GluN2A, and GluN2B 2AT chimera but rescued by GluN2A 2BT chimera GluN2B -/- neurons were nucleofected at plating with YFP-tagged GluN2B, GluN2B RQ-SD CaMKII binding mutant, GluN2A, GluN2B with a GluN2A tail chimera (GluN2B 2AT), or GluN2A with a GluN2B tail chimera (GluN2A 2BT). In the GluN2B rescued cells, in response to chemical LTP, CFP-CaMKIIα translocates from a diffuse dendritic distribution to discrete puncta. The vast majority of CFP-CaMKIIα puncta are co-localized with PSD-95 (arrowheads) but a small number appear extra synaptic (arrows). In the RS-QD mutant rescued cells, while there was a significant degree of CaMKII translocation between control and stimulated, compared to GluN2B, it is impaired similarly with GluN2A, and GluN2B 2AT but GluN2A 2BT completely rescues translocation (ANOVA p < 0.0001 and Bonferroni’s multiple comparison test * p < 0.05, ** p < 0.01, *** p < 0.001 compared to mock stimulated, ††† p < 0.0001 daggers comparing stimulated with GluN2B stimulated). The translocation and synaptic localization of CFP-CaMKIIα is impaired both when compared to unstimulated and compared to GluN2B stimulated for RQ-SD and GluN2A. Synaptic CFP puncta were defined as those with a minimum three pixel overlap with PSD-95. Scale bar 10 µm.  146  4.5 Discussion  Central to the induction of experimental long term potentiation (LTP) is the influx of Ca2+ through NMDA receptor channels leading to an elevation in spine Ca2+ concentration (Lynch et al., 1983). Most forms of experimentally induced NMDA receptor mediated LTP requires the activation of calcium/calmodulin-dependent protein kinase II (CaMKII) (Lisman et al., 2002). Both global cell stimulation (Shen and Meyer, 1999) with glutamate and glycine and chemical LTP (cLTP) protocols that saturates the neuronal synaptic microenvironment with GluN1 subunit co-agonist glycine and the removal of Mg2+ block (Sharma et al., 2006) induces CaMKII aggregation and translocation from the dendritic shaft to spines and excitatory synapses. Here, we show that CaMKII translocation after bath glutamate and glycine and cLTP stimulation is lost in GluN2B -/- hippocampal neurons while the translocation response to local glutamate and glycine stimulation is intact. This finding that CaMKII can translocate to excitatory synapses in the absence of GluN2B is surprising and the precise mechanism for this propagating accumulation (Rose et al., 2009) remains to be determined. However, GluN2A containing NMDA receptors are still present and are electrophysiologically active and can mediate membrane depolarization which may be sufficient to activate L-type Ca2+ channels and trigger propagating calcium spikes distally along the dendrite (Cai et al., 2004; Spruston, 2008). While it is difficult to reconcile the targeting to CaMKII to excitatory synapses in the absence of the cytoplasmic tail of GluN2B, densin-180 is an abundant post-synaptic density protein that binds to CaMKII independent of CaMKII autophosphorylation in a non-competitive manner with GluN2B (Apperson et al., 1996; Strack et al., 2000b) although autophosphorylation of CaMKII vastly increases the affinity between  147  CaMKII and densin-180 that can form ternary complexes with α-actinin (Walikonis et al., 2001) which may facilitate the slow phase of the spreading translocation of CaMKII (Rose et al., 2009). It has recently been shown that densin-180 also binds to Cav1 L-type voltage gated calcium channels (VGCC) and localizes them to dendritic spines and that activated CaMKII binding to densin-180 facilitates Cav1.3 Ca2+ currents during repetitive but not sustained depolarizing stimuli (Jenkins et al., 2010). In a molecular replacement strategy, we were fortunate to be able to rescue GluN2B -/hippocampal neurons in culture with YFP-tagged wild-type GluN2B to levels indistinguishable from GluN2B +/+ neurons. While the identity of the transfected construct and stimulation state of the neurons on each coverslip was blinded when images of neurons were being acquired, the selection of cells to be imaged was based on qualitative assessment of the expression level of the YFP channel. Cells were chosen if the YFP puncta density and intensity were within the range comparable to the YFP-GluN2B construct, which in turn resembles the endogenous expression level of GluN2B in wild-type neurons. In the post hoc analysis, there was no significant difference in the puncta density and average puncta size of control PSD-95 immunostaining and no differences in the puncta density, synaptic localization, or fluorescent intensity of the YFPtagged GluN2 construct expression. Therefore, a variety of GluN2 constructs including a double R1300Q/S1303D CaMKII binding GluN2B mutant, wild-type GluN2A, and cytoplasmic tail swap chimeras were also able to restore synaptic NMDA receptors to levels comparable to the wild-type to GluN2B -/- neurons. While cells rescued with the YFP-GluN2B construct regained CaMKII translocation responses to both bath stimulation and cLTP induction, as expected the GluN2B RQ-SD CaMKII binding mutant had impaired CaMKII aggregation in response to both bath and cLTP induction 148  and most of the aggregates that did form did not localize properly to excitatory synapses or GluN2 clusters. A recent report showed that while the phosphomimetic S1303D mutation inhibited CaMKII binding in vitro, the high availability of adenosine triphosphate (ATP) in vivo should promote S1303 phosphorylation thus inhibiting GluN2B/CaMKII interactions. Paradoxically, ATP appears to promote CaMKII binding to GluN2B through a net positive effect from a number of different mechanisms (O'Leary et al., 2011). Specifically, while ATP promotes S1303 phosphorylation (Omkumar et al., 1996; O'Leary et al., 2011) and CaMKII T305/6 autophosphorylation (Colbran and Soderling, 1990) which increases its dissociation rate from GluN2B, ATP also enhances CaMKII T286 autophosphorylation which when fully phosphorylated, the holoenzyme becomes constitutively active. This may partially explain the formation of aggregates in cells rescued with the GluN2B RS-QD mutant. If sufficient Ca2+ can enter the cell through the functioning pore of GluN2B RS-QD, contingent on sufficient calcium/calmodulin, some CaMKII may be fully autophosphorylated at T286 even without binding to GluN2B that locks CaMKII into a persistent active Ca2+/calmodulin trapping state and suppression of T305/6 autophosphorylation (Bayer et al., 2001). Likewise, there was a small degree of CaMKII aggregation in stimulated cells rescued by the GluN2B 2AT chimera. In this case, there may be sufficient Ca2+ entry through the GluN2B pore to fully autophosphorylate some CaMKII despite the lack of elements in the GluN2B tail that promotes CaMKII association at synaptic sites. Conversely, signalling through NMDA receptors results in a rapid and reversible calcium-dependent decrease in intracellular pH (Irwin et al., 1994) which may promote CaMKII self-association (Hudmon et al., 1996). CaMKII transfected into heterologous cells lacking neuron-specific proteins can still form intracellular freely diffusible annular aggregates of 149  CaMKII within 5 minutes when treated with ionomycin (Hudmon et al., 2005), a calciumpermeable ionophore that additionally induces cell cytoplasmic acidification (Llopis et al., 1998). It is possible that the stimulation protocols employed here in which NMDA receptors were activated for 3 and 10 minutes, for bath and cLTP induction respectively, may be sufficient to induce pH mediated CFP-CaMKIIα aggregation. Indeed, the longer NMDA receptor activation period during the cLTP stimulation over bath stimulation may account for the impaired, but significantly higher levels of CFP-CaMKIIα aggregation in response to stimulation compared with mock stimulation seen in cells rescued with GluN2B RS-QD, GluN2A, and GluN2B 2AT compared to only the GluN2B RS-QD rescue in response to bath stimulation. Despite these alternate modes of CaMKII aggregation, our results support the idea that the differences in permeation properties between GluN2B and GluN2A play a relatively minor role in the induction of CaMKII aggregation and that the cytoplasmic tail of GluN2B is the major factor in controlling the synaptic accumulation of CaMKII, localizing it to optimal subcellular locations to sense incoming Ca2+ flux and interact with its molecular targets. Since most forms of experimentally induced NMDA receptor-mediated LTP requires functional CaMKII, CaMKII translocation may be a hallmark of LTP induction. While bath application of NMDA to hippocampal slices produces long-term synaptic depression (LTD) (Lee et al., 1998) that like LTP is also likely dependent on CaMKII (Lee et al., 1998; Margrie et al., 1998). In our bath stimulation experiments, the bath application of glutamate and glycine stimulate both synaptic and extra-synaptic receptor populations may result in the production of LTD (Rusakov et al., 2004). However, it is unclear whether CaMKII translocates after the induction of global LTD. The CaMKII T286D phosphomimetic constitutively active mutant potentiates synaptic strength if T305/T306 are not phosphorylated, but depresses synaptic 150  strength if they are phosphorylated (Pi et al., 2010). Since T305/T306 phosphorylation inhibits synaptic accumulation of CaMKII (Shen et al., 2000), these results indirectly suggest that LTD may not involve the aggregation of CaMKII. Our results are consistent with some recently published work, particularly if CaMKII translocation can be used as a surrogate marker for the induction of LTP. In hippocampal slice cultures, the RNA interference knockdown of GluN2B or pharmacological treatment with Ro256981 blocks high frequency stimulation induction of LTP which was not rescued by the overexpression of GluN2A. Additionally, replacement of GluN2B with a chimeric GluN2B construct with a GluN2A tail fails to rescue LTP but conversely, a GluN2A chimera with a GluN2B tail was able to rescue LTP induction (Foster et al., 2010). While our study was in progress, a GluN2B L1298A/R1300Q double point mutant CaMKII binding deficient knock in mouse was developed that showed impaired LTP and long-term recall of Morris water maze memory training (Halt et al., 2012). Consistent with our results seen with the molecular replacement of GluN2B with GluN2B RS-QD, hippocampal cultures prepared from the GluN2B L1298/R1300Q knock in mouse also showed impaired CaMKII translocation to synapses in response to glutamate treatment. cLTP treatment also failed to translocate CaMKII to synapses in forebrain slices from these GluN2B knockin mice, and they were deficient in stimulated AMPA receptor GluA1 Ser831 phosphorylation. Taken together, these results suggest CaMKII interaction with GluN2B is necessary for multiple aspects of LTP and memory consolidation and for CaMKII translocation to synapses in some but not all activity paradigms, as local dendritic stimulation and associated calcium waves can circumvent this requirement.  151  Chapter 5: Conclusion  5.1 Summary of Findings and Future Directions  5.1.1 NMDA Receptors Mediate Synaptic Competition in Culture  Synaptic activity mediated by NMDA receptors sculpts wiring of the nervous system, regulating functional and structural connectivity (Constantine-Paton et al., 1990; Katz and Shatz, 1996). While this is studied most intensely in the developing visual system (Huberman et al., 2008), here we developed a cell culture paradigm of NMDA receptor-dependent synaptic competition using fluorescent protein labelled GluN1 -/- (KO) and GluN1 +/+ (WT) hippocampal mouse neurons in culture. Unexpectedly, the number of synapses formed onto KO cells in pure KO cultures was not different than the number of synapses formed onto WT cells in pure WT cultures. Furthermore, we also did not observe differences in synapse density between KO and WT cells when they were grown together in equal proportion. It is only when KO and WT cells are grown in defined ratios that activity-dependent differences in synapse density was observed. Specifically, only when sparse NMDA receptor competent cells (WT) are grown with a majority of KO cells do we observed increased synapse density on WT neurons at DIV 14. Although altered network activity in these cultures could affect differential gene expression resulting in enhanced synapse density, we propose a model also involving retrograde factors but that are generated by NMDA receptor-competent synapses that is detected by input neurons that summate the total retrograde signal and compare that with a target range. Only if the total retrograde signal does not reach a threshold following initial synapse development does further  152  synapse development or reduction in the elimination of synapses occur, contingent on the availability of NMDA receptor competent cells to provide a ‘reward’ signal. Cell culture models testing rules and mechanisms of synaptic competition in defined circuits may contribute to a better understanding of the role of activity in sculpting nervous system connectivity during development. Targeted genetic modifications could be further combined with microfluidic chambers in which subsets of neurons can be pharmacologically manipulated (Taylor et al., 2010) to aid in defining signalling pathways and temporal parameters mediating activity-regulated neuronal connectivity. Unresolved issues include the identification of the biochemical mechanism behind the increased synapse density onto minority WT neurons when grown with a majority of KO neurons. One approach to explore possible mechanisms would be to visualize and quantitatively compare, both early during the peak of synaptogenesis and at later time points when synapses are more mature, the expression levels and localization of cell surface synaptic organizing complexes between these two cell populations such as neuroligins, LRRTMs, TrKC, NGLs, EphBs, ephrins, SynCAMs, NCAM, and cadherins. Another avenue of inquiry would be to resolve whether the increased synapse density is due primarily to an increased rate of formation or through enhanced stabilization of synapses through decreased rates of synapse elimination through time-lapse imaging.  153  5.1.2 Glutamate Binding to GluN2B Controls Surface Trafficking of NMDA Receptors  Ionotropic glutamate receptors are multimeric protein complexes that require quality control mechanisms to ensure correct assembly before exit from the endoplasmic reticulum (ER). In addition to ER retention signals that are masked upon receptor subunit multimerization, additional studies have suggested that some ionotropic glutamate receptors undergo further quality control inspection for ligand binding prior to release from the ER. Here, we used a range of GluN2B ligand binding point mutants with reduced glutamate efficacies to assess the role of glutamate binding in surface trafficking of NMDA receptors. In both heterologous cells coexpressing GluN1 and in primary neuron cultures, the surface expression of GluN2B mutants correlated with their apparent affinity for glutamate. Similar surface expression levels were found in the presence of dominant negative dynamin to inhibit endocytosis, suggesting that glutamate binding is required for forward trafficking of assembled NMDA receptors to the cell surface as opposed to enhanced endocytosis of ligand binding deficient receptors. Co-expression of wild-type GluN2B did not enhance surface expression of the mutant GluN2B suggesting that the ligand binding sites on both GluN2B subunits in the NMDA receptor tetramer must be occupied for export and that this is limited by the least avid ligand binding site. Prior studies on AMPA and kainate receptors were reported in a more binary fashion in the sense that severe ligand binding mutants were studied and found to be strongly and similarly impaired in surface expression. Here, we used a panel of GluN2B ligand mutants covering a spectrum of glutamate efficacies and we observed a correlation between glutamate efficacy and surface expression both in heterologous cells and in primary neurons. These results suggest that the rate of receptor release for forward trafficking is dependent on the rate of ligand binding and  154  that ligand binding appears to control forward trafficking for all ionotropic glutamate receptor classes; glutamate binding to AMPA and kainate receptors, and glycine binding to GluN1, and now glutamate binding to GluN2B. However, further investigation could be directed at confirming that the mutant glutamate binding subunits associate with either co-transfected GluN1 in heterologous cells and endogenous GluN1 in primary mouse neurons as efficiently as the wild-type GluN2B subunit by native gel electrophoresis, quantitative co-immunoprecipitation, or by Förster/Fluorescence resonance energy transfer (FRET). Additionally, the residues that were chosen for mutational analysis are conserved across GluN2A, C, and D subunits and confirmation of the generalizability of these findings could be extended to the other GluN2 subunits. Interestingly, GluN3 subunits when co-expressed with GluN1assemble into excitatory glycine receptors that are unaffected by glutamate or NMDA, and is inhibited by D-serine, a co-activator of conventional NMDA receptors (Chatterton et al., 2002). Similar mutations could be made to the glycine binding site of GluN3 subunits to determine if the requirements for glycine binding to GluN1 subunits (Kenny et al., 2009) is also generalizable to GluN3 concomitant with partial rescue by cell-permeable dichlorokynurenic acid, a cell permeable competitive antagonist of the glycine site of GluN1.  5.1.3 Intracellular Determinants of GluN2 Control Synaptic Recruitment of CaMKIIα  Both global cell stimulation through NMDA receptors and selective activation of synaptic NMDA receptors through a chemical LTP induction protocol enhances synaptic levels of CaMKII by inducing aggregation and translocation from the dendritic shaft to spines. We find 155  here that the translocation of CaMKII is lost in GluN2B -/- hippocampal neurons in response to these stimulation protocols, but the spreading translocation of CaMKII in response to local stimulation remains intact. The translocation of CaMKII is restored upon rescue of GluN2B -/neurons with YFP tagged GluN2B wild-type (WT) construct and by a chimeric GluN2A with a GluN2B tail, but not in cells rescued with a CaMKII binding deficient double point mutant, wildtype GluN2A, nor a chimeric GluN2B with a GluN2A tail. These findings provide further evidence for the importance of elements in the cytoplasmic tail of GluN2B that facilitate the optimal activation of CaMKII in response to NMDA receptor mediated activity. While the loss of CaMKII aggregation and translocation in GluN2B -/- neurons was expected as these cells have highly impaired localization of the obligate NMDA receptor subunit GluN1 to synapses, the intact CaMKII translocation response to local stimulation was surprising. Since this effect requires functional voltage gated calcium channels (VGCC) in the absence of GluN2B containing NMDA receptors, further investigations could be pursued to determine the importance of the source of Ca2+ for the activation of CaMKII. For instance, similar experiments could be performed with the GluN1 N598R mutant that is impermeable to calcium ions (Behe et al., 1995) in conjunction with pharmacological strategies such as the inhibition of VGCCs with nifedipine and blockade of intracellular calcium release with ryanodine. Additionally, under conditions where there was only partial CaMKII aggregation, these aggregates further fail to localize properly to excitatory synapses. 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