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Functional regulation of N-methyl-D-aspartate receptor subtypes and their involvement in hippocampal… Vasuta, Oana Cristina 2009

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  FUNCTIONAL REGULATION OF NMDA RECEPTOR SUBTYPES AND THEIR INVOLVEMENT IN HIPPOCAMPAL PLASTICITY   by  Oana Cristina Vasuta   BSc, University of Iasi, 2002 MSc, University of Bucharest, 2004     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)    December 2009  © Oana Cristina Vasuta 2009  ii  ABSTRACT   Regulation of NMDAR activity by desensitization is important in physiological and pathological states. We previously reported that desensitization decreases during hippocampal neuronal development, correlating with NMDAR composition, synaptic localization and association with PSD-95. To determine if PSD-95-induced changes in NMDAR desensitization occur because of direct binding to NR2 subunits or due to recruitment of regulatory proteins, we tested the effects of various PSD-95 constructs on NMDAR currents in HEK293 cells and neurons.  In HEK cells, wt PSD-95 significantly reduced wt NMDAR desensitization without altering currents of NMDARs containing NR2A-S1462A, a mutation that abolishes PSD-95 binding.  Moreover, PDZ1-2 domain was sufficient for this effect in neurons with low endogenous PSD-95 levels.  Moreover, other PSD-95 family members with highly homologous PDZ1-2 domains significantly reduced NMDAR desensitization.  In mature neurons, disruption of PSD-95/NMDAR interaction through PKC activation, or through interference peptides, increased desensitization to levels found in immature neurons.   We conclude that direct binding of PSD-95 increases stability of NMDAR responses to agonist exposure. Desensitization is a property that shapes synaptic responses, and modulates the calcium signal mediated by the two predominant NMDARs subtypes in hippocampus, with possible consequences for their functioning. Further, we examined the involvement of NR2 subtypes in synaptic plasticity in hippocampal dentate gyrus of juvenile mice. Exercise was used as a means to alter expression of the NR2 subunits in this region. We compared two groups of animals: Controls, which were housed in conditions of minimal enrichment, and iii  Runners, which had access to an exercise wheel. NMDAR-dependent LTP expression was significantly greater in Runners than in Controls; in the presence of NR2B subunit antagonists, it was significantly reduced in both groups. NR2A subunit antagonist blocked LTP in slices from Runners and produced a slight depression in Control animals. LTD could not be prevented by either of the NR2B specific antagonists. Strikingly, eliminating NR2A subunit-containing receptor activity prevented LTD in Runners, but not in Control animals. Overall, these results indicate that interplay between subtype, subcellular localization and size of NMDAR subpopulations accounts for their diverse role in synaptic plasticity induction, and that exercise increases the contribution of NR2A to plasticity.           iv  CONTENTS ABSTRACT ............................................................................................................................. ii CONTENTS ............................................................................................................................ iv LIST OF FIGURES .................................................................................................................. vii LIST OF ABBREVIATIONS ...................................................................................................... viii ACKNOWLEDGEMENTS ......................................................................................................... xi DEDICATION .........................................................................................................................xii CO-AUTHORSHIP STATEMENT .............................................................................................. xiii 1 INTRODUCTION .............................................................................................................. 1 1.1 The hippocampal NMDA receptor family ............................................................................1 1.1.1 General overview ................................................................................................................................... 1 1.1.2 Structure and assembly of NMDA receptor complexes ......................................................................... 3 1.1.2.1 Structure....................................................................................................................................... 3 1.1.2.2 Assembly ...................................................................................................................................... 5 1.1.3 Expression of hippocampal NMDA receptors ........................................................................................ 6 1.1.3.1 Regional variability of expression ............................................................................................... 10 1.1.3.2 Temporal variability of expression ............................................................................................. 13 1.1.4 Properties ............................................................................................................................................ 15 1.1.5 Cytoskeletal and signalling proteins associated with NMDARs ........................................................... 19 1.1.5.1 PSD-95 complex and other MAGUKs .......................................................................................... 19 1.1.5.2 Other interacting molecules ....................................................................................................... 22 1.1.6 NMDAR function in cellular physiology ............................................................................................... 23 1.1.6.1 Fast excitatory transmission ....................................................................................................... 23 1.1.6.2 Structural development of neural processes ............................................................................. 26 1.1.6.3 Synaptic plasticity ....................................................................................................................... 27 1.1.6.4 Neuronal survival and excitotoxicity .......................................................................................... 28 1.1.6.5 Insights on the NMDAR function from NMDAR subunit mutant mice ....................................... 30 1.2 NMDAR desensitization ................................................................................................... 31 1.2.1 Definition and classification ................................................................................................................. 31 1.2.2 Structural determinants of NMDAR desensitization ........................................................................... 34 1.2.2.1 Structural determinants at the N-terminal domain ................................................................... 35 1.2.2.2 Structural determinants at the C-terminal domain .................................................................... 36 1.2.3 Proved and hypothesised roles for NMDAR desensitization ............................................................... 37 1.3 NMDAR-dependent synaptic plasticity in hippocampus .................................................... 39 1.3.1 Definition and forms of synaptic plasticity .......................................................................................... 39 1.3.1.1 Transient and short-term forms of plasticity ............................................................................. 40 1.3.1.2 Long-term plasticity: early and late LTP/LTD ............................................................................. 42 v  1.3.1.3 Metaplasticity ............................................................................................................................. 43 1.3.1.4 Homeostatic plasticity ................................................................................................................ 43 1.3.2 Induction .............................................................................................................................................. 44 1.3.2.1 Classical patterns ........................................................................................................................ 45 1.3.2.2 Spike-timing protocols ............................................................................................................... 46 1.3.2.3 Calcium signalling through NMDARs in plasticity induction ...................................................... 46 1.3.3 Expression ............................................................................................................................................ 47 1.3.3.1 Presynaptic locus of expression ................................................................................................. 48 1.3.3.2 Postsynaptic locus of expression ................................................................................................ 48 1.3.4 Particularities of plasticity in hippocampal dentate gyrus ................................................................... 50 1.4 Theme and hypothesis ..................................................................................................... 53 1.5 Bibliography .................................................................................................................... 55 2 NMDA Receptor Desensitization Regulated by Direct Binding to PDZ1-2 Domains of PSD- 95............ ............................................................................................................................ 73 2.1 Materials and methods .................................................................................................... 75 2.1.1 Primary neuronal cultures and transfection ........................................................................................ 75 2.1.2 HEK293 cell culture and transfection................................................................................................... 75 2.1.3 Drug treatments .................................................................................................................................. 76 2.1.4 Electrophysiology ................................................................................................................................. 76 2.1.5 Co-immunoprecipitation and western blot analysis ............................................................................ 78 2.1.6 Materials .............................................................................................................................................. 79 2.1.7 Data analysis ........................................................................................................................................ 80 2.2 Results ............................................................................................................................. 80 2.2.1 PSD-95 regulates NMDAR glycine-independent desensitization ......................................................... 80 2.2.2 PDZ1-2 domains of PSD-95 are sufficient to alter NMDAR desensitization ......................................... 83 2.2.3 PKC uncouples PSD-95 from NMDARs and increases receptor desensitization in mature hippocampal neurons 87 2.2.4 PKC increases NMDAR desensitization by uncoupling PSD-95 from recombinant NMDA receptors expressed in HEK cells ........................................................................................................................................ 90 2.3 Discussion ........................................................................................................................ 91 2.3.1 Role of PSD-95 direct binding to NMDARs on current desensitization................................................ 91 2.3.2 Effects of PSD-95/NMDAR interaction on neuronal NMDAR function ................................................ 94 2.3.3 Phorbol ester-induced PKC activation and regulation of neuronal NMDAR function ......................... 95 2.3.4 PSD-95/NMDAR association may regulate synaptic plasticity and excitotoxicity ............................... 96 2.4 Bibliography .................................................................................................................. 106 3 Effects of Exercise on NMDA Receptor Subunit Contributions to Bidirectional Synaptic Plasticity in the Mouse Dentate Gyrus ................................................................................ 113 3.1 Materials and methods .................................................................................................. 115 3.1.1 Subjects .............................................................................................................................................. 115 vi  3.1.2 Slice preparation ................................................................................................................................ 115 3.1.3 Field recordings and stimulation procedures .................................................................................... 116 3.1.4 Data analyses ..................................................................................................................................... 117 3.2 Results ........................................................................................................................... 117 3.2.1 Voluntary exercise enhances NMDA receptor-dependent LTP induction in the DG ......................... 117 3.2.2 Exercise alters the contribution of NR2 subunits to LTP.................................................................... 118 3.2.3 LTD in the DG is NMDA receptor-dependent in both Control and Runner animals .......................... 119 3.2.4 Exercise enhances the contribution of NR2A, but not NR2B subunits to LTD ................................... 120 3.3 Discussion ...................................................................................................................... 122 3.4 Bibliography .................................................................................................................. 136 4 Discussion and Conclusions ........................................................................................ 141 4.1 The role of NR2 subpopulations of NMDARs in desensitization and plasticity .................. 141 4.1.1 The role of NMDAR desensitization in plasticity mechanisms ........................................................... 142 4.1.2 Charge transfer, NR2 subunits and plasticity induction mechanisms ............................................... 144 4.1.3 Synaptic plasticity induced through the activation of basal-level versus modified NMDARs ........... 146 4.1.3.1 The involvement of the NR2 subunits in LTP ........................................................................... 148 4.1.3.2 The involvement of the NR2 subunits in LTD ........................................................................... 149 4.1.3.3 NR2A subunit-dependent plasticity is enhanced by exercise .................................................. 150 4.2 Role of NMDAR-mediated excitability and signalling in hippocampal neurophysiology .... 151 4.2.1 The role of NMDARs in normal hippocampal physiology .................................................................. 152 4.2.1.1 Hippocampal excitability .......................................................................................................... 152 4.2.1.2 Oscillatory activity .................................................................................................................... 153 4.2.1.3 NMDAR – mediated plasticity and its role in memory formation and learning ....................... 155 4.2.1.4 Hippocampal place cells, NMDARs, and memory formation ................................................... 157 4.2.2 The role of NMDARs in hippocampal pathology ................................................................................ 158 4.3 Final conclusions and future directions ........................................................................... 161 4.1 Bibliography .................................................................................................................. 164 APPENDIX.......................................................................................................................... 185 vii   LIST OF FIGURES FIGURE 2.1 EXPRESSION OF PSD-95 REGULATES GLYCINE-INDEPENDENT DESENSITIZATION OF RECOMBINANT NMDARS.....................................................................................................................97 FIGURE 2.2 OVEREPRESSION OF PSD-95 IN IMMATURE NEURONS REDUCES GLYCINE- INDEPENDENT DESENSITIZATION...........................................................................................................98 FIGURE 2.3 PKC ACTIVATION UNCOUPLES NR2A FROM PSD-95 IN MATURE NEURONS..........99 FIGURE 2.4 PKC ACTIVATION ALTERS DESENSITIZATION IN MATURE NEURONS.......................................................................................................................................................100 FIGURE 2.5 PKC ACTIVATION ALTERS DESENSITIZAT ION OF RECOMBINANT NMDA RECEPTORS IN HEK CELLS THAT EXPRESS PSD-95................................101 FIGURE 3.1 SELECTIVE EFFECT OF NMDA RECEPTOR SUBTYPES BLOCKADE ON LTP IN CONTROL VERSUS RUNNER  ANIMALS............................................................................126 FIGURE 3.2 SELECTIVE EFFECT OF NMDA RECEPTOR SUBTYPES BLOCKADE ON LTD IN CONTROL ANIMALS VERSUS RUNNERS..........................................................................127 FIGURE 3.3 LTD IN NR2A KNOCKOUT ANIMALS................................................................................128 FIGURE 3.4 THE EFFECT OF DL-TBOA ON PLASTICITY INDUCTION.............................................129 FIGURE 3.5 SUMMARY OF THE EFFECTS OF EXERCISE IN CONTROL AND RUNNER GROUPS FOLLOWING ON LTP AND LTD....................................................................130 FIGURE 4. 1 SUMMERY OF PLASTICITY DATA...................................................................................193  viii  LIST OF ABBREVIATIONS • 2-BP, 2-bromo-palmitate • ACSF, Artificial cerebrospinal fluid • AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid • ANOVA, analysis of variance • AP2, clathrin adaptor protein • GRIP, glutamate receptor interacting protein • ABP, AMPAR-binding protein • PICK, protein interacting with C kinase 1 • APV, 2-amino-5-phosphonovaleric acid • ATD, amino-terminal domain • BAPTA, 1,2-bisethane-N,N,N',N'-tetraacetic acid • BDNF, brain-derived neurotrophic factor • BSS, balanced salt solution • CA1, cornu ammonis region 1 • CA3, cornu ammonis region 3 • CamKII, calmodulin-dependent kinase II • cDNA, complementary deoxyribonucleic acid • CNS, Central nervous system • GTP, guanosine triphosphate • HFS, high-frequency stimulation • NSF, N-ethylmaleimide-sensitive factor • LFS, low frequency stimulation • DG, dentate gyrus • CREB, cAMP response element binding ix  • DIV, days in vitro • DMSO, dimethyl sulfoxide • DNQX, 6,7-dinitroquinoxaline-2,3-dione • EDTA, ethylenediaminetetraacetic acid • EPSC, excitatory postsynaptic current • FBS, fetal bovine serum • fEPSP, field excitatory postsynaptic potential • GABA, γ-aminobutyric acid • GAP43, growth associated protein-43 • GFP, green fluorescent protein • GK, guanylate-like kinase • GluR, glutamate receptor • HBSS, Hank’s balanced salt solution • HEK293, human embryonic kidney 293 • iGluRs, ionotropic glutamate receptors • IP, immunoprecipitation • Ip, peak current • Iss, steady-state current • KO, knock-out • LTD, Long-term depression • LTP, Long-term potentiation • MAGUK, membrane-associated guanylate kinase • MEM, minimum essential medium • mEPSC, miniature excitatory postsynaptic current • mGluR, metabotropic glutamate receptor • mRNA, messenger ribonucleic acid x  • NBM, neurobasal media • NMDA, N-methyl-D-aspartate • NMDAR, N-methyl-D-aspartate receptor • nNOS, neuronal nitric oxide synthase • PBS, phosphate-buffered saline • PDL, poly-D lysine • PDZ, PSD-95/Discs large/zona occludens-1 • PKC, protein kinase C • PSD, postsynaptic density • PSD-95, post-synaptic density 95 kDa • lkSAP, synapse-associated protein • SH3, Src homology 3 • shRNA, short hairpin RNA • TPA, 12-O-tetradecanoylphorbol-13-acetate • TTX, tetrodotoxin • VH, holding potential • wt, wild-type        xi  ACKNOWLEDGEMENTS  I am grateful to my PhD supervisor Dr. Lynn Raymond for invaluable help and guidance during this work. I have learned from her how to be precise in communication, critical while judging scientific data, careful and thorough in experimentation. I am highly indebted to her for giving me the opportunity to continue my PhD program in her laboratory, and I am happy I have spent a few years with her. Many thanks go to my PhD co-supervisor Dr. Brian Christie, who guided my first project and provided valuable advice and help during my first couple of years in the program. The members of Raymond and Christie laboratory helped with comments and suggestions on technical issues, and I am particularly grateful to Rachel James, Lily Zhang, Ashley George, Lavan Sornarajah and Austen Milnerwood. The committee members, Dr. Ann Marie Craig, Dr. Brian MacVicar and Dr. Anthony Phillips offered advice and insightful comments during meetings. I was lucky to have their expert guiding through the program. Finally, I am thankful to my husband Victor for his patience and for morally supporting me throughout.           xii  DEDICATION     I dedicate this work to my son Ilie                 xiii  CO-AUTHORSHIP STATEMENT I am a co-first author of the manuscript reproduced in Chapter Two, entitled “NMDA receptor desensitization regulated by direct binding to PDZ1-2 domains of PSD- 95”, and published in Journal of Neurophysiology in 2008. As a co-author, I (CV) have contributed equally to Lavan Sornarajah (LS) to the work towards this publication. More specifically, I have performed the research and have taken part in identification of research programs designed to test:  1) the effect of PSD-95 on recombinant NR2A-containing NMDARs and NR2B- NMDARs expressed in HEK cells (Figure 2.1 a-c, CV and LS); 2) the level of desensitization in the NR2A S1462A – containing recombinant NMDARs, when they were coexpressed with PSD-95 (Figure 2.1d, CV); 3) the effect of other MAGUK-family members, like SAP102 and PSD-93 on recombinant NMDAR desensitization (part of Figure 2.1e, CV); 4) the effect of PSD-95 overexpression in immature neurons (Figure 2.2, LS and CV) 5) the effect of PKC (activated by TPA treatments) on recombinant NMDAR desensitization (Figure 2.5, CV). I have analysed the data obtained in the above-mentioned projects, and have participated, together with LS, in manuscript preparation. I have not participated in the design of the experiments presented in the Figure 2.3 and Figure 2.4, nor have I taken part in performing the research or analysing the data. The authors of this work have been Lily Zhang (Figure 2.3) and Lavan Sornarajah (Figure 2.4). For the second manuscript, reproduced in Chapter three and entitled “Effects of exercise on NMDA receptor subunit contributions to bidirectional synaptic plasticity in the mouse dentate gyrus” I have performed the research, have analysed the data, and have xiv  taken part in identification of research programs designed to test all hypotheses except for the experiments presented in Figure 3.3 and Figure 3.4. I have prepared Figures 3.1 and 3.2 based on data obtained primarily by myself, but also by Rachel James and Charlotte Count. I have summarised the results in Figures 3.5, 4.1 and 4.2, and have prepared the manuscript together with my research supervisor, Dr. Brian Christie.             1  Chapter One  1 INTRODUCTION 1.1 The hippocampal NMDA receptor family 1.1.1  General overview Excitation in the brain is mediated by the neurotransmitter glutamate, an amino acid found in abundance in the brain (as it is in all animal tissues), where it takes part in cellular metabolic processes, apart from its role in synaptic transmission. Excitation may also be mediated by another amino acid, aspartate, acting on N- methyl-D aspartate receptors (NMDARs) only (which have a weak affinity for it); released at hippocampal synapses (Szerb, 1988, Nadler et al., 1990) this amino acid mediates a signal that is dependent on the level of synaptic activity. Glutamate, released from the presynaptic axon varicosities onto soma and dendrites, depolarises hippocampal neurons (Biscoe and Straughan, 1966). The evidence for this role is the immunocytochemical data showing staining of the presynaptic sites at asymmetrical (excitatory) synapses, by antibodies raised against glutamate. This role is mediated by the action of glutamate on ionotropic receptors; upon binding of the transmitter, these allow sodium, potassium and calcium fluxes, which modify the membrane potential of the postsynaptic neuron. Apart from depolarisation, glutamate contributes to neuronal signalling through the activation of metabotropic receptors. 2  The ionotropic glutamate receptors are comprised of three classes: the NMDA receptors (NMDARs), α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (AMPARs), and kainate receptors. These mediate currents with strikingly different properties, which makes them play different roles in synaptic and cellular function. The NMDARs differ from the other ionotropic glutamate receptors in that they mediate a slower mode of transmission, and exhibit relatively high calcium permeability as well as voltage-dependent block by Mg2+ in the pore (McBain and Mayer, 1994, Dingledine et al., 1999). The latter properties underlie NMDA receptors’ role as coincident detectors of activity in the two components of the synapses, and are involved in the induction of both synaptic plasticity, and in events related to neuronal survival and death (Bliss and Collingridge, 1993, Cull-Candy et al., 2001, Hardingham and Bading, 2003). Ever since they were first described, in the mid 20th century, evidence regarding NMDA receptors’ properties, patterns of expression, and physiological importance has increased enormously. However this information cannot yet precisely explain the various, complex and sometimes apparently opposing, roles of this receptor population. The present understanding about the structure, expression patterns, properties and roles of the NMDA receptors is owing to the development of NMDAR specific agonists and antagonists, cloning of the NMDAR subunits (cDNA cloning techniques), knock-in and knock-out studies, and development of antibodies against NMDAR subunits. A series of synthetic agonists and antagonists for this receptor have been produced, and they provide the opportunity to gain detailed understanding of the role the NMDA 3  receptors play in brain function. General antagonists such as 2-amino-phosphonovaleric acid (APV), memantine, and MK801, are some of the most extensively used in studies characterizing NMDAR structure, properties and function. Pharmacological agents like haloperidol (Vicini et al., 1998), or ifenprodil (Williams, 1993) bind with higher affinity to NR2B-containing receptors, while NVP-AA007 shows a preference for NR2A-containing NMDARs.  1.1.2  Structure and assembly of NMDA receptor complexes 1.1.2.1 Structure Each subunit in the tetrameric NMDAR complex is a protein macromolecule, with topology consisting of a large amino-terminal domain (ATD) (~50 kDa), a carboxy- terminal (C-terminal) domain, and four hydrophobic domains (M1-M4). M2 forms a re- entrant loop that lines the pore of the receptor, similar to the P-loop of voltage-gated channels. ATD is a polypeptide made of ~ 400 amino acids; that which is part of the NR1 subunit forms dimers in solution. It was determined that zinc (Fayyazuddin et al., 2000, Low et al., 2000, Paoletti et al., 2000) and ifenprodil (Perin-Dureau et al., 2002) bind to the receptor though interaction with this domain, modulating gating properties. The ligand binds to a clamshell-shaped domain known as S1-S2, and composed of the segment situated between the ATD and M1 (S1 domain), along with the M3-M4 loop (S2 domain). The agonist-binding core for the NR1 has been excised from the pore region, expressed, and crystallised (Ivanovic et al., 1998, Furukawa and Gouaux, 2003), and its 4  examination proved that glycine binds in a cleft between the two lobes of the construct, and that the sequence of the S1S2 polypeptide is interrupted by two alpha-helixes that span the membrane. The S1S2 region in the NR2 subunits binds the agonist glutamate. The C- terminal region is large in the NMDAR subunits, and it interacts with various cytoskeletal proteins. There are seven different NR subunits that may contribute to formation of a functional receptor: NR1 (with eight variants – NR1-1a to NR1-4a, and NR1-1b to NR1-4b -- produced by alternative splicing, amongst which NR-1a is the most abundant), NR2 (NR2A-NR2D), and NR3 (NR3A-NR3B). The amino acid sequence identity between the NR1 and the NR2 subunits (the most predominant ones in the hippocampus), is about 27%, while the NR2 subunits share 40-50% identity between themselves (Kutsuwada et al., 1992, Monyer et al., 1992, Ishii et al., 1993). The seven different subunits are encoded by separate genes. NR1 is encoded by a single gene, but it undergoes transcriptional modifications resulting in eight isoforms. NR2 subunits are encoded by four different genes, so they further subdivide in four different types, known as NR2A-NR2D. These subunits undergo posttranslational modifications as well, like serine/threonine, or tyrosine phosporylation, glycosylation and proteolysis (McBain and Mayer, 1994). Elsewhere in the brain, the existence of the “orphan” delta subunits has been reported (Lomeli et al., 1993), but not in hippocampus. Also the  NR3 subunit  expression in hippocampus is limited to the period of embryogenesis (Ciabarra et al., 1995); very low levels are to be found in adult CA1 region.  5  1.1.2.2 Assembly NMDA receptors found in the hippocampal formation are heterotetrameric complexes formed of two obligatory subunits NR1, and two of the NR2 subunits (Benveniste and Mayer, 1991, Clements and Westbrook, 1991, Behe et al., 1995). The receptor is organised as a dimer of dimers, and the NR1-NR1-NR2-NR2 arrangement is due to interactions at the amino-terminal domain (Meddows et al., 2001, Furukawa et al., 2005) and at the S1 segment (Regalado et al., 2001, Furukawa et al., 2005). The assembly of the subunits into a functional tetrameric complex occurs before insertion in the plasma membrane. The precise rules that give specificity to the assembly, as well as the factors that influence and govern it are of high importance, as the final composition of the receptor population accounts for its roles. Specific sets of rules dictating the correct assembly and composition apply for the developing, and for the mature nervous system. Most hippocampal NMDARs contain either NR2A or NR2B subunits, evident from studies comparing certain characteristics of neuronal responses mediated by NMDARs, with the heterologously-expressed pure NR2A- or NR2B- containing populations. The characteristics that allow distinguishing among the two diheteromers are pharmacological sensitivity to certain drugs, agonist sensitivity, and the decay time of EPSCs. Diheteromers’ pattern of expression has been clarified in a series of studies (Kutsuwada et al., 1992; Monyer et al., 1994; Ohno et al., 1992; Tovar and Westbrook, 1999; Vicini et al., 1998). 6  The possibility of triheteromers’ existence came about when receptors with kinetics resembling NR2A diheteromers, but pharmacology (block by haloperidol) similar to the NR2B diheteromers, were discovered (Vicini et al., 1998). Other studies reported decay time constants mediated by NMDARs that were intermediate between pure NR2A of NR2B –mediated responses (Dalby and Mody, 2003). The possibility of NR2A and NR2B assembly in a triheteromeric complex was demonstrated in a study using heterologous systems of expression (Vicini et al., 1998; see also (Brimecombe et al., 1997; Chazot et al., 1994; Cheffings and Colquhoun, 2000; Chen et al., 1997), and their presence in neurons was found to constitute the majority of NMDARs in a study using immunocytochemical techniques (Luo et al., 1997). Triheteromers’ existence in vivo was not quite demonstrated (note: the existence of NR1/2B/2D in hippocampus in vivo was though (Pina-Crispo & Gibb 2002). A recent study suggests a relatively small proportion of NR1/NR2A/NR2B triheteromers in hippocampus, as 60–70% of NR2A and 70–85% of NR2B subunits were associated in NR1/NR2A or NR1/NR2B di-heteromeric complexes (Al-Hallaq et al., 2007).  1.1.3  Expression of hippocampal NMDA receptors NMDARs are expressed in pyramidal, granule and inhibitory hippocampal neurons, mainly in the somato-dendritic membrane, as proven by conventional immunocytochemical, and genetic techniques (Laurie and Seeburg, 1994b, Monyer et al., 1994). Here, they are concentrated at synaptic junctions: EM gold localization of NR1 and NR2A/B subunit studies reveal enrichment of the gold particles at asymetrical synapses 7  (Racca et al., 2000, Sassoe-Pognetto and Ottersen, 2000). There is a 100-fold higher density of ionotropic receptors at synaptic versus extrasynaptic membranes (Bekkers and Stevens, 1989, Nusser et al., 1998). Some functional studies also detected NMDARs presynaptically in hippocampus (Breukel et al., 1998) and in entorhinal cortex (Woodhall et al., 2001). At glutamatergic synapses, NMDARs colocalize with AMPARs; they can localize outside of synapses as well, at peri-and extrasynaptic sites. In the newly formed synapses, NMDAR  represent the only ionotropic glutamate receptor; these are known as “silent” synapses (Isaac et al., 1995, Nusser et al., 1998, Takumi et al., 1998, Petralia et al., 1999) because glutamate binding to NMDA receptors does not trigger ion influx in the absence of postsynaptic membrane depolarization, as a result of NMDAR block by Mgo2+, at resting membrane potentials. Based on the shape and size of the postsynaptic density (PSD), hippocampal synapses expressed in CA1/CA3 pyramidal neurons can be classified as perforated (where PSD has a complex shape and is relatively large), and nonperforated (with disk-like, rather small PSDs) (Harris et al., 1992). NMDARs seem to be enriched at perforated synapses, which makes these relatively more powerful in information transmission (Ganeshina et al., 2004). Additional physiological evidence points to a correlation between spine morphology and receptor distribution, and finds NMDARs to be enriched in thinner, filopodia-like spines (Matsuzaki et al., 2001). As for the precise location within the spines, NMDARs are likely to occupy a disk-like space near the center of the postsynaptic density (whereas other ionotropic glutamate receptors are evenly distributed throughout PSD (Racca et al., 2000). 8  Based on data obtained by immunogold techniques, it is believed that the NMDAR content at hippocampal synapses is linearly related to the diameter of the PSD (Takumi et al., 1999). Out of the 10s to 100s of glutamate receptors expressed on one spine (Kennedy and Ehlers, 2006), an average PSD contains approximately 20 NMDARs (Sheng and Hoogenraad, 2007); with respect to the total number of synaptically expressed receptors per cell, we shall consider that synapses occupy only approximately 1-2% of cell membranes (Rusakov et al., 1998). Within these limits, modifications in the size of NMDAR subpopulations might still occur. This could be a critical mechanism involved in cases where the amount of charge transferred through NMDARs is a key factor, as is the case for plasticity and excitotoxicity. Mechanisms of homeostatic regulation have been described (Turrigiano and Nelson, 2004), in which alterations in network activity levels lead to scaling up or down the NMDAR component in order to maintain neuronal functioning within normal limits. Chronic block of activity for example induces a general up-regulation of surface NMDARs (Rao and Craig, 1997). The expression of both NR2A and NR2B is enhanced; the synaptic NMDAR number is increased, as is the spine density (Mu et al., 2003) in a PKA-dependent manner (Crump et al., 2001). Excessive neuronal activity, on the other hand, promotes splicing of NR1 C2 cassette, slowing surface delivery of newly synthesized NMDARs and favoring endocytosis of NMDARs, to scale down synaptic activity (Mu et al., 2003). In disease states, a readjustment of NMDAR population sizes was also reported. For instance in addiction, an increased number of synaptic receptors was noted, as a result of acute 9  cocaine administration (Borgland et al., 2006), however, acute ethanol administration in hippocampus leads to selective internalization of NR2A, resulting in a pure NR2B population (Suvarna et al., 2005). In Alzheimer disease, amyloid beta protein triggers NMDAR internalization (Mattson, 2004), while in schizophrenia, neuregulin (genetically linked to the disease), also induces rapid internalization of receptors from the surface by a clathrin-dependent mechanism (Hahn et al., 2006). NMDAR losses were also reported to occur in putamen, in Huntington disease (Young et al., 1988), but this condition also displays increased signalling through NR2B type NMDAR in the striatum of presymptomatic transgenic Huntington’s Disease mice (Li et al., 2004). Receptor density at the synapse was suggested to be constant, so that if more receptors must be expressed, then the synapse itself needs to undergo enlargement (Sabatini et al., 2001), as an invariable number of receptor “slots” are available for occupancy. However, the synaptic density of NR2A relative to the NR2B subunit may change with activity (Aoki et al., 2003; Fujisawa and Aoki, 2003; Mu et al., 2003). One of the subtype-specific trafficking mechanisms involves the YEKL motif in the C-terminal of NR2B subunits, that can bind AP2, triggering this particular subunit’s internalization (Roche et al., 2001). Other factors that affect NMDAR numbers are mGluR activity, which triggers removal of both NR2A and NR2B from synapses (Philpot and Bear, 2002). As well, if glycine binds to the receptors, without receptor activation, it can prime them for internalization (Nong et al., 2003). 10  An input-specific fine-tuning of NMDAR composition is expected to occur later in adulthood, when patterned activity is modulating the efficiency of neurotransmission and/or the neuronal resistance to damaging insults through the NMDARs. This fine tuning occurs within well-defined limits, and may be part of the mechanism by which subjective experience shapes in particular ways neurons belonging to the same region of the nervous system, in a fashion that confers each its uniqueness. More specific qualitative and quantitative aspects of NMDAR patterns of expression in various brain regions and at different times in development are still under investigation, but some are considered below.  1.1.3.1 Regional variability of expression Some patterns of NMDAR expression were characterized, which are similar in all mammalian nervous systems, and probably under genetic control; for instance, NR2A, NR2B and NR2D (but not NR2C) subunits are specific to hippocampus and cortex, while NR2B and NR2D (but not NR2A and NR2C) - to cerebellar stellate cells. In hippocampus, the one gene encoding for the NR1 subunit and the four genes encoding for the NR2 subunits are variably expressed (as they are throughout the brain). The gene that encodes NR3B is not expressed in this region, whereas the one encoding for the NR3A is sparsely expressed. Knock out studies proved that the NR1 subunit is essential for the formation of NMDARs (Forrest et al., 1994). Indeed the messenger ribonucleic acid (mRNA) for the 11  eight different NR1 isoforms are expressed throughout hippocampus (Laurie and Seeburg, 1994b), with NR1a, NR1-1 and NR1-2 predominating in all pyramidal neurons. Low levels of NR1-4 have been detected in CA3 pyramids. The NR2A/NR2B subunits are expressed abundantly in all cell layers of the hippocampus, while the NR2C mRNA has not been detected in this region by in situ hybridization methods. NR2D subunit is restricted to interneurons in stratum radiatum of CA1 and CA3, where it may form heterodimers with NR2B (Standaert et al., 1996). From the NR3 group, only the NR3A subunit is sparsely expressed in CA1 and subiculum (Ciabarra et al., 1995), where it predominantly forms diheteromers with NR1 but a small proportion of NR1/NR2 complexes also contain NR3A in mature hippocampus (Al-Hallaq RA et al., 2002). NMDARs are located mostly on the ~30,000 spines covering a typical hippocampal pyramidal neuron. Within the cell, these are specifically distributed on the various dendritic domains; thus some regions, like the soma and the first 100 um of the apical dendrite, contain no spines at all, whereas 50% of the total number of spines is located on apical branches, and 35% on the basal dendrites; the apical tuft (which contains ~10% of all spines), displays excitatory synapses on shafts too (Andersen et al., 1980, Bannister and Larkman, 1995, Megias et al., 2001) The NR2A/NR2B are selectively targeted at certain synapses in hippocampal pyramidal cells: for example, NR2A are preferentially expressed at commissural/associational synapses, while the NR2B subunits – at the fimbrial-CA3 pyramidal cell synapses (Ito et al., 1997). In CA1, NR2B subunits are enriched at the 12  apical, but not basal dendrites (Kawakami et al., 2003). Within the same synapse, NR2B subunits may be localized more at extrasynaptic sites, and NR2A more centrally (Tovar and Westbrook, 1999). NR2 subunits’ expression is selectively dependent on various factors; it is found that the synaptic incorporation of the NR2B subunits is independent of the level of synaptic activity, whereas that of the NR2A is dependent on this parameter; moreover, the level of synaptic NR subunits regulates the further expression of the NR2A subunits (Barria and Malinow, 2002). Once the formation steps required for the normal structure and function of the neurons had been achieved, the synaptic/extrasynaptic populations may undergo subtle changes, either during basal turnover, or in an activity-dependent manner. Because of an elaborate macromolecular signaling complex that links NMDARs at the membrane, it was initially thought that these receptors are quite static (at least in comparison to AMPARs). However, NMDAR populations were shown to be able to move from extrasynaptic to synaptic sites (Tovar and Westbrook, 2002). The NMDAR subtypes can undergo lateral diffusion, with NR2A being slower than the NR2B (Groc et al., 2004; Groc et al., 2006). It was suggested that activity does not affect NMDAR mobility in mature neurons – as opposed to AMPARs’ (Groc et al., 2004)    13  1.1.3.2 Temporal variability of expression Initial studies reported that NMDA-binding sites are 50-130% more numerous early than later in development; also, the senescent rat hippocampus was found to display a low density of NMDA-binding sites (Bonhaus et al., 1990), while the responses mediated by NMDARs are more prominent in the developing brain, compared to the mature one. More recent, immunocytochemical studies find the number of NMDARs to be relatively invariant (as opposed to AMPAR numbers, which vary considerably) (Nusser et al., 1998, Racca et al., 2000). Functional NMDARs have been observed at different stages in development, depending on brain region, starting with the embryonic day 16 in cortex (LoTurco et al., 1991), and postnatal day one in cerebellum (Farrant et al., 1994). Recent in situ hybridization studies suggest indeed that NR1a, NR1-1 and NR1-2 are constantly expressed in hippocampal pyramidal neurons throughout development. A few splice variants of the NR1 have been detected mainly during embryogenesis, as is the case of NR3A subunit (Ciabarra et al., 1995); NR2B is enriched at this time too, whereas NR2A does not form functional receptors until later in development (Watanabe et al., 1992, Monyer et al., 1994). A small (<10%) proportion of NR2-containing NMDARs also include NR3A in mature hippocampus (Al-Hallaq et al., 2002). Indeed, the subunit composition of NMDAR populations is not constant in time, but the ratio of NR2A/NR2B may change dynamically as the organism matures. This change occurs similarly in all cells of a certain type early in development. Hippocampal neurons express NR2B-containing receptors early in development, mostly on non-synaptic plasma membrane. At a certain time-point, a shift in composition, from NR2B to NR2A subunit is registered in many 14  forebrain regions (Sheng 1994; Flint et al., 1997; Stocca and Vicini, 1998; Barria and Malinow, 2002b; van Zundert et al., 2004; Watanabe et al., 1993; Williams et al., 1992; Zhu and Malinow, 2002). This time-point is both genetically programmed, and influenced by experience, as it can be postponed in the visual cortex by sensory deprivation (Mierau et al., 2004; Philpot et al., 2001). Activity may be involved indirectly in the switch of receptor composition, by modulation of intracellular factors that further affect NMDARs, like the PSD-95 family of proteins or CamKII (Li et al., 1998). Other studies do not support this temporal succession of NR2B and NR2A subunits in the synapse as being the absolute rule, and show that the two subunits are segregated, many spines containing either one or the other, but not both subunits (Janssen et al., 2005). Studies assessing the proportion of NR2 subunits in cultured cells’ development using RNase protection assay, determined that the levels of NR2B exceed by 6 times at 8 DIV, by 4 times at 14 DIV, and by 2 times at 22 DIV, those of NR2A in cortical cultures (Sinor et al., 2000). The mRNA for NR2B is detected at first week in vitro, and following that, its expression remains constant; the NR2A is very low during the first 2 weeks, but doubles by the 3rd week, and doubles again by week 4. Another study using cortical neuronal cultures shows that indeed synaptic receptors are composed of NR2B subunits at 3 DIV, while the NR2A, barely detectable at 7 DIV, increased to mature levels at 21 DIV (Li et al., 1998). The NR2 subunit switch occurs at synapses in vivo as well around the same postnatal age (Flint et al., 1997, Stocca and Vicini, 1998).   15  1.1.4  Properties NMDAR-mediated currents have very slow kinetics compared with the other ionotropic glutamate receptors. Indeed, both the rate at which the responses to application of agonist rise (activation), and decline, after removal of agonist (deactivation) are slower than those seen in AMPAR and kainate receptors.  NMDARs continue to mediate an ion flux for hundreds of milliseconds after the glutamate pulse is terminated. The activation time was measured at around 7 ms. Deactivation time course, dictated by the dissociation rate constant of glutamate for its binding site, is longer than that of the glutamate presence in the synaptic cleft; the time constants are anywhere between 200 ms and 1-2 seconds, a lengthy time period, allowing the receptors to act as long-lasting indicators of presynaptic activity. The slow kinetics of NMDAR EPSCs are due to channel gating kinetics, and not by a prolonged presence of glutamate within the synaptic cleft, since the agonist transient rises fast, reaching a concentration of ~ 1.1 mM at peak, and then decays, all in about 1.2  ms (Clements et al., 1992). The mechanisms of NMDAR activation are extremely complex, as suggested by single channel studies (Gibb and Colquhoun, 1991, Edmonds and Colquhoun, 1992, Gibb and Colquhoun, 1992); more specifically, a pattern of bursts, clusters and superclusters was observed to describe the kinetic scheme underlining transitions from closed to open states. It is possible that NMDARs rapidly open (showing high open probably), with short latency after binding the agonist, and then slowly deactivate.  Or, channels may open with lower probability, so that the majority do not open for the first time during the peak response; thus, the prolonged decay time of the EPSCs is due to the different time-to-first opening of 16  each channel, following agonist binding. This would presume there is a higher number of NMDARs at the synapse, in order to obtain the expected EPSC amplitude. Further experiments reported that NMDARs activated by a few ms pulse of glutamate (1mM) open for the first time within the first 20 ms after agonist binding (Jahr, 1992). Also, the estimated open probability was 0.3 (high, (Jahr, 1992), suggesting that only a few receptors need to participate to the peak of the EPSC of an amplitude similar to that observed experimentally; more precisely, as few as one to four receptors  with a high open- probability need be open simultaneously for EPSC generation. These results have largely been obtained by the examination of NMDARs in outside-out patches. Conversely, the peak open probability was found to be low in studies of autaptic transmission, where a value of 0.04 was reported (meaning that about 40 receptors must participate in the generation of a 5 pA – miniature EPSC at the resting membrane voltage of ~-70mV). Deactivation time constants are determined by a variety of factors.  A recent study showed that decay constants are dependent on the temperature used during experimentation, and the values are lower (25-50 ms instead of hundreds of ms) at physiological conditions (Feldmeyer et al., 2002). In terms of molecular determinants, the rate of deactivation is modulated by a tyrosine residue (Tyr 535) on the NR1 subunit (Furukawa et al., 2005, Inanobe et al., 2005). The open time was linked to the transmembrane domain in the receptor structure, as mutations in this region alter it (they also alter desensitization rates) (Ren et al., 2003).The two most common heterodimers present in hippocampus deactivate with different kinetics: the decay times are ~ 50-150 ms for NR1/NR2A, and in the range of several hundred ms for NR1/NR2B receptors (Vicini et al., 1998). 17  Some NMDA receptors enter desensitized states in the presence of agonist; the time course of desensitization is slow, and the extent of desensitization less complete, compared to other ionotropic glutamate receptors (Iss/Ip ratio is 0.3-0.7; compared to the values for the GluR1-4 and GluR5-7 subunits, 0.006-0.01). While NR2C subunit-containing receptors do not desensitise at all, the hippocampal subunits show different characteristics, with NR2B subunits desensitizing less than the NR2A. Desensitization is further discussed in the next chapter. The open pr obability was estimated to vary (due to modulation by intracellular factors, like calcium) between 0.05-0.3 (Jahr, 1992, Rosenmund et al., 1995b) – see the discussion above). The peak open probability is 2-5 fold higher for NR2A - than for NR2B - containing receptors (Chen et al., 1999, Erreger et al., 2005). The receptors have a relatively high a ffinity for glutamate, EC50 =1-10uM  – compared to AMPARs’ EC50 = 100-500µM, a property that modulates channel behaviour (Lester and Jahr, 1992); because of the high affinity for the agonist, the peak synaptic glutamate concentrations are normally high enough to fully activate NMDA receptors. Extracellular ions like zinc can decrease the glutamate EC50 (Zheng et al., 2001). NMDARs  contain a second agonist-binding site on the NR1 subunit for either glycine or serine, which must be occupied before the glutamate can activate the receptor (Johnson and Ascher, 1987, Dingledine et al., 1988). Glycine levels seem to be high enough endogenously, so that this site is always occupied; otherwise serine can substitute for glycine (Schell et al., 1995, Baranano et al., 2001). The affinity of the NR1 subunit for glycine depends on the identity of the NR2 that coassembles to form the functional 18  receptor (Laurie and Seeburg, 1994a).  NR2B-containing receptors exhibit a ~10-fold lower EC50 for both glutamate and glycine than NR1/NR2A (Benke et al., 1999; Dingledine et al., 1999) making NR1/NR2B receptors more sensitive to agonist activation. NMDARs exhibit high single channel conductance, estimated at 40-50 pS (Jahr and Stevens, 1987, Gibb and Colquhoun, 1992), compared with that of the other ionotropic glutamate receptors. This parameter is not different for the two main subtypes, NR1/NR2A and NR1/NR2B (Clark et al., 1997). NMDARs are calcium permeable (Ascher and Nowak, 1988), with estimates of calcium fractional permeability in the range of 0.08 – 0.11 (Schneggenburger et al., 1993, Garaschuk et al., 1996). Moreover, NMDAR activation triggers calcium-dependent calcium release from intracellular stores (Emptage et al., 1999). The high calcium permeability is related, among other determinants, to an asparagine residue situated in the second transmembrane domain (Burnashev, 1996). Calcium permeability is another parameter that is not affected by NR2A/2B subunit composition (Schneggenburger, 1996). Magnesium ions block the channel of the receptor at negative potentials below -40 mV (Mayer et al., 1984, Nowak et al., 1984) so ion fluxes occur only at depolarised potentials, or in conditions where extracellular fluid lacks magnesium. Among the structural determinants of magnesium block is the asparagine residue mentioned above to have a role in calcium permeability.  Because of these biophysical characteristics, NMDARs open only when both the pre-and post synaptic neurons are activated, making them the proposed “coincidence detectors” for Hebb’s postulate. The NR2A and NR2B subunits are equally blocked by magnesium, which binds to these subunits with an affinity 19  of EC50 = 1-20 μM (Mori et al., 1992, Ishii et al., 1993). The NR2C subunits have a lower affinity for this ion. In addition to magnesium, NMDARs are modulated by a series of intra-and extracellular factors: glycine, zinc, polyamines, histamine, pH, kinases, phosphatases, neurosteroids, and calmodulin. Some of these modulators (eg zinc, protons) affect the behaviour of the two subtypes differently (McBain and Mayer, 1994, Dingledine et al., 1999).  1.1.5 Cytoskeletal and signalling proteins associated with NMDARs Because of the relatively long C-terminal tail of the NR subunits, NMDARs interact with a variety of intracellular proteins, many of which regulate their function. Indeed, the C-terminal domains are formed of ~100 amino acids for the NR1 subunits, and ~ 600 amino acids for the NR2A/B.  1.1.5.1 PSD-95 complex and other MAGUKs C-terminal segments of NMDAR subunits interact directly through their PSD- 95/Discs large/Zona occludens-1 (PDZ) binding motifs with the membrane-associated guanylate kinase (MAGUK) family of proteins. These protein-protein interactions occur through both the NR1 and NR2 subunits, and have been clearly described (Kornau et al., 1995, Niethammer et al., 1996, Bassand et al., 1999). 20  MAGUKs, a large family of synaptic scaffold proteins, are comprised of three PDZ domains, a SH3 domain, and a guanylate kinase domain. Some members of this family are post-synaptic density 95 kDa (PSD-95), synapse-associated protein 102 kDa (SAP102), synapse-associated protein 97 kDa, and PSD-93; they are expressed at specific points during development, and are targeted to specific membrane domains. Due to slight variations in amino acid composition, NMDAR subunits preferentially bind to certain MAGUKs; also, because each of the scaffold proteins is targeted more or less to certain subcellular locations and its expression peaks at a certain time in development, there is a space-and-time specific restriction for the interaction as well. Which MAGUK protein binds which specific population of NMDARs is a matter of great importance for receptor function. The Wenthold laboratory reported in a series of papers that NR2B expression coincides in time with that of SAP102, whereas that of NR2A – with PSD-95. Furthermore, the two pairs were shown to specifically interact, and traffic together in the same vesicles, helped by specific motor proteins. Consistent with this, NR2A expression is up-regulated in cerebellar neurons overexpressing PSD-95, while that of NR2B subunits is depressed (Losi et al., 2003). In the developing sensory cortex, a clear dependence of NR2A expression on PSD-95 was reported too: PSD-95 is up- regulated by activity, and this MAGUK is able to further up-regulate the expression of NR2A subunit (Yoshii et al., 2003).  In adult animals (P42), the two NR2 subunits were found to equally interact with each MAGUK-family member (Al-Hallaq et al., 2007). By genetically deleting  the subunit C-terminus, it was shown that NR2A-scaffold binding is not essential in receptor trafficking and synaptic targeting (Sprengel et al., 1998, Mohrmann et al., 2002), whereas that of NR2B is absolutely crucial for this function. 21  Consistent with this is the report on enhanced internalization of NR2B-containing receptors that lack the C  terminus  in cultures (Roche et al., 2001). Much of the information about the role of MAGUK proteins in regulating NMDAR function comes from knock-down and overexpression studies. Due to the scaffolding role, and the direct interaction between the two, it was expected NMDAR expression/activity was down- or up-regulated, respectively, following these manipulations. Surprisingly however, both conditions failed to produce an effect on NMDAR-mediated currents (Migaud et al., 1998). Even when a massive knock down of three different MAGUK proteins was tested, the effect on synaptic NMDAR expression was small (Elias et al., 2006). Studies using a mutant form of the NR2A –containing NMDAR,  with truncated cytoplasmic tail (Sprengel et al., 1998) report the same observation, as do studies using interfering peptides (Sheng 1999). These results remain to be reconciled with the ones in heterologous systems, reporting that PSD-95 increases NMDAR opening rate and insertion in the membrane (Lin et al., 2004). In conclusion, there is an unresolved controversy regarding the precise effect of MAGUKs on NMDAR expression and function:  PSD-95 promotes NMDAR clustering (El-Husseini et al., 2000a, El-Husseini et al., 2000b), and surface expression (Roche et al., 2001, Lavezzari et al., 2004, Lin et al., 2004); but also the clustering and trafficking of NMDAR to the synapses was independent of interaction with PSD-95 in other studies (Sheng 2004, Waites 2005; Shin 2003). In general it is widely accepted that association of NMDARs with scaffold proteins can: 1) alter important properties of NMDAR, like their desensitization (Li et al., 2002, Li et al., 2003a); 2) bring into receptor proximity certain kinases and phosphatases, which are 22  able to regulate receptor properties, as well as trafficking (Sheng, 2004); and 3) affect receptor expression in plasma membrane, conferring distinct degrees of stability to synaptic / non-synaptic sites (Sans et al., 2000, Roche et al., 2001, Prybylowski et al., 2005). Indeed, the PSD-95 family of proteins may be responsible for stabilizing NMDAR- mediated synaptic responses; the peak current through extrasynaptic NR2B undergoes more extensive rundown, compared to synaptic NR2A receptors, and this is not due to intrinsic subunit-specific properties, but to subcellular receptor localization (Li et al 2002). NMDAR desensitization is another property regulated by receptor interaction with PSD-95, being highly dependent on receptor localization. Its specific regulation during development (younger neurons being able to desensitize more than the mature ones) is probably due to the increased expression of PSD-95 in adulthood (Li et al., 2003a). While it is clear that MAGUKs play a central role in altering desensitization, the mechanism remains unknown. A direct interaction of the scaffold molecule with the receptor may regulate desensitization of the NMDARs, or this role may be undertaken by enzymes clustered by scaffolds in the receptor’s proximity.  1.1.5.2 Other interacting molecules NMDAR subunits directly bind to a variety of cytoskeletal and signalling proteins. NR1 C-terminal tails bind the following intracellular proteins: 1) actinin, important in linking the receptor to actin, the major dendritic cytoskeleton; 2) calmodulin, affecting the 23  receptor’s open probability in a calcium-dependent manner; 3) yotiao links the receptor to kinases and phosphatases (protein phosphatase 1, PKA; Sheng and Pak, 2000); 4) neurofilament; and 5) dopamine receptors. Both NR2A and NR2B subunits directly interact with enzymes like phospholipase C, through binding to their src domain (Gurd and Bissoon, 1997). Calcium-calmodulin-dependent kinase II (CamKII), an enzyme with a crucial role in synaptic plasticity, binds preferentially to the NR2B subunit of the NMDARs (Strack and Colbran, 1998). Activated by calcium, this enzyme undergoes autophosphorylation, which allows it to bind the NR2B C-terminal region, entering thus in an autoactive state. Once in the PSD, the enzyme phosphorylates GluR1 and possibly other proteins involved in AMPAR trafficking.  1.1.6  NMDAR function in cellular physiology At a cellular level, NMDAR–mediated activity impacts on excitatory synaptic transmission, on the structural development of certain neuronal-specific processes (like dendrites and axons), and on the mechanisms modulating the efficiency of physiological synaptic communication.  1.1.6.1 Fast excitatory transmission Depending on the polarization level of the membrane they are inserted in, NMDARs contribute several different components to the synaptic EPSCs. At membrane potentials more hyperpolarized than ~-40mV, NMDARs have a minor contribution to the 24  measured synaptic EPSC due to their block by magnesium ions. They add a second component to the dual nature of the EPSCs recorded while the postsynaptic membrane is charged to a potential of -40 to 0 mV. This component, an inward current, is due to the sodium and calcium ions flowing into the cell. The characteristics of this inward current differ from that mediated by AMPARs, in that it is of smaller amplitude, but of longer duration. Further depolarization of the membrane, above 0 mV, makes NMDARs active in mediating an outward current, which contributes to the repolarization of the postsynaptic membrane. Thus, NMDARs play an insignificant role during monosynaptic excitatory synaptic transmission activated by low-frequency presynaptic activation, as this normally cannot raise the synaptic potential to levels close to -40mV, unless back-propagating action potentials are contributing. At depolarised potentials due to high-frequency stimulation, and/or a strong backpropagating component, NMDARs contribute largely to EPSC duration, mainly during the -40 - 0 mV interval, due to their slow deactivation kinetics. It is worth noting that the amplitude and duration of the EPSC are considered to be a measure of synaptic “strength” (Lester et al., 1990). Indeed, NMDAR emerge then as the main mediator of long-duration depolarization that occurs during high-frequency activity (Collingridge et al., 1988), as the temporal integration of synaptic inputs is largely controlled by the time course of NMDA channel currents (Popescu et al. 2004; Erreger et al. 2005). The time course has important implications for the function of the synapse because of the profile of calcium influx mediated by these receptors, which is crucial for synaptic plasticity. One mechanism by which the time course of NMDA currents can be modulated is by changes in the rate of desensitization. 25  The impact NMDAR composition has on neurotransmission is, in part, a result of differences in current kinetics mediated by the different subunits (see above, 1.3).  NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) show different profiles depending on the stage of development (Carmignoto and Vicini, 1992, Hestrin, 1992), possessing longer durations in younger animals than in adults, with no apparent change in their amplitude distributions. For example, the decay time-constant may differ almost threefold between 23-33 days old - compared with 10-15 days old animals (Hestrin, 1992). The decrease in NMDAR - mediated EPSC duration is delayed when the animals are reared in the dark, a condition that prolongs developmental plasticity (Carmignoto and Vicini, 1992). These changes in characteristics of the EPSC during development are due to the switch in subunit composition from NR2B- to NR2A - rich receptors, as it was later proved (Flint et al., 1997, Stocca and Vicini, 1998, Vicini et al., 1998). The physiological significance of NR2–specific biophysical properties makes the NR2B subunits more efficient at EPSC summation, the slow decay bringing neurons more rapidly to firing threshold (Kumar and Huguenard, 2003; Lei and McBain, 2002). On the other hand, NR2A subunits make receptors more effective as coincidence detectors. Also, NMDAR populations that contain the NR2A subunit mediate more charge transfer during high-frequency stimulation than NR2B populations, whereas the latter exceed the NR2A in charge transfer during low-frequency stimulation (Erreger et al., 2005). This observation may explain the relatively different roles of the NMDARs in plasticity induction. The impact NMDAR localization has on receptor function arises from their differential activation by neurotransmitter, distinct regulation of their biophysical 26  properties by location-dependent enzymes and scaffold proteins, and distinct trafficking mechanisms, that affect population size and composition. Glutamate escaping from the cleft (during high frequency presynaptic firing or during aberrant excessive stimulation) could tonically activate the NMDARs present in extrasynaptic membrane, regulating thus cellular excitability through non-synaptic mechanisms (Sah et al., 1989, Blanton and Kriegstein, 1992). It was suggested that this activation may affect regenerative electrical properties in neurons, facilitating the coupling between dendritic and somatic electrical events. As discussed above, the synaptic population of NMDARs was found to be richer in NR2A subunits, consistent with the up-regulation of this subunit later in development, while the NR2B subunit is prominently expressed at earlier stages (Li et al., 1998). The extrasynaptic population of NMDAR, richer in NR2B-subunit, was proposed to have a role in sensing global glutamate signals, and being able to detect glutamate arising from multiple synapses (Kullmann and Asztely, 1998); thus the NR2B-containing receptors have been proposed to mediate a form of “cross-talk” between hippocampal synapses (Scimemi et al., 2004). Another role for the extrasynaptic population may be to provide a reserve pool of receptors, easily transported to and from PSD (Tovar and Westbrook, 2002).  1.1.6.2 Structural development of neural processes The initial observation that NMDARs may be involved in the regulation of neuronal development  and growth was made in the 1990s, when it was reported that 27  growth cones are dependent on calcium signal mediated by these receptors (Komuro and Rakic, 1993). Genetic manipulations of NMDAR subunits show the receptor’s role in dendritic arbour development (Lee et al., 2005). Formation of dendritic spines is dependent on NMDAR activation too, as it is triggered by LTP protocols in vitro (Engert and Bonhoeffer, 1999, Maletic-Savatic et al., 1999). The cytoskeletal changes important for emergence of new spines may be linked to several proteins, whose activation is mediated by NMDARs.  Amongst these are a Rap-specific GTPase (which has been shown to cause an enlargement of spines in transfected neurons) (Pak et al., 2001), and cortactin, a synaptic actin-binding protein, whose overexpression leads to spine elongation (Hering and Sheng, 2003). NMDAR activation during a brief temporal window in development is required for experience-dependent modifications of synaptic connectivity in visual cortex  (Olson and Freeman, 1980, Daw et al., 1992). It is also required in the formation of ocular dominance columns and segregation of retino-geniculate fibers (Bear et al., 1990), as it is in the process of synapse elimination (Rabacchi et al., 1992).  1.1.6.3 Synaptic plasticity The efficiency of synaptic transmission can be modified to become stronger or weaker through the activation of NMDARs, among other mechanisms. This can be 28  achieved either by patterned presynaptic stimulation, or by coincident activation of the pre- and post-synaptic elements. The calcium permeability through the NMDARs is the property that distinguishes them as major players in the induction of synaptic plasticity. Further, the different kinetics, as well as the different subcellular location of the NMDAR subtypes, may explain their divergent role in strengthening, and weakening synaptic transmission, when receptors are activated in a certain fashion. Of similar importance in plasticity induction is another NMDAR –specific property: their blockade by magnesium, which occurs in a voltage specific manner, as discussed in 1.4. The colocalization of NMDAR and AMPARs at individual synapses results in the former playing the role of highly specialized coincidence detectors (Lisman et al., 1997, 2002; Nicoll and Malenka, 1995). Synaptic potentiation only occurs when both the pre-and postsynaptic neurons are active, and this co-activation can be only detected by the NMDARs. More details on the mechanisms of NMDAR-induced plasticity will be discussed in subchapter 3 of the introduction.  1.1.6.4 Neuronal survival and excitotoxicity It is still through the calcium signal they mediate, that NMDARs play a role in activating cascades impacting on neuronal fate. Neuronal vulnerability to excitotoxic events is mediated by NMDARs, among other factors. These receptors can induce cell 29  death when overactivated or completely blocked, but also trigger protective mechanisms, beneficial for neuronal health in other conditions. While investigating the vulnerability to excitotoxicity mediated by NMDARs, the initial focus was on location-specific subpopulations. In a series of four studies, the synaptic population was found to mediate pro-survival effects, while activation of the extrasynaptic receptors triggered cell-death pathways through a CREB shut-off mechanism (Hardingham, 2006; Hardingham and Bading, 2002; Soriano et al., 2006). However, the calcium-mediated activation of synaptically-located nNOS enzyme may also play a central role in toxicity (Aarts et al., 2002b; Sattler et al., 1999). The differential regulation of synaptic/extrasynaptic receptors, in terms of rundown and desensitization (which limit calcium influx) (Li et al., 2002; Li et al., 2003) may further prove important for toxicity. Recently, the subtype-specific populations were also investigated with regards to involvement in excitotoxicity. Surprisingly, a complete dependence on subunit composition, and not on subcellular location, was found, at least for neurons at a certain time-point in development (Liu et al., 2007). Another study reports the same dependence for the same period in development that does not apply though for mature neurons, where death occurs through activation of NMDARs regardless of the subtype (von Engelhardt et al., 2007).    30  1.1.6.5 Insights on the NMDAR function from NMDAR subunit mutant mice Various mutant mice have been created, lacking either NR1, or any of the NR2 subunits, as well as conditional knock-out mice lacking these proteins either in a specific hippocampal region or for a specific period of time. The examination of synaptic function, plasticity properties and behaviour of these mice suggests some of the functions the NMDA receptor has in the normal physiological conditions.  The NR1 subunit knock-out mouse model was created in 1994 (Forrest et al., 1994), and it proved the essential function of this subunit in formation of functional channels, as the mice died a few hours after birth. Another mouse model, showing only 5% of normal expression levels of the NR1 protein (Mohn et al., 1999), displays behavioural deficits similar to those seen in the animal models of schizophrenia (increased motor activity, stereotypy, social deficits). The regional NR1 knockout, restricted to the hippocampal CA1, provides information about its essential role in releasing the NR2 subunits from the endoplasmic reticulum, and delivering functional receptors to the plasma membrane (Fukaya et al., 2003). Moreover, LTP was impaired, as was the ability to acquire new information in spatial memory tasks. The ability to consolidate information was tested in a temporally- restricted knock-out, and was found to be impaired as well, so NMDARs expressed in CA1 are important in plasticity, spatial memory acquisition and consolidation. The hippocampal CA3-specific knock-out mouse displayed impaired LTP too (Nakazawa et al., 2002), but only at commissural-associational synapses; in memory tests, it showed deficits when some of the spatial cues were missing, which led to the proposed role of this region in 31  “pattern completion”, a role dependent on the NMDARs. Mice with point mutations on the NR1 subunit were also created, in order to investigate particular properties, like glycine binding (Kew et al., 2000) or calcium permeability (Single et al., 2000). The NR2B subunits are sufficient for plasticity induction when NR2A are lacking, as revealed by investigations of the NR2A knock-out (Sakimura et al., 1995); spatial learning was also slightly impaired. NR2B knock-out mice die shortly after birth, confirming this subunit is crucial early in development; LTD was abolished, and no synaptic NMDAR-mediated currents could be recorded in mice kept alive for a few days (Kutsuwada et al., 1996).  1.2 NMDAR desensitization 1.2.1  Definition and classification NMDAR-mediated current decreases in the continuous present of the agonist, a phenomenon called desensitization. The conformational change underlying the desensitized state  can occur either when the receptor-channel is in the open state, or when it is closed (Lester et al., 1993, Lin and Stevens, 1994, Colquhoun and Hawkes, 1995, Colquhoun, 1998), and is due to certain conformational changes preventing ion flow through the channel pore, even though the agonist is bound to the extracellular ligand- binding site. Physiologically, the relaxation of the NMDAR-mediated current in the presence of the agonist may be characterised by slow kinetics, and it is measured as the steady-state-to-peak ratio for the macroscopic currents obtained by prolonged application 32  of the agonist. The recovery of the receptors from the desensitized states is also slow, requiring seconds; this parameter is estimated by paired-pulse applications of glutamate at different time intervals (Lerma 1992). Desensitization has initially been described to occur in conditions of low glycine concentrations (Mayer et al., 1989, Benveniste et al., 1990). The two agonists, glutamate and glycine cooperate negatively when glycine levels are low, such that the receptor enters a desensitized state. Since the glycine affinity is influenced by subunit composition, (see 1.4) this form of desensitization may also be different for the two receptor subtypes. In normal physiological conditions the levels of this amino acid are high, so this type of modulation may be relevant only under conditions where glycine levels are down- regulated. The rate of onset of the glycine-sensitive component of desensitization is affected by polyamines like spermine; more specifically, it becomes slower and is reduced in extent in the presence of spermine  (Benveniste and Mayer, 1993). NMDARs continue to show a diminished response to the continuous presence of glutamate even when glycine levels have been restored. Further research isolated a calcium-dependent component (Rosenmund et al., 1995a), but a certain level of desensitization still remains in conditions of calcium exclusion, which has later been found to depend on modulation at the C-terminus of the receptor, as well as on zinc (binding at the N-terminus) (Sather et al., 1992a, Tong and Jahr, 1994). Because the calcium- dependent component has been termed “inactivation”, we will refer to the last form as “glycine-insensitive desensitization”. 33  Indeed, NMDAR have been shown to “inactivate” i n a calcium-dependent manner, in conditions of saturating glycine (Vyklicky et al., 1990, Legendre et al., 1993, Rosenmund and Westbrook, 1993, Krupp et al., 1996). The increase in intracellular calcium triggers a series of events that lead to the action of intracellular second messengers (activated by elevated intracellular calcium) on the C-terminal region of the NMDAR subunits. The source of calcium could be the NMDAR themselves, but not necessarily. Consistent with this phenomenon, other effects of calcium on NMDARs include reducing the single channel conductance (Ascher and Nowak, 1988), and decreasing receptor activity  (Mayer et al., 1987). The time course of inactivation is very slow and requires NMDAR activation over a period of several seconds. It was reported absent in outside-out patches, suggesting an essential intracellular calcium-sensitive constituent (Rosenmund and Westbrook, 1993). Actin in its filamentous form regulates NMDAR activity in a calcium-dependent manner, and its depolymerisation induces receptor rundown. Also, postsynaptic calcium-dependent enzymes like calcineurin, as well as regulatory proteins like calmodulin, modulate the extent of inactivation, both for whole-cell and synaptic currents. This type of desensitization was shown to depend on the NR2 subunit identity (Krupp et al., 1996), being substantial for the NR2A, but not significant in the NR2B subunits. NR2C subunits do not desensitize in this manner. The third type of desensitization, glycine–insensitive, initially described in outside- out membrane patches (Sather et al., 1992b, Tong and Jahr, 1994, Tong et al., 1995), was believed to appear only when the intracellular contents were fully dialysed. Later, this form 34  of desensitization was recorded in whole-cell preparations as well.  Polyamines like spermine and histamine have a modulatory effect on glycine-insensitive desensitization (Lerma, 1992). Desensitization may also be modulated by arachidonic acid, which has been shown to make NMDAR responses more transient. The glycine – insensitive desensitization in the NR2A subunits in particular can be described by two kinetic components: a slow one, with a time constant of ∼1–2 s, and a fast one, with a time constant of 200-300 ms (Chen et al.1997; Krupp et al. 1998; Villarroel et al. 1998; Zheng et al. 2001). It was argued that the fast component, caused by an allosteric interaction between the zinc binding-site in the N-terminus and the glutamate binding pre- M1 region should be excluded from the category “glycine-insensitive desensitization”, being only an “apparent” form (Hu and Zheng, 2005).  1.2.2   Structural determinants of NMDAR desensitization Some of the molecular determinants of the desensitized conformations have been elucidated. They have been localised both at the N-terminal and at the C-terminal regions of the receptor, depending of the type of desensitization. The ATD of the NR1 subunit is particularly relevant for the glycine-dependent form (Krupp et al., 1998, Villarroel et al., 1998, Zheng et al., 2001), while that on the NR2 subunit for the glycine-insensitive form (Krupp et al., 1998, Hu and Zheng, 2005, Jackson et al., 2006). Modulation at the C- terminal domain is a determinant both for calcium-dependent inactivation (Ehlers et al., 1996) and for glycine - insensitive desensitization (Krupp et al., 2002, Li et al., 2003a). 35   1.2.2.1 Structural determinants at the N-terminal domain The binding sites on the NR1 and NR2 subunits interact allosterically when glycine levels are sub-saturating, with a negative effect on current flow. Two sites on the N-terminus of NR2A subunits were shown to collaborate to control the degree of glycine-insensitive desensitization. More specifically, the sites are comprised of four amino acids located upstream of the M1 region (two of which being identified as Ala 555 and Ser 556, (Villarroel et al., 1998)), and a number of ~190 amino acids located in a region preceding the S1 (Krupp et al., 1998, Villarroel et al., 1998). Recent studies performed in the nominal absence of zinc could not replicate these results, and the authors argued that the aforementioned sites play a role in zinc-dependent modulation, and not in glycine-sensitive desensitization (Hu and Zheng, 2005). They propose, on the other hand, several residues in the lurcher motif of either NR1 or NR2A are critical for this form of desensitization of NR1/NR2A receptors. The lurcher motif is a short stretch of highly conserved amino acid residues located in the extracellular regions of the NMDAR. Indeed, the lurcher motif has been linked to desensitization before, when single point mutation [NR1a(A653T)] of NR1 has been reported to alter desensitization of NMDA receptors (Kohda et al., 2000). Moreover, Villarroel and colleagues have described an NR2A chimera that lacked the slow component of desensitization giving insight on the molecular determinant of the “proper” glycine-insensitive desensitization. This chimera was created by introducing a triple mutation in the pre M1 region (F553Y, P566A and A567S). 36  As for the zinc-mediated modulation of the NMDAR currents, additional data shed more light on the allosteric binding of the ion at the N-terminal domain of the NR2A (Zheng et al., 2001), and that this binding can increase the fast component in the kinetics of the current decline (Legendre and Westbrook, 1990, Chen et al., 1997, Paoletti et al., 1997, Low et al., 2000, Erreger and Traynelis, 2008). Recently, Erreger and colleagues demonstrated that the affinity of zinc for its binding site depends on glutamate, and not on glycine binding; it also does not depend on the channel pore opening. They predict, based on modelling data, that zinc regulation affects EPSC amplitude (Erreger and Traynelis, 2005); recordings in outside-out patches show no effect on mean single-channel open duration, open channel probability, or single-channel current amplitude (Erreger and Traynelis, 2008).  1.2.2.2 Structural determinants at the C-terminal domain Calcium-dependent inactivation is the effect of intracellular calcium elevation (Rosenmund et al., 1995a), which activates either the enzyme calcineurin, or/and calmodulin. Calcineurin acts by dephosphorylating the NR2 subunits; calmodulin, a regulatory protein, binds to the C0 cassette of NR1, displacing α-actinin and disrupting NR1 association with cytoskeleton, reducing thus the NMDAR-mediated current. Mutation studies show that the C-terminus of the NR1 subunit contains two calmodulin-binding sites, which seem to be relevant for the regulation of calcium-dependent inactivation (Ehlers et al., 1996, Zhang et al., 1998, Krupp et al., 1999). Regulation of the NMDAR subunits at the C-terminus, with relevance for glycine- insensitive desensitization, can occur either through the action of enzymes or through the 37  direct interaction of the C-terminal tail with synaptic scaffold molecules. There are several phosphorylation sites on the NR1 and NR2 subunits; a previously unidentified site for PKC phosphorylation on the NR1 and /or NR2A subunits has been suggested to play a role in regulating glycine-insensitive desensitization. Even though calcium was present in the recording conditions in this experiment, the authors argued that calcium-dependent inactivation could not have been affected by their manipulations. The phosphorylation site Ser 1303 on the NR2B subunits is the locus of CamKII- mediated regulation of these subunits’ activity. This enzyme has recently been shown to increase the extent and/or rate of desensitization of NR1/NR2B-mediated macroscopic currents in HEK293 cells; when tested on NR2A-containing subunits, the effect was the opposite (Sessoms-Sikes et al., 2005). Modulation of desensitization at the C-terminus in the absence of the enzymatic activity, by the direct effect of scaffolding molecules on the NMDAR conformation has been suggested by a previous study, but not directly tested.  1.2.3  Proved and hypothesised roles for NMDAR desensitization  The role of NMDAR desensitization in synaptic function was suggested by Jahr in 1995, when the first synaptic NMDAR-mediated response was shown to desensitize in a glycine-independent, calcium-dependent manner. The authors used autaptic cultures, which permit the recording of synaptic (or “autaptic”) EPSCs. The NMDAR-isolated response shows desensitization in the response to a second pulse of glutamate, after a first stimulation has opened all synaptic receptors 1.5 s in advance. More precisely, 38  desensitization is measured as the ratio between the test EPSC evoked 1.5 s after a complete activation of synaptic receptors in the presence APV, and another test EPSC, evoked 1.5 s after a complete activation of synaptic receptors in control solution (no antagonist). This study showed for the first time, the shaping of the EPSC by NMDAR desensitization. NMDAR desensitization may play a role in plasticity too, as hypothesised in the above–discussed studies, which proved desensitization shapes synaptic responses, and consequently the calcium signal. The calcium profile is crucial in the induction phase, and dictates the direction of plasticity. Thus it was speculated that, during high-frequency stimulation, the degree of desensitization may be important, since the recovery of receptors from this state takes several seconds. Plus, calcineurin was shown to be essential in modulating the form of desensitization described above; on the other hand, other studies implicated calcineurin as a determinant of homosynaptic LTD, so the two phenomena may share the same mechanism. The potential role of NMDAR desensitization in plasticity was hypothesised also in studies that described the effect of CamKII on NR2B-containing receptors. This enzyme has been linked before to plasticity mechanism, as it was proved essential for LTP induction, as it was suggested to have a role in distinguishing distinct patterns of intracellular calcium rise (De Koninck and Schulman 1998). CamKII levels, barely detectable immediately after birth, increase by 10-fold at P16 , while NR2B levels decrease in development, so there might be a clearly defined temporal window for the interaction of 39  the two, and this may partially explain the plasticity levels that can be elicited at different points in development. The role of NMDAR desensitization which received most attention is that in death- related mechanisms / neuronal survival. That is because excessive and prolonged elevations in glutamate, required to induce NMDAR desensitization, have been detected in brain trauma and cerebral ischemia. The intracellular signalling cascades activated by this excessive glutamate lead to various forms of neuronal damage and apoptosis. Desensitization of the NMDAR has been reported to have a neuroprotective effect (Villarroel et al., 1997), while situations where desensitization is decreased, for example, late in adulthood (Li et al., 2003b), may render neurons more prone to apoptosis.  1.3 NMDAR-dependent synaptic plasticity in hippocampus 1.3.1 Definition and forms of synaptic plasticity A form of enhancement in synaptic communication was first described in 1939 by Larrabee & Bronk, in sympathetic ganglia. It occurred due to high – frequency stimulation of the presynaptic fibers, and it only lasted a few minutes (5-7min).  Longer enhancement was observed a few decades later, when Bliss and Lomo applied repeated high-frequency stimulation episodes (tetani), to fibers innervating the dentate gyrus of the rabbit in vivo (Bliss and Lomo, 1970, 1973). They named this phenomenon long-term potentiation (LTP), but the reverse was also described later, when 40  patterned presynaptic activity can lead to weakening of synaptic efficiency (Dudek and Bear, 1993). Moreover, synapses can be modified in a graded manner, their efficacy ranging anywhere from -50 to +50 % of its base level. The polarity of the change is dictated mostly by the amplitude and time course of the calcium transient, essential to plasticity induction, as will be discussed below. Many brain regions display various forms of plasticity: the prefrontal, sensory and motor cortex (Artola and Singer, 1987; Sakamoto et al., 1987; Laroche et al., 1990), cerebellum (Crepel and Jaillard, 1991), amygdala, and nucleus accumbens (Kombian and Malenka, 1994). However, the hippocampus was the region where this phenomenon was most extensively studied, due to the ease in stimulating and recording specific components, conferred by its famous laminar structure. Synaptic plasticity in the hippocampus can occur both at inhibitory and excitatory synapses, onto either principal cells or interneurons. Various forms of plasticity have been described so far, based on their mechanism of induction, ranging from NMDAR-dependent or mGluR-dependent to classical or spike- timing depending plasticity. Essential aspects regarding hippocampal, NMDAR-dependent plasticity at excitatory synapses, will be considered below.  1.3.1.1 Transient and short-term forms of plasticity Basic synaptic transmission is normally mediated by AMPAR currents, of certain characteristics. Amongst these, the amplitude is considered a measure of “synaptic 41  strength”. This parameter is modifiable – transiently or “permanently” in certain conditions, and the modification is taken to reflect the level of synaptic plasticity. One of such conditions that are linked with transient modifications in synaptic responses is the paired-pulse. The postsynaptic response to a second pulse, delivered a few hundreds of ms after an initial one, is increased. This phenomenon was described in hippocampus amongst other regions (like neuromuscular junction (Magleby, 1979), and was called “facilitation”; if the time between the two pulses is in the order of seconds though, the synapse shows “depression”. Amongst the mechanisms of expression of facilitation, it can be mentioned that more neurotransmitter is released upon the second pulse, due to the high levels of presynaptic calcium, which invaded during the initial pulse (Wu and Saggau, 1994).  Synapses showing high levels of facilitation are considered to have a low initial probability of release, and vice-versa. Depression, on the other hand can result from depletion of vesicular content, and from feedback mechanisms which activate presynaptic GluRs or GABARs. Repeated synaptic stimulation induces a short-lived type of facilitation called post- tetanic potentiation, with duration in the order of minutes; it is probably due to increased calcium concentration in the presynapse (Wu and Saggau, 1994).     42  1.3.1.2 Long-term plasticity: early and late LTP/LTD Experimentally, LTP and LTD are measured either at a population or single-cell level. In the first case, field potentials are taken to reflect neurotransmission, and in the latter, the AMPAR-mediated EPSCs. In terms of time course, LTP is quickly observable after the induction phase (in ~2 seconds in CA1; but it takes minutes in dentate gyrus (DG), peaks after another 30 seconds, and then slightly declines to a stable value, lasting, variably, tens of minutes to hours, days or even years (Abraham, 2003). It was noticed that after inhibiting cellular protein synthesis, hippocampal plasticity could not last longer than a few hours (Frey et al., 1988); the same happens with transcription inhibitors. Protein kinases (like PKC, CamKII) on the other hand, must be active approximately an hour after induction, otherwise plasticity decays soon to baseline levels (Malenka et al., 1989; Malinow et al., 1989). Based on these criteria, LTP is believed to be comprised of three distinct phases, each sensitive to certain blockers but immune to others: short-term plasticity, which is observable for an hour after induction and cannot be washed out with either kinase – or protein synthesis inhibitors; early LTP, lasting from the 1st – to the 5th hour after induction, and dependent on kinases’ activity, and late LTP, lasting longer than ~ 5 hours, and dependent on transcription and protein synthesis. LTD follows the same rules of categorization.   43  1.3.1.3 Metaplasticity Not all synapses respond identically to plasticity induction protocols. It was suggested, initially by theoretical analyses that the prior synaptic activity each synapse had been engaged in, may affect the way it will later respond to patterned activity. The Bienenstock-Munro-Cooper model put forward the idea that, depending on the frequency of stimulation, the synaptic weight falls below (LTD) or above (LTP) the value for basal neurotransmission. This in turn, more importantly, is not a static value, but a function of postsynaptic activity, and could be shifted to the right or left following certain manipulations. Later it was experimentally demonstrated that stimuli that normally cannot elicit plasticity changes themselves, affect the response to LTP protocols if delivered a while before them. For example, weak trains, or low frequency-activation of NMDARs, can prevent subsequent induction of LTP (Coan et al., 1989; Huang et al., 1992). Also, LTD could not be induced immediately after unsilencing the so-called “silent synapses” (Montgomery and Madison, 2002).  1.3.1.4 Homeostatic plasticity Aside from conferring a certain degree of flexibility, plasticity could also be seen as a means of stability in a network of neurons, based on the observations that neuronal activity levels can homeostatically regulate the properties of neural circuits to maintain firing rates within certain boundaries (Turrigiano and Nelson, 2004).  44  1.3.2  Induction One of the most striking characteristics of LTP / LTD is its very rapid onset after the induction protocols have been applied. These protocols, initially described as repeated tetani, could alternatively take the form of coincident pre-and postsynaptic depolarization. Apart from influencing the direction of plasticity, specifically patterned activity affects the magnitude and the duration of plasticity. Crucial to the induction of either form of plasticity, is the activation of certain postsynaptic receptors, and the downstream events they trigger. NMDAR was the first class to be recognized, and indeed hippocampal plasticity is essentially dependent on its activation. Plasticity is defined as NMDAR-dependent if it is prevented by antagonists of NMDARs, like AP5, applied during the induction phase (when applied later they have no effect on plasticity, as demonstrated by Collingridge in the early eighties (Collingridge et al., 1983b) . Alternatively, other classes of glutamate receptors can induce non-NMDAR – dependent types of plasticity. mGluRs have been linked to LTD, especially in adults, where this forms prevails over the NMDAR-dependent type. But these receptors can induce LTP too, especially onto CA1 interneurons. Also, a kainate receptor –dependent type of LTP has been described in CA3.    45  1.3.2.1 Classical patterns The most popular protocol to induce LTP is the famous train of 100 Hz pulses repeated 4 times, which when applied to a bundle of fibres, induces synaptic enhancement in large populations of neurons. This was the pattern first described to produce a long- lasting effect, as previous attempts using lower frequencies rendered short-time plasticity only. Alternative forms of high-frequency stimulation protocols are the brief trains of 400 Hz pulses (Douglas and Goddard, 1975), the primed-burst stimulation (Larson et al., 1986), and the theta-burst stimulation (Rose and Dunwiddie, 1986). For the last two, a priming stimulus or a brief train, respectively are succeeded after a 200 ms interval by a burst or a train of stimuli. The initial stimulation in these cases activates the feedforward interneurons, leading to presynaptic GABABR-mediated reduction in GABA release, maximal right around 200ms after stimulation (Davies et al., 1991). The number of stimuli in a train was found to prevail in importance regarding the magnitude of LTP, over the actual pattern of stimulation (Hernandez et al., 2005), however the minimal patterns are probably more likely to occur naturally than the long trains of hundreds of stimuli. The first studies on LTD induction used one train of 100 pulses at 1Hz, but this didn’t prove to be an entirely satisfactory paradigm; more efficiently, prolonged trains of 900 pulses reliably produce LTD, as described later by Dudek and Bear (Dudek and Bear, 1993).  46  1.3.2.2 Spike-timing protocols LTP can be induced in single cells as well, when both pre-and postsynaptic neurons are depolarised within a narrow temporal window. Normally, the afferent stimulus must precede the brief depolarizing postsynaptic pulse by ~20 ms in order for LTP to be induced (Levy and Steward, 1983, Gustafsson and Wigstrom, 1986). If the order is reversed, depression of the synapse occurs, making the temporal requirements for the pre-and postsynaptic activation very straightforward (they are not obvious with classical patterns of induction). Physiologically, postsynaptic depolarization of a certain synapse which is not strongly activated by presynaptic firing may occur with the back-propagation of action potentials due to activation of other synapses (Stuart and Sakmann, 1994).  1.3.2.3 Calcium signalling through NMDARs in plasticity induction The essential role of calcium in plasticity induction was proved in 1983 by Lynch and colleagues, who used calcium chelators in their recordings, and reported that LTP is prevented by such conditions (Lynch et al., 1983). The calcium signal is mediated by the NMDARs, which are highly permeable to the influx of this ion (see above), and further amplified postsynaptically by calcium released from internal stores. This was proved by preventing the refill of these stores with thapsigargin, which blocks the induction of LTP (Harvey and Collingridge, 1992). Another source of elevated intracellular calcium is the voltage-gated calcium channel, particularly important in non-NMDAR forms of plasticity. 47  Specific characteristics of the calcium transient are important in dictating the direction of plasticity: in conditions of low extracellular calcium, a protocol that normally induces LTP becomes one that induces LTD (Mulkey and Malenka, 1992); the same happens when calcium entry during tetanus is reduced by the use of low concentrations of AP5. From these observations, it can be concluded that the concentration of intracellular calcium is the crucial parameter in the induction of the two forms of plasticity. How exactly lower calcium leads to LTD, while higher levels – to LTP, was speculated by Lisman (Lisman, 1989, Harvey and Collingridge, 1992)), who pointed out that the affinity of protein phosphatases for calcium is much higher than that of the kinases, and so the activation of each  class preferentially may trigger the different cascades of event with the opposite outcome.  1.3.3 Expression Following above-mentioned protocols of activation, synapses communicate more efficiently, a fact obvious in the larger amplitude of the AMPAR-mediated current. This may be due to modification in the presynaptic neuron, like increased release of the neurotransmitter, and in the postsynaptic neuron, through modifying existing receptors, or inserting new ones. The different characteristics and loci of expression, specific for the distinct phases of plasticity will be discussed below. Generally, they are influenced by the age of the animal, the brain region tested, and other factors.  48  1.3.3.1 Presynaptic locus of expression In LTP, the probability of release was found to be increased in a number of studies (Stevens and Wang, 1994, Bolshakov et al., 1997), consistent with diminished paired-pulse facilitation during LTP (Schulz et al., 1994). Also, modifications of the fusion pore – such that more neurotransmitter is released - could alternatively account for LTP expression (Choi et al., 2000). Finally, the presynaptic boutons were increased in certain conditions associated with LTP (Antonova et al., 2001). On the other hand, in LTD, the rate of neurotransmitter release is decreased, as concluded in imaging studies investigating vesicular turnover (Stanton et al., 2001).  1.3.3.2 Postsynaptic locus of expression Postsynaptically, both modifications in the properties of existing AMPA receptors and insertion of new ones, can account for the increased basal transmission in LTP. AMPAR sensitivity to glutamate is modified in LTP, as evident in studies using ligands for this receptor, where the sensitivity was found greatly increased (Davies et al., 1989, Montgomery et al., 2001, Bagal et al., 2005). The AMPAR properties support modifications mainly through enzymatic phosphorylation  (Lee et al., 2000). Single channel conductance for example, was found to be increased after CamKII-mediated phosphorylation of the Ser831 site on AMPARs (Derkach et al., 1999). The change in conductance may be, in some cases, sufficient to account for LTP (Benke et al., 1998). There are in fact a number of kinases involved in different phases of LTP, and at different 49  points in development: while CamKII is activated in adults, PKC and PKA are essential at earlier developmental stages, before the postnatal day nine (Yasuda et al., 2003). At around two weeks of age, either kinase is sufficient for LTP, but a blockage of all three prevents LTP expression (Wikstrom et al., 2003). The number of AMPARs may increase following LTP-induction protocols, especially of those containing the GluR1 subunit (Shi et al., 1999, Hayashi et al., 2000, Lu et al., 2001). The insertion occurs either at functional, or at “silent” synapses (Pickard et al., 2001). For LTD, the postsynaptic mechanisms of expression involve dephosphorylation of GluR1 by phosphatases like PP1 and PP2, and internalization of GluR2-containing AMPARs. For the latter, a large number of studies employed various techniques, to conclude that GluR2 internalization at extrasynaptic sites precedes synaptic AMPAR downregulation (Ashby et al., 2004). Central to the expression of LTD seems to be the specific interaction on the GluR2 subunit with N-ethylmaleimide-sensitive factor (NSF) which triggers its internalization by exchanging places with adaptor protein 2 (AP2) (Collingridge et al., 2004). GluR2 subunits directly interact with scaffolding proteins like glutamate receptor interacting protein/ AMPAR-binding protein (GRIP/ABP) and protein interacting with C kinase 1 (PICK) and these may also have an alternative role in internalization. Specifically, PICK1 has been linked to LTD (Kim et al., 2001), through a mechanism that involves calcium binding. However, GluR2 knock-out animals display NMDAR-dependent LTD; this is probably mediated though the dephosphorylation of the GluR1 subunit (discussed above). 50  1.3.4  Particularities of plasticity in hippocampal dentate gyrus The dentate gyrus (DG) is a C-shaped structure, extending, in rodents, from a dorso-medial position, close to the septal complex, to a ventro-lateral position, close to amygdala. Along with the hippocampus proper (CA1, CA2 and CA3), subiculum and parasubiculum, it is part of the hippocampal formation. One important particularity of the hippocampal formation is the unidirectionality of its inputs and outputs, such that excitation flows from one sub-structure to another without feeding-back. DG, a structure that appeared much later in evolution compared to the other hippocampal components, receives excitation from entorhinal cortex through perforant path fibers, and excites in turn the CA3 cells, through mossy fibers. There are three layers to the DG, the middle one, ~ 70 um, being formed of densely packed granule cells, the principal cells of this structure. Some particularities of granule cells are: 1) their shape and dendritic arbour: they lack basal dendrites, and have instead a large apical tuft that is cone-shaped; 2) they are smaller than the pyramidal cells; and 3) they burst-fire very rarely, at about ~0.5 Hz, displaying a high threshold for activation (Jung & McNaughton, 1993); however, during the theta oscillations, they can fire sustained bursts (Munoz 1990). Granule cells extend their dendrites in the molecular layer, and their axons, which form the mossy fibers, towards CA3. The third layer of the DG is the polymorphic layer, which contains another type of cell, the mossy cell. The organization of dentate gyrus makes it, as it makes the other hippocampal structures, ideal for experimentation. Among the characteristics that make the study of 51  plasticity easy in DG are the compact cell layer, made up of dense packing of cell bodies, the parallel position of the apical dendrites, making them easy to be activated synchronously, and, stratification of afferent fibers, which synapse on specific locations of the dendritic tree. Plasticity occurs in DG at the synapses between perforant path fibers and granule cells. In fact, plasticity had been discovered and described for the first time precisely at these synapses, in rabbit. Because the fibers coming from the entorhinal cortex split in two types, lateral and medial, with different characteristics, plasticity in DG is also of two types. The basal responses mediated by the two inputs are different in the rising phase of the field EPSPs (being slower for the lateral input, as the lateral fibers terminate more distally), and paired-pulse plasticity (lateral input displaying facilitation, while medial input- depression).  LTP at medial perforant path synapses is a typical form of NMDAR-dependent plasticity; LTD has also been described for this path. Plasticity at the lateral perforant path, on the other hand, is NMDAR-independent, and sensitive to opioids, in a frequency-dependent manner (Xie & Lewis, 1991). Also, pairing protocols are not effective in inducing this kind of plasticity (Colino & Malenka 1993). An important particularity of dentate gyrus is that it is one of the few structures supporting neurogenesis in adulthood. About 9000 new granule cells are born every day, with an essential role in certain hippocampal functions (Shors et al., 2001). A small fraction of them survives to develop into mature granule cells, to become integrated into the DG cellular network, and to establish similar connectivity (Hastings and Gould, 1999, 52  Song et al., 2002). The threshold for LTP induction was reported to be lower for the immature granule cells (Schmidt-Hieber et al., 2004), due to both expression of T-type calcium channels, and increased input resistance. Neurogenesis is regulated by genetic (Kempermann, 1997, 2000) and epigenetic factors, amongst which exercise / running has been shown to dramatically enhance it (van Praag, 1999).              53  1.4 Theme and hypothesis Previous work in our laboratory suggested that synaptic and extrasynaptic NMDARs are differentially regulated (Li et al, 2002), and this may have important consequences for receptor functions. Our purpose was to investigate the mechanisms for this differential modulation, as well as if there are subunit differences pertaining to it. The property we brought under scrutiny was desensitization, being highly modulated for the various NMDARs subtypes (location and subunit–wise), as well as having tremendous impact on the calcium influx, crucial to NMDAR functions. We have investigated how the interaction of NMDARs with the MAGUK family of proteins regulates desensitization. Predominantly, we investigated the effect of PSD-95, a scaffold protein with a certain profile of expression during development, and having certain affinities with NMDAR subtypes. Our hypotheses for the first chapter were: 1) PSD-95 regulates desensitization directly, and not through recruiting neuronal-specific factors in receptors’ proximity; 2) a direct interaction of PSD-95 with the receptor is sufficient to regulate desensitization, both in recombinant and neuronal receptors. Moreover, we proposed to elucidate which domains of PDS-95 are required for regulation and to test the effect of uncoupling the receptors from PSD-95 on desensitization (using three different means: TPA, 2BP, and NR2B-9C peptide treatments). 54  Having elucidated the extent of desensitization each subpopulation of NMDARs displays, and how PSD-95 modulates it, we proceeded further to elucidate how these subpopulations are involved in plasticity induction in the rodent hippocampus. Our hypotheses for this chapter were that the level of potentiation and depression induced through the activation of NR2 subpopulations would show differences that may possibly be explained by the profile of calcium signal mediated by these receptors. Both the amount of the intracellular calcium and its subcellular location are important for dictating the direction, and magnitude of plasticity. Further, the amount and localization of calcium signal are a direct consequence of the NMDAR subtype, localization, and various properties. Amongst these, desensitization is a limiting factor. We have evaluated these characteristics of plasticity in mouse dentate gyrus - its direction and magnitude – after having isolated the NR2A and NR2B subpopulations of NMDARS, and activated them with typical plasticity induction paradigms. We have also used a behavioural manipulation, exercise, in order to modify the size of the NR2B subpopulation.      55  1.5 Bibliography  Abraham WC (How long will long-term potentiation last? Philos Trans R Soc Lond B Biol Sci 358:735-744.2003). Al-Hallaq RA, Conrads TP, Veenstra TD, Wenthold RJ (NMDA di-heteromeric receptor populations and associated proteins in rat hippocampus. J Neurosci 27:8334-8343.2007). Al-Hallaq RA, Jarabek BR, Fu Z, Vicini S, Wolfe BB, Yasuda RP (Association of NR3A with the N-methyl-D-aspartate receptor NR1 and NR2 subunits. Mol Pharmacol 62:1119- 1127.2002). Andersen P, Silfvenius H, Sundberg SH, Sveen O (A comparison of distal and proximal dendritic synapses on CA1 pyramids in guinea-pig hippocampal slices in vitro. J Physiol 307:273-299.1980). Antonova I, Arancio O, Trillat AC, Wang HG, Zablow L, Udo H, Kandel ER, Hawkins RD (Rapid increase in clusters of presynaptic proteins at onset of long-lasting potentiation. Science 294:1547-1550.2001). Ascher P, Nowak L (The role of divalent cations in the N-methyl-D-aspartate responses of mouse central neurones in culture. J Physiol 399:247-266.1988). Ashby MC, De La Rue SA, Ralph GS, Uney J, Collingridge GL, Henley JM (Removal of AMPA receptors (AMPARs) from synapses is preceded by transient endocytosis of extrasynaptic AMPARs. J Neurosci 24:5172-5176.2004). Bagal AA, Kao JP, Tang CM, Thompson SM (Long-term potentiation of exogenous glutamate responses at single dendritic spines. Proc Natl Acad Sci U S A 102:14434-14439.2005). Bannister NJ, Larkman AU (Dendritic morphology of CA1 pyramidal neurones from the rat hippocampus: II. Spine distributions. J Comp Neurol 360:161-171.1995). Baranano DE, Ferris CD, Snyder SH (Atypical neural messengers. Trends Neurosci 24:99- 106.2001). Barria A, Malinow R (Subunit-specific NMDA receptor trafficking to synapses. Neuron 35:345- 353.2002). 56  Bassand P, Bernard A, Rafiki A, Gayet D, Khrestchatisky M (Differential interaction of the tSXV motifs of the NR1 and NR2A NMDA receptor subunits with PSD-95 and SAP97. Eur J Neurosci 11:2031-2043.1999). Bear MF, Kleinschmidt A, Gu QA, Singer W (Disruption of experience-dependent synaptic modifications in striate cortex by infusion of an NMDA receptor antagonist. J Neurosci 10:909-925.1990). Behe P, Stern P, Wyllie DJ, Nassar M, Schoepfer R, Colquhoun D (Determination of NMDA NR1 subunit copy number in recombinant NMDA receptors. Proc R Soc Lond B Biol Sci 262:205-213.1995). Bekkers JM, Stevens CF (NMDA and non-NMDA receptors are co-localized at individual excitatory synapses in cultured rat hippocampus. Nature 341:230-233.1989). Benke TA, Luthi A, Isaac JT, Collingridge GL (Modulation of AMPA receptor unitary conductance by synaptic activity. Nature 393:793-797.1998). Benveniste M, Clements J, Vyklicky L, Jr., Mayer ML (A kinetic analysis of the modulation of N-methyl-D-aspartic acid receptors by glycine in mouse cultured hippocampal neurones. J Physiol 428:333-357.1990). Benveniste M, Mayer ML (Kinetic analysis of antagonist action at N-methyl-D-aspartic acid receptors. Two binding sites each for glutamate and glycine. Biophys J 59:560- 573.1991). Benveniste M, Mayer ML (Multiple effects of spermine on N-methyl-D-aspartic acid receptor responses of rat cultured hippocampal neurones. J Physiol 464:131-163.1993). Biscoe TJ, Straughan DW (Micro-electrophoretic studies of neurones in the cat hippocampus. J Physiol 183:341-359.1966). Blanton MG, Kriegstein AR (Properties of amino acid neurotransmitter receptors of embryonic cortical neurons when activated by exogenous and endogenous agonists. J Neurophysiol 67:1185-1200.1992). Bliss TV, Collingridge GL (A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361:31-39.1993). Bliss TV, Lomo T (Plasticity in a monosynaptic cortical pathway. J Physiol 207:61P.1970). 57  Bliss TV, Lomo T (Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. J Physiol 232:331- 356.1973). Bolshakov VY, Golan H, Kandel ER, Siegelbaum SA (Recruitment of new sites of synaptic transmission during the cAMP-dependent late phase of LTP at CA3-CA1 synapses in the hippocampus. Neuron 19:635-651.1997). Bonhaus DW, Perry WB, McNamara JO (Decreased density, but not number, of N-methyl-D- aspartate, glycine and phencyclidine binding sites in hippocampus of senescent rats. Brain Res 532:82-86.1990). Borgland SL, Taha SA, Sarti F, Fields HL, Bonci A (Orexin A in the VTA is critical for the induction of synaptic plasticity and behavioral sensitization to cocaine. Neuron 49:589- 601.2006). Burnashev N (Calcium permeability of glutamate-gated channels in the central nervous system. Curr Opin Neurobiol 6:311-317.1996). Carmignoto G, Vicini S (Activity-dependent decrease in NMDA receptor responses during development of the visual cortex. Science 258:1007-1011.1992). Chen N, Luo T, Raymond LA (Subtype-dependence of NMDA receptor channel open probability. J Neurosci 19:6844-6854.1999). Chen N, Moshaver A, Raymond LA (Differential sensitivity of recombinant N-methyl-D- aspartate receptor subtypes to zinc inhibition. Mol Pharmacol 51:1015-1023.1997). Choi S, Klingauf J, Tsien RW (Postfusional regulation of cleft glutamate concentration during LTP at 'silent synapses'. Nat Neurosci 3:330-336.2000). Ciabarra AM, Sullivan JM, Gahn LG, Pecht G, Heinemann S, Sevarino KA (Cloning and characterization of chi-1: a developmentally regulated member of a novel class of the ionotropic glutamate receptor family. J Neurosci 15:6498-6508.1995). Clements JD, Lester RA, Tong G, Jahr CE, Westbrook GL (The time course of glutamate in the synaptic cleft. Science 258:1498-1501.1992). Clements JD, Westbrook GL (Activation kinetics reveal the number of glutamate and glycine binding sites on the N-methyl-D-aspartate receptor. Neuron 7:605-613.1991). 58  Collingridge GL, Herron CE, Lester RA (Frequency-dependent N-methyl-D-aspartate receptor- mediated synaptic transmission in rat hippocampus. J Physiol 399:301-312.1988). Collingridge GL, Isaac JT, Wang YT (Receptor trafficking and synaptic plasticity. Nat Rev Neurosci 5:952-962.2004). Collingridge GL, Kehl SJ, McLennan H (The antagonism of amino acid-induced excitations of rat hippocampal CA1 neurones in vitro. J Physiol 334:19-31.1983). Colquhoun D (Binding, gating, affinity and efficacy: the interpretation of structure-activity relationships for agonists and of the effects of mutating receptors. Br J Pharmacol 125:924-947.1998). Colquhoun D, Hawkes AG (Desensitization of N-methyl-D-aspartate receptors: a problem of interpretation. Proc Natl Acad Sci U S A 92:10327-10329.1995). Cull-Candy S, Brickley S, Farrant M (NMDA receptor subunits: diversity, development and disease. Curr Opin Neurobiol 11:327-335.2001). Davies CH, Starkey SJ, Pozza MF, Collingridge GL (GABA autoreceptors regulate the induction of LTP. Nature 349:609-611.1991). Davies SN, Lester RA, Reymann KG, Collingridge GL (Temporally distinct pre- and post- synaptic mechanisms maintain long-term potentiation. Nature 338:500-503.1989). Daw NW, Fox K, Sato H, Czepita D (Critical period for monocular deprivation in the cat visual cortex. J Neurophysiol 67:197-202.1992). Derkach V, Barria A, Soderling TR (Ca2+/calmodulin-kinase II enhances channel conductance of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate type glutamate receptors. Proc Natl Acad Sci U S A 96:3269-3274.1999). Dingledine R, Boland LM, Chamberlin NL, Kawasaki K, Kleckner NW, Traynelis SF, Verdoorn TA (Amino acid receptors and uptake systems in the mammalian central nervous system. Crit Rev Neurobiol 4:1-96.1988). Dingledine R, Borges K, Bowie D, Traynelis SF (The glutamate receptor ion channels. Pharmacol Rev 51:7-61.1999). Douglas RM, Goddard GV (Long-term potentiation of the perforant path-granule cell synapse in the rat hippocampus. Brain Res 86:205-215.1975). 59  Dudek SM, Bear MF (Bidirectional long-term modification of synaptic effectiveness in the adult and immature hippocampus. J Neurosci 13:2910-2918.1993). Edmonds B, Colquhoun D (Rapid decay of averaged single-channel NMDA receptor activations recorded at low agonist concentration. Proc Biol Sci 250:279-286.1992). Ehlers MD, Zhang S, Bernhadt JP, Huganir RL (Inactivation of NMDA receptors by direct interaction of calmodulin with the NR1 subunit. Cell 84:745-755.1996). El-Husseini AE, Schnell E, Chetkovich DM, Nicoll RA, Bredt DS (PSD-95 involvement in maturation of excitatory synapses. Science 290:1364-1368.2000a). El-Husseini AE, Topinka JR, Lehrer-Graiwer JE, Firestein BL, Craven SE, Aoki C, Bredt DS (Ion channel clustering by membrane-associated guanylate kinases. Differential regulation by N-terminal lipid and metal binding motifs. J Biol Chem 275:23904- 23910.2000b). Elias GM, Funke L, Stein V, Grant SG, Bredt DS, Nicoll RA (Synapse-specific and developmentally regulated targeting of AMPA receptors by a family of MAGUK scaffolding proteins. Neuron 52:307-320.2006). Emptage N, Bliss TV, Fine A (Single synaptic events evoke NMDA receptor-mediated release of calcium from internal stores in hippocampal dendritic spines. Neuron 22:115-124.1999). Engert F, Bonhoeffer T (Dendritic spine changes associated with hippocampal long-term synaptic plasticity. Nature 399:66-70.1999). Erreger K, Dravid SM, Banke TG, Wyllie DJ, Traynelis SF (Subunit-specific gating controls rat NR1/NR2A and NR1/NR2B NMDA channel kinetics and synaptic signalling profiles. J Physiol 563:345-358.2005). Erreger K, Traynelis SF (Allosteric interaction between zinc and glutamate binding domains on NR2A causes desensitization of NMDA receptors. J Physiol 569:381-393.2005). Erreger K, Traynelis SF (Zinc inhibition of rat NR1/NR2A N-methyl-D-aspartate receptors. J Physiol 586:763-778.2008). Farrant M, Feldmeyer D, Takahashi T, Cull-Candy SG (NMDA-receptor channel diversity in the developing cerebellum. Nature 368:335-339.1994). 60  Fayyazuddin A, Villarroel A, Le Goff A, Lerma J, Neyton J (Four residues of the extracellular N-terminal domain of the NR2A subunit control high-affinity Zn2+ binding to NMDA receptors. Neuron 25:683-694.2000). Feldmeyer D, Lubke J, Silver RA, Sakmann B (Synaptic connections between layer 4 spiny neurone-layer 2/3 pyramidal cell pairs in juvenile rat barrel cortex: physiology and anatomy of interlaminar signalling within a cortical column. J Physiol 538:803- 822.2002). Flint AC, Maisch US, Weishaupt JH, Kriegstein AR, Monyer H (NR2A subunit expression shortens NMDA receptor synaptic currents in developing neocortex. J Neurosci 17:2469- 2476.1997). Forrest D, Yuzaki M, Soares HD, Ng L, Luk DC, Sheng M, Stewart CL, Morgan JI, Connor JA, Curran T (Targeted disruption of NMDA receptor 1 gene abolishes NMDA response and results in neonatal death. Neuron 13:325-338.1994). Fukaya M, Kato A, Lovett C, Tonegawa S, Watanabe M (Retention of NMDA receptor NR2 subunits in the lumen of endoplasmic reticulum in targeted NR1 knockout mice. Proc Natl Acad Sci U S A 100:4855-4860.2003). Furukawa H, Gouaux E (Mechanisms of activation, inhibition and specificity: crystal structures of the NMDA receptor NR1 ligand-binding core. Embo J 22:2873-2885.2003). Furukawa H, Singh SK, Mancusso R, Gouaux E (Subunit arrangement and function in NMDA receptors. Nature 438:185-192.2005). Ganeshina O, Berry RW, Petralia RS, Nicholson DA, Geinisman Y (Differences in the expression of AMPA and NMDA receptors between axospinous perforated and nonperforated synapses are related to the configuration and size of postsynaptic densities. J Comp Neurol 468:86-95.2004). Garaschuk O, Schneggenburger R, Schirra C, Tempia F, Konnerth A (Fractional Ca2+ currents through somatic and dendritic glutamate receptor channels of rat hippocampal CA1 pyramidal neurones. J Physiol 491 ( Pt 3):757-772.1996). Gibb AJ, Colquhoun D (Glutamate activation of a single NMDA receptor-channel produces a cluster of channel openings. Proc Biol Sci 243:39-45.1991). 61  Gibb AJ, Colquhoun D (Activation of N-methyl-D-aspartate receptors by L-glutamate in cells dissociated from adult rat hippocampus. J Physiol 456:143-179.1992). Gurd JW, Bissoon N (The N-methyl-D-aspartate receptor subunits NR2A and NR2B bind to the SH2 domains of phospholipase C-gamma. J Neurochem 69:623-630.1997). Gustafsson B, Wigstrom H (Hippocampal long-lasting potentiation produced by pairing single volleys and brief conditioning tetani evoked in separate afferents. J Neurosci 6:1575- 1582.1986). Hardingham GE, Bading H (The Yin and Yang of NMDA receptor signalling. Trends Neurosci 26:81-89.2003). Harris KM, Jensen FE, Tsao B (Three-dimensional structure of dendritic spines and synapses in rat hippocampus (CA1) at postnatal day 15 and adult ages: implications for the maturation of synaptic physiology and long-term potentiation. J Neurosci 12:2685- 2705.1992). Harvey J, Collingridge GL (Thapsigargin blocks the induction of long-term potentiation in rat hippocampal slices. Neurosci Lett 139:197-200.1992). Hastings NB, Gould E (Rapid extension of axons into the CA3 region by adult-generated granule cells. J Comp Neurol 413:146-154.1999). Hayashi Y, Shi SH, Esteban JA, Piccini A, Poncer JC, Malinow R (Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction. Science 287:2262-2267.2000). Hering H, Sheng M (Activity-dependent redistribution and essential role of cortactin in dendritic spine morphogenesis. J Neurosci 23:11759-11769.2003). Hernandez RV, Navarro MM, Rodriguez WA, Martinez JL, Jr., LeBaron RG (Differences in the magnitude of long-term potentiation produced by theta burst and high frequency stimulation protocols matched in stimulus number. Brain Res Brain Res Protoc 15:6- 13.2005). Hestrin S (Developmental regulation of NMDA receptor-mediated synaptic currents at a central synapse. Nature 357:686-689.1992). Hu B, Zheng F (Molecular determinants of glycine-independent desensitization of NR1/NR2A receptors. J Pharmacol Exp Ther 313:563-569.2005). 62  Inanobe A, Furukawa H, Gouaux E (Mechanism of partial agonist action at the NR1 subunit of NMDA receptors. Neuron 47:71-84.2005). Isaac JT, Nicoll RA, Malenka RC (Evidence for silent synapses: implications for the expression of LTP. Neuron 15:427-434.1995). Ishii T, Moriyoshi K, Sugihara H, Sakurada K, Kadotani H, Yokoi M, Akazawa C, Shigemoto R, Mizuno N, Masu M, et al. (Molecular characterization of the family of the N-methyl-D- aspartate receptor subunits. J Biol Chem 268:2836-2843.1993). Ito I, Futai K, Katagiri H, Watanabe M, Sakimura K, Mishina M, Sugiyama H (Synapse- selective impairment of NMDA receptor functions in mice lacking NMDA receptor epsilon 1 or epsilon 2 subunit. J Physiol 500 ( Pt 2):401-408.1997). Ivanovic A, Reilander H, Laube B, Kuhse J (Expression and initial characterization of a soluble glycine binding domain of the N-methyl-D-aspartate receptor NR1 subunit. J Biol Chem 273:19933-19937.1998). Jackson MF, Konarski JZ, Weerapura M, Czerwinski W, MacDonald JF (Protein kinase C enhances glycine-insensitive desensitization of NMDA receptors independently of previously identified protein kinase C sites. J Neurochem 96:1509-1518.2006). Jahr CE (High probability opening of NMDA receptor channels by L-glutamate. Science 255:470-472.1992). Jahr CE, Stevens CF (Glutamate activates multiple single channel conductances in hippocampal neurons. Nature 325:522-525.1987). Johnson JW, Ascher P (Glycine potentiates the NMDA response in cultured mouse brain neurons. Nature 325:529-531.1987). Kawakami R, Shinohara Y, Kato Y, Sugiyama H, Shigemoto R, Ito I (Asymmetrical allocation of NMDA receptor epsilon2 subunits in hippocampal circuitry. Science 300:990- 994.2003). Kew JN, Koester A, Moreau JL, Jenck F, Ouagazzal AM, Mutel V, Richards JG, Trube G, Fischer G, Montkowski A, Hundt W, Reinscheid RK, Pauly-Evers M, Kemp JA, Bluethmann H (Functional consequences of reduction in NMDA receptor glycine affinity in mice carrying targeted point mutations in the glycine binding site. J Neurosci 20:4037- 4049.2000). 63  Kim CH, Chung HJ, Lee HK, Huganir RL (Interaction of the AMPA receptor subunit GluR2/3 with PDZ domains regulates hippocampal long-term depression. Proc Natl Acad Sci U S A 98:11725-11730.2001). Kohda K, Wang Y, Yuzaki M (Mutation of a glutamate receptor motif reveals its role in gating and delta2 receptor channel properties. Nat Neurosci 3:315-322.2000). Komuro H, Rakic P (Modulation of neuronal migration by NMDA receptors. Science 260:95- 97.1993). Kornau HC, Schenker LT, Kennedy MB, Seeburg PH (Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science 269:1737- 1740.1995). Krupp JJ, Vissel B, Heinemann SF, Westbrook GL (Calcium-dependent inactivation of recombinant N-methyl-D-aspartate receptors is NR2 subunit specific. Mol Pharmacol 50:1680-1688.1996). Krupp JJ, Vissel B, Heinemann SF, Westbrook GL (N-terminal domains in the NR2 subunit control desensitization of NMDA receptors. Neuron 20:317-327.1998). Krupp JJ, Vissel B, Thomas CG, Heinemann SF, Westbrook GL (Interactions of calmodulin and alpha-actinin with the NR1 subunit modulate Ca2+-dependent inactivation of NMDA receptors. J Neurosci 19:1165-1178.1999). Krupp JJ, Vissel B, Thomas CG, Heinemann SF, Westbrook GL (Calcineurin acts via the C- terminus of NR2A to modulate desensitization of NMDA receptors. Neuropharmacology 42:593-602.2002). Kullmann DM, Asztely F (Extrasynaptic glutamate spillover in the hippocampus: evidence and implications. Trends Neurosci 21:8-14.1998). Kutsuwada T, Kashiwabuchi N, Mori H, Sakimura K, Kushiya E, Araki K, Meguro H, Masaki H, Kumanishi T, Arakawa M, et al. (Molecular diversity of the NMDA receptor channel [see comments]. Nature 358:36-41.1992). Kutsuwada T, Sakimura K, Manabe T, Takayama C, Katakura N, Kushiya E, Natsume R, Watanabe M, Inoue Y, Yagi T, Aizawa S, Arakawa M, Takahashi T, Nakamura Y, Mori H, Mishina M (Impairment of suckling response, trigeminal neuronal pattern formation, 64  and hippocampal LTD in NMDA receptor epsilon 2 subunit mutant mice. Neuron 16:333-344.1996). Larson J, Wong D, Lynch G (Patterned stimulation at the theta frequency is optimal for the induction of hippocampal long-term potentiation. Brain Res 368:347-350.1986). Laurie DJ, Seeburg PH (Ligand affinities at recombinant N-methyl-D-aspartate receptors depend on subunit composition. Eur J Pharmacol 268:335-345.1994a). Laurie DJ, Seeburg PH (Regional and developmental heterogeneity in splicing of the rat brain NMDAR1 mRNA. J Neurosci 14:3180-3194.1994b). Lavezzari G, McCallum J, Dewey CM, Roche KW (Subunit-specific regulation of NMDA receptor endocytosis. J Neurosci 24:6383-6391.2004). Lee HK, Barbarosie M, Kameyama K, Bear MF, Huganir RL (Regulation of distinct AMPA receptor phosphorylation sites during bidirectional synaptic plasticity. Nature 405:955- 959.2000). Lee LJ, Lo FS, Erzurumlu RS (NMDA receptor-dependent regulation of axonal and dendritic branching. J Neurosci 25:2304-2311.2005). Legendre P, Rosenmund C, Westbrook GL (Inactivation of NMDA channels in cultured hippocampal neurons by intracellular calcium. J Neurosci 13:674-684.1993). Legendre P, Westbrook GL (The inhibition of single N-methyl-D-aspartate-activated channels by zinc ions on cultured rat neurones. J Physiol (Lond) 429:429-449.1990). Lerma J (Spermine regulates N-methyl-D-aspartate receptor desensitization. Neuron 8:343- 352.1992). Lester RA, Clements JD, Westbrook GL, Jahr CE (Channel kinetics determine the time course of NMDA receptor-mediated synaptic currents. Nature 346:565-567.1990). Lester RA, Jahr CE (NMDA channel behavior depends on agonist affinity. J Neurosci 12:635- 643.1992). Lester RA, Tong G, Jahr CE (Interactions between the glycine and glutamate binding sites of the NMDA receptor. J Neurosci 13:1088-1096.1993). Levy WB, Steward O (Temporal contiguity requirements for long-term associative potentiation/depression in the hippocampus. Neuroscience 8:791-797.1983). 65  Li B, Chen N, Luo T, Otsu Y, Murphy TH, Raymond LA (Differential regulation of synaptic and extra-synaptic NMDA receptors. Nat Neurosci 5:833-834.2002). Li B, Otsu Y, Murphy TH, Raymond LA (Developmental decrease in NMDA receptor desensitization associated with shift to synapse and interaction with postsynaptic density- 95. J Neurosci 23:11244-11254.2003a). Li JH, Wang YH, Wolfe BB, Krueger KE, Corsi L, Stocca G, Vicini S (Developmental changes in localization of NMDA receptor subunits in primary cultures of cortical neurons. Eur J Neurosci 10:1704-1715.1998). Li L, Fan M, Icton CD, Chen N, Leavitt BR, Hayden MR, Murphy TH, Raymond LA (Role of NR2B-type NMDA receptors in selective neurodegeneration in Huntington disease. Neurobiol Aging 24:1113-1121.2003b). Li L, Murphy TH, Hayden MR, Raymond LA (Enhanced striatal NR2B-containing N-methyl-D- aspartate receptor-mediated synaptic currents in a mouse model of Huntington disease. J Neurophysiol 92:2738-2746.2004). Lin F, Stevens CF (Both open and closed NMDA receptor channels desensitize. J Neurosci 14:2153-2160.1994). Lin Y, Skeberdis VA, Francesconi A, Bennett MV, Zukin RS (Postsynaptic density protein-95 regulates NMDA channel gating and surface expression. J Neurosci 24:10138- 10148.2004). Lisman J (A mechanism for the Hebb and the anti-Hebb processes underlying learning and memory. Proc Natl Acad Sci U S A 86:9574-9578.1989). Lomeli H, Sprengel R, Laurie DJ, Kohr G, Herb A, Seeburg PH, Wisden W (The rat delta-1 and delta-2 subunits extend the excitatory amino acid receptor family. FEBS Lett 315:318- 322.1993). Losi G, Prybylowski K, Fu Z, Luo J, Wenthold RJ, Vicini S (PSD-95 regulates NMDA receptors in developing cerebellar granule neurons of the rat. J Physiol 548:21-29.2003). LoTurco JJ, Blanton MG, Kriegstein AR (Initial expression and endogenous activation of NMDA channels in early neocortical development. J Neurosci 11:792-799.1991). 66  Low CM, Zheng F, Lyuboslavsky P, Traynelis SF (Molecular determinants of coordinated proton and zinc inhibition of N-methyl-D-aspartate NR1/NR2A receptors. Proc Natl Acad Sci U S A 97:11062-11067.2000). Lu W, Man H, Ju W, Trimble WS, MacDonald JF, Wang YT (Activation of synaptic NMDA receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons. Neuron 29:243-254.2001). Lynch G, Larson J, Kelso S, Barrionuevo G, Schottler F (Intracellular injections of EGTA block induction of hippocampal long-term potentiation. Nature 305:719-721.1983). Maletic-Savatic M, Malinow R, Svoboda K (Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science 283:1923-1927.1999). Matsuzaki M, Ellis-Davies GC, Nemoto T, Miyashita Y, Iino M, Kasai H (Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons. Nat Neurosci 4:1086-1092.2001). Mayer ML, MacDermott AB, Westbrook GL, Smith SJ, Barker JL (Agonist- and voltage-gated calcium entry in cultured mouse spinal cord neurons under voltage clamp measured using arsenazo III. J Neurosci 7:3230-3244.1987). Mayer ML, Vyklicky L, Jr., Clements J (Regulation of NMDA receptor desensitization in mouse hippocampal neurons by glycine. Nature 338:425-427.1989). Mayer ML, Westbrook GL, Guthrie PB (Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones. Nature 309:261-263.1984). McBain CJ, Mayer ML (N-methyl-D-aspartic acid receptor structure and function. Physiol Rev 74:723-760.1994). Meddows E, Le Bourdelles B, Grimwood S, Wafford K, Sandhu S, Whiting P, McIlhinney RA (Identification of molecular determinants that are important in the assembly of N-methyl- D-aspartate receptors. J Biol Chem 276:18795-18803.2001). Megias M, Emri Z, Freund TF, Gulyas AI (Total number and distribution of inhibitory and excitatory synapses on hippocampal CA1 pyramidal cells. Neuroscience 102:527- 540.2001). Migaud M, Charlesworth P, Dempster M, Webster LC, Watabe AM, Makhinson M, He Y, Ramsay MF, Morris RG, Morrison JH, O'Dell TJ, Grant SG (Enhanced long-term 67  potentiation and impaired learning in mice with mutant postsynaptic density-95 protein. Nature 396:433-439.1998). Mohn AR, Gainetdinov RR, Caron MG, Koller BH (Mice with reduced NMDA receptor expression display behaviors related to schizophrenia. Cell 98:427-436.1999). Mohrmann R, Kohr G, Hatt H, Sprengel R, Gottmann K (Deletion of the C-terminal domain of the NR2B subunit alters channel properties and synaptic targeting of N-methyl-D- aspartate receptors in nascent neocortical synapses. J Neurosci Res 68:265-275.2002). Montgomery JM, Pavlidis P, Madison DV (Pair recordings reveal all-silent synaptic connections and the postsynaptic expression of long-term potentiation. Neuron 29:691-701.2001). Monyer H, Burnashev N, Laurie DJ, Sakmann B, Seeburg PH (Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12:529-540.1994). Monyer H, Sprengel R, Schoepfer R, Herb A, Higuchi M, Lomeli H, Burnashev N, Sakmann B, Seeburg PH (Heteromeric NMDA receptors: molecular and functional distinction of subtypes. Science 256:1217-1221.1992). Mori H, Masaki H, Yamakura T, Mishina M (Identification by mutagenesis of a Mg(2+)-block site of the NMDA receptor channel. Nature 358:673-675.1992). Mu Y, Otsuka T, Horton AC, Scott DB, Ehlers MD (Activity-dependent mRNA splicing controls ER export and synaptic delivery of NMDA receptors. Neuron 40:581-594.2003). Mulkey RM, Malenka RC (Mechanisms underlying induction of homosynaptic long-term depression in area CA1 of the hippocampus. Neuron 9:967-975.1992). Nadler JV, Martin D, Bustos GA, Burke SP, Bowe MA (Regulation of glutamate and aspartate release from the Schaffer collaterals and other projections of CA3 hippocampal pyramidal cells. Prog Brain Res 83:115-130.1990). Nakazawa K, Quirk MC, Chitwood RA, Watanabe M, Yeckel MF, Sun LD, Kato A, Carr CA, Johnston D, Wilson MA, Tonegawa S (Requirement for hippocampal CA3 NMDA receptors in associative memory recall. Science 297:211-218.2002). Niethammer M, Kim E, Sheng M (Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases. J Neurosci 16:2157-2163.1996). 68  Nowak L, Bregestovski P, Ascher P, Herbet A, Prochiantz A (Magnesium gates glutamate- activated channels in mouse central neurones. Nature 307:462-465.1984). Nusser Z, Lujan R, Laube G, Roberts JD, Molnar E, Somogyi P (Cell type and pathway dependence of synaptic AMPA receptor number and variability in the hippocampus. Neuron 21:545-559.1998). Olson CR, Freeman RD (Profile of the sensitive period for monocular deprivation in kittens. Exp Brain Res 39:17-21.1980). Pak DT, Yang S, Rudolph-Correia S, Kim E, Sheng M (Regulation of dendritic spine morphology by SPAR, a PSD-95-associated RapGAP. Neuron 31:289-303.2001). Paoletti P, Ascher P, Neyton J (High-affinity zinc inhibition of NMDA NR1-NR2A receptors. J Neurosci 17:5711-5725.1997). Paoletti P, Perin-Dureau F, Fayyazuddin A, Le Goff A, Callebaut I, Neyton J (Molecular organization of a zinc binding n-terminal modulatory domain in a NMDA receptor subunit. Neuron 28:911-925.2000). Perin-Dureau F, Rachline J, Neyton J, Paoletti P (Mapping the binding site of the neuroprotectant ifenprodil on NMDA receptors. J Neurosci 22:5955-5965.2002). Petralia RS, Esteban JA, Wang YX, Partridge JG, Zhao HM, Wenthold RJ, Malinow R (Selective acquisition of AMPA receptors over postnatal development suggests a molecular basis for silent synapses. Nat Neurosci 2:31-36.1999). Pickard L, Noel J, Duckworth JK, Fitzjohn SM, Henley JM, Collingridge GL, Molnar E (Transient synaptic activation of NMDA receptors leads to the insertion of native AMPA receptors at hippocampal neuronal plasma membranes. Neuropharmacology 41:700- 713.2001). Prybylowski K, Chang K, Sans N, Kan L, Vicini S, Wenthold RJ (The synaptic localization of NR2B-containing NMDA receptors is controlled by interactions with PDZ proteins and AP-2. Neuron 47:845-857.2005). Rabacchi S, Bailly Y, Delhaye-Bouchaud N, Mariani J (Involvement of the N-methyl D- aspartate (NMDA) receptor in synapse elimination during cerebellar development. Science 256:1823-1825.1992). 69  Racca C, Stephenson FA, Streit P, Roberts JD, Somogyi P (NMDA receptor content of synapses in stratum radiatum of the hippocampal CA1 area. J Neurosci 20:2512-2522.2000). Regalado MP, Villarroel A, Lerma J (Intersubunit cooperativity in the NMDA receptor. Neuron 32:1085-1096.2001). Ren H, Honse Y, Karp BJ, Lipsky RH, Peoples RW (A site in the fourth membrane-associated domain of the N-methyl-D-aspartate receptor regulates desensitization and ion channel gating. J Biol Chem 278:276-283.2003). Roche KW, Standley S, McCallum J, Dune Ly C, Ehlers MD, Wenthold RJ (Molecular determinants of NMDA receptor internalization. Nat Neurosci 4:794-802.2001). Rose GM, Dunwiddie TV (Induction of hippocampal long-term potentiation using physiologically patterned stimulation. Neurosci Lett 69:244-248.1986). Rosenmund C, Feltz A, Westbrook GL (Calcium-dependent inactivation of synaptic NMDA receptors in hippocampal neurons. J Neurophysiol 73:427-430.1995a). Rosenmund C, Feltz A, Westbrook GL (Synaptic NMDA receptor channels have a low open probability. J Neurosci 15:2788-2795.1995b). Rosenmund C, Westbrook GL (Rundown of N-methyl-D-aspartate channels during whole-cell recording in rat hippocampal neurons: role of Ca2+ and ATP [published erratum appears in J Physiol (Lond) 1994 Mar 15;475(3):547-8]. J Physiol (Lond) 470:705-729.1993). Sah P, Hestrin S, Nicoll RA (Tonic activation of NMDA receptors by ambient glutamate enhances excitability of neurons. Science 246:815-818.1989). Sakimura K, Kutsuwada T, Ito I, Manabe T, Takayama C, Kushiya E, Yagi T, Aizawa S, Inoue Y, Sugiyama H, et al. (Reduced hippocampal LTP and spatial learning in mice lacking NMDA receptor epsilon 1 subunit. Nature 373:151-155.1995). Sans N, Petralia RS, Wang YX, Blahos J, 2nd, Hell JW, Wenthold RJ (A developmental change in NMDA receptor-associated proteins at hippocampal synapses. J Neurosci 20:1260- 1271.2000). Sassoe-Pognetto M, Ottersen OP (Organization of ionotropic glutamate receptors at dendrodendritic synapses in the rat olfactory bulb. J Neurosci 20:2192-2201.2000). 70  Sather W, Dieudonne S, MacDonald JF, Ascher P (Activation and desensitization of N-methyl- D-aspartate receptors in nucleated outside-out patches from mouse neurones. J Physiol 450:643-672.1992a). Sather W, Dieudonne S, MacDonald JF, Ascher P (Activation and desensitization of N-methyl- D-aspartate receptors in nucleated outside-out patches from mouse neurones. J Physiol 450:643-672.1992b). Schell MJ, Molliver ME, Snyder SH (D-serine, an endogenous synaptic modulator: localization to astrocytes and glutamate-stimulated release. Proc Natl Acad Sci U S A 92:3948- 3952.1995). Schmidt-Hieber C, Jonas P, Bischofberger J (Enhanced synaptic plasticity in newly generated granule cells of the adult hippocampus. Nature 429:184-187.2004). Schneggenburger R (Simultaneous measurement of Ca2+ influx and reversal potentials in recombinant N-methyl-D-aspartate receptor channels. Biophys J 70:2165-2174.1996). Schneggenburger R, Zhou Z, Konnerth A, Neher E (Fractional contribution of calcium to the cation current through glutamate receptor channels. Neuron 11:133-143.1993). Schulz PE, Cook EP, Johnston D (Changes in paired-pulse facilitation suggest presynaptic involvement in long-term potentiation. J Neurosci 14:5325-5337.1994). Scimemi A, Fine A, Kullmann DM, Rusakov DA (NR2B-containing receptors mediate cross talk among hippocampal synapses. J Neurosci 24:4767-4777.2004). Sessoms-Sikes S, Honse Y, Lovinger DM, Colbran RJ (CaMKIIalpha enhances the desensitization of NR2B-containing NMDA receptors by an autophosphorylation- dependent mechanism. Mol Cell Neurosci 29:139-147.2005). Sheng S (The promise and challenge toward the clinical application of maspin in cancer. Front Biosci 9:2733-2745.2004). Shi SH, Hayashi Y, Petralia RS, Zaman SH, Wenthold RJ, Svoboda K, Malinow R (Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation. Science 284:1811-1816.1999). Shors TJ, Miesegaes G, Beylin A, Zhao M, Rydel T, Gould E (Neurogenesis in the adult is involved in the formation of trace memories. Nature 410:372-376.2001). 71  Single FN, Rozov A, Burnashev N, Zimmermann F, Hanley DF, Forrest D, Curran T, Jensen V, Hvalby O, Sprengel R, Seeburg PH (Dysfunctions in mice by NMDA receptor point mutations NR1(N598Q) and NR1(N598R). J Neurosci 20:2558-2566.2000). Song HJ, Stevens CF, Gage FH (Neural stem cells from adult hippocampus develop essential properties of functional CNS neurons. Nat Neurosci 5:438-445.2002). Sprengel R, Suchanek B, Amico C, Brusa R, Burnashev N, Rozov A, Hvalby O, Jensen V, Paulsen O, Andersen P, Kim JJ, Thompson RF, Sun W, Webster LC, Grant SG, Eilers J, Konnerth A, Li J, McNamara JO, Seeburg PH (Importance of the intracellular domain of NR2 subunits for NMDA receptor function in vivo. Cell 92:279-289.1998). Standaert DG, Landwehrmeyer GB, Kerner JA, Penney JB, Jr., Young AB (Expression of NMDAR2D glutamate receptor subunit mRNA in neurochemically identified interneurons in the rat neostriatum, neocortex and hippocampus. Brain Res Mol Brain Res 42:89-102.1996). Stanton PK, Heinemann U, Muller W (FM1-43 imaging reveals cGMP-dependent long-term depression of presynaptic transmitter release. J Neurosci 21:RC167.2001). Stevens CF, Wang Y (Changes in reliability of synaptic function as a mechanism for plasticity. Nature 371:704-707.1994). Stocca G, Vicini S (Increased contribution of NR2A subunit to synaptic NMDA receptors in developing rat cortical neurons. J Physiol 507:13-24.1998). Strack S, Colbran RJ (Autophosphorylation-dependent targeting of calcium/ calmodulin- dependent protein kinase II by the NR2B subunit of the N-methyl- D-aspartate receptor. J Biol Chem 273:20689-20692.1998). Stuart GJ, Sakmann B (Active propagation of somatic action potentials into neocortical pyramidal cell dendrites. Nature 367:69-72.1994). Szerb JC (Changes in the relative amounts of aspartate and glutamate released and retained in hippocampal slices during stimulation. J Neurochem 50:219-224.1988). Takumi Y, Bergersen L, Landsend AS, Rinvik E, Ottersen OP (Synaptic arrangement of glutamate receptors. Prog Brain Res 116:105-121.1998). Takumi Y, Ramirez-Leon V, Laake P, Rinvik E, Ottersen OP (Different modes of expression of AMPA and NMDA receptors in hippocampal synapses. Nat Neurosci 2:618-624.1999). 72  Tong G, Jahr CE (Regulation of glycine-insensitive desensitization of the NMDA receptor in outside-out patches. J Neurophysiol 72:754-761.1994). Tong G, Shepherd D, Jahr CE (Synaptic desensitization of NMDA receptors by calcineurin. Science 267:1510-1512.1995). Tovar KR, Westbrook GL (The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro. J Neurosci 19:4180-4188.1999). Vicini S, Wang JF, Li JH, Zhu WJ, Wang YH, Luo JH, Wolfe BB, Grayson DR (Functional and pharmacological differences between recombinant N-methyl-D-aspartate receptors. J Neurophysiol 79:555-566.1998). Villarroel A, Regalado MP, Lerma J (Desensitization of NMDA receptors as a mechanism of neuroprotection. Methods Find Exp Clin Pharmacol 19 Suppl A:51-53.1997). Villarroel A, Regalado MP, Lerma J (Glycine-independent NMDA receptor desensitization: localization of structural determinants. Neuron 20:329-339.1998). Vyklicky L, Jr., Benveniste M, Mayer ML (Modulation of N-methyl-D-aspartic acid receptor desensitization by glycine in mouse cultured hippocampal neurones. J Physiol 428:313- 331.1990). Watanabe M, Inoue Y, Sakimura K, Mishina M (Developmental changes in distribution of NMDA receptor channel subunit mRNAs. Neuroreport 3:1138-1140.1992). Wikstrom MA, Matthews P, Roberts D, Collingridge GL, Bortolotto ZA (Parallel kinase cascades are involved in the induction of LTP at hippocampal CA1 synapses. Neuropharmacology 45:828-836.2003). Yasuda H, Barth AL, Stellwagen D, Malenka RC (A developmental switch in the signaling cascades for LTP induction. Nat Neurosci 6:15-16.2003). Yoshii A, Sheng MH, Constantine-Paton M (Eye opening induces a rapid dendritic localization of PSD-95 in central visual neurons. Proc Natl Acad Sci U S A 100:1334-1339.2003). Zhang S, Ehlers MD, Bernhardt JP, Su CT, Huganir RL (Calmodulin mediates calcium- dependent inactivation of N-methyl-D- aspartate receptors. Neuron 21:443-453.1998). Zheng F, Erreger K, Low CM, Banke T, Lee CJ, Conn PJ, Traynelis SF (Allosteric interaction between the amino terminal domain and the ligand binding domain of NR2A. Nat Neurosci 4:894-901.2001). 73  Chapter Two1 2 NMDA Receptor Desensitization Regulated by Direct Binding to PDZ1-2 Domains of PSD-95  N-methyl-D-aspartic acid (NMDA) – type glutamate receptors are expressed at most CNS excitatory synapses and play key roles in synaptogenesis, induction of synaptic plasticity, and excitotoxic neuronal death. These receptors are tetrameric complexes of two NR1 with two NR2, or one NR2 and one regulatory NR3, subunits (Cull-Candy et al., 2001).  NR2 subunits are encoded by four different genes – NR2A, B, C, D – that are tightly regulated temporally and spatially (Monyer et al., 1994), and these subunits determine differences in NMDAR pharmacological and physiological properties (Cull- Candy et al., 2001).  Both NR2A and NR2B subunits are highly expressed in the mature forebrain where NMDARs containing NR1/NR2B predominate at non-synaptic sites in the neuronal plasma membrane and NR2A-containing NMDARs are enriched at the synapse (Stocca and Vicini, 1998, Tovar and Westbrook, 1999, Barria and Malinow, 2002). Differences in subcellular distribution are not absolute, however, and determining effects of subunit composition on NMDAR localization and signaling in neurons is complicated by expression of tri-heteromeric NR1/NR2A/NR2B complexes (Luo et al., 1997, Thomas et al., 2006). Additionally, subcellular localization of NMDARs affects their functional regulation (Li et al., 2002, Li et al., 2003a).  i. 21  A version of this chapter has been published. Sornarajah L*, Vasuta OC*, Zhang L, Sutton C, Li B, El- Husseini A, Raymond LA (2008) NMDA receptor desensitization regulated by direct binding to PDZ1-2 domains of PSD-95 Journal of Neurophysiology 99(6):3052-62 (used with permission).  74  Desensitization of glutamate receptors -- the decay of current with sustained agonist exposure – protects neurons from excitotoxicity (Zorumski et al., 1990). NMDAR desensitization also shapes neuronal responses to repeated stimulation (Tong et al., 1995). Previously, we reported that NMDAR desensitization is regulated by receptor subcellular localization in hippocampal pyramidal neurons (Li et al., 2003a). Interestingly, synaptic NMDARs showed dramatically less desensitization than non-synaptic NMDARs, despite enrichment of NR2A subunits at the synapse and extrasynaptic localization of NR2B- containing receptors. This result was unexpected since NR1/NR2A desensitizes more extensively than NR1/NR2B when expressed in non-neuronal cells (Krupp et al., 1996, Dingledine et al., 1999). NMDAR association with PSD-95 in hippocampal neurons also correlated with decreased desensitization (Li et al., 2003a); however, it was not clear whether this effect was secondary to PSD-95’s role as a scaffolding protein, anchoring protein kinases and phosphatases in close proximity to NMDARs (Kim and Sheng, 2004), or if its binding to NMDARs directly altered receptor desensitization. Palmitoylation at two cysteines near the N-terminus of PSD-95 targets this protein to the synapse (Craven et al., 1999) where it participates in a complex of nearly 200 proteins associated with NMDARs (Kim and Sheng, 2004, Collins et al., 2006). Expression of PSD-95 with NMDARs in non-neuronal cells results in receptor clustering (Kim and Sheng, 1996), although PSD-95 is not required for NMDAR targeting or clustering at neuronal synapses (El-Husseini et al., 2000a) In neurons, studies suggest a role for PSD-95 in regulating NMDAR surface expression (Roche et al., 2001, Lin et al., 2004)  and stabilizing NR2B-containing NMDARs at synapses (Prybylowski et al., 2005), but evidence for a direct role in regulating NMDAR function is lacking. Here we show for the 75  first time that direct binding of the PDZ1-2 domains of PSD-95 to NR2A- or NR2B- containing receptors reduces NMDAR desensitization.  Additionally, PSD-95 binding to NMDARs and its effect on current desensitization is regulated by PKC activation.  2.1 Materials and methods 2.1.1  Primary neuronal cultures and transfection Hippocampal cultures were prepared from 17- to 18-day-old rat embryos as described previously (Li et al., 2002) of ~300-400 cells/mm2 and grown in B-27 supplemented Neurobasal medium in a humidified atmosphere with 5% CO2. Medium was refreshed twice every week by replacing half the volume. Neurons were transfected with 1.2 µg of plasmid DNA per well 24-well plate, using a calcium phosphate kit (Clonetech, Mountain View, CA). Neurons, plated on coverslips, were transfected at 4 days in vitro (Cottrell et al., 2004) and used for patch clamp recording 2-3 days after transfection.  2.1.2  HEK293 cell culture and transfection HEK293 cells (CRL 1573; American Type Culture Collection, Rockville, MD) were maintained as we described previously (Chen et al., 1997). Cells were transfected using the calcium phosphate precipitation method (Chen et al., 1997) with a total of 12 µg of plasmid DNA per 10 cm plate. Cells were transfected with a 1:1:2 ratio of cDNAs encoding NR1-1a, NR2 (A or B or  NR2A-S1462A), and either green fluorescent protein 76  (GFP), PSD-95-GFP, PSD-95 PDZ1-2-GFP, growth-associated protein 43 (GAP-43) first 12 amino acids (GAP12), GAP12 PDZ1-2-GFP, Prenylated PSD-95-GFP, SAP-102, or PSD-93. To minimize NMDAR-mediated death, cells were bathed in medium supplemented with 100 µM memantine following transfection until the time of recording.  2.1.3  Drug treatments Cultured hippocampal neurons were treated with 12-O-tetradecanoylphorbol-13- acetate (TPA), RO-320432 (RO), or 2-bromopalmitate (2-BP) by direct addition to the medium.  Cultures were then returned to the incubator for time periods ranging from 10 min to 6 hours, as indicated in the Results section.  At the end of the drug incubation period, cells were collected for biochemical experiments, or else the medium was replaced by external recording solution and cells were moved on glass cover slips to the recording chamber for electrophysiology, where recordings were made within five minutes of removal of the drug.  2.1.4  Electrophysiology Cultured neurons were used for recording at 4-7 DIV (“immature”), or >13 DIV (“mature”). Recordings from HEK293 cells were made 12-24 hours following the end of transfection. Conventional whole-cell patch clamp recording was conducted as previously described (Hamill et al., 1981). Electrodes were fabricated from borosilicate glass (Warner 77  Instruments, Hamden, CT) using a Narashige (Tokyo, Japan) PP-83 electrode puller. Open tip resistance was 5-6 MΩ for electrodes containing (in mM):  115 Cs-methanesulfonate, 10 HEPES, 20 K2-creatine phosphate, 4 MgATP, 10 BAPTA, as well as 50 U/ml creatine phosphokinase, pH 7.26, 310 mOsm.  HEK293 cells were superfused with external recording solution, containing (in mM):  145 NaCl, 5.4 KCl, 0.2 CaCl2, 10 HEPES and 11 glucose, pH 7.3.   For neurons, the external solution contained (in mM):  167 NaCl, 2.4 KCl, 10 HEPES, 10 glucose, 0.2 CaCl2, pH 7.3 (325 mOsm), and tetrodotoxin (TTX) (300 nM) and glycine (50 µM) were added just before use.  Agonist (1 mM NMDA) was dissolved in the same solution used to bathe the cells and gravity-fed to the cells through one side of a theta-tube (Chen et al., 1997). All other drugs, dissolved in the external bathing solution, were included in both the control and agonist side of the theta-tube.  Computer-controlled solenoid-driven valves were used to rapidly switch between the two solutions. Agonist was applied for 10 sec at 1- min intervals. For experiments in cultured hippocampal neurons, large, pyramidal-shaped neurons were selected for recording.  In experiments with cultured HEK293 cells, smaller cells (<30 micron diameter, with capacitance of <30 pF) were preferentially selected for recording in order to optimize the rate of agonist exchange, keeping the 10-90% rise-time to peak less than 100 ms. In all recordings, the peak and steady-state current amplitudes were stable over periods of more than 15 minutes.  All recordings were made in voltage-clamp mode at a holding potential of -70 mV. Data were acquired using the Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA). Currents were filtered at 1 kHz and digitized at 10 kHz. 78  pClamp 8.1 software (Axon Instruments) was used for data acquisition and analysis. Series resistance and cell capacitance were regularly monitored and recordings were abandoned if series resistance exceeded 20 MΩ.  2.1.5  Co-immunoprecipitation and western blot analysis Batches of 14-17 DIV hippocampal neuronal cultures in 10-cm dishes were treated with drugs or vehicle as described in Results, then each dish was collected in 1 ml Harvest buffer containing 1 mM EGTA, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 20 µg/ml pepstatin A, and 20 µg/ml leupeptin in PBS. Cell suspensions were processed as described previously (Li et al., 2003a). Briefly, after centrifugation, pellets were lysed and solubilized in Harvest buffer containing 0.1% SDS and 0.8% Triton X-100 (0.5 ml final volume). One-tenth of the lysate was reserved for input loading, and the remainder was incubated with protein-A and protein-G beads then briefly centrifuged to remove nonspecifically bound proteins. The supernatant was incubated with 10 µg of rabbit polyclonal anti-NR2A antibody (Upstate Biotechnology) for 1 hr at 4°C, then Protein-A and protein-G beads were added for another 1-hr incubation period. Beads were washed three times with 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 1% Triton X-100. Proteins were eluted from the beads and denatured by boiling in loading buffer for 5 min, then loaded to SDS-PAGE. Proteins from each group of different treatment conditions (vehicle, 2-BP, TPA, and TPA with 2-BP; or vehicle, RO, and TPA) were loaded to the same gel. After transfer, membranes were probed with antibodies against NR2A (the same as used for immunoprecipitation, at 1 μg/ml) and PSD-95 (mouse 79  monocolonal; 5 µg/ml; Chemicon). Bands were visualized using Enhanced Chemiluminescence (Amersham) and densities were quantified by densitometric analysis (Li et al., 2003a). The PSD-95 to NR2A band-density ratio was calculated to determine the amount of PSD-95 co-immunoprecipitated with NR2A.  2.1.6  Materials All chemicals were purchased from Sigma (St. Louis, MO).  PSD-95 constructs were described previously (Craven et al., 1999, El-Husseini et al., 2000a, Schnell et al., 2002, Christopherson et al., 2003, Prange et al., 2004).  NR2A-S1462A and wild-type rat NR2A (on which the mutant NR2A was made) cDNAs were generous gifts from Dr. Robert Wenthold and were previously described (Prybylowski et al., 2005).  NR1-1a (Dingledine et al., 1999)(Dingledine et al., 1999)(Dingledine et al., 1999)(Dingledine et al., 1999)(Dingledine et al., 1999)(Dingledine et al., 1999)(Dingledine et al., 1999)(Dingledine et al., 1999) NR2B and NR2A (ε1) cDNAs were described previously (Chen et al., 1997). NR2A (ε1) was used for all experiments in HEK cells except those comparing effects of PSD-95 on wild-type versus mutant S1462A NR2A, when the corresponding rat cDNA construct was used for wild-type NR2A.  Tissue culture reagents were obtained from Invitrogen. Stock solutions of 100 mM NMDA and 100 mM glycine were each stored in individual aliquots for up to 6 weeks at -200C.  TPA was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1mM and diluted to a final concentration of 100 nM.  A 200 mM stock solution of 2-BP in ethanol was diluted to a final concentration of 100 μM.  RO was dissolved in DMSO for a stock concentration of 2mM and used at 1μM.  Stock 80  solutions of TPA, 2-BP and RO were stored as aliquots at -200C and thawed only once.  2.1.7  Data analysis Results are presented as mean ± SE. Sets of different results were compared using one-way ANOVA followed by Bonferroni post-test or Student's t-test as appropriate, and significant differences were determined at the 95% confidence intervals unless otherwise indicated.  Three to 10 responses of each cell were averaged for estimation of steady-state to peak current ratio (Iss/Ip).  2.2 Results  2.2.1  PSD-95 regulates NMDAR glycine-independent desensitization NMDAR desensitization attenuates receptor activation during sustained exposure to agonists (Tong et al., 1995). Glycine-independent desensitization can be isolated in cultured hippocampal neurons by recording NMDAR currents in response to 10-sec applications of saturating concentrations of agonist – 1 mM NMDA with 50 µM glycine – in low external calcium and a high intracellular concentration of BAPTA  (Mayer et al., 1989, Tong and Jahr, 1994), as well as strychnine (50 uM) to block the chloride channels. Previously, we demonstrated a developmental decrease in this form of NMDAR desensitization recorded from cultured hippocampal neurons, which could be explained by 81  receptor localization to the synapse; however, the reduction in desensitization also correlated with co-association of NMDARs with PSD-95 (Li et al., 2003a). PSD-95 recruits many scaffolding and signaling molecules to the post-synaptic density directly through its various domains or indirectly through macromolecular MAGUK-AKAP complexes (Colledge et al., 2000, Sheng and Pak, 2000, Lim et al., 2002, Kim and Sheng, 2004). To assess whether the previously reported effect of PSD-95 on NMDAR desensitization (Li et al., 2003a) occurred as a result of neuronal-specific mechanisms, we compared  NMDAR currents from HEK293 cells transfected with either GFP (control) or PSD-95, in combination with different subtypes of NMDARs.  HEK293 cells expressing GFP and NR1/NR2A showed significantly greater desensitization compared to cells co-expressing GFP and NR1/NR2B (Fig. 2.1A, B), as previously reported (Krupp et al., 1996, Dingledine et al., 1999). Strikingly, immature neurons that predominantly express NR1 and NR2B subunits exhibit more extensive NMDAR desensitization than recombinant NR1/NR2B in HEK293 cells (compare Figs. 2.2A,B with 1A,B), whereas mature hippocampal neurons that express largely NR2A-containing receptors (Li et al., 2002) show significantly less desensitization compared with either NMDARs in immature neurons or NR1/NR2A expressed in non-neuronal cells (compare Figs. 2.4A,B with 2.1A,B).  Co-expression of PSD-95 significantly reduced the extent of NMDA-evoked current desensitization in both NR1/NR2A- and NR1/NR2B-expressing HEK293 cells (Fig. 2.1A,B).  In contrast, expression of PSD-95 did not alter the mean NMDA-evoked peak current density (69 ± 9 pA/pF, n=17 for NR1/NR2A + GFP and 63 ± 8 pA/pF, n=10 for NR1/NR2A + PSD-95; P>0.05 by unpaired t-test).  Moreover, the extent of desensitization showed no correlation with peak current density for individual 82  cells as illustrated in Fig. 2.1C, suggesting that the number of active, surface NMDARs does not influence desensitization of whole-cell NMDAR current.  Notably, the steady- state to peak ratio for NMDAR current in PSD-95-expressing cells was similar for NR1/NR2A and NR1/NR2B, and also resembled the ratio found in mature cultured hippocampal neurons (compare Figs. 2.1A,B with 2.4A,B). Previous studies have shown that nanomolar concentrations of zinc accelerate NMDAR desensitization in transfected HEK cells (Chen et al., 1997, Zheng et al., 2001, Erreger et al., 2005, Erreger and Traynelis, 2008), and that PSD-95 expression in Xenopus oocytes reduces the effect of zinc on NR1/NR2A channel function (Yamada et al., 2002) However, when we recorded NMDAR currents from NR1/NR2A-transfected HEK cells under similar conditions to those used in Fig. 2.1A,B except for the addition of 1 µM TPEN, which chelates any contaminating zinc in the recording solution  (Paoletti et al., 1997) we also found a significant difference in steady-state to peak ratio between cells co- transfected with PSD-95 compared with GFP alone.  The Iss/Ip for current evoked by 1 mM glutamate in the presence of 100 µM glycine, nominal zero calcium, and 1 µM TPEN in the bathing solution (and all other conditions the same as described for data in Fig. 1) was 61 ± 4% and 22 ± 6% for PSD-95- and GFP-expressing cells, respectively (n=7 and 6 different cells, respectively; P<0.001 by unpaired t-test).  The more profound desensitization observed in the absence of PSD-95 in these experiments was typical of the current responses evoked by saturating glutamate compared to NMDA in NR1/NR2A- transfected HEK293 cells. The lack of apparent zinc effect on NMDAR desensitization in our experiments may be a result of minimal zinc contamination in the recording solutions. 83  Calcium salts could be a major source of zinc contamination, and our recording solutions contained nominal zero, or very low, added Ca2+. The data shown in Fig. 2.1A,B demonstrate that the role of PSD-95 in regulating NMDAR desensitization is independent of neuronal- and synapse-specific proteins. Therefore, we hypothesized that direct binding of PSD-95 to NMDARs is required to alter glycine-independent desensitization.  NMDARs bind PSD-95 through the C-terminal PDZ- binding motif (ESDV) of the NR2 subunit (Kornau et al., 1995).  To test our hypothesis, we compared NMDAR desensitization in NR1-transfected HEK cells co-expressing wild- type NR2A (using the rat cDNA) versus NR2A-S1462A (containing a mutation that eliminates binding to PDZ domains, made on the wild-type rat cDNA construct) with or without PSD-95.  Consistent with our hypothesis, PSD-95 had no effect on desensitization of NMDARs composed of NR1/NR2A-S1462A (Fig. 2.1D).  Importantly, these mutant receptors showed similar desensitization to wild-type NR1/NR2A in the absence of PSD- 95, and wild-type NR1/NR2A desensitization was significantly reduced by co-transfection with PSD-95 (Fig. 2.1D).  2.2.2 PDZ1-2 domains of PSD-95 are sufficient to alter NMDAR desensitization PSD-95 contains three N-terminal PDZ domains, a central Src homology 3 (SH3) domain, and a C-terminal guanylate kinase-like (Aoki et al.) domain, which can each recruit various proteins to the membrane (Cho et al., 1992, Kim et al., 1995, Kornau et al., 84  1995).  PDZ domains 1 and 2 are required for binding the NR2 C-terminus (Kornau et al., 1995).  Deletion mutagenesis studies established that the N-terminal region of PSD-95, containing palmitoylated Cys-3 and Cys-5, regulates both membrane targeting and multimerization (Craven et al., 1999, Hsueh and Sheng, 1999, Christopherson et al., 2003). In order to isolate which domains of PSD-95 are required for regulating NMDAR desensitization, we utilized PSD-95 constructs with deletions of specific domains and/or with mutations that eliminated PSD-95 multimerization.  In HEK293 cells expressing NR1/NR2A, co-transfection of PSD-95 truncated after domain PDZ2 (PSD-95 PDZ1-2- GFP) resulted in significantly reduced NMDA-evoked current desensitization compared to cells co-expressing only GFP, and an Iss/Ip ratio similar to that found with full-length wild-type PSD-95  (Fig. 1E). Although the PSD-95 PDZ1-2 construct lacks many of the domains involved in recruiting kinases and phosphatases to the membrane, it can multimerize, allowing proteins such as GluR1 to co-cluster via free PDZ domains (Schnell et al., 2002).  To assess the role of PSD-95 multimerization in the regulation of NMDAR desensitization we used two different PSD-95 mutants that targeted to the membrane but remained in monomeric form:  a variant of the truncated PSD-95 PDZ1-2-GFP construct, in which the N-terminal 13 amino acids were replaced with the first 12 amino acids of GAP-43 (GAP12 PDZ1-2-GFP); and a variant of the C3,5S mutant of full-length PSD-95, in which the prenylation motif of paralemmin was added to the C-terminus (Prenyl-PSD- 95-GFP).  The Iss/Ip of NMDA-evoked currents recorded from HEK293 cells expressing these multimerization-deficient constructs was not significantly different from that observed in cells expressing full-length wild-type PSD-95-GFP, indicating that multimerization does not play a role in regulating glycine-independent NMDAR 85  desensitization (Fig. 1E).  Although mutating the N-terminus of PSD-95 should block multimerization, these constructs were GFP-tagged and GFP may form oligomers (Jain et al., 2001).  However, we transfected the same constructs without the GFP tag and found no significant difference in the Iss/Ip (data not shown).  As an additional control, we recorded NMDAR current from cells expressing the first 12 amino acids of GAP-43 fused to GFP only (GAP12-GFP) together with NR1/NR2A. This construct had no effect on NMDAR desensitization (Fig. 1E; Iss/Ip was not significantly different from that found in cells co- expressing GFP and NR1/NR2A), confirming that it is the PDZ domains of the GAP12 PDZ1-2-GFP protein that are responsible for altering NMDAR current desensitization. Together, these data strongly support the idea that PSD-95 regulation of NMDAR desensitization occurs through direct binding of PDZ1-2 domains to the NMDAR and does not require domains involved in scaffolding other proteins in proximity to NMDARs. Two closely related family members of PSD-95, synapse-associated protein 102 (SAP-102) and postsynaptic density protein 93 (PSD-93), which both contain PDZ1-2 domains that are highly homologous to those in PSD-95 (Kim and Sheng, 2004), are also abundantly expressed in hippocampal neurons and may, in some cases, compensate for effects mediated by PSD-95 (Elias et al., 2006).  We found that both SAP-102 and PSD-93 significantly reduced NR1/NR2A desensitization in transfected HEK293 cells (Fig. 1E) albeit to a smaller extent than PSD-95; this may be, in part, a result of lower affinity of these family members for NR2A (Sans et al. 2000).   Notably, SAP-102 had no effect on desensitization of NR1/NR2B in transfected HEK cells (Iss/Ip was 61 ± 4%, n=23 for NR1/NR2B+GFP and 60 ± 3%, n=7 for NR1/NR2B+SAP-102; P>0.05 by unpaired t-test), and both NR1/NR2B and SAP-102 are highly expressed in immature hippocampal neurons 86  prior to upregulation of NR2A and PSD-95 (Sans et al., 2000). SAP102 may be a less effective interactor with NMDARs as well because of lack of anchoring at the membrane. To determine whether expression of PSD-95 PDZ1-2-GFP, including only the N- terminal region through PDZ1 and PDZ2, in immature cultured hippocampal neurons is sufficient to reduce NMDA-evoked current desensitization to levels found in mature neurons, we transfected GFP, PSD-95-GFP, or PSD-95 PDZ1-2-GFP plasmids into neurons at 4 DIV and compared NMDA-evoked currents recorded at 6 DIV. Expression of PSD-95-GFP in immature neurons resulted in a decrease in NMDA-evoked current desensitization compared to GFP-transfected controls and an Iss/Ip similar to that observed in mature neurons (compare Figs. 2A,B with 4A,B).   Notably, there was no difference in peak current density between neurons transfected with GFP and PSD-95 (51 ± 7 pA/pF, n=7 and 46 ± 9 pA/pF, n=12 for GFP- and PSD-95-transfected neurons, respectively). Transfection of PSD-95 PDZ1-2-GFP also significantly reduced NMDAR desensitization compared to GFP-transfected neurons, although the effect was smaller than that observed for full-length PSD-95-GFP (Fig. 2A,B). From these data, our observations in HEK293 cells, and our previously published results (Li et al., 2003a), we conclude that binding of PSD-95 PDZ1-2 domains to NMDARs plays a critical role in reducing receptor desensitization over the course of neuronal development.    87   2.2.3  PKC uncouples PSD-95 from NMDARs and increases receptor desensitization in mature hippocampal neurons The above results suggested that NMDAR desensitization in mature neurons might be altered by signaling proteins that regulate the association of NMDARs with PSD-95. Treatment of mature hippocampal neurons with phorbol esters to activate PKC increases diffuse staining of NMDAR clusters throughout dendrites, indicating a shift from synaptic to non-synaptic localization; under these conditions, PSD-95 staining remained punctate and clustered at the synapse suggesting that NMDARs and PSD-95 become uncoupled after PKC activation (Fong et al., 2002). To confirm that treatment with phorbol esters results in dissociation of NMDARs from PSD-95, we treated neurons with 100 nM TPA for 10, 30 or 60 minutes and examined the interaction between NMDARs and PSD-95 by co-immunoprecipitation with an anti-NR2A antibody. TPA significantly decreased the amount of PSD-95 co- immunoprecipitated with NR2A compared with control after a 60-min treatment (PSD- 95/NR2A ratio scaled to control was 77 ± 13%, 67 ± 15%, and 51 ± 13% after 10-, 30- and 60-min TPA treatments, respectively; n=5 independent experiments with all four conditions done in parallel; P <0.01 for control vs. 60-min TPA).  Although phorbol esters activate PKC over the course of minutes, prolonged treatment can reduce PKC activity in many cell types (Wagey et al., 2001). To confirm that the effect of a 60-min TPA treatment was not a result of down-regulation of PKC activity, we treated neurons with 1 μM RO, which inhibits PKC activity by binding to the catalytic region ATP binding cassette 88  (Birchall et al., 1994). Consistent with our hypothesis that NR2A and PSD-95 co- association was reduced because of PKC activation by TPA, treatment with RO for 10 minutes had no effect on the co-immunoprecipitation of NR2A and PSD-95 (PSD- 95/NR2A ratio scaled to control was 98 ± 19% and 64 ± 12% after 10-min RO and 60-min TPA treatments, respectively; n=5 independent experiments with all three conditions done in parallel; P<0.05 for control vs. 60-min TPA). Previous studies have shown that inhibition of PSD-95 palmitoylation by treatment with 2-BP shifts PSD-95 away from synapses and reduces co-localization with NMDARs (El-Husseini Ael et al., 2002).  Indeed, 6-hr treatment with 100 μM 2-BP caused a significant reduction in co-immunoprecipitation of PSD-95 with NR2A (Fig. 3B). Co- treatment of neurons with 2-BP (6 hours) and TPA (60 min) resulted in a reduction in the association of PSD-95 with NR2A that was not significantly different than the decrease found for either treatment alone (Fig. 3A,B). Together, these results indicate that PKC activation by TPA results in a partial uncoupling of NR2A and PSD-95 that is similar, and not additive, to that produced by 2-BP. To determine whether PKC-induced dissociation of PSD-95 from NMDARs could alter desensitization, we recorded NMDA-evoked current from cultured mature hippocampal neurons after a 60-min treatment with 100 nM TPA or control solution. TPA treatment caused a marked increase in desensitization to levels similar to those found in immature neurons (compare Fig. 4A,B with Fig. 2A,B), whereas shorter treatments with TPA (10 min) or inhibition of PKC with RO – both treatments that did not affect coupling between PSD-95 and NR2A – did not alter NMDAR desensitization (Fig. 4B). As a 89  control, incubation with an inactive phorbol ester, 4α-TPA (100 nM), for 1 hour did not alter NMDAR current desensitization (Fig. 4B). Consistent with the similar reduction in co-association of NR2A and PSD-95 produced by treatment with TPA or 2-BP or the two together, these treatments also produced a similar, non-additive, reduction in NMDAR current Iss/Ip (Fig. 4A,B).  Both treatments result in dissociation of NMDARs from PSD- 95:  TPA treatment results in extrasynaptic localization of NMDARs while PSD-95 distribution remains unaltered at synaptic sites (Fong et al., 2002); 2-BP treatment results in removal of PSD-95 from synaptic sites while NMDAR distribution is unaltered at the synapse (El-Husseini Ael et al., 2002).  The increase in desensitization that results from either treatment suggests binding of PSD-95 to NMDARs is critical in regulating this process. Previous work indicated that PKC activation increases NMDAR peak current and surface expression in Xenopus oocytes (Lan et al., 2001).  However, we found no change in peak NMDAR current density after a 60-min treatment with 100nM TPA (Fig. 4C), consistent with a previous study using a biochemical approach to show that NMDAR surface expression in cultured hippocampal neurons was unchanged following an identical TPA treatment (Fong et al., 2002). TPA has also been shown to enhance NMDAR currents in hippocampal slices (Chen and Huang, 1992) and cultures (Xiong et al., 1998).  To further test whether TPA directly alters channel gating under our experimental conditions, we determined whether 60-min TPA had any effect on NMDAR current in immature hippocampal neurons that express low levels of PSD-95.  TPA did not significantly change the NMDAR Iss/Ip, peak current or current density compared with the control treatment (control vs. 60-min TPA showed: Iss/Ip of 34 ± 9% vs. 29 ± 11% and peak current density 90  of 31 ± 10 pA/pF vs. 37 ± 13 pA/pF; n=15 for control n=7 for 60-min TPA; recordings were made from neurons at 4-5 DIV).  2.2.4  PKC increases NMDAR desensitization by uncoupling PSD-95 from recombinant NMDA receptors expressed in HEK cells The effect of prolonged treatment with phorbol esters, resulting in dissociation of NMDARs from PSD-95, on recombinant NMDAR desensitization was also assessed in HEK cells expressing NR1 and NR2A along with either GFP or PSD-95-GFP. The cells were treated with TPA (100 nM) for 10 or 60 minutes, followed by the assessment of NMDAR desensitization. TPA significantly decreased the steady-state-to-peak ratio in cells expressing PSD-95 after a 60-min treatment, compared with control (Iss/Ip was 31 ± 4%, n=8 for 60-min treatment with TPA; and 59 ± 4%, n=8 for control cells, treated with DMSO; P<0.001 by one-way ANOVA followed by Bonferroni post-test). The 10-min treatment with TPA slightly increased NMDAR desensitization, though not significantly, compared to control (Iss/Ip was 49 ± 3%, n=8; P>0.05 by one-way ANOVA). The treatment with TPA had no significant effect on desensitization in HEK cells expressing NR1/NR2A and GFP (Iss/Ip was 41 ± 3%, n=4 for 60- min TPA treatment, 34 ± 3%, n=6 for 10-min TPA treatment, and 37 ± 7%, n=4 for control cells, treated with DMSO; P>0.05 by one-way ANOVA).   91  2.3 Discussion 2.3.1  Role of PSD-95 direct binding to NMDARs on current desensitization Our findings indicate direct binding of PSD-95 PDZ1-2 domains to the NMDAR NR2A/B C-terminus reduces glycine-independent desensitization in HEK293 cells and neurons. This is somewhat surprising, since NMDAR desensitization gating is largely determined by amino acids in the lurcher motif of the channel vestibule (Hu and Zheng, 2005).  However, there is precedence for changes in NMDAR C-terminal domains affecting function of distant domains:  src-mediated phosphorylation of NR2A C-terminal residues relieves zinc inhibition, which is determined by the amino-terminal domain (ATD) (Zheng et al., 1998). It is important to note that we measured desensitization by calculating the ratio of steady-state to peak NMDA-evoked current during a 10s NMDA application.  For technical reasons (cells were attached to the coverslip, limiting the rate of agonist application and thereby slowing the rise time to peak) we were unable to accurately measure the rate of onset of desensitization and therefore could not determine whether PSD-95 binding affected this rate specifically.  Since entry to the desensitized state occurs through only one (C1) of three possible agonist-bound closed states (Auerbach and Zhou, 2005), it is also possible that PSD-95 binding enhances the rate of exit and/or reduces the rate of entry to this particular closed state, favoring occupation of alternate agonist-bound closed states (C2 and C3), and/or alters the dwell time in open states. 92  Expression in HEK293 cells of wild-type PSD-95 or multimerization-deficient truncates containing just the N-terminus and PDZ1-2 domains is sufficient to reduce NMDAR current desensitization to levels similar to mature hippocampal neurons, indicating other neuronal-specific proteins and signaling pathways are not required. However, neuronal-specific mechanisms also contribute to regulating NMDAR desensitization.  Hippocampal neurons expressing predominantly NR2B-containing receptors and low levels of PSD-95 in early development show more extensive NMDAR desensitization than NR1/NR2B-transfected HEK293 cells.  Neuronal NR1/NR2B desensitization in the absence of PSD-95 binding can be enhanced by NR2B Ser1303 phosphorylation (Sessoms-Sikes et al., 2005) and reduced by tyrosine phosphatase inhibition (Li et al., 2003a), and occurrence of these processes may be cell-type specific. Conversely, mature neurons exhibit minimal desensitization while expressing high levels of NR2A-containing receptors, which desensitize extensively in HEK293 cells. This may be explained by direct interaction with PSD-95:  NMDARs in mature neurons are largely synaptic where they interact with PSD-95, while PSD-95 co-expression with NR1/NR2A in HEK293 cells is sufficient to reduce desensitization. Previous work indicated PSD-95 expression enhances NMDAR surface expression (Lin et al., 2004).  In contrast, another study reported that PSD-95 over-expression in immature cerebellar granule cells reduced whole-cell NMDAR current density (Losi et al., 2003) .We found similar NMDA-evoked peak current density in HEK cells co-expressing NR1/NR2A with GFP or PSD-95; also, there was no correlation between peak current density and Iss/Ip in individual cells.  Moreover, in hippocampal neurons NMDAR peak current density was unaltered following PSD-95 over-expression or dissociation of 93  NMDARs and PSD-95.  We conclude that regulation of NMDAR desensitization by PSD- 95 is not related to changes in receptor surface expression.  Previously, PSD-95 was shown to stabilize NR2B-containing receptors at the synapse, whereas trafficking of NR2A- or NR2B-containing receptors to the cell surface occurred independently of PSD-95 (Prybylowski et al., 2005)..  Together with our results, these studies suggest the larger role for PSD-95 regulation of neuronal NMDARs is in maintaining synaptic receptors and stabilizing their response to glutamate, rather than modulating surface delivery.  In immature neurons, we observed a significant reduction in NMDAR desensitization with expression of PSD-95 PDZ1-2-GFP compared to GFP-transfected controls, but the effect was attenuated compared with expression of full-length PSD-95, whereas similar effects were seen for both constructs expressed in HEK cells.  The attenuated effect in neurons may be explained by inefficient synaptic targeting or clustering of PSD-95 PDZ1-2-GFP, resulting in less effective interaction with synaptically- localized NMDARs; synaptic targeting of PSD-95 is determined in part by the C-terminal 13–25 amino acids (Craven et al., 1999) and the PDZ3 domain interaction with postsynaptic protein CRIPT, which links PSD-95 to the microtubule cytoskeleton (Passafaro et al., 1999).    94  2.3.2  Effects of PSD-95/NMDAR interaction on neuronal NMDAR function Previous studies investigated the role of PSD-95 in modulating neuronal NMDAR current. Consistent with our results, in mice expressing truncated C-terminus NR2A (NR2AΔC/ΔC, which is unable to bind PSD-95), whole-cell recordings from hippocampal neurons revealed enhanced NMDAR current desensitization (Steigerwald et al., 2000). Interestingly, dramatic reductions in hippocampal CA1 long-term potentiation (LTP) were reported in these mice (Sprengel et al., 1998, Steigerwald et al., 2000). Increased desensitization should slow the late component of the NMDAR EPSC decay time course and potentially reduce EPSC amplitude (Lester and Jahr, 1992).  Both changes were observed in the NR2AΔC/ΔC mice, while the former was also observed in hippocampal slices from a PSD-95 knock-out mouse (Beique et al., 2006); however, effects of the mutations on synaptic NMDAR subunit composition (Beique et al., 2006) or peri-synaptic localization (Steigerwald et al., 2000) make EPSC changes difficult to interpret.  Hippocampal slices from a different PSD-95 knock-out mouse, or in which PSD- 95 was acutely knocked-down using short hairpin RNAs (shRNA), showed no change in NMDAR-mediated EPSC amplitude, but decay rate was not examined (Nakagawa et al., 2004, Elias et al., 2006).The authors suggested other PSD-95 family members (e.g., SAP- 102, PSD-93) could compensate for PSD-95 function. As we have shown that SAP-102 and PSD-93 also regulate NR2A-containing NMDAR desensitization, such compensation may contribute to normalizing EPSC characteristics at a subset of synapses.  Although acute disruption of PSD-95/NMDAR interactions, utilizing peptides that competitively 95  bind to PSD-95 PDZ1-2 domains, in hippocampal slices did not alter NMDAR-mediated EPSC amplitude (Lim et al., 2003), the marginal efficacy of these peptides in uncoupling NMDAR/PSD-95 synaptic complexes may contribute to lack of effect on NMDAR EPSCs.  2.3.3  Phorbol ester-induced PKC activation and regulation of neuronal NMDAR function   Although previous studies have identified sites of PKC phosphorylation on NR1 (Tingley et al., 1997), we found that PKC activity can regulate neuronal NMDAR desensitization by uncoupling PSD-95 from NMDARs.  A previous study using different techniques also showed dissociation of PSD-95 and NMDARs following 60-min TPA treatment in hippocampal neurons, without changes in NMDAR surface expression (Fong et al., 2002).  Several lines of evidence indicate the effect of 60-min TPA treatment on NMDAR desensitization is mediated by disrupting PSD-95/NMDAR binding rather than by direct phosphorylation of NMDARs or associated proteins.  Brief (10-min) TPA treatments that did not uncouple NMDARs from PSD-95 in mature neurons also had no effect on desensitization, and 60-min TPA did not alter NMDAR desensitization in immature neurons expressing low PSD-95 levels or in mature neurons pre-treated with 2- BP to disrupt NMDAR/PSD-95 interaction.  Moreover, in transfected HEK cells NR1/NR2A desensitization was significantly increased by 60-min (but not 10-min) TPA treatment in cells co-expressing PSD-95 but not GFP, matching the results in neurons. Interestingly, a previous study showed that in HEK293 cells expressing NR1/ NR2A or NR1/NR2B in the absence of PSD-95, acute PKC activation also enhanced glycine- 96  independent desensitization, and this effect persisted after deletion of the C-terminal tail of NR1 or NR2A (Jackson et al., 2006). Differences in the time course of phorbol ester- mediated effects may result in PKC regulation of NMDAR desensitization by distinct mechanisms. A recent study reported that an increase in peak NMDAR current mediated by PKC in Xenopus oocytes is occluded by PSD-95 co-expression and depends on phosphorylation of NR2A Serine-1462 (Liao et al., 2000, Lin et al., 2006).  These results suggest that lack of NMDAR peak current increase in mature neurons following phorbol ester treatment may be explained by high levels of PSD-95/NMDAR co-association (Li et al., 2003a). However, when we uncoupled PSD-95 from NMDARs with 2-BP and subsequently activated PKC, NMDAR peak current density was unchanged.  Moreover, TPA does not alter NMDAR current density in immature neurons with low PSD-95 levels.  While the outcomes of NMDAR regulation by PKC may be different in neurons and Xenopus oocytes, our data are in agreement with the major finding of Lin and colleagues (Lin et al. 2006), that PKC and PSD-95 converge in modulating NMDAR function.  2.3.4  PSD-95/NMDAR association may regulate synaptic plasticity and excitotoxicity NMDAR desensitization limits calcium influx during repeated synaptic stimulation that can induce plasticity, or with prolonged exposure to glutamate such as may occur during ischemia.  Here, we have demonstrated a novel mechanism by which NMDAR 97  function can be regulated that may impact these processes.  PSD-95 binding to NR2 subunits to reduce NMDAR desensitization may contribute to the shift in stimulation frequencies required to induce NMDAR-dependent synaptic plasticity that occur during brain development (Philpot et al., 2003), with consequences for new learning. As well, the effect of NMDAR/PSD-95 binding on the integrated calcium current in response to sustained glutamate insults may alter neuronal sensitivity to excitotoxicity. Since palmitoylation of PSD-95 is itself activity-dependent (El-Husseni et al., 2002), and this process targets PSD-95 to synapses and promotes its interaction with NMDARs, regulation of NMDAR desensitization by PSD-95 binding may be a highly dynamic mechanism for modulating neuronal signaling.    98   Figure 2. 1  99   Figure 2. 2    100   Figure 2. 3 101   Figure 2. 4 102    Figure 2. 5  103  Figure legends Figure 2.1 Expression of PSD-95 Regulates Glycine-Independent Desensitization of Recombinant NMDARs.  A.  Representative traces of NMDAR currents in HEK cells expressing NMDAR subunits with GFP or PSD-95-GFP.  Currents were normalized for comparison of desensitization. B. Expression of PSD-95 significantly reduced the extent of NMDA-evoked current desensitization in both NR1/NR2A- and NR1/NR2B-expressing cells. NR1/2A + GFP, n=23; NR1/2A + PSD-95-GFP, n=15; NR1/2B + GFP, n=23; NR1/2B + PSD-95, n=13; *P<0.05, **P<0.01, ***P<0.001; one-way ANOVA followed by Bonferroni post-test. C.  Iss/Ip shows no correlation with peak current density in NR1/NR2A-expressing HEK cells co-transfected with either GFP (closed squares, n=17) or wild-type PSD-95 (open circles, n=10).  D.  Mutation of the PDZ ligand in (Prybylowski et al.) NR2A eliminates effect of PSD-95 on NMDAR desensitization.  NR1/NR2A + GFP, n=10; NR1/NR2A + PSD-95, n=15; NR1/NR2A-S1462A + GFP, n=8; NR1/NR2A- S1462A + PSD-95, n=21.  *P<0.05 compared to all other conditions; one-way ANOVA followed by Bonferroni post-test.  E.  PDZ1-2 domains are sufficient to alter NMDAR desensitization; multimerization of PSD-95 and protein targeting to the membrane are not required. NR1/2A +:  GFP, n=23 (same data shown in B); PSD-95-GFP, n=15 (same data shown in B); PSD PDZ1-2-GFP, n=11; GAP12 PDZ1-2, n=7; Prenyl PSD-95, n=8; GAP12, n=8; SAP-102, n=12; PSD-93, n=10.  *P<0.05 and **P<0.01 by One-way ANOVA followed by Bonferroni post-test compared to NR1/2A + GFP; #P<0.05 and ##P<0.01 by one-way ANOVA followed by Bonferroni post-test compared to NR1/2A + PSD-95. 104   Figure 2.2 Overepression of PSD-95 in Immature Neurons Reduces Glycine- Independent Desensitization.  A.  Representative traces of NMDAR responses in immature neurons overexpressing GFP, PSD-95 PDZ1-2-GFP or PSD-95-GFP.  B.  Overexpression of PSD-95 PDZ1-2-GFP or PSD-95-GFP in immature hippocampal neurons results in decreased desensitization compared to GFP-transfected controls. GFP n=7; PSD-95 PDZ1- 2-GFP, n=6; PSD-95-GFP, n=12; *P<0.05, **P<0.01 by one-way ANOVA followed by Bonferroni post-test.  Figure 2.3  PKC Activation Uncouples NR2A from PSD-95 in Mature Neurons.  A. Representative blot of co-immunoprecipitation.  PSD-95 band density was significantly reduced in IP lanes with TPA alone or TPA plus 2-BP treatments compared to control.  B. 1-hr TPA treatment of mature neurons significantly uncoupled PSD-95 from NR2A similarly to 6-hr 2-BP treatments.  Combined 1-hr TPA and 6-hr 2-BP treatment did not produce additive reduction in NR2A/PSD-95 association.  n=8 independent experiments with all four conditions done in parallel; *P<0.05, **P<0.01 by one-way ANOVA followed by Bonferroni post-test.  Figure 2.4 PKC Activation Alters Desensitization in Mature Neurons.  A. Representative traces of responses from mature neurons under control conditions, treated with TPA for 1 hour, and treated with TPA for one hour following 6-hr 2-BP treatment.  B. 105  1-hr TPA treatment reduced Iss/Ip of mature (>14 DIV) neurons.  Shorter 10-min TPA treatments, 10-min incubation with a PKC inhibitor (RO), or 1-hr treatment with an inactive phorbol ester (4αTPA) did not alter desensitization of mature neurons.  Combined TPA and 2-BP treatments did not further reduce Iss/Ip. Control, n=23; 1-hr TPA, n=9; 10- min TPA, n=7; 10-min RO, n= 9; 60-min 4αTPA, n=8; 2-BP + TPA, n=9. **P<0.01 compared to all other conditions by one-way ANOVA followed by Bonferroni post-test. C. Change in desensitization not due to increased peak current. Mature neurons displayed no changes in NMDAR current density with any treatment.  Control, n=12; 60-min TPA, n=9; 10-min TPA, n=7; 10-min RO, n=9; 60-min 4αTPA, n=8; 2-BP + TPA, n=9. P>0.05 by one-way ANOVA followed by Bonferroni post-test.  Figure 2.5 PKC activation alters desensitization of recombinant NMDA receptors in HEK cells that express PSD-95. A.  Representative traces of NMDAR currents in HEK cells expressing NR1 and NR2A subunits, with PSD-95-GFP or GFP, in control conditions or treated for 10 or 60 minutes with TPA.  Current amplitudes were normalized for comparison of desensitization. B. Treatment with TPA for 60 min increased the extent of NMDA-evoked current desensitization in NR1/NR2A- and PSD-95- expressing cells. Control, n=8; 10-min TPA, n=8; 60-min TPA, n=8, **P<0.01, ***P<0.001; one-way ANOVA followed by Bonferroni post-test. Treatment with the phorbol ester, either for 10 or 60 minutes, did not induce a significant effect in GFP-expressing cells. Control, n=4; 10-min TPA, n=6; 60-min TPA, n=4. 106  2.4 Bibliography Aoki C, Fujisawa S, Mahadomrongkul V, Shah PJ, Nader K, Erisir A (NMDA receptor blockade in intact adult cortex increases trafficking of NR2A subunits into spines, postsynaptic densities, and axon terminals. Brain Res 963:139-149.2003). Auerbach A, Zhou Y (Gating reaction mechanisms for NMDA receptor channels. J Neurosci 25:7914-7923.2005). Barria A, Malinow R (Subunit-specific NMDA receptor trafficking to synapses. Neuron 35:345- 353.2002). Beique JC, Lin DT, Kang MG, Aizawa H, Takamiya K, Huganir RL (Synapse-specific regulation of AMPA receptor function by PSD-95. Proc Natl Acad Sci U S A 103:19535- 19540.2006). Birchall AM, Bishop J, Bradshaw D, Cline A, Coffey J, Elliott LH, Gibson VM, Greenham A, Hallam TJ, Harris W, et al. (Ro 32-0432, a selective and orally active inhibitor of protein kinase C prevents T-cell activation. J Pharmacol Exp Ther 268:922-929.1994). Chen L, Huang LY (Protein kinase C reduces Mg2+ block of NMDA-receptor channels as a mechanism of modulation. Nature 356:521-523.1992). Chen N, Moshaver A, Raymond LA (Differential sensitivity of recombinant N-methyl-D- aspartate receptor subtypes to zinc inhibition. Molecular pharmacology 51:1015- 1023.1997). Cho KO, Hunt CA, Kennedy MB (The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein. Neuron 9:929-942.1992). Christopherson KS, Sweeney NT, Craven SE, Kang R, El-Husseini Ael D, Bredt DS (Lipid- and protein-mediated multimerization of PSD-95: implications for receptor clustering and assembly of synaptic protein networks. J Cell Sci 116:3213-3219.2003). Colledge M, Dean RA, Scott GK, Langeberg LK, Huganir RL, Scott JD (Targeting of PKA to glutamate receptors through a MAGUK-AKAP complex. Neuron 27:107-119.2000). Collins MO, Husi H, Yu L, Brandon JM, Anderson CN, Blackstock WP, Choudhary JS, Grant SG (Molecular characterization and comparison of the components and multiprotein 107  complexes in the postsynaptic proteome. Journal of neurochemistry 97 Suppl 1:16- 23.2006). Cottrell JR, Borok E, Horvath TL, Nedivi E (CPG2: a brain- and synapse-specific protein that regulates the endocytosis of glutamate receptors. Neuron 44:677-690.2004). Craven SE, El-Husseini AE, Bredt DS (Synaptic targeting of the postsynaptic density protein PSD-95 mediated by lipid and protein motifs. Neuron 22:497-509.1999). Cull-Candy S, Brickley S, Farrant M (NMDA receptor subunits: diversity, development and disease. Curr Opin Neurobiol 11:327-335.2001). Dingledine R, Borges K, Bowie D, Traynelis SF (The glutamate receptor ion channels. Pharmacological reviews 51:7-61.1999). El-Husseini AE, Schnell E, Chetkovich DM, Nicoll RA, Bredt DS (PSD-95 involvement in maturation of excitatory synapses. Science 290:1364-1368.2000). El-Husseini Ael D, Schnell E, Dakoji S, Sweeney N, Zhou Q, Prange O, Gauthier-Campbell C, Aguilera-Moreno A, Nicoll RA, Bredt DS (Synaptic strength regulated by palmitate cycling on PSD-95. Cell 108:849-863.2002). Elias GM, Funke L, Stein V, Grant SG, Bredt DS, Nicoll RA (Synapse-Specific and Developmentally Regulated Targeting of AMPA Receptors by a Family of MAGUK Scaffolding Proteins. Neuron 52:307-320.2006). Erreger K, Dravid SM, Banke TG, Wyllie DJ, Traynelis SF (Subunit-specific gating controls rat NR1/NR2A and NR1/NR2B NMDA channel kinetics and synaptic signalling profiles. J Physiol 563:345-358.2005). Erreger K, Traynelis SF (Zinc inhibition of rat NR1/NR2A N-methyl-D-aspartate receptors. J Physiol 586:763-778.2008). Fong DK, Rao A, Crump FT, Craig AM (Rapid synaptic remodeling by protein kinase C: reciprocal translocation of NMDA receptors and calcium/calmodulin-dependent kinase II. J Neurosci 22:2153-2164.2002). Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391:85-100.1981). 108  Hsueh YP, Sheng M (Requirement of N-terminal cysteines of PSD-95 for PSD-95 multimerization and ternary complex formation, but not for binding to potassium channel Kv1.4. J Biol Chem 274:532-536.1999). Hu B, Zheng F (Molecular determinants of glycine-independent desensitization of NR1/NR2A receptors. The Journal of pharmacology and experimental therapeutics 313:563- 569.2005). Jackson MF, Konarski JZ, Weerapura M, Czerwinski W, MacDonald JF (Protein kinase C enhances glycine-insensitive desensitization of NMDA receptors independently of previously identified protein kinase C sites. J Neurochem 96:1509-1518.2006). Jain RK, Joyce PB, Molinete M, Halban PA, Gorr SU (Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells. Biochem J 360:645-649.2001). Kim E, Niethammer M, Rothschild A, Jan YN, Sheng M (Clustering of Shaker-type K+ channels by interaction with a family of membrane-associated guanylate kinases. Nature 378:85- 88.1995). Kim E, Sheng M (Differential K+ channel clustering activity of PSD-95 and SAP97, two related membrane-associated putative guanylate kinases. Neuropharmacology 35:993- 1000.1996). Kim E, Sheng M (PDZ domain proteins of synapses. Nat Rev Neurosci 5:771-781.2004). Kornau HC, Schenker LT, Kennedy MB, Seeburg PH (Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science 269:1737- 1740.1995). Krupp JJ, Vissel B, Heinemann SF, Westbrook GL (Calcium-dependent inactivation of recombinant N-methyl-D-aspartate receptors is NR2 subunit specific. Mol Pharmacol 50:1680-1688.1996). Lan JY, Skeberdis VA, Jover T, Grooms SY, Lin Y, Araneda RC, Zheng X, Bennett MV, Zukin RS (Protein kinase C modulates NMDA receptor trafficking and gating. Nat Neurosci 4:382-390.2001). Lester RA, Jahr CE (NMDA channel behavior depends on agonist affinity. J Neurosci 12:635- 643.1992). 109  Li B, Chen N, Luo T, Otsu Y, Murphy TH, Raymond LA (Differential regulation of synaptic and extra-synaptic NMDA receptors. Nat Neurosci 5:833-834.2002). Li B, Otsu Y, Murphy TH, Raymond LA (Developmental decrease in NMDA receptor desensitization associated with shift to synapse and interaction with postsynaptic density- 95. J Neurosci 23:11244-11254.2003). Liao GY, Kreitzer MA, Sweetman BJ, Leonard JP (The postsynaptic density protein PSD-95 differentially regulates insulin- and Src-mediated current modulation of mouse NMDA receptors expressed in Xenopus oocytes. J Neurochem 75:282-287.2000). Lim IA, Hall DD, Hell JW (Selectivity and promiscuity of the first and second PDZ domains of PSD-95 and synapse-associated protein 102. J Biol Chem 277:21697-21711.2002). Lim IA, Merrill MA, Chen Y, Hell JW (Disruption of the NMDA receptor-PSD-95 interaction in hippocampal neurons with no obvious physiological short-term effect. Neuropharmacology 45:738-754.2003). Lin Y, Jover-Mengual T, Wong J, Bennett MV, Zukin RS (PSD-95 and PKC converge in regulating NMDA receptor trafficking and gating. Proc Natl Acad Sci U S A 103:19902- 19907.2006). Lin Y, Skeberdis VA, Francesconi A, Bennett MV, Zukin RS (Postsynaptic density protein-95 regulates NMDA channel gating and surface expression. J Neurosci 24:10138- 10148.2004). Losi G, Prybylowski K, Fu Z, Luo J, Wenthold RJ, Vicini S (PSD-95 regulates NMDA receptors in developing cerebellar granule neurons of the rat. J Physiol 548:21-29.2003). Luo J, Wang Y, Yasuda RP, Dunah AW, Wolfe BB (The majority of N-methyl-D-aspartate receptor complexes in adult rat cerebral cortex contain at least three different subunits (NR1/NR2A/NR2B). Mol Pharmacol 51:79-86.1997). Mayer ML, Vyklicky L, Jr., Clements J (Regulation of NMDA receptor desensitization in mouse hippocampal neurons by glycine. Nature 338:425-427.1989). Monyer H, Burnashev N, Laurie DJ, Sakmann B, Seeburg PH (Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12:529-540.1994). 110  Nakagawa T, Futai K, Lashuel HA, Lo I, Okamoto K, Walz T, Hayashi Y, Sheng M (Quaternary structure, protein dynamics, and synaptic function of SAP97 controlled by L27 domain interactions. Neuron 44:453-467.2004). Paoletti P, Ascher P, Neyton J (High-affinity zinc inhibition of NMDA NR1-NR2A receptors. J Neurosci 17:5711-5725.1997). Passafaro M, Sala C, Niethammer M, Sheng M (Microtubule binding by CRIPT and its potential role in the synaptic clustering of PSD-95. Nat Neurosci 2:1063-1069.1999). Philpot BD, Espinosa JS, Bear MF (Evidence for altered NMDA receptor function as a basis for metaplasticity in visual cortex. J Neurosci 23:5583-5588.2003). Prange O, Wong TP, Gerrow K, Wang YT, El-Husseini A (A balance between excitatory and inhibitory synapses is controlled by PSD-95 and neuroligin. Proc Natl Acad Sci U S A 101:13915-13920.2004). Prybylowski K, Chang K, Sans N, Kan L, Vicini S, Wenthold RJ (The synaptic localization of NR2B-containing NMDA receptors is controlled by interactions with PDZ proteins and AP-2. Neuron 47:845-857.2005). Prybylowski KL, Grossman SD, Wrathall JR, Wolfe BB (Expression of splice variants of the NR1 subunit of the N-methyl-D-aspartate receptor in the normal and injured rat spinal cord. Journal of neurochemistry 76:797-805.2001). Roche KW, Standley S, McCallum J, Dune Ly C, Ehlers MD, Wenthold RJ (Molecular determinants of NMDA receptor internalization. Nat Neurosci 4:794-802.2001). Sans N, Petralia RS, Wang YX, Blahos J, 2nd, Hell JW, Wenthold RJ (A developmental change in NMDA receptor-associated proteins at hippocampal synapses. J Neurosci 20:1260- 1271.2000). Schnell E, Sizemore M, Karimzadegan S, Chen L, Bredt DS, Nicoll RA (Direct interactions between PSD-95 and stargazin control synaptic AMPA receptor number. Proc Natl Acad Sci U S A 99:13902-13907.2002). Sessoms-Sikes S, Honse Y, Lovinger DM, Colbran RJ (CaMKIIalpha enhances the desensitization of NR2B-containing NMDA receptors by an autophosphorylation- dependent mechanism. Mol Cell Neurosci 29:139-147.2005). 111  Sheng M, Pak DT (Ligand-gated ion channel interactions with cytoskeletal and signaling proteins. Annu Rev Physiol 62:755-778.2000). Sprengel R, Suchanek B, Amico C, Brusa R, Burnashev N, Rozov A, Hvalby O, Jensen V, Paulsen O, Andersen P, Kim JJ, Thompson RF, Sun W, Webster LC, Grant SG, Eilers J, Konnerth A, Li J, McNamara JO, Seeburg PH (Importance of the intracellular domain of NR2 subunits for NMDA receptor function in vivo. Cell 92:279-289.1998). Steigerwald F, Schulz TW, Schenker LT, Kennedy MB, Seeburg PH, Kohr G (C-Terminal truncation of NR2A subunits impairs synaptic but not extrasynaptic localization of NMDA receptors. J Neurosci 20:4573-4581.2000). Stocca G, Vicini S (Increased contribution of NR2A subunit to synaptic NMDA receptors in developing rat cortical neurons. J Physiol 507:13-24.1998). Thomas CG, Miller AJ, Westbrook GL (Synaptic and extrasynaptic NMDA receptor NR2 subunits in cultured hippocampal neurons. J Neurophysiol 95:1727-1734.2006). Tingley WG, Ehlers MD, Kameyama K, Doherty C, Ptak JB, Riley CT, Huganir RL (Characterization of protein kinase A and protein kinase C phosphorylation of the N- methyl-D-aspartate receptor NR1 subunit using phosphorylation site-specific antibodies. J Biol Chem 272:5157-5166.1997). Tong G, Jahr CE (Regulation of glycine-insensitive desensitization of the NMDA receptor in outside-out patches. J Neurophysiol 72:754-761.1994). Tong G, Shepherd D, Jahr CE (Synaptic desensitization of NMDA receptors by calcineurin. Science 267:1510-1512.1995). Tovar KR, Westbrook GL (The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro. J Neurosci 19:4180-4188.1999). Wagey R, Hu J, Pelech SL, Raymond LA, Krieger C (Modulation of NMDA-mediated excitotoxicity by protein kinase C. J Neurochem 78:715-726.2001). Xiong ZG, Raouf R, Lu WY, Wang LY, Orser BA, Dudek EM, Browning MD, MacDonald JF (Regulation of N-methyl-D-aspartate receptor function by constitutively active protein kinase C. Mol Pharmacol 54:1055-1063.1998). 112  Yamada Y, Iwamoto T, Watanabe Y, Sobue K, Inui M (PSD-95 eliminates Src-induced potentiation of NR1/NR2A-subtype NMDA receptor channels and reduces high-affinity zinc inhibition. J Neurochem 81:758-764.2002). Zheng F, Erreger K, Low CM, Banke T, Lee CJ, Conn PJ, Traynelis SF (Allosteric interaction between the amino terminal domain and the ligand binding domain of NR2A. Nat Neurosci 4:894-901.2001). Zheng F, Gingrich MB, Traynelis SF, Conn PJ (Tyrosine kinase potentiates NMDA receptor currents by reducing tonic zinc inhibition. Nature neuroscience 1:185-191.1998). Zorumski CF, Thio LL, Clark GD, Clifford DB (Blockade of desensitization augments quisqualate excitotoxicity in hippocampal neurons. Neuron 5:61-66.1990).                    113   Chapter Three2 3 Effects of Exercise on NMDA Receptor Subunit Contributions to Bidirectional Synaptic Plasticity in the Mouse Dentate Gyrus   NMDA receptors are heteromeric complexes that primarily contain both NR1 and NR2 subunits, and rarely NR3 subunits (Prybylowski and Wenthold, 2004). There are at least eight different splice variants of the NR1 subunit (NR1A-H), and all of the NR1 splice variants can be found in the hippocampus, although their expression is both regionally and developmentally regulated (Laurie et al., 1995). The NR2 subunits are expressed as one of four different gene products (NR2A-D), and are instrumental in determining NMDA receptor properties (Dingledine et al., 1999, Cull-Candy and Leszkiewicz, 2004). NR2A and NR2B subunit expression also varies within the hippocampus with greater NR2B expression in CA1 than DG in adult animals (Coultrap et al., 2005). Therefore, NMDA receptor composition not only varies throughout development but also within the different regions of the hippocampus, suggesting distinct roles of NMDA receptors depending on age and hippocampal location.  2 2 A version of this chapter has been published. Vasuta C, Caunt C, James R, Samadi S, Schibuk E, Kannangara T, Titterness AK, Christie BR (2007) Effects of exercise on NMDA receptor subunit contributions to bidirectional synaptic plasticity in the mouse dentate gyrus. Hippocampus 2007 17(12):1201-8.  114  Recently it has been proposed that NMDA receptor NR2 subunits have the capacity to determine the direction of change in synaptic plasticity in pyramidal cells (Liu et al., 2004, Massey et al., 2004, Weitlauf C et al., 2005, Fox et al., 2006). The “subunit hypothesis” states that NR2A subunits control the induction of LTP, while NR2B subunits mediate LTD (long-term depression) (Sakimura et al., 1995, Liu et al., 2004, Yang et al., 2005). These results are not uncontested in the CA1 region however (Hendricson et al., 2002, Kohr et al., 2003, Berberich et al., 2005, Morishita et al., 2006), and it may be that different brain regions utilize subunits in distinct ways (Weitlauf C et al., 2005). As subunit composition varies within the hippocampus (Coultrap et al., 2005) the contribution of NR2A and NR2B to LTP and LTD might be different in DG from CA1. A goal of this study is to elucidate the role of NR2A and NR2B containing NMDA receptors to LTP and LTD in DG. NMDA receptor subunit expression is not only regulated by age (Monyer et al., 1994, Wenzel et al., 1997)  but also experience (Fox et al., 1999). We have previously shown that voluntary exercise enhances NR2B mRNA in DG (Farmer et al., 2004) and increases the capacity for the DG to express LTP in vitro and in vivo in adult animals (van Praag et al., 1999, Farmer et al., 2004, Christie et al., 2005) even after neonatal teratogen exposure (Christie et al., 2005). Exercise also increases neurogenesis and synaptogenesis in the DG (van Praag et al., 1999, van Praag et al., 2002, Eadie et al., 2005, Redila et al., 2006) and it seems likely that exercise exerts its effects on multiple levels. The mechanisms behind exercise-induced enhancements of LTP in DG, however, remain to be determined, as well as the effects of exercise on LTD. 115   3.1 Materials and methods  3.1.1  Subjects Experiments were performed primarily on C57BL/6 mice, 3 to 5 weeks of age obtained from Charles River Laboratories (QC, Canada). Additional experiments were conducted using NR2A knockout animals (Townsend et al., 2003) bred in our facility. Animals were housed in cages containing minimal enrichment (small opaque tubes, paper towels) and separated into Control and Runner groups.  The only difference between the two groups was that Runners also had access to a running wheel in the cage for 7-10 days before they were used for experiments. On average, animals run about 4 km a day under these conditions. Both groups were maintained on a 12 h light/dark cycle with constant ambient temperature (21 ± 1°C) and humidity (50 ± 7%).  Food and water were available ad libitum and all testing was performed in the dark phase of the light cycle. All animal procedures were conducted in accordance with UBC and Canadian animal care policies.  3.1.2  Slice preparation Mice were anesthetized with halothane, decapitated and the brain was rapidly removed and placed in cold sucrose based artificial cerebro-spinal fluid (ACSF) containing (in mM): 110 sucrose, 60 NaCl, 3 KCl, 1.25 NaH2PO4, 28.0 NaHCO3, 0.5 CaCl2, 7 MgCl2, 116  and 5 dextrose, 0.6 ascorbate. The solution was continuously bubbled with 95% O2-5% CO2. Transverse hippocampal slices (400 μm) were cut using a Vibratome Series 1000 (Pelco). After cutting, each slice was placed in the incubation chamber (Isotemp 202) in oxygenated ACSF containing (in mM) 125.0 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25.0 NaHCO3, 2 CaCl2, 1.3 MgCl2, and 10 dextrose. Slices were incubated at 25 + 1 oC for a minimum of one hour post-dissection.  3.1.3  Field recordings and stimulation procedures The slices were transferred to a recording chamber and perfused with ACSF containing bicuculline (1.0 µM) to block GABAA-mediated inhibition. Stimulating and recording electrodes were placed under visual guidance in the medial aspect of the dentate molecular layer using an Olympus BX50wi microscope (10X objective). Field potentials were evoked using a single 0.10 ms biphasic pulse applied to perforant-path fibers using sharpened tungsten electrodes (A-M systems, WA) or twisted bipolar nichrome wires (66 um). For each animal, the stimulus intensity was set at 60% of the intensity necessary to evoke a maximum fEPSP response (20-400 µA).  Individual fEPSPs were evoked and recorded every 15 s and a stable 15 minute baseline was required in all experiments. After baseline recording, we applied one of two conditioning protocols: High frequency stimulation (HFS: four bursts of 50 pulses at 100 Hz, 30 s between bursts) to induce LTP, and low frequency stimulation (LFS: 900 pulses at 1 Hz) to induce LTD. After application of the conditioning protocol, fEPSPs were again recorded for one hour. Ifenprodil (3 µM) and Ro25-6981 (0.5 µM or 1 µM) were obtained from Sigma-Aldrich (USA). NVP- 117  AAM077 was a gift from Novartis Pharma AG (Basel, Switzerland). DL-TBOA was obtained from Tocris Cookson (Bristol, UK).  3.1.4  Data analyses The slope of the initial phase of the EPSP waveform, the duration and the peak amplitude of the voltage deflection were measured for each evoked fEPSP. Computed results were processed for statistical analysis using Clampfit (Axon Instruments, Molecular Devices), and GraphPad (Prism) or Statistica (Statsoft). Data are presented as means ± SEM, and statistical significance was evaluated by performing t-tests.  3.2   Results 3.2.1  Voluntary exercise enhances NMDA receptor-dependent LTP induction in the DG To examine synaptic plasticity, we activated perforant path fibers to the DG granule cells of two experimental groups of mice, the Control and the Runner animals. LTP was induced using 100 Hz conditioning stimuli identical to those used in similar studies in CA1 and cortical pyramidal cells (Liu et al., 2004; Massey et al., 2004; Weitlauf et al., 2005). We found that the 100 Hz stimulation induced a robust LTP in slices obtained from control animals (26.7 + 9.0%, n=14; t(20)=3.1, p<0.01). As reported previously (van Praag et al., 1999), slices obtained from animals that were allowed access to a running wheel showed 118  significantly more LTP (56.0 + 6.6%, n=17; t(16)=8.46; p<0.01) than Control animals (t(29)=2.75; p<0.05; Figure 1). LTP induction in both groups could be blocked by the non- specific NMDA antagonist APV (Control: -12 + 20%; t(7)=1.7, p=0.13, N=5; Runner: -12 + 17%; t(4)=0.74, p=0.49, N=5).  3.2.2  Exercise alters the contribution of NR2 subunits to LTP We next examined a role for individual NR2 subunits subtypes in LTP using selective NR2-specific antagonists. As is shown in Figure 1, we found that the NR2B subunit antagonist ifenprodil (3 µM) completely blocked LTP in Control animals (1.8 + 6.8%, n=18; t(17)=0.27; p=0.79) but some residual LTP was still present in Runners (17.0 + 7.9%, n=22; t(20)=2.17; p=0.042). However, the LTP in Runners in this condition was severely attenuated when compared to that from Runners tested in normal ACSF (t(37)=3.64; p<0.01). To confirm the results obtained with ifenprodil, we also used the specific NR2B antagonist RO25-6981. At a concentration of 0.5 µM, Ro25-6981 also produced a complete block of LTP in slices obtained from Control animals (-13.6 + 4.4%, n=8; t(7)=3.10, p<0.05), and unmasked LTD. In contrast to ifenprodil, Ro25-6981 (0.5 µM) completely blocked LTP in Runners (-2.7 + 14%, n=7; t(6)=0.50, p=0.64). A higher concentration of RO25-6981 (1 µM) produced identical results (data not shown). The antagonist NVP-AAM077 (0.4 µM), which has a higher affinity for NR2A containing NMDA receptors than those containing NR2B subunits (Frizelle et al., 2006), also completely blocked LTP in Control slices, and revealed a slight, insignificant 119  depression (-17.0 + 8.0%, n=9; t(8)=2.1, p=0.065), similar to that observed with APV. In contrast to the results with the NR2B antagonist ifenprodil, NVP completely blocked LTP in Runners (3.6 + 2.7%, n=11; t(10)=1.3, p=0.21) although it did not significantly alter basal synaptic transmission (data not shown). Taken together, these results indicate that both NR2B and NR2A subunits contribute to LTP in Controls and Runners. However, subtle differences in the role of NR2A subunits with LTP were unmasked by voluntary exercise. Specifically, NVP produced LTD following HFS in Control animals but not Runners, suggesting that although LTP was blocked by an NR2A antagonist in both groups, there were slight differences in the contribution of NR2A subunits to synaptic plasticity following exercise.  3.2.3  LTD in the DG is NMDA receptor-dependent in both Control and Runner animals To evaluate the role of NR2 subunits in LTD in the DG, and how exercise affected LTD induction in this region, we applied low-frequency stimuli (1 Hz, 900 pulses) identical to that used in previous studies (Liu et al., 2004, Massey et al., 2004, Weitlauf C et al., 2005). The application of these stimuli caused robust LTD in Control slices (-26.3 ± 6.0%, n=16; t(15)=4.4, p<0.01) and in Runners (-25.6%  ± 2.3%, n=12; t(8)=11, p<0.01), as shown in Figure 2. This LTD was NMDA receptor-dependent, as significant LTD was not observed in either Controls (-8 + 3.4%, n=4; t(3)=2.37, p=0.10) or Runners (0.9 + 4.2%, n=6; t(5)=0.22, p=0.83) in the presence of APV (50 µM). 120  3.2.4  Exercise enhances the contribution of NR2A, but not NR2B subunits to LTD In order to determine if NR2B subunits contributed to induction and expression of LTD in the DG, we first used the specific NR2B antagonist ifenprodil (3 µM). Administration of the LFS in the presence of ifenprodil did not prevent LTD in Control  (- 42.7 + 10.6%, n=9; t(8)=4.0, p<0.01) or Runner  (-31.3 + 4.5%; t(6)=6.97, p<0.01) slices (Figure 2). To verify these results we also used R025-6981, another specific NR2B antagonist. Control animals exhibited significant LTD in the presence of either 0.5 µM (- 35.3 +6.7%, n=11; t(10)=5.2, p<0.01) or 1.0 µM (-34.4 + 7.1%, n=13; t(12)=4.8, p<0.01) Ro25-6981 in the bath. Similarly, LTD was not blocked by 0.5 µM of Ro25-6981 in Runners (-43.2 + 8.6%; t(7)=5.0, p<0.01). These results suggest that NR2B subunits are not critical for LTD in the DG of control animals, or following voluntary exercise. Because NR2A subunits are prevalent in the DG (Pandis et al., 2006), and since NR2B antagonists had no effect on the LTD induced here, we used the compound NVP- AAM077 (0.4 µM) to determine if NR2A subunits contribute to the LTD we observed. Control animals exhibited significant LTD in the presence of NVP-AAM077 (-50.3 + 8.0%, n=9; t(8)=6.3, p<0.01), and there was significantly more LTD than was obtained in Control animals in ACSF (t(20)=2.5, p=0.02). Interestingly, a completely different effect was observed in Runners. NVP-AAM077 completely blocked LTD in slices from these animals (3.96 + 11.6%; t(6)=0.34, p=0.74) such that there was significantly less LTD than that in normal ACSF (t(14)=2.8, p<0.01). 121  Since NVP-AAM077 may have had some non-specific effects at sites other than NR2A subunits, we also conducted similar studies in two NR2A knock out animals that were allowed to exercise. Robust LTD was observed in NR2A KO Runners in normal ACSF (-44.9 + 9.3%, n=4; t(3)=4.8, p<0.05) that was not blocked by  NVP-AAM077 (-72.1 + 9.14%, n=5; t(4)=4.8, p<0.01; Figure 3). Indeed, there actually was a trend for more LTD being observed with NVP (t(7)=2.1, p=0.078). In one slice we also tested the efficacy of the NR2B antagonist Ifenprodil (3 µM), but as in all other cases, it failed to block LTD induction (-58%). Therefore, the contribution of NR2A subunits to LTD is enhanced following voluntary exercise. The experiments thus far indicate that NR2A subunits are critically involved in LTD in animals that exercise. It remains unclear however, whether the population of receptors that these subunits form is increased or/and whether the subcellular localization of these subunits is modified by exercise. In previous studies, DL-TBOA has been used to block glutamate uptake to determine whether extrasynaptically located receptors are involved in LTD induction in other brain areas (Massey et al., 2004). Using identical procedures in the DG, we found that prior treatment with DL-TBOA (10 µM) produced significant LTD in both Control (-49.9 + 6.1%, n=7) and Runner (-54.3 + 4.7%, n=5) slices (Figure 4). We were unable to block the LTD when Ifenprodil was included with the DL- TBOA in either Control (-43.7 +4.5%; n=3) or Runner (-52.3 + 6.8%; n=4) slices. In Runners however, the co-application of DL-TBOA and NVP-AAM077 blocked LTD induction (-12 + 5.8%, n=4); an effect not observed in control animals (-41 + 2.2%, n=3).  122  3.3 Discussion Our results show that long-term plasticity in the DG of mice can be dramatically influenced by a single behavioral manipulation, voluntary exercise. Perforant path evoked responses in the DG of slices taken from animals that exercised over a period of 7-10 days exhibited significantly more LTP than those taken from control animals (see Figure 5). Continuing this trend, LTP in the DG was completely blocked by NR2B antagonists in Control slices, but not in slices from Runners, where, although reduced, a significant degree of LTP remained. When NVP-AAM077, an antagonist with a higher selectivity for NR2A subunits over NR2B subunits was used, LTP was completely blocked both in Control and Runner slices. These results suggest that voluntary exercise can regulate LTP by affecting the contribution of NMDA receptor subunits to synaptic plasticity in the DG. In contrast to the results with LTP, the magnitude of LTD was relatively unaffected by exercise (see Figure 2).  In addition, neither NR2B- nor NR2A-specific antagonists blocked LTD in Control slices, despite the fact that the non-specific NMDAR antagonist APV completely blocked LTD in both Control and Runner slices. In Runners, LTD was also unaffected by ifenprodil, however NVP-AAM077 completely prevented LTD induction in both normal ACSF and following exposure of slices to DL-TBOA. Therefore, whereas NR2B subunits may contribute to LTD in CA1 (Liu et al., 2004, Bartlett et al., 2007), they are not instrumental for LTD in the DG. On the other hand, voluntary exercise appears to increase the role of NR2A-containing NMDA receptors to LTD in the DG, again indicating that voluntary exercise robustly regulates NMDA receptor subunit contribution to hippocampal synaptic plasticity. The only alternative explanation we can 123  devise for these results is that exercise induces a requirement for both NR2A and NR2B subunits in LTD, and that only NVP-AAM077 is capable of providing an adequate blockade of both these subunits in animals that exercise. This hypothesis does not seem as attractive given that none of the specific subunit antagonists that we used blocked LTD in control animals. There are precedents for the modification of bidirectional synaptic plasticity by behavioral manipulations. Recently the effects of enriched environments on bidirectional synaptic plasticity have also been examined in the CA1 region of the hippocampus. Although this region does not exhibit a profound change in neurogenesis and synaptogenesis with exercise, both LTD and LTP can be enhanced by environmental enrichment (Duffy et al., 2001, Artola et al., 2006). Similarly, exposure to stress can also modify bidirectional synaptic plasticity. Acute stress decreases the capacity for LTP induction in the hippocampus, but this effect can also be reversed by enriched environments (Artola et al., 2006, McDermott et al., 2006, Yang et al., 2006, Yang et al., 2007). Conversely, the capacity for LTD is increased significantly by stress, suggesting that stress may shift the capacity for bidirectional synaptic plasticity in favor of depression over potentiation (Kim et al., 1996, Xu et al., 1997, Manahan-Vaughan, 2000, Xiong et al., 2004, Yang et al., 2004, 2005, Artola et al., 2006, Yang et al., 2006, Yang et al., 2007). The main difference between these previous results and those found here, is that exercise increased the capacity for LTP in the DG, a finding reported previously (van Praag et al., 1999, Farmer et al., 2004, Christie et al., 2005), without significantly altering the capacity for the expression of LTD in the DG. Further investigation into the NMDA subunits active in the induction of both of these forms of plasticity revealed that both NR2A and NR2B 124  subunits appear to play a large role in LTP in both Runner and Control animals. Neither subunit appeared to be critical for LTD in the DG of control animals; however LTD in animals that exercised was dependent upon the activation of NR2A subunits. Only in the NR2A Knock-out animals was this not case, suggesting that if NR2A subunits are not present, some other subunit is utilized to allow LTD induction. The fact that LTD is conserved in these animals also suggests that LTD is a critical process for “normal” neuronal functioning.  Taken together, these results suggest to us that NR2A and NR2B subunits can contribute to LTP and LTD in the DG, and that behavioural manipulations such as voluntary exercise alter some factor or condition that regulates the specific role played by these subunits. In the case of LTP, it is possible that a strong depolarization can recruit NR2A and NR2B subunits, both of which contribute to LTP since removing either subunit population appears to lower the amount of calcium entering into the cell to a point that is less than optimal for LTP induction (Berberich et al., 2005, Berberich et al., 2006). For LTD on the other hand, the total charge crossing the membrane does not provide the most parsimonious explanation for our results because: 1) NR2A subunit antagonists did specifically block LTD in animals that engaged in exercise; 2) we were also unable to define a role for NR2B subunits in LTD in either group of animals examined. This suggests that either NR2B subunits do not play a role in LTD in the DG at all, or their contribution is so minor, that removing them under these conditions does not alter calcium influx sufficiently. Despite increasing the concentration of the NR2B antagonist Ro25- 125  6981 significantly (0.5 to 1.0 µM), we were unable reduce the amount of LTD induced by the LFS. This seems to go against reports that the NR2B subunits are the major charge carrier during LFS (Erreger et al., 2005) and thus are primarily responsible for LTD induction (Liu et al., 2004, Massey et al., 2004), however these previous results were obtained in pyramidal cells where NR2B specific antagonists can block LTD but not LTP in vivo (Fox et al., 2006). Thus, NMDA subunits may contribute quite differently to synaptic plasticity in different areas of the brain, but this is only one factor that can possibly help explaining the discrepancy in the findings reported by various laboratories.  The dentate gyrus is unique in the hippocampal formation in that it exhibits significant neurogenesis, increased dendritic complexity, and synaptogenesis in response to voluntary exercise (van Praag et al., 1999, Eadie et al., 2005, Redila and Christie, 2006). Thus, exercise has the capacity to increase the number of new cells in the DG that can contribute to synaptic plasticity, as well as altering the number of synapses, in both existing and new granule cells, that can contribute to bidirectional plasticity. Given the short time frame that these animals were allowed to engage in exercise, it is likely that the majority of the effect observed here was due to alterations in synaptic structure rather than due to the increase in cell proliferation/neurogenesis that accompanies running. In this instance, the time frame was not long enough for these new cells to mature and become functionally integrated into the existing neuronal network (van Praag et al., 2002, Overstreet-Wadiche and Westbrook, 2006). 126  The biggest surprise in this work was that we did not see an increase in the role of NR2B subunits in synaptic plasticity as we would have expected given previous results (Farmer et al., 2004). Rather, there appeared to be a general facilitation of LTP irrespective of drug condition, and an NR2A specific alteration in LTD induction in animals that exercise. To test whether this change in LTD induction might involve the movement of specific receptor subunit types to extrasynaptic sites, we decreased glutamate transport with DL-TBOA to increase extrasynaptic glutamate levels (Massey et al., 2004, Bartlett et al., 2007). These experiments were performed to investigate whether NR2A subunits at synaptic sites might act as the main contributors to LTD induction in DG granule cells. Although this proved to be a robust way to induce LTD, the overall amount of LTD was not significantly different from that induced normally, and once again, only NR2A subunits and not NR2B subunits appeared to be involved in the induction of LTD. If the NR2A subunit is not present, it is possible that some other subunit can assume the compensatory role necessary for at least LTD induction to occur - see Figure 3 (Farmer et al., 2004).  Due to the different results obtained in Control and Runner animals in the present experiments and the limitations of using pharmacological agents in general (Neyton and Paoletti, 2006), we are unable to state unequivocally that LTD induction is determined by either the total amount of charge entering the cell, or by some strict form of subunit mediated control. We do believe however, that these results seem to support the hypothesis that behavioral manipulations can actually alter the capacity of different subunits to 127  regulate this form of plasticity; possibly by altering access to different biochemical pathways (Li et al., 2006a, b). For example, given that both the LFS and HFS paradigms used here appear to work via NMDA receptors, it may be that LFS activity preferentially leads to some form of phosphatase activity, while the HFS leads to some form of kinase activity (Mulkey and Malenka, 1992). Unraveling these signaling pathways will provide a unique insight into how different forms of sensory stimulation and perception can alter the functional capacity of neuronal systems in the brain during cognitive processing.             128  -20 -10 0 10 20 30 40 50 60 -50 0 50 100 Ifenprodil NVP ACSF Time (min) EP SP  s lo pe  (%  c ha ng e) 0.5 mV 5 ms  -20 -10 0 10 20 30 40 50 60 -50 0 50 100 ACSF NVP Ifenprodil Time (min) EP SP  s lo pe  (%  c ha ng e) 0.5 mV 5 ms  Figure 3. 1  A. B. 129  -20 -10 0 10 20 30 40 50 60 -100 -50 0 50 Ifenprodil NVP ACSF Time (min) EP SP  s lo pe  (%  c ha ng e) 0.5 mV 5 ms  -20 -10 0 10 20 30 40 50 60 -100 -50 0 50 Ifenprodil NVP ACSF Time (min) EP SP  s lo pe  (%  c ha ng e) 0.5 mV 5 ms  Figure 3. 2  A. B. 130    Figure 3. 3      131   Figure 3. 4 132   Figure 3. 5           133  Figure legends Figure 3.1  A) In slices taken from Control animals, robust LTP is normally obtained when the HFS stimuli (4x50 pulses at 100Hz) are administered (Black Squares). Inclusion of the NR2B antagonist Ifenprodil (3 µM) in the ACSF significantly attenuated LTP expression, though the initial STP was not significantly different (Dark Grey Squares). In contrast, when NVP-AAM077 (0.4 µM) was included in the ACSF, the HFS stimuli now produced no STP and resulted in a small depression. B) In Runners, robust LTP was also attained in normal ACSF (Black Squares). Ifenprodil again attenuated LTP expression, thought a significant degree of LTP was still present at 60 minutes. NVP- AAM077 again blocked both STP and LTP, but did not result in a depression being observed in slices from Runners. Scale Bars: 5 mV, 5 msec.  Figure 3.2 A) The application of 900 pulses at 1 Hz reliably produced LTD in the DG of slices taken from Control animals, irrespective of whether Ifenprodil (3 µM) or NVP-AAM077 (0.4 µM) was present in the ACSF. B) In slices taken from Runners, reliable LTD was again observed in both normal ACSF and with Ifenprodil. Slices exposed to NVP-AAM077 failed to show LTD however. Scale Bars: 5 mV, 5 msec.  Figure 3.3  In experiments performed in NR2A Knockout animals that were allowed to exercise, robust LTD was observed in the DG following LFS in normal ACSF (Black bar). Unlike in WT animals, the inclusion of NVP-AAM077 failed to block this 134  LTD in the NR2A Knockout Runners, meaning that compensatory mechanisms may have played a role in the occurrence of this form of plasticity, mediated by other subunits than the NR2A.  Figure 3.4 A) The inclusion of DL-TBOA (10 µM) in slices from Control animals invariably produced a reduction in the size of EPSPs recorded in ACSF. Following application of the LFS stimuli, and wash-out of the drug, a persistent and robust LTD was observed. Similar results were obtained when ifenprodil was also included in the ACSF with DL-TBOA. Inclusion of NVP-AAM077 reduced the amount of depression observed with DL-TBOA, but also resulted in robust LTD following LFS. B) In Runners, robust LTD was obtained with DL-TBOA alone, and when it was presented in combination with ifenprodil. In contrast, DL-TBOA + NVP-AAM077 failed to produce significant LTD following the application of the LFS. C) Bar graph comparing the last five minutes of post- conditioning baseline (55-60 minutes) for all groups tested.  Figure 3.5  Summary of effects of exercise in both Control and Runner animals following High Frequency Stimulation (HFS) and Low-Frequency Stimulation (LFS). The main effect of exercise on slices exposed to HFS is to produce an increase in the final response slope, as compared to Control slices, irrespective of the antagonist used and even when LTP Is not induced. In contrast, the effects on LTD appear to be to selectively alter 135  the role of the NMDA NR2A subunit such that it is now critical for the induction of LTD in the DG.                136  3.4 Bibliography Artola A, von Frijtag JC, Fermont PC, Gispen WH, Schrama LH, Kamal A, Spruijt BM (Long- lasting modulation of the induction of LTD and LTP in rat hippocampal CA1 by behavioural stress and environmental enrichment. Eur J Neurosci 23:261-272.2006). Bartlett TE, Bannister NJ, Collett VJ, Dargan SL, Massey PV, Bortolotto ZA, Fitzjohn SM, Bashir ZI, Collingridge GL, Lodge D (Differential roles of NR2A and NR2B-containing NMDA receptors in LTP and LTD in the CA1 region of two-week old rat hippocampus. Neuropharmacology 52:60-70.2007). Berberich S, Jensen V, Hvalby O, Seeburg PH, Kohr G (The role of NMDAR subtypes and charge transfer during hippocampal LTP induction. Neuropharmacology.2006). Berberich S, Punnakkal P, Jensen V, Pawlak V, Seeburg PH, Hvalby O, Kohr G (Lack of NMDA receptor subtype selectivity for hippocampal long-term potentiation. J Neurosci 25:6907-6910.2005). Christie BR, Swann SE, Fox CJ, Froc D, Lieblich SE, Redila V, Webber A (Voluntary exercise rescues deficits in spatial memory and long-term potentiation in prenatal ethanol-exposed male rats. Eur J Neurosci 21:1719-1726.2005). Coultrap SJ, Nixon KM, Alvestad RM, Valenzuela CF, Browning MD (Differential expression of NMDA receptor subunits and splice variants among the CA1, CA3 and dentate gyrus of the adult rat. Brain Res Mol Brain Res 135:104-111.2005). Cull-Candy SG, Leszkiewicz DN (Role of distinct NMDA receptor subtypes at central synapses. Sci STKE 2004:re16.2004). Dingledine R, Borges K, Bowie D, Traynelis SF (The glutamate receptor ion channels. Pharmacol Rev 51:7-61.1999). Duffy SN, Craddock KJ, Abel T, Nguyen PV (Environmental enrichment modifies the PKA- dependence of hippocampal LTP and improves hippocampus-dependent memory. Learn Mem 8:26-34.2001). Eadie BD, Redila VA, Christie BR (Voluntary exercise alters the cytoarchitecture of the adult dentate gyrus by increasing cellular proliferation, dendritic complexity, and spine density. J Comp Neurol 486:39-47.2005). 137  Erreger K, Dravid SM, Banke TG, Wyllie DJ, Traynelis SF (Subunit-specific gating controls rat NR1/NR2A and NR1/NR2B NMDA channel kinetics and synaptic signalling profiles. J Physiol 563:345-358.2005). Farmer J, Zhao X, van Praag H, Wodtke K, Gage FH, Christie BR (Effects of voluntary exercise on synaptic plasticity and gene expression in the dentate gyrus of adult male Sprague- Dawley rats in vivo. Neuroscience 124:71-79.2004). Fox CJ, Russell KI, Wang YT, Christie BR (Contribution of NR2A and NR2B NMDA subunits to bidirectional synaptic plasticity in the hippocampus in vivo. Hippocampus 16:907- 915.2006). Fox K, Henley J, Isaac J (Experience-dependent development of NMDA receptor transmission. Nat Neurosci 2:297-299.1999). Frizelle PA, Chen PE, Wyllie DJ (Equilibrium constants for NVP-AAM077 acting at recombinant NR1/NR2A and NR1/NR2B NMDA receptors: implications for studies of synaptic transmission. Mol Pharmacol.2006). Hendricson AW, Miao CL, Lippmann MJ, Morrisett RA (Ifenprodil and ethanol enhance NMDA receptor-dependent long-term depression. J Pharmacol Exp Ther 301:938-944.2002). Kim JJ, Foy MR, Thompson RF (Behavioral stress modifies hippocampal plasticity through N- methyl-D-aspartate receptor activation. Proc Natl Acad Sci U S A 93:4750-4753.1996). Kohr G, Jensen V, Koester HJ, Mihaljevic AL, Utvik JK, Kvello A, Ottersen OP, Seeburg PH, Sprengel R, Hvalby O (Intracellular domains of NMDA receptor subtypes are determinants for long-term potentiation induction. J Neurosci 23:10791-10799.2003). Laurie DJ, Putzke J, Zieglgansberger W, Seeburg PH, Tolle TR (The distribution of splice variants of the NMDAR1 subunit mRNA in adult rat brain. Brain Res Mol Brain Res 32:94-108.1995). Li S, Tian X, Hartley DM, Feig LA (Distinct roles for Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1) and Ras-GRF2 in the induction of long-term potentiation and long-term depression. J Neurosci 26:1721-1729.2006a). Li S, Tian X, Hartley DM, Feig LA (The environment versus genetics in controlling the contribution of MAP kinases to synaptic plasticity. Curr Biol 16:2303-2313.2006b). 138  Liu L, Wong TP, Pozza MF, Lingenhoehl K, Wang Y, Sheng M, Auberson YP, Wang YT (Role of NMDA receptor subtypes in governing the direction of hippocampal synaptic plasticity. Science 304:1021-1024.2004). Manahan-Vaughan D (Long-term depression in freely moving rats is dependent upon strain variation, induction protocol and behavioral state. Cereb Cortex 10:482-487.2000). Massey PV, Johnson BE, Moult PR, Auberson YP, Brown MW, Molnar E, Collingridge GL, Bashir ZI (Differential roles of NR2A and NR2B-containing NMDA receptors in cortical long-term potentiation and long-term depression. J Neurosci 24:7821-7828.2004). McDermott CM, Hardy MN, Bazan NG, Magee JC (Sleep deprivation-induced alterations in excitatory synaptic transmission in the CA1 region of the rat hippocampus. J Physiol 570:553-565.2006). Monyer H, Burnashev N, Laurie DJ, Sakmann B, Seeburg PH (Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12:529-540.1994). Morishita W, Lu W, Smith GB, Nicoll RA, Bear MF, Malenka RC (Activation of NR2B- containing NMDA receptors is not required for NMDA receptor-dependent long-term depression. Neuropharmacology.2006). Mulkey RM, Malenka RC (Mechanisms underlying induction of homosynaptic long-term depression in area CA1 of the hippocampus. Neuron 9:967-975.1992). Neyton J, Paoletti P (Relating NMDA receptor function to receptor subunit composition: limitations of the pharmacological approach. J Neurosci 26:1331-1333.2006). Overstreet-Wadiche LS, Westbrook GL (Functional maturation of adult-generated granule cells. Hippocampus.2006). Pandis C, Sotiriou E, Kouvaras E, Asprodini E, Papatheodoropoulos C, Angelatou F (Differential expression of NMDA and AMPA receptor subunits in rat dorsal and ventral hippocampus. Neuroscience 140:163-175.2006). Prybylowski K, Wenthold RJ (N-Methyl-D-aspartate receptors: subunit assembly and trafficking to the synapse. J Biol Chem 279:9673-9676.2004). Redila VA, Christie BR (Exercise-induced changes in dendritic structure and complexity in the adult hippocampal dentate gyrus. Neuroscience 137:1299-1307.2006). 139  Redila VA, Olson AK, Swann SE, Mohades G, Webber AJ, Weinberg J, Christie BR (Hippocampal cell proliferation is reduced following prenatal ethanol exposure but can be rescued with voluntary exercise. Hippocampus.2006). Sakimura K, Kutsuwada T, Ito I, Manabe T, Takayama C, Kushiya E, Yagi T, Aizawa S, Inoue Y, Sugiyama H, et al. (Reduced hippocampal LTP and spatial learning in mice lacking NMDA receptor epsilon 1 subunit. Nature 373:151-155.1995). Townsend M, Yoshii A, Mishina M, Constantine-Paton M (Developmental loss of miniature N- methyl-D-aspartate receptor currents in NR2A knockout mice. Proc Natl Acad Sci U S A 100:1340-1345.2003). van Praag H, Christie BR, Sejnowski TJ, Gage FH (Running enhances neurogenesis, learning, and long-term potentiation in mice. Proc Natl Acad Sci U S A 96:13427-13431.1999). van Praag H, Schinder AF, Christie BR, Toni N, Palmer TD, Gage FH (Functional neurogenesis in the adult hippocampus. Nature 415:1030-1034.2002). Weitlauf C, Honse Y, Auberson YP, Mishina M, Lovinger DM, Winder DG (Activation of NR2A-Containing NMDA Receptors Is Not Obligatory for NMDA Receptor-Dependent Long-Term Potentiation. Journal of Neuroscience 25:8386-8390.2005). Wenzel A, Fritschy JM, Mohler H, Benke D (NMDA receptor heterogeneity during postnatal development of the rat brain: differential expression of the NR2A, NR2B, and NR2C subunit proteins. J Neurochem 68:469-478.1997). Xiong W, Wei H, Xiang X, Cao J, Dong Z, Wang Y, Xu T, Xu L (The effect of acute stress on LTP and LTD induction in the hippocampal CA1 region of anesthetized rats at three different ages. Brain Res 1005:187-192.2004). Xu L, Anwyl R, Rowan MJ (Behavioural stress facilitates the induction of long-term depression in the hippocampus. Nature 387:497-500.1997). Yang CH, Huang CC, Hsu KS (Behavioral stress modifies hippocampal synaptic plasticity through corticosterone-induced sustained extracellular signal-regulated kinase/mitogen- activated protein kinase activation. J Neurosci 24:11029-11034.2004). Yang CH, Huang CC, Hsu KS (Behavioral stress enhances hippocampal CA1 long-term depression through the blockade of the glutamate uptake. J Neurosci 25:4288- 4293.2005). 140  Yang J, Han H, Cao J, Li L, Xu L (Prenatal stress modifies hippocampal synaptic plasticity and spatial learning in young rat offspring. Hippocampus 16:431-436.2006). Yang J, Hou C, Ma N, Liu J, Zhang Y, Zhou J, Xu L, Li L (Enriched environment treatment restores impaired hippocampal synaptic plasticity and cognitive deficits induced by prenatal chronic stress. Neurobiol Learn Mem 87:257-263.2007).                   141  Chapter Four 4 Discussion and Conclusions  4.1 The role of NR2 subpopulations of NMDARs in desensitization and plasticity  Our research provides evidence that NMDAR desensitization is regulated by the interaction of the receptor with MAGUK family members. Since these interact differentially with each NMDAR subtype, their modulation of desensitization shows specificity. Thus, the NR2A subytpe NMDARs desensitize generally more than the NR2B- containing NMDARs. Depending on the time in development, synaptic NMDARs are either NR2B-rich (early) or NR2A-rich (for the mature synapses). Also, the expression of MAGUKs increases in development, with PSD-95 being localized synaptically, and SAP102 moving outside the PSD over the course of development. Consequently, the synaptic / extrasynaptic subpopulations of NMDARs show complex profiles for desensitization. In mature neurons, even though synapses contain the extensively - desensitizing NR2A subunit, their coupling with PSD-95 decreases their level of desensitization. Interestingly, extrasynaptic NMDARs show a high level of desensitization, as would be expected for receptors that may be less tightly associated with MAGUKs (since PSD-95 is enriched at synapses, and SAP-102 is not anchored to membranes and therefore may be more transiently associated with NR2 subunits). On the other hand, 142  studies indicate a predominance of the NR2B subtype at extrasynaptic sites, and these receptors show low levels of desensitization in HEK293 cells; clearly there are other neuronal factors regulating NR2B-type NMDAR desensitization in neurons.  In addition, prominent desensitization would be displayed by the small fraction of NR2A-containing receptors that are expressed extrasynaptically, and which account for up to approximately 25% of the total currents gated by the extrasynaptic pool of NMDARs (Liu et al., 2007).  4.1.1 The role of NMDAR desensitization in plasticity mechanisms Some of the functional implications of modulation in the level of desensitization are linked to the role of NMDARs in excitotoxicity and plasticity. That is, because on one hand, desensitization limits calcium signal, and on the other hand, the profile of calcium influx is crucial in both plasticity and cell-death mechanisms. Moreover, desensitization was shown to shape synaptic responses, in a study that used micro-island (autaptic) hippocampal cultures as the system to test the behaviour of synaptic NMDARs (Tong et al., 1995). Because the recovery from desensitization takes as long as several seconds, the amount of calcium (important for plasticity), can be strongly regulated by the mechanisms involved in modulation of desensitization, such as those involving PSD-95 and other MAGUKs, which have been central to our investigation. The association of the synaptic receptors with PSD-95 in mature neurons makes them undergo reduced desensitization, allowing them to remain active during periods of repetitive stimulation such as occur with LTP induction. Previous work has linked the 143  activation of this particular pool of receptors, the synaptic one, to the induction of LTP (Massey et al., 2004); others report that it is the subunit composition of NMDARs (mainly NR2A subunit) that accounts for the role in LTP induction, rather than its localization (Liu et al., 2004). Hippocampal synaptic plasticity in transgenic mice expressing either mutants of PSD-95 or of NMDAR subunits that interfere with NR2 / PSD-95 binding has been assessed in previous studies. In one case, hippocampal LTP has been induced in mice in which the NMDARs do not bind to PSD-95 because of a C-terminal truncation in the NR2A subunit (NR2AΔC/ΔC) (Sprengel et al., 1998); another study investigated plasticity in mice that express the PDZ1-2 fragment only of the PSD-95 (Migaud et al., 1998). Mutant mice expressing NMDAR with a truncated C-terminus  (NR2AΔC/ΔC) show smaller amplitudes of EPSCs, more desensitization and slower recovery from desensitization (Steigerwald et al., 2000); all these characteristics point to less calcium influx during repetitive stimulation compared to control mice. LTP in these mice is decreased significantly (Sprengel et al., 1998). These data are consistent with our findings described in Chapter two: in the absence of PSD-95-mediated regulation of NR2A- containing NMDARs, a high level of desensitization is expected, so less calcium entry, and ultimately decreased magnitude of LTP. In the mutant mice developed by Migaud et al, PDZ1-2 still binds the receptor. Although the desensitization has not been directly assessed in these mice, we would expect the steady-state to peak ratio to have a high value (equivalent to a low level of desensitization), based on our results; due to this regulation, calcium levels mediated by the receptors would be high. Consistently, LTP was increased 144  in these mutant mice, and regulation of desensitization may partially account for it. That is, because the high calcium load mediated by receptors that undergo low desensitization (due to PDZ1-2 binding in this case) is expected to trigger LTP of large magnitude. In contrast to these findings, acute dissociation of the NMDAR - PSD-95 interaction in slices, using antibodies and interference peptides, had no effect on NMDAR- mediated plasticity, or on NMDAR-mediated EPSCs (Lim et al., 2003). This may be due, at least in part, to difficulties in the diffusion of these components into distant regions of the dendritic arbour.  4.1.2  Charge transfer, NR2 subunits and plasticity induction mechanisms Two of the most important features of the calcium influx, which dictates the direction of plasticity, is its amount and its timing (Lisman, 1989), and it has long been regarded that a prolonged, but modest increase in intracellular calcium would result in LTD, while a large, fast influx would lead to LTP (Malenka et al., 1992; Hansel et al., 1997). Simulation studies, on the other hand, predict that NR2A-type NMDARs mediate a larger calcium signal during high-frequency stimulation (100Hz). Activated at low frequency (1 Hz) however, NR2B – containing receptors become the major mediators of calcium charge (Erreger et al., 2005); these subunits have been linked to the induction of LTD. In our second project, we have determined the differential contribution of NR2A and NR2B subunits to different forms of plasticity, and have confirmed that the basal 145  levels (in the absence of experience-driven plasticity) of NR2A subunit-containing receptors are essential for LTP induction. LTD, on the other hand, could be induced through the activation of either NR2A- or NR2B –containing receptors. This last result is consistent with studies reporting that, while NR2B subunits could mediate LTD (Liu et al., 2004), these are not absolutely essential for this process, and alternatives exist (Morishita et al., 2007). Because the type of NR2 subunit does not constitute a strict determinant for the direction of plasticity, we have utilized a behavioural paradigm previously shown to modulate the size of NMDAR supopulations, and have carried out experiments aimed at differentially activating the synaptic/extrasynaptic pools in plasticity induction. For this, we have used the running paradigm in order to alter NMDAR subunit expression - based on a previous study (Farmer et al., 2004) -  and then examined its effect on plasticity induction. Also, in some experiments, we have allowed activation of the extrasynaptic pool of NMDARs by using the drug TBOA during the synaptic stimulation induction protocol; the drug prevents glutamate uptake, making the neurotransmitter available for the distally- located receptors. The precise location of calcium influx subcellularly, as well as the NMDAR subunit composition, could each contribute to determining the direction of synaptic plasticity.  NMDARs activate different signalling cascades at synaptic versus extrasynaptic sites (Hardingham et al., 2002, Hardingham and Bading, 2003), and different NMDAR subtypes (NR1/NR2A versus NR1/NR2B) also exhibit differential interactions with scaffolding and signalling proteins (Al-Hallaq et al 2007). It was reported that in adult 146  animals the NR2A subunit is more abundant at the synapse, while NR2B predominates at extrasynaptic sites (Li et al., 1998, Stocca and Vicini, 1998, Tovar and Westbrook, 1999). As we discussed above, calcium influx profiles through the two subunits also differ, which can affect the downstream pathways in distinct ways. It also has to be considered that different signalling molecules are available at the two locations – synaptic and extrasynaptic. Lastly, specific behavioural paradigms may engage distinct NMDAR populations (in terms of subcellular localization and/or subunit composition). Consistent with this idea, a recent paper shows that environmental enrichment gates certain signalling cascades relevant for plasticity, i.e. makes a MAP-kinase available in NMDAR-dependent LTP (Li et al., 2006a).  4.1.3 Synaptic plasticity induced through the activation of basal-level versus modified NMDARs We report here a modification in the threshold for synaptic changes following exercise, which is consistent with previous reports of a sensory activity – dependent enhancement in plasticity at the synapses in visual cortex (Clothiaux et al., 1991, Carmignoto and Vicini, 1992)  and barrel cortex (Benuskova et al., 1994).  In our experiments, we observe a shift after exercise, towards greater ease to potentiate the synapses using high frequency stimulation (Figure 4.1, left). The increased magnitude of LTP in animals that exercise is consistent with previous findings (van Praag et al., 1999). 147  One explanation for the effects of exercise on NMDAR-mediated plasticity is that receptor composition has been modified following this paradigm; another explanation, is the enhanced receptor coupling with PSD-95. To test the first hypothesis, we assessed synaptic plasticity in conditions of acute pharmacological block, which selectively eliminated either one or the other of the two major subtypes of NMDARs from participating in plasticity. When ifenprodil was used, the HFS protocol resulted in increased LTP in Runners compared to Controls, similar to the trend described above when the receptor population was intact (Figure 4.1). LTD induced in the absence of the NR2B subpopulation, was characterized by a magnitude that was no different from that induced in ACSF (no antagonist), both in Controls and in Runners (Figure 4.1). It must be mentioned however, the possibily that triheteromers composed of both NR2A and NR2B subunits, may be expressed too, and may contribute to these effects. These observations taken together suggest that exercise does not exert its effects on synaptic plasticity by modulating the participation of NR2B-containing receptors to plasticity. When the other major population of NMDARs, the one comprised of NR2A subunits, was blocked by NVP-AAM077, the expression of plasticity, in general, was modest (Figure 4.1). This result can be explained by the presumably large proportion of the total synaptic NMDAR population consisting of NR2A-type receptors in adult animals (Sans et al., 2000), which, when blocked, would render the synapses poor in ready-to-be active NMDARs. The fact that LTP could not be induced in NVP-AAM077 either in the Control or in the Runner group supports the idea that this subunit is necessary for this form of plasticity. The only case in which plasticity could be induced when this subunit was blocked was LTD in Controls (Figure 4.1). 148  Based on results from previous studies that have assessed the effects of exercise on NMDAR gene expression in dentate gyrus (Farmer et al., 2004), we had expected NR2B subunit expression would be increased after physical activity. However, our electrophysiological data revealed that it is the NR2A subunit that shows an enhanced contribution to plasticity after exercise. This result is not incompatible to the previous one cited above, in that increases in mRNA levels for the NR2B (shown by Farmer et al 2004) do not necessarily correlate with increased protein expression, or expression of the subunit at subcellular locations relevant for plasticity.  4.1.3.1   The involvement of the NR2 subunits in LTP LTP could not be induced in Controls when either the NR2A or NR2B population was blocked (Figure 4.1, insert, left), suggesting that a concurrent activity of the two is needed for LTP. Alternatively, the activation of a large population of NMDARs, indifferent of the subtype is necessary, so that blocking any fraction of NMDARs would diminish the overall NMDAR population to a size insufficient for LTP induction. Studies in CA1 report that the NR2A subpopulation is sufficient for LTP (Liu et al., 2004); however, others find that block of the NR2B subpopulation can prevent LTP induction (Berberich et al., 2006), suggesting the population formed by either of the two NMDAR subtypes alone is insufficient in this particular region, to allow LTP induction. Exercise augments the magnitude of synaptic potentiation induced in ACSF, and it seems that the NR2A population can partially account for it, since a significant LTP is recorded in ifenprodil (Figure 4.1, insert, right). Whereas there is a parallel shift towards synaptic 149  potentiation with exercise in both antagonists, exercise modulates the NR2A population so that it is now sufficient to trigger significant LTP by itself, while the NR2B-type NMDAR population alone remains insufficient to induce LTP.  4.1.3.2  The involvement of the NR2 subunits in LTD In contrast to LTP, LTD requires the availability of any NMDAR subgroup for induction, since a significant magnitude was recorded in the presence of either NR2B-or NR2A-selective antagonists. Each subpopulation is sufficient for this form of plasticity, and they do not have an additive effect, since the amplitude of LTD elicited when both are active is similar to that elicited when either one is active (Figure 4.1, insert, left). A similar magnitude of NMDAR-induced synaptic depression is recorded in Runners and Controls. This LTD could equally be triggered by the activation of all NMDARs, or only of the subgroup composed of NR2A subunits, since bath-applied ifenprodil had no effect on LTD. The elimination of the NR2A-population of receptors renders the synapses incapable to undergo depression in Runners (Figure 4.1, insert, right). Hence, the speculation emerges, that exercise increases the availability of NR2A population of NMDARs, either by an increase in subunit expression, or by a shift to subcellular locations relevant for LTD.   150  4.1.3.3  NR2A subunit-dependent plasticity is enhanced by exercise Our result that exercise shifts the direction of plasticity towards potentiation, and that the NR2A population is involved in this shift, comes in agreement with previously proposed scenarios regarding this subunit’s behaviour during high-frequency stimulation (Malenka et al., 1992, Erreger et al., 2005). The increased LTP following exercise is due to the high charge transfer mediated by the NR2A subunits during induction. Though they are not major charge carriers during low-frequency stimulation, NR2A subunits form a sufficient fraction to induce LTD in Controls, while in Runners they are solely responsible for it, a result that strengthens the idea that NR2A subunits form the major population of receptors available for synaptic activation after exercise in addition, the data supports the idea that the NR2B subunits’s involvement in plasticity is diminished after exercise. Our conclusions are limited by the difficulties in inferring subunit expression in individual neurons, based on a population response. Whole –cell recordings would help address more accurately these types of questions, as well as more direct methods assessing protein levels, including immunoprecipitation followed by western blot and/or immunocytochemistry. Finally, limitations interpreting our results come from the incomplete block and imperfect selectivity displayed by the NR2 –“specific” inhibitors, as discussed in Chapter three. Apart from modulating the size/ availability of NR2 populations of NMDARs, exercise may also reorganize them within the cell. Previous studies suggest an activity- 151  dependent translocation of NR2A from extrasynaptic to synaptic sites. This process is apparent in development, and it was linked with activity-dependent upregulation of PSD- 95 (Yoshii et al., 2003), which appears to be necessary for synaptic localization of NR2A (Sans et al., 2000, Elias et al., 2008). If running activates similar mechanisms, our findings may be explained by the shift of the population composed of NR2A to the synapse, where it exerts the reported effects on plasticity. In the NR2A knock-out animals however, plasticity could still be induced, probably through different mediators, made available by compensation. Further experiments, involving patch clamp recording from individual cells, as well as use of biochemical and immunocytochemical techniques, will help resolve these questions.  4.2 Role of NMDAR-mediated excitability and signalling in hippocampal neurophysiology  NMDAR signalling has been linked to a series of neural phenomena, and malfunctions of the brain, spanning from synaptic plasticity, to epileptic discharges and psychosis. All of these functions of the NMDAR could be inferred from the immediate, evident roles that they accomplish at the cellular level, which spring, in turn directly from their properties and structure characteristics (discussed in Introduction). Desensitization is one such property that shapes the behaviour of NMDARs, and may have a certain impact on their involvement in higher functions specific to the 152  hippocampal formation. Also, plasticity induction, in which NMDARs play a central role at least for certain types of synapses, is very important in phenomena linked to memory formation, but it has also been found to be disrupted in a number of disorders of the brain. The role of hippocampal NMDARs in higher-order functions specific to this brain structure will be considered below.   4.2.1 The role of NMDARs in normal hippocampal physiology 4.2.1.1 Hippocampal excitability The hippocampal formation receives its main excitatory input from the entorhinal cortex; this input travels through all the structures of the hippocampus, one after the other in a strictly unique direction. Thus, entorhinal perforant path fibers excite DG granule cells, which send their fibers (called mossy fibers) to CA3 pyramidal neurons; these, in turn, extend their axons, the Shaffer collaterals, towards CA1, where they excite the principal cells there, the CA1 pyramids. Further, CA1 neurons send their excitatory output to subiculum, which projects back to entorhinal cortex. This unidirectionality in the flow of excitaton is a hallmark of hippocampal neurophysiology. NMDARs are constituents of all the above-mentioned excitatory synapses, taking part in mediating the flow of information whenever the synapses are strongly activated to allow these receptors to be part of synaptic communication. More specifically, these receptors mediate ion fluxes whenever the presynaptic element releases neurotransmitter, 153  and at the same time, the postsynaptic membrane exhibits a voltage of ~ -40 - -10 mV (in which case the sodium and calcium ions flow inwardly), or higher than 0 mV (in which case, a potassium flux, joining the sodium and calcium conductances, is outward). Such is the general contribution of these receptors to synaptic communication; it undergoes slight variations though, due to the difference in subtypes and regulation by various factors, as discussed in introduction. The role of NMDAR desensitization in modulating hippocampal excitability can only be inferred, as thorough studies have not been conducted to test it. This property does shape synaptic responses; more specifically, the response to a second pulse delivered 1-2 seconds after an initial one has been shown to be decreased (Tong et al., 1995).  4.2.1.2  Oscillatory activity Computer simulation studies predict that NMDARs are involved in the generation of rhythmic activity (Traven et al., 1993). At the hippocampal level, certain types of oscillatory activity have been described: theta rhythms, for example, occur when the animal is engaged in “voluntary” behaviour (as opposed to reflex-type of movements). These rhythms could be either sensitive or insensitive to atropine, and this relates to their involvement in either attention or movement generation (Kramis et al., 1975). It is unclear how NMDARs contribute to the generation and propagation of the theta activity in hippocampus. Sparse reports regarding the effect of certain NMDAR antagonists exist though: ketamine, for example has the ability to eliminate a certain component of theta 154  waves, namely the one related to the translation of movements (Buzsaki, 2002; Buzsaki et al., 2003). However, even though the hippocampus is the structure linked to memory formation, and theta waves occur specifically here, some authors found theta oscillations to relate to motor behaviour, rather than to learning (Black et al., 1970, Black and Young, 1972). Other components of the EEGs recorded in hippocampus are beta, gamma and ripple waves, each linked to certain behavioural correlates, as well as irregular large and small amplitude activity. Specific NMDAR expression in interneurons regulates oscillation, but it remains largely unclear how NMDARs participate in the generation of these types of electrical activity, or how they shape and modulate it. Repeated activity, characterised by certain frequencies, shows regional variability within the hippocampus. While granule cells of the dentate gyrus are rather silent, displaying rare bursts of firing, CA3 and CA1 pyramidal cells burst more robustly (Wong and Prince, 1981). On the other hand NMDAR composition is also variable in these regions, with CA1 being richer in NR2B subunits compared to DG, in adult animals (Coultrap et al., 2005). While the role of NMDARs in generating this bursting activity is unclear, these receptors would certainly be activated during such bursts, resulting in altered downstream calcium-dependent signalling pathways.   155  4.2.1.3  NMDAR – mediated plasticity and its role in memory formation and learning NMDAR–mediated plasticity occurs at certain synapses in the hippocampus. These are the synapses between the medial perforant path and granule cells in DG, and the synapses between the Shaffer collaterals and CA1 pyramidal cells. The other hippocampal synapses display non-NMDAR forms of plasticity (being opioid-dependent, as is the case for lateral perforant path- granule cell synapses; or kainate receptor dependent, as is the case of mossy fibers – CA3 pyramidal cell synapses). The proof that NMDARs are crucial in certain forms of plasticity in the hippocampus comes from a landmark study that used the NMDAR antagonist APV during various phases of the LTP protocol (Collingridge et al., 1983a). This study proved unequivocally that inhibition of NMDARs during the induction phase prevents LTP and LTD. Aside from the profound amnesia associated with damage to the medial temporal lobe (Milner, 1972), synaptic plasticity and its relationship to memory formation adds another proof that the hippocampus represents the brain structure where memory traces are encoded. Indeed, plasticity is believed to represent the cellular correlate of learning and memory (Bliss and Collingridge, 1993). There are certain correlations between plasticity and memory aspects, on one hand, and between NMDAR activity and memory, on the other hand. Some of these will be considered below. 156  The initial studies on the relationship between learning and plasticity were correlational, with a focus on the age correlation between the persistence of LTP and the rate of learning. For example, older rats show a lesser ability to perform in Morris water- maze tests compared to younger subjects (de Toledo-Morrell and Morrell, 1985). LTP is easily established earlier in development, reaching a maximum at two weeks of age, and decreases afterwards (Harris and Teyler, 1984). LTP persistence is also associated with the rate of learning (Barnes, 1979), and age-related decline in memory tasks correspond to decline in LTP (Chapman et al., 1999, Lynch, 2004). Four theoretical criteria have been put forward, which remain to be proven experimentally, in order to definitively answer the question regarding the relationship between NMDAR-mediated plasticity and memory (Martin, 2000). These criteria are 1) detectability: synaptic plasticity must be detected in association with memory formation in certain brain regions, and at certain synapses; 2) anterograde alteration: preventing synaptic plasticity in a certain area should prevent the formation of memory specific to that area; 3) retrograde alteration and 4) mimicry: the artificial induction of synaptic plasticity shall result in an “apparent memory” for an event that did not actually occur. All criteria but the last have been met experimentally up to now (Green and Greenough, 1986, Matthies et al., 1986, Morris et al., 1986, Laroche et al., 1989, Richter-Levin et al., 1995, Kentros et al., 1998, Izquierdo et al., 1999, Brun et al., 2001, Sacchetti et al., 2001, Gruart et al., 2006). One of the most compelling examples of a link between NMDAR signalling and memory is in the function of hippocampal place cells, as outlined below.  157  4.2.1.4 Hippocampal place cells, NMDARs, and memory formation Hippocampal CA1 place cells, discovered in 1971 by O’Keefe and Dostrovsky, have the ability to signal the animal’s location in its environment (O'Keefe and Dostrovsky, 1971). More specifically, these complex-spike cells are active during a rodent’s explorative behaviour, and fire in specific patterns not due to any sensory input, but in relation to the abstract notion of space. Because place cells can maintain their firing fields for minutes during tasks that test working memory, because place field characteristics are changed by experience, and because place cell firing during sleep recapitulates patterns experienced during the waking period, it is believed that they have a role in memory. Moreover, the behaviour of place cells in familiar and novel environments depends on NMDAR activity. Indeed, it has been considered that NMDARs are the elements that confer mnemonic properties to place cells  (Nakazawa et al., 2004). Mutation studies proved that place fields are non-functional in cases where NMDARs, or other effectors acting downstream of NMDARs, have been genetically modified (McHugh et al., 1996, Rotenberg et al., 1996, Tonegawa et al., 1996). More specifically, the temporal firing characteristics, essential to the place cells functioning, are altered in such cases (McHugh et al., 1996). Also, NMDARs seem to affect especially the long-term stability of the place fields, rather than their initial formation and short-term maintenance (Kentros et al., 1998). Together, these data provide strong evidence for a link between NMDAR function and hippocampal spatial memory.  158  4.2.2  The role of NMDARs in hippocampal pathology NMDARs are involved in pathological states either through hyper-activation, or through hypo-activation. The conditions in which evidence suggests a central role for this receptor will be considered below, and the specific involvement of NR2 subunits will be stressed, when it is the case they play a differential role. It is easier to induce epileptiform activity in hippocampus, as compared to other brain regions (Jung, 1951). The NMDARs play an important role in this type of activity, as their block in hippocampal CA1 reduces the amplitude of paroxysmal discharge and attenuates spike firing occurring in conditions of reduced inhibition (Avoli et al., 2002). Up-regulation of glutamatergic receptors, in general, has been characterized in epilepsy, among other factors involved. NMDAR expression is selectively increased in DG in relation to the manifestation of this disease (Brines et al., 1997, Mathern et al., 1997), and the receptors mediate prolonged currents (Izokawa, 1997). In kindling studies, an increased NMDAR-mediated transmission at the entorhinal – granule cell synapses is considered a major cause of epileptogenesis (Mody and Heinemann, 1987). Other pathological conditions that occur due to calcium overload in hippocampal neurons, as a result of the NMDAR hyperactivity are: stroke (Obrenovitch and Urenjak, 1997, Dirnagl et al., 1999), head trauma (Obrenovitch and Urenjak, 1997), and neurodegenerative diseases (Olney et al., 1997). Indeed, when the brain is injured, in conditions such as thrombotic stroke, or anoxic insult during cardiac arrest, excessive activation of NMDA receptors triggers mechanisms leading to cell death (Meldrum and Garthwaite, 1990). It could be speculated that in these conditions, desensitization of 159  NMDARs plays an important role, since this property modulates calcium load that enters the cell, especially during excessive receptor activation. Moreover, the interaction of PSD- 95 – NMDARs, which we have shown to regulate desensitization, has been investigated in this disease with respect to its possible role in mediating toxicity; this interaction has also been proposed as a target for treatment (Aarts et al., 2002). Huntington’s di sease ( HD), a progressive degenerative condition of the striatal and cortical neurons (mainly), shows loss of hippocampal CA1 pyramidal cells too, at late stages (Spargo et al., 1993). The mutated huntingtin protein, when overexpressed in cell lines, increases the NMDAR-mediated current (Chen et al., 1999, Li et al., 2004) and expression in striatal neurons (from transgenic HD mice) leads to augmentation of the NR2B subunit surface expression (Fan et al., 2007). While hippocampal LTD is enhanced in animal models of HD, LTP shows deficits in CA1 (Usdin et al., 1999, Spencer and Murphy, 2000); it is not known whether these changes in synaptic plasticity correlate with altered surface and/or synaptic expression of NMDAR subtypes in hippocampal CA1 neurons. As with other neurodegenerative conditions the increase in glutamatergic synaptic transmission occurs in Alzheimer’s d isease as well. The weak NMDAR antagonist memantine has been used in the treatment of patients suffering for a severe form of the disease, as it improves somewhat cognitive deficits (Winblad and Poritis, 1999). On the other hand, accumulation of the amyloid – beta in aggregates, specific to this disease, has the effect of diminishing glutamatergic transmission, by inducing NMDAR internalization (Mattson, 2004). It is believed NR2B subunits are more affected, through a mechanism linked to their specific interaction with calcineurin (Snyder et al., 2005). 160  Hypoactivity of NMDA receptors has been suggested to be an underlying cause for schizophrenia (Coyle et al., 2003), among other mechanisms. That is because certain NMDAR antagonists, like ketamine or phencyclidine produce effects that are very similar to schizophrenic symptoms when administered to healthy individuals. These symptoms include psychosis, apathy, withdrawal, bizarre delusions and altered sensory experiences. Genetic reduction of NMDAR expression to ~ 5% of the normal levels induces schizophrenia-like behaviours in animals. The NR2 subunits of NMDARs may be differentially involved in this disease through their specific interaction with growth factors such as neuregulin, which has genetic links to schizophrenia. Neuroregulin 1, when overactive, prevents NR2A phosphorylation in patients suffering from this disease (Hahn et al., 2006). This may interfere with the normal functioning of the NMDARs, leading to the above-mentioned symptoms. LTP at hippocampal synapses may also be altered in schizophrenia (Roberts and Greene, 2003), which may contribute to the cognitive problems encountered in patients suffering from this disease. Also, LTP induction in CA1 is prevented by drugs like phencyclidine and ketamine (Anis et al., 1983, Stringer et al., 1983). Finally, neuregulin-1 also has an effect on NMDAR-dependent plasticity; more specifically it enhances depotentiation (Kwon et al., 2005). Taken together, these data are consistent with a role for NMDAR hypoactivity in cognitive and psychiatric symptoms associated with schizophrenia.    161  4.3 Final conclusions and future directions NMDA receptors expressed in mammalian hippocampal neurons mediate synaptic transmission, various forms of synaptic plasticity, and are involved in the physiology and pathology of this brain region. The mechanisms by which these receptors accomplish their diverse functions are largely unknown; however, evidence so far leads to the hypothesis that calcium permeability of NMDARs is a critical factor. Modulating certain aspects of this calcium signal, such as its magnitude, temporal characteristics, and location in the cell, by changes in NMDA receptor desensitization, subunit composition, or subcellular expression, is relevant for determining which downstream signalling pathways become activated. Even though each of these characteristics has been studied with respect to their involvement in receptor function, evidence so far is not conclusive. Most probably, interplay between modulation of gating properties, as well as receptor population size, subunit composition, subcellular localization, and interaction with signalling proteins, is responsible for the different, even opposing outcomes of NMDAR activation. One aspect regarding NMDARs that remains to be clarified is the mechanism that regulates its pattern of expression, both early in development, and later, in mature neurons. While early the expression is expected to be less prone to external modulation, and more constitutive in nature, NMDAR patterns of expression in mature neurons and/or at mature synapses may be shaped by activity/experience in distinct ways. Indeed, the synthesis and synaptic targeting of NMDAR were found to be activity dependent, being different for inactive, basal, and highly active synapses (reviewed in (Perez-Otano and Ehlers, 2004, Turrigiano and Nelson, 2004). Further, alterations in NMDARs levels have important 162  effects on homeostatic forms of plasticity (Turrigiano and Nelson, 2004), as well as on metaplasticity (Philpot et al., 2001) but their precise involvement in each of these functional aspects remains to be clarified. The contribution of glycine-independent desensitization of NMDAR to plasticity remains to be determined, as our studies regarding this issue are only suggestive. Results from studies conducted in our laboratory elucidated the distinct ways this property is regulated for synaptic and extrasynaptic receptors. Throughout development, neurons show a decreasing ability to modulate the calcium influx through desensitization, and this decrease in desensitization with age is not solely dependent on subunit changes (Li et al., 2003a), but on the direct interaction of the receptor with MAGUK family of proteins, as revealed in this thesis (Chapter two). Lastly, the NR2 subtypes of NMDAR have initially been proposed as factors that lead to bidirectional synaptic plasticity. However, the initial finding, that activation of NR2A-containing receptors results in the induction of LTP, whereas that of NR2B receptors - in LTD, does not hold for other brain regions. The inconsistent results regarding this issue need reconciliation, and plasticity induction needs further examination taking into account interplay between the size, composition and location of the NMDAR populations. Investigating this idea in a simple system, such as the autaptic cultures, or organotypic slice cultures, where these subpopulations could be differentially activated in a combinatorial way, may prove to be useful.  163    Figure 4. 1 Summary of the plasticity data discussed in subchapter 4.1.3. LTP and LTD, in each drug condition, are compared in the two groups of animals tested (Controls, light grey bars and Runners, dark grey bars). Insert: Same data presented in the main graph, separately illustrating the magnitude of plasticity induced in the three conditions tested (ACSF, ifenprodil and NVP), in the two groups of animals (Controls on the left and Runners on the right). NM DA R po pu lat ion NR 2A  po pu lat ion NR 2B  po pu lat ion NM DA R po pu lat ion NR 2A  po pu lat ion NR 2B  po pu lat ion -75 -50 -25 0 25 50 75 EP SP  s lo pe  (%  c ha ng e) NM DA R po pu lat ion NR 2A  po pu lat ion NR 2B  po pu lat ion NM DA R po pu lat ion NR 2A  po pu lat ion NR 2B  po pu lat ion -75 -50 -25 0 25 50 75 EP SP  s lo pe  (%  c ha ng e) AC SF Ife np ro di l NV P AC SF Ife np ro di l NV P -60 -40 -20 0 20 40 60 80 C R LTDLTP E P S P  s lo p e  ( %  c h a n g e ) 164  4.1 Bibliography Aarts M, Liu Y, Liu L, Besshoh S, Arundine M, Gurd JW, Wang YT, Salter MW, Tymianski M (Treatment of ischemic brain damage by perturbing NMDA receptor- PSD-95 protein interactions. Science 298:846-850.2002). Abraham WC (How long will long-term potentiation last? Philos Trans R Soc Lond B Biol Sci 358:735-744.2003). Al-Hallaq RA, Conrads TP, Veenstra TD, Wenthold RJ (NMDA di-heteromeric receptor populations and associated proteins in rat hippocampus. J Neurosci 27:8334-8343.2007). Al-Hallaq RA, Jarabek BR, Fu Z, Vicini S, Wolfe BB, Yasuda RP (Association of NR3A with the N-methyl-D-aspartate receptor NR1 and NR2 subunits. Mol Pharmacol 62:1119- 1127.2002). Andersen P, Silfvenius H, Sundberg SH, Sveen O (A comparison of distal and proximal dendritic synapses on CAi pyramids in guinea-pig hippocampal slices in vitro. J Physiol 307:273-299.1980). Anis NA, Berry SC, Burton NR, Lodge D (The dissociative anaesthetics, ketamine and phencyclidine, selectively reduce excitation of central mammalian neurones by N- methyl-aspartate. Br J Pharmacol 79:565-575.1983). Antonova I, Arancio O, Trillat AC, Wang HG, Zablow L, Udo H, Kandel ER, Hawkins RD (Rapid increase in clusters of presynaptic proteins at onset of long-lasting potentiation. Science 294:1547-1550.2001). Aoki C, Fujisawa S, Mahadomrongkul V, Shah PJ, Nader K, Erisir A (NMDA receptor blockade in intact adult cortex increases trafficking of NR2A subunits into spines, postsynaptic densities, and axon terminals. Brain Res 963:139-149.2003). Artola A, von Frijtag JC, Fermont PC, Gispen WH, Schrama LH, Kamal A, Spruijt BM (Long- lasting modulation of the induction of LTD and LTP in rat hippocampal CA1 by behavioural stress and environmental enrichment. Eur J Neurosci 23:261-272.2006). Ascher P, Nowak L (The role of divalent cations in the N-methyl-D-aspartate responses of mouse central neurones in culture. J Physiol 399:247-266.1988). Ashby MC, De La Rue SA, Ralph GS, Uney J, Collingridge GL, Henley JM (Removal of AMPA receptors (AMPARs) from synapses is preceded by transient endocytosis of extrasynaptic AMPARs. J Neurosci 24:5172-5176.2004). Auerbach A, Zhou Y (Gating reaction mechanisms for NMDA receptor channels. J Neurosci 25:7914-7923.2005). Avoli M, D'Antuono M, Louvel J, Kohling R, Biagini G, Pumain R, D'Arcangelo G, Tancredi V (Network and pharmacological mechanisms leading to epileptiform synchronization in the limbic system in vitro. Prog Neurobiol 68:167-207.2002). Bagal AA, Kao JP, Tang CM, Thompson SM (Long-term potentiation of exogenous glutamate responses at single dendritic spines. Proc Natl Acad Sci U S A 102:14434-14439.2005). Bannister NJ, Larkman AU (Dendritic morphology of CA1 pyramidal neurones from the rat hippocampus: II. Spine distributions. J Comp Neurol 360:161-171.1995). Baranano DE, Ferris CD, Snyder SH (Atypical neural messengers. Trends Neurosci 24:99- 106.2001). 165  Barnes CA (Memory deficits associated with senescence: a neurophysiological and behavioral study in the rat. J Comp Physiol Psychol 93:74-104.1979). Barria A, Malinow R (Subunit-specific NMDA receptor trafficking to synapses. Neuron 35:345- 353.2002). Bartlett TE, Bannister NJ, Collett VJ, Dargan SL, Massey PV, Bortolotto ZA, Fitzjohn SM, Bashir ZI, Collingridge GL, Lodge D (Differential roles of NR2A and NR2B-containing NMDA receptors in LTP and LTD in the CA1 region of two-week old rat hippocampus. Neuropharmacology 52:60-70.2007). Bassand P, Bernard A, Rafiki A, Gayet D, Khrestchatisky M (Differential interaction of the tSXV motifs of the NR1 and NR2A NMDA receptor subunits with PSD-95 and SAP97. Eur J Neurosci 11:2031-2043.1999). Bear MF, Kleinschmidt A, Gu QA, Singer W (Disruption of experience-dependent synaptic modifications in striate cortex by infusion of an NMDA receptor antagonist. J Neurosci 10:909-925.1990). Behe P, Stern P, Wyllie DJ, Nassar M, Schoepfer R, Colquhoun D (Determination of NMDA NR1 subunit copy number in recombinant NMDA receptors. Proc R Soc Lond B Biol Sci 262:205-213.1995). Beique JC, Lin DT, Kang MG, Aizawa H, Takamiya K, Huganir RL (Synapse-specific regulation of AMPA receptor function by PSD-95. Proc Natl Acad Sci U S A 103:19535- 19540.2006). Bekkers JM, Stevens CF (NMDA and non-NMDA receptors are co-localized at individual excitatory synapses in cultured rat hippocampus. Nature 341:230-233.1989). Benke TA, Luthi A, Isaac JT, Collingridge GL (Modulation of AMPA receptor unitary conductance by synaptic activity. Nature 393:793-797.1998). Benuskova L, Diamond ME, Ebner FF (Dynamic synaptic modification threshold: computational model of experience-dependent plasticity in adult rat barrel cortex. Proc Natl Acad Sci U S A 91:4791-4795.1994). Benveniste M, Clements J, Vyklicky L, Jr., Mayer ML (A kinetic analysis of the modulation of N-methyl-D-aspartic acid receptors by glycine in mouse cultured hippocampal neurones. J Physiol 428:333-357.1990). Benveniste M, Mayer ML (Kinetic analysis of antagonist action at N-methyl-D-aspartic acid receptors. Two binding sites each for glutamate and glycine. Biophys J 59:560- 573.1991). Benveniste M, Mayer ML (Multiple effects of spermine on N-methyl-D-aspartic acid receptor responses of rat cultured hippocampal neurones. J Physiol 464:131-163.1993). Berberich S, Jensen V, Hvalby O, Seeburg PH, Kohr G (The role of NMDAR subtypes and charge transfer during hippocampal LTP induction. Neuropharmacology.2006). Berberich S, Punnakkal P, Jensen V, Pawlak V, Seeburg PH, Hvalby O, Kohr G (Lack of NMDA receptor subtype selectivity for hippocampal long-term potentiation. J Neurosci 25:6907-6910.2005). Birchall AM, Bishop J, Bradshaw D, Cline A, Coffey J, Elliott LH, Gibson VM, Greenham A, Hallam TJ, Harris W, et al. (Ro 32-0432, a selective and orally active inhibitor of protein kinase C prevents T-cell activation. J Pharmacol Exp Ther 268:922-929.1994). Biscoe TJ, Straughan DW (Micro-electrophoretic studies of neurones in the cat hippocampus. J Physiol 183:341-359.1966). 166  Black AH, Young GA (Electrical activity of the hippocampus and cortex in dogs operantly trained to move and to hold still. J Comp Physiol Psychol 79:128-141.1972). Black AH, Young GA, Batenchuk C (Avoidance training of hippocampal theta waves in flaxedilized dogs and its relation to skeletal movement. J Comp Physiol Psychol 70:15- 24.1970). Blanton MG, Kriegstein AR (Properties of amino acid neurotransmitter receptors of embryonic cortical neurons when activated by exogenous and endogenous agonists. J Neurophysiol 67:1185-1200.1992). Bliss TV, Collingridge GL (A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361:31-39.1993). Bliss TV, Lomo T (Plasticity in a monosynaptic cortical pathway. J Physiol 207:61P.1970). Bliss TV, Lomo T (Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. J Physiol 232:331- 356.1973). Bolshakov VY, Golan H, Kandel ER, Siegelbaum SA (Recruitment of new sites of synaptic transmission during the cAMP-dependent late phase of LTP at CA3-CA1 synapses in the hippocampus. Neuron 19:635-651.1997). Bonhaus DW, Perry WB, McNamara JO (Decreased density, but not number, of N-methyl-D- aspartate, glycine and phencyclidine binding sites in hippocampus of senescent rats. Brain Res 532:82-86.1990). Borgland SL, Taha SA, Sarti F, Fields HL, Bonci A (Orexin A in the VTA is critical for the induction of synaptic plasticity and behavioral sensitization to cocaine. Neuron 49:589- 601.2006). Brines ML, Sundaresan S, Spencer DD, de Lanerolle NC (Quantitative autoradiographic analysis of ionotropic glutamate receptor subtypes in human temporal lobe epilepsy: up-regulation in reorganized epileptogenic hippocampus. Eur J Neurosci 9:2035-2044.1997). Brun VH, Ytterbo K, Morris RG, Moser MB, Moser EI (Retrograde amnesia for spatial memory induced by NMDA receptor-mediated long-term potentiation. J Neurosci 21:356- 362.2001). Burnashev N (Calcium permeability of glutamate-gated channels in the central nervous system. Curr Opin Neurobiol 6:311-317.1996). Carmignoto G, Vicini S (Activity-dependent decrease in NMDA receptor responses during development of the visual cortex. Science 258:1007-1011.1992). Chapman PF, White GL, Jones MW, Cooper-Blacketer D, Marshall VJ, Irizarry M, Younkin L, Good MA, Bliss TV, Hyman BT, Younkin SG, Hsiao KK (Impaired synaptic plasticity and learning in aged amyloid precursor protein transgenic mice. Nat Neurosci 2:271- 276.1999). Chen L, Huang LY (Protein kinase C reduces Mg2+ block of NMDA-receptor channels as a mechanism of modulation. Nature 356:521-523.1992). Chen N, Luo T, Raymond LA (Subtype-dependence of NMDA receptor channel open probability. J Neurosci 19:6844-6854.1999). Chen N, Moshaver A, Raymond LA (Differential sensitivity of recombinant N-methyl-D- aspartate receptor subtypes to zinc inhibition. Mol Pharmacol 51:1015-1023.1997). Cho KO, Hunt CA, Kennedy MB (The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein. Neuron 9:929-942.1992). 167  Choi S, Klingauf J, Tsien RW (Postfusional regulation of cleft glutamate concentration during LTP at 'silent synapses'. Nat Neurosci 3:330-336.2000). Christie BR, Swann SE, Fox CJ, Froc D, Lieblich SE, Redila V, Webber A (Voluntary exercise rescues deficits in spatial memory and long-term potentiation in prenatal ethanol-exposed male rats. Eur J Neurosci 21:1719-1726.2005). Christopherson KS, Sweeney NT, Craven SE, Kang R, El-Husseini Ael D, Bredt DS (Lipid- and protein-mediated multimerization of PSD-95: implications for receptor clustering and assembly of synaptic protein networks. J Cell Sci 116:3213-3219.2003). Ciabarra AM, Sullivan JM, Gahn LG, Pecht G, Heinemann S, Sevarino KA (Cloning and characterization of chi-1: a developmentally regulated member of a novel class of the ionotropic glutamate receptor family. J Neurosci 15:6498-6508.1995). Clements JD, Lester RA, Tong G, Jahr CE, Westbrook GL (The time course of glutamate in the synaptic cleft. Science 258:1498-1501.1992). Clements JD, Westbrook GL (Activation kinetics reveal the number of glutamate and glycine binding sites on the N-methyl-D-aspartate receptor. Neuron 7:605-613.1991). Clothiaux EE, Bear MF, Cooper LN (Synaptic plasticity in visual cortex: comparison of theory with experiment. J Neurophysiol 66:1785-1804.1991). Colledge M, Dean RA, Scott GK, Langeberg LK, Huganir RL, Scott JD (Targeting of PKA to glutamate receptors through a MAGUK-AKAP complex. Neuron 27:107-119.2000). Collingridge GL, Herron CE, Lester RA (Frequency-dependent N-methyl-D-aspartate receptor- mediated synaptic transmission in rat hippocampus. J Physiol 399:301-312.1988). Collingridge GL, Isaac JT, Wang YT (Receptor trafficking and synaptic plasticity. Nat Rev Neurosci 5:952-962.2004). Collingridge GL, Kehl SJ, McLennan H (The antagonism of amino acid-induced excitations of rat hippocampal CA1 neurones in vitro. J Physiol 334:19-31.1983a). Collingridge GL, Kehl SJ, McLennan H (Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus. J Physiol 334:33- 46.1983b). Collins MO, Husi H, Yu L, Brandon JM, Anderson CN, Blackstock WP, Choudhary JS, Grant SG (Molecular characterization and comparison of the components and multiprotein complexes in the postsynaptic proteome. Journal of neurochemistry 97 Suppl 1:16- 23.2006). Colquhoun D (Binding, gating, affinity and efficacy: the interpretation of structure-activity relationships for agonists and of the effects of mutating receptors. Br J Pharmacol 125:924-947.1998). Colquhoun D, Hawkes AG (Desensitization of N-methyl-D-aspartate receptors: a problem of interpretation. Proc Natl Acad Sci U S A 92:10327-10329.1995). Cottrell JR, Borok E, Horvath TL, Nedivi E (CPG2: a brain- and synapse-specific protein that regulates the endocytosis of glutamate receptors. Neuron 44:677-690.2004). Coultrap SJ, Nixon KM, Alvestad RM, Valenzuela CF, Browning MD (Differential expression of NMDA receptor subunits and splice variants among the CA1, CA3 and dentate gyrus of the adult rat. Brain Res Mol Brain Res 135:104-111.2005). Coyle JT, Tsai G, Goff D (Converging evidence of NMDA receptor hypofunction in the pathophysiology of schizophrenia. Ann N Y Acad Sci 1003:318-327.2003). 168  Craven SE, El-Husseini AE, Bredt DS (Synaptic targeting of the postsynaptic density protein PSD-95 mediated by lipid and protein motifs. Neuron 22:497-509.1999). Cull-Candy S, Brickley S, Farrant M (NMDA receptor subunits: diversity, development and disease. Curr Opin Neurobiol 11:327-335.2001). Cull-Candy SG, Leszkiewicz DN (Role of distinct NMDA receptor subtypes at central synapses. Sci STKE 2004:re16.2004). Davies CH, Starkey SJ, Pozza MF, Collingridge GL (GABA autoreceptors regulate the induction of LTP. Nature 349:609-611.1991). Davies SN, Lester RA, Reymann KG, Collingridge GL (Temporally distinct pre- and post- synaptic mechanisms maintain long-term potentiation. Nature 338:500-503.1989). Daw NW, Fox K, Sato H, Czepita D (Critical period for monocular deprivation in the cat visual cortex. J Neurophysiol 67:197-202.1992). de Toledo-Morrell L, Morrell F (Electrophysiological markers of aging and memory loss in rats. Ann N Y Acad Sci 444:296-311.1985). Derkach V, Barria A, Soderling TR (Ca2+/calmodulin-kinase II enhances channel conductance of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate type glutamate receptors. Proc Natl Acad Sci U S A 96:3269-3274.1999). Dingledine R, Boland LM, Chamberlin NL, Kawasaki K, Kleckner NW, Traynelis SF, Verdoorn TA (Amino acid receptors and uptake systems in the mammalian central nervous system. Crit Rev Neurobiol 4:1-96.1988). Dingledine R, Borges K, Bowie D, Traynelis SF (The glutamate receptor ion channels. Pharmacol Rev 51:7-61.1999). Dirnagl U, Iadecola C, Moskowitz MA (Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci 22:391-397.1999). Douglas RM, Goddard GV (Long-term potentiation of the perforant path-granule cell synapse in the rat hippocampus. Brain Res 86:205-215.1975). Dudek SM, Bear MF (Bidirectional long-term modification of synaptic effectiveness in the adult and immature hippocampus. J Neurosci 13:2910-2918.1993). Duffy SN, Craddock KJ, Abel T, Nguyen PV (Environmental enrichment modifies the PKA- dependence of hippocampal LTP and improves hippocampus-dependent memory. Learn Mem 8:26-34.2001). Eadie BD, Redila VA, Christie BR (Voluntary exercise alters the cytoarchitecture of the adult dentate gyrus by increasing cellular proliferation, dendritic complexity, and spine density. J Comp Neurol 486:39-47.2005). Edmonds B, Colquhoun D (Rapid decay of averaged single-channel NMDA receptor activations recorded at low agonist concentration. Proc Biol Sci 250:279-286.1992). Ehlers MD, Zhang S, Bernhadt JP, Huganir RL (Inactivation of NMDA receptors by direct interaction of calmodulin with the NR1 subunit. Cell 84:745-755.1996). El-Husseini AE, Schnell E, Chetkovich DM, Nicoll RA, Bredt DS (PSD-95 involvement in maturation of excitatory synapses. Science 290:1364-1368.2000a). El-Husseini AE, Topinka JR, Lehrer-Graiwer JE, Firestein BL, Craven SE, Aoki C, Bredt DS (Ion channel clustering by membrane-associated guanylate kinases. Differential regulation by N-terminal lipid and metal binding motifs. J Biol Chem 275:23904- 23910.2000b). 169  El-Husseini Ael D, Schnell E, Dakoji S, Sweeney N, Zhou Q, Prange O, Gauthier-Campbell C, Aguilera-Moreno A, Nicoll RA, Bredt DS (Synaptic strength regulated by palmitate cycling on PSD-95. Cell 108:849-863.2002). Elias GM, Elias LA, Apostolides PF, Kriegstein AR, Nicoll RA (Differential trafficking of AMPA and NMDA receptors by SAP102 and PSD-95 underlies synapse development. Proc Natl Acad Sci U S A 105:20953-20958.2008). Elias GM, Funke L, Stein V, Grant SG, Bredt DS, Nicoll RA (Synapse-specific and developmentally regulated targeting of AMPA receptors by a family of MAGUK scaffolding proteins. Neuron 52:307-320.2006). Emptage N, Bliss TV, Fine A (Single synaptic events evoke NMDA receptor-mediated release of calcium from internal stores in hippocampal dendritic spines. Neuron 22:115-124.1999). Engert F, Bonhoeffer T (Dendritic spine changes associated with hippocampal long-term synaptic plasticity. Nature 399:66-70.1999). Erreger K, Dravid SM, Banke TG, Wyllie DJ, Traynelis SF (Subunit-specific gating controls rat NR1/NR2A and NR1/NR2B NMDA channel kinetics and synaptic signalling profiles. J Physiol 563:345-358.2005). Erreger K, Traynelis SF (Allosteric interaction between zinc and glutamate binding domains on NR2A causes desensitization of NMDA receptors. J Physiol 569:381-393.2005). Erreger K, Traynelis SF (Zinc inhibition of rat NR1/NR2A N-methyl-D-aspartate receptors. J Physiol 586:763-778.2008). Fan MM, Fernandes HB, Zhang LY, Hayden MR, Raymond LA (Altered NMDA receptor trafficking in a yeast artificial chromosome transgenic mouse model of Huntington's disease. J Neurosci 27:3768-3779.2007). Farmer J, Zhao X, van Praag H, Wodtke K, Gage FH, Christie BR (Effects of voluntary exercise on synaptic plasticity and gene expression in the dentate gyrus of adult male Sprague- Dawley rats in vivo. Neuroscience 124:71-79.2004). Farrant M, Feldmeyer D, Takahashi T, Cull-Candy SG (NMDA-receptor channel diversity in the developing cerebellum. Nature 368:335-339.1994). Fayyazuddin A, Villarroel A, Le Goff A, Lerma J, Neyton J (Four residues of the extracellular N-terminal domain of the NR2A subunit control high-affinity Zn2+ binding to NMDA receptors. Neuron 25:683-694.2000). Feldmeyer D, Lubke J, Silver RA, Sakmann B (Synaptic connections between layer 4 spiny neurone-layer 2/3 pyramidal cell pairs in juvenile rat barrel cortex: physiology and anatomy of interlaminar signalling within a cortical column. J Physiol 538:803- 822.2002). Flint AC, Maisch US, Weishaupt JH, Kriegstein AR, Monyer H (NR2A subunit expression shortens NMDA receptor synaptic currents in developing neocortex. J Neurosci 17:2469- 2476.1997). Fong DK, Rao A, Crump FT, Craig AM (Rapid synaptic remodeling by protein kinase C: reciprocal translocation of NMDA receptors and calcium/calmodulin-dependent kinase II. J Neurosci 22:2153-2164.2002). Forrest D, Yuzaki M, Soares HD, Ng L, Luk DC, Sheng M, Stewart CL, Morgan JI, Connor JA, Curran T (Targeted disruption of NMDA receptor 1 gene abolishes NMDA response and results in neonatal death. Neuron 13:325-338.1994). 170  Fox CJ, Russell KI, Wang YT, Christie BR (Contribution of NR2A and NR2B NMDA subunits to bidirectional synaptic plasticity in the hippocampus in vivo. Hippocampus 16:907- 915.2006). Fox K, Henley J, Isaac J (Experience-dependent development of NMDA receptor transmission. Nat Neurosci 2:297-299.1999). Frizelle PA, Chen PE, Wyllie DJ (Equilibrium constants for NVP-AAM077 acting at recombinant NR1/NR2A and NR1/NR2B NMDA receptors: implications for studies of synaptic transmission. Mol Pharmacol.2006). Fukaya M, Kato A, Lovett C, Tonegawa S, Watanabe M (Retention of NMDA receptor NR2 subunits in the lumen of endoplasmic reticulum in targeted NR1 knockout mice. Proc Natl Acad Sci U S A 100:4855-4860.2003). Furukawa H, Gouaux E (Mechanisms of activation, inhibition and specificity: crystal structures of the NMDA receptor NR1 ligand-binding core. Embo J 22:2873-2885.2003). Furukawa H, Singh SK, Mancusso R, Gouaux E (Subunit arrangement and function in NMDA receptors. Nature 438:185-192.2005). Ganeshina O, Berry RW, Petralia RS, Nicholson DA, Geinisman Y (Differences in the expression of AMPA and NMDA receptors between axospinous perforated and nonperforated synapses are related to the configuration and size of postsynaptic densities. J Comp Neurol 468:86-95.2004). Garaschuk O, Schneggenburger R, Schirra C, Tempia F, Konnerth A (Fractional Ca2+ currents through somatic and dendritic glutamate receptor channels of rat hippocampal CA1 pyramidal neurones. J Physiol 491 ( Pt 3):757-772.1996). Gibb AJ, Colquhoun D (Glutamate activation of a single NMDA receptor-channel produces a cluster of channel openings. Proc Biol Sci 243:39-45.1991). Gibb AJ, Colquhoun D (Activation of N-methyl-D-aspartate receptors by L-glutamate in cells dissociated from adult rat hippocampus. J Physiol 456:143-179.1992). Green EJ, Greenough WT (Altered synaptic transmission in dentate gyrus of rats reared in complex environments: evidence from hippocampal slices maintained in vitro. J Neurophysiol 55:739-750.1986). Gruart A, Munoz MD, Delgado-Garcia JM (Involvement of the CA3-CA1 synapse in the acquisition of associative learning in behaving mice. J Neurosci 26:1077-1087.2006). Gurd JW, Bissoon N (The N-methyl-D-aspartate receptor subunits NR2A and NR2B bind to the SH2 domains of phospholipase C-gamma. J Neurochem 69:623-630.1997). Gustafsson B, Wigstrom H (Hippocampal long-lasting potentiation produced by pairing single volleys and brief conditioning tetani evoked in separate afferents. J Neurosci 6:1575- 1582.1986). Hahn CG, Wang HY, Cho DS, Talbot K, Gur RE, Berrettini WH, Bakshi K, Kamins J, Borgmann-Winter KE, Siegel SJ, Gallop RJ, Arnold SE (Altered neuregulin 1-erbB4 signaling contributes to NMDA receptor hypofunction in schizophrenia. Nat Med 12:824-828.2006). Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391:85-100.1981). Hardingham GE, Bading H (The Yin and Yang of NMDA receptor signalling. Trends Neurosci 26:81-89.2003). 171  Hardingham GE, Fukunaga Y, Bading H (Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nat Neurosci 5:405-414.2002). Harris KM, Jensen FE, Tsao B (Three-dimensional structure of dendritic spines and synapses in rat hippocampus (CA1) at postnatal day 15 and adult ages: implications for the maturation of synaptic physiology and long-term potentiation. J Neurosci 12:2685- 2705.1992). Harris KM, Teyler TJ (Developmental onset of long-term potentiation in area CA1 of the rat hippocampus. J Physiol 346:27-48.1984). Harvey J, Collingridge GL (Thapsigargin blocks the induction of long-term potentiation in rat hippocampal slices. Neurosci Lett 139:197-200.1992). Hastings NB, Gould E (Rapid extension of axons into the CA3 region by adult-generated granule cells. J Comp Neurol 413:146-154.1999). Hayashi Y, Shi SH, Esteban JA, Piccini A, Poncer JC, Malinow R (Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction. Science 287:2262-2267.2000). Hendricson AW, Miao CL, Lippmann MJ, Morrisett RA (Ifenprodil and ethanol enhance NMDA receptor-dependent long-term depression. J Pharmacol Exp Ther 301:938-944.2002). Hering H, Sheng M (Activity-dependent redistribution and essential role of cortactin in dendritic spine morphogenesis. J Neurosci 23:11759-11769.2003). Hernandez RV, Navarro MM, Rodriguez WA, Martinez JL, Jr., LeBaron RG (Differences in the magnitude of long-term potentiation produced by theta burst and high frequency stimulation protocols matched in stimulus number. Brain Res Brain Res Protoc 15:6- 13.2005). Hestrin S (Developmental regulation of NMDA receptor-mediated synaptic currents at a central synapse. Nature 357:686-689.1992). Hsueh YP, Sheng M (Requirement of N-terminal cysteines of PSD-95 for PSD-95 multimerization and ternary complex formation, but not for binding to potassium channel Kv1.4. J Biol Chem 274:532-536.1999). Hu B, Zheng F (Molecular determinants of glycine-independent desensitization of NR1/NR2A receptors. J Pharmacol Exp Ther 313:563-569.2005). Inanobe A, Furukawa H, Gouaux E (Mechanism of partial agonist action at the NR1 subunit of NMDA receptors. Neuron 47:71-84.2005). Isaac JT, Nicoll RA, Malenka RC (Evidence for silent synapses: implications for the expression of LTP. Neuron 15:427-434.1995). Ishii T, Moriyoshi K, Sugihara H, Sakurada K, Kadotani H, Yokoi M, Akazawa C, Shigemoto R, Mizuno N, Masu M, et al. (Molecular characterization of the family of the N-methyl-D- aspartate receptor subunits. J Biol Chem 268:2836-2843.1993). Ito I, Futai K, Katagiri H, Watanabe M, Sakimura K, Mishina M, Sugiyama H (Synapse- selective impairment of NMDA receptor functions in mice lacking NMDA receptor epsilon 1 or epsilon 2 subunit. J Physiol 500 ( Pt 2):401-408.1997). Ivanovic A, Reilander H, Laube B, Kuhse J (Expression and initial characterization of a soluble glycine binding domain of the N-methyl-D-aspartate receptor NR1 subunit. J Biol Chem 273:19933-19937.1998). 172  Izquierdo I, Schroder N, Netto CA, Medina JH (Novelty causes time-dependent retrograde amnesia for one-trial avoidance in rats through NMDA receptor- and CaMKII-dependent mechanisms in the hippocampus. Eur J Neurosci 11:3323-3328.1999). Jackson MF, Konarski JZ, Weerapura M, Czerwinski W, MacDonald JF (Protein kinase C enhances glycine-insensitive desensitization of NMDA receptors independently of previously identified protein kinase C sites. J Neurochem 96:1509-1518.2006). Jahr CE (High probability opening of NMDA receptor channels by L-glutamate. Science 255:470-472.1992). Jahr CE, Stevens CF (Glutamate activates multiple single channel conductances in hippocampal neurons. Nature 325:522-525.1987). Jain RK, Joyce PB, Molinete M, Halban PA, Gorr SU (Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells. Biochem J 360:645-649.2001). Johnson JW, Ascher P (Glycine potentiates the NMDA response in cultured mouse brain neurons. Nature 325:529-531.1987). Jung R ([Electrophysiology of the convulsive seizure in animal and man.]. Med Hyg (Geneve) 9:371.1951). Kawakami R, Shinohara Y, Kato Y, Sugiyama H, Shigemoto R, Ito I (Asymmetrical allocation of NMDA receptor epsilon2 subunits in hippocampal circuitry. Science 300:990- 994.2003). Kentros C, Hargreaves E, Hawkins RD, Kandel ER, Shapiro M, Muller RV (Abolition of long- term stability of new hippocampal place cell maps by NMDA receptor blockade. Science 280:2121-2126.1998). Kew JN, Koester A, Moreau JL, Jenck F, Ouagazzal AM, Mutel V, Richards JG, Trube G, Fischer G, Montkowski A, Hundt W, Reinscheid RK, Pauly-Evers M, Kemp JA, Bluethmann H (Functional consequences of reduction in NMDA receptor glycine affinity in mice carrying targeted point mutations in the glycine binding site. J Neurosci 20:4037- 4049.2000). Kim CH, Chung HJ, Lee HK, Huganir RL (Interaction of the AMPA receptor subunit GluR2/3 with PDZ domains regulates hippocampal long-term depression. Proc Natl Acad Sci U S A 98:11725-11730.2001). Kim E, Niethammer M, Rothschild A, Jan YN, Sheng M (Clustering of Shaker-type K+ channels by interaction with a family of membrane-associated guanylate kinases. Nature 378:85- 88.1995). Kim E, Sheng M (Differential K+ channel clustering activity of PSD-95 and SAP97, two related membrane-associated putative guanylate kinases. Neuropharmacology 35:993- 1000.1996). Kim E, Sheng M (PDZ domain proteins of synapses. Nature reviews 5:771-781.2004). Kim JJ, Foy MR, Thompson RF (Behavioral stress modifies hippocampal plasticity through N- methyl-D-aspartate receptor activation. Proc Natl Acad Sci U S A 93:4750-4753.1996). Kohda K, Wang Y, Yuzaki M (Mutation of a glutamate receptor motif reveals its role in gating and delta2 receptor channel properties. Nat Neurosci 3:315-322.2000). Kohr G, Jensen V, Koester HJ, Mihaljevic AL, Utvik JK, Kvello A, Ottersen OP, Seeburg PH, Sprengel R, Hvalby O (Intracellular domains of NMDA receptor subtypes are determinants for long-term potentiation induction. J Neurosci 23:10791-10799.2003). 173  Komuro H, Rakic P (Modulation of neuronal migration by NMDA receptors. Science 260:95- 97.1993). Kornau HC, Schenker LT, Kennedy MB, Seeburg PH (Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science 269:1737- 1740.1995). Kramis R, Vanderwolf CH, Bland BH (Two types of hippocampal rhythmical slow activity in both the rabbit and the rat: relations to behavior and effects of atropine, diethyl ether, urethane, and pentobarbital. Exp Neurol 49:58-85.1975). Krupp JJ, Vissel B, Heinemann SF, Westbrook GL (Calcium-dependent inactivation of recombinant N-methyl-D-aspartate receptors is NR2 subunit specific. Mol Pharmacol 50:1680-1688.1996). Krupp JJ, Vissel B, Heinemann SF, Westbrook GL (N-terminal domains in the NR2 subunit control desensitization of NMDA receptors. Neuron 20:317-327.1998). Krupp JJ, Vissel B, Thomas CG, Heinemann SF, Westbrook GL (Interactions of calmodulin and alpha-actinin with the NR1 subunit modulate Ca2+-dependent inactivation of NMDA receptors. J Neurosci 19:1165-1178.1999). Krupp JJ, Vissel B, Thomas CG, Heinemann SF, Westbrook GL (Calcineurin acts via the C- terminus of NR2A to modulate desensitization of NMDA receptors. Neuropharmacology 42:593-602.2002). Kullmann DM, Asztely F (Extrasynaptic glutamate spillover in the hippocampus: evidence and implications. Trends Neurosci 21:8-14.1998). Kutsuwada T, Kashiwabuchi N, Mori H, Sakimura K, Kushiya E, Araki K, Meguro H, Masaki H, Kumanishi T, Arakawa M, et al. (Molecular diversity of the NMDA receptor channel [see comments]. Nature 358:36-41.1992). Kutsuwada T, Sakimura K, Manabe T, Takayama C, Katakura N, Kushiya E, Natsume R, Watanabe M, Inoue Y, Yagi T, Aizawa S, Arakawa M, Takahashi T, Nakamura Y, Mori H, Mishina M (Impairment of suckling response, trigeminal neuronal pattern formation, and hippocampal LTD in NMDA receptor epsilon 2 subunit mutant mice. Neuron 16:333-344.1996). Kwon OB, Longart M, Vullhorst D, Hoffman DA, Buonanno A (Neuregulin-1 reverses long- term potentiation at CA1 hippocampal synapses. J Neurosci 25:9378-9383.2005). Lan JY, Skeberdis VA, Jover T, Grooms SY, Lin Y, Araneda RC, Zheng X, Bennett MV, Zukin RS (Protein kinase C modulates NMDA receptor trafficking and gating. Nat Neurosci 4:382-390.2001). Laroche S, Doyere V, Bloch V (Linear relation between the magnitude of long-term potentiation in the dentate gyrus and associative learning in the rat. A demonstration using commissural inhibition and local infusion of an N-methyl-D-aspartate receptor antagonist. Neuroscience 28:375-386.1989). Larson J, Wong D, Lynch G (Patterned stimulation at the theta frequency is optimal for the induction of hippocampal long-term potentiation. Brain Res 368:347-350.1986). Laurie DJ, Putzke J, Zieglgansberger W, Seeburg PH, Tolle TR (The distribution of splice variants of the NMDAR1 subunit mRNA in adult rat brain. Brain Res Mol Brain Res 32:94-108.1995). Laurie DJ, Seeburg PH (Ligand affinities at recombinant N-methyl-D-aspartate receptors depend on subunit composition. Eur J Pharmacol 268:335-345.1994a). 174  Laurie DJ, Seeburg PH (Regional and developmental heterogeneity in splicing of the rat brain NMDAR1 mRNA. J Neurosci 14:3180-3194.1994b). Lavezzari G, McCallum J, Dewey CM, Roche KW (Subunit-specific regulation of NMDA receptor endocytosis. J Neurosci 24:6383-6391.2004). Lee HK, Barbarosie M, Kameyama K, Bear MF, Huganir RL (Regulation of distinct AMPA receptor phosphorylation sites during bidirectional synaptic plasticity. Nature 405:955- 959.2000). Lee LJ, Lo FS, Erzurumlu RS (NMDA receptor-dependent regulation of axonal and dendritic branching. J Neurosci 25:2304-2311.2005). Legendre P, Rosenmund C, Westbrook GL (Inactivation of NMDA channels in cultured hippocampal neurons by intracellular calcium. J Neurosci 13:674-684.1993). Legendre P, Westbrook GL (The inhibition of single N-methyl-D-aspartate-activated channels by zinc ions on cultured rat neurones. J Physiol (Lond) 429:429-449.1990). Lerma J (Spermine regulates N-methyl-D-aspartate receptor desensitization. Neuron 8:343- 352.1992). Lester RA, Clements JD, Westbrook GL, Jahr CE (Channel kinetics determine the time course of NMDA receptor-mediated synaptic currents. Nature 346:565-567.1990). Lester RA, Jahr CE (NMDA channel behavior depends on agonist affinity. J Neurosci 12:635- 643.1992). Lester RA, Tong G, Jahr CE (Interactions between the glycine and glutamate binding sites of the NMDA receptor. J Neurosci 13:1088-1096.1993). Levy WB, Steward O (Temporal contiguity requirements for long-term associative potentiation/depression in the hippocampus. Neuroscience 8:791-797.1983). Li B, Chen N, Luo T, Otsu Y, Murphy TH, Raymond LA (Differential regulation of synaptic and extra-synaptic NMDA receptors. Nat Neurosci 5:833-834.2002). Li B, Otsu Y, Murphy TH, Raymond LA (Developmental decrease in NMDA receptor desensitization associated with shift to synapse and interaction with postsynaptic density- 95. J Neurosci 23:11244-11254.2003a). Li JH, Wang YH, Wolfe BB, Krueger KE, Corsi L, Stocca G, Vicini S (Developmental changes in localization of NMDA receptor subunits in primary cultures of cortical neurons. Eur J Neurosci 10:1704-1715.1998). Li L, Fan M, Icton CD, Chen N, Leavitt BR, Hayden MR, Murphy TH, Raymond LA (Role of NR2B-type NMDA receptors in selective neurodegeneration in Huntington disease. Neurobiol Aging 24:1113-1121.2003b). Li L, Murphy TH, Hayden MR, Raymond LA (Enhanced striatal NR2B-containing N-methyl-D- aspartate receptor-mediated synaptic currents in a mouse model of Huntington disease. J Neurophysiol 92:2738-2746.2004). Li S, Tian X, Hartley DM, Feig LA (Distinct roles for Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1) and Ras-GRF2 in the induction of long-term potentiation and long-term depression. J Neurosci 26:1721-1729.2006a). Li S, Tian X, Hartley DM, Feig LA (The environment versus genetics in controlling the contribution of MAP kinases to synaptic plasticity. Curr Biol 16:2303-2313.2006b). Liao GY, Kreitzer MA, Sweetman BJ, Leonard JP (The postsynaptic density protein PSD-95 differentially regulates insulin- and Src-mediated current modulation of mouse NMDA receptors expressed in Xenopus oocytes. J Neurochem 75:282-287.2000). 175  Lim IA, Hall DD, Hell JW (Selectivity and promiscuity of the first and second PDZ domains of PSD-95 and synapse-associated protein 102. J Biol Chem 277:21697-21711.2002). Lim IA, Merrill MA, Chen Y, Hell JW (Disruption of the NMDA receptor-PSD-95 interaction in hippocampal neurons with no obvious physiological short-term effect. Neuropharmacology 45:738-754.2003). Lin F, Stevens CF (Both open and closed NMDA receptor channels desensitize. J Neurosci 14:2153-2160.1994). Lin Y, Jover-Mengual T, Wong J, Bennett MV, Zukin RS (PSD-95 and PKC converge in regulating NMDA receptor trafficking and gating. Proc Natl Acad Sci U S A 103:19902- 19907.2006). Lin Y, Skeberdis VA, Francesconi A, Bennett MV, Zukin RS (Postsynaptic density protein-95 regulates NMDA channel gating and surface expression. J Neurosci 24:10138- 10148.2004). Lisman J (A mechanism for the Hebb and the anti-Hebb processes underlying learning and memory. Proc Natl Acad Sci U S A 86:9574-9578.1989). Liu L, Wong TP, Pozza MF, Lingenhoehl K, Wang Y, Sheng M, Auberson YP, Wang YT (Role of NMDA receptor subtypes in governing the direction of hippocampal synaptic plasticity. Science 304:1021-1024.2004). Liu Y, Wong TP, Aarts M, Rooyakkers A, Liu L, Lai TW, Wu DC, Lu J, Tymianski M, Craig AM, Wang YT (NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo. J Neurosci 27:2846-2857.2007). Lomeli H, Sprengel R, Laurie DJ, Kohr G, Herb A, Seeburg PH, Wisden W (The rat delta-1 and delta-2 subunits extend the excitatory amino acid receptor family. FEBS Lett 315:318- 322.1993). Losi G, Prybylowski K, Fu Z, Luo J, Wenthold RJ, Vicini S (PSD-95 regulates NMDA receptors in developing cerebellar granule neurons of the rat. J Physiol 548:21-29.2003). LoTurco JJ, Blanton MG, Kriegstein AR (Initial expression and endogenous activation of NMDA channels in early neocortical development. J Neurosci 11:792-799.1991). Low CM, Zheng F, Lyuboslavsky P, Traynelis SF (Molecular determinants of coordinated proton and zinc inhibition of N-methyl-D-aspartate NR1/NR2A receptors. Proc Natl Acad Sci U S A 97:11062-11067.2000). Lu W, Man H, Ju W, Trimble WS, MacDonald JF, Wang YT (Activation of synaptic NMDA receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons. Neuron 29:243-254.2001). Luo J, Wang Y, Yasuda RP, Dunah AW, Wolfe BB (The majority of N-methyl-D-aspartate receptor complexes in adult rat cerebral cortex contain at least three different subunits (NR1/NR2A/NR2B). Mol Pharmacol 51:79-86.1997). Lynch G, Larson J, Kelso S, Barrionuevo G, Schottler F (Intracellular injections of EGTA block induction of hippocampal long-term potentiation. Nature 305:719-721.1983). Lynch MA (Long-term potentiation and memory. Physiol Rev 84:87-136.2004). Malenka RC, Lancaster B, Zucker RS (Temporal limits on the rise in postsynaptic calcium required for the induction of long-term potentiation. Neuron 9:121-128.1992). Maletic-Savatic M, Malinow R, Svoboda K (Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science 283:1923-1927.1999). 176  Manahan-Vaughan D (Long-term depression in freely moving rats is dependent upon strain variation, induction protocol and behavioral state. Cereb Cortex 10:482-487.2000). Massey PV, Johnson BE, Moult PR, Auberson YP, Brown MW, Molnar E, Collingridge GL, Bashir ZI (Differential roles of NR2A and NR2B-containing NMDA receptors in cortical long-term potentiation and long-term depression. J Neurosci 24:7821-7828.2004). Mathern GW, Kuhlman PA, Mendoza D, Pretorius JK (Human fascia dentata anatomy and hippocampal neuron densities differ depending on the epileptic syndrome and age at first seizure. J Neuropathol Exp Neurol 56:199-212.1997). Matsuzaki M, Ellis-Davies GC, Nemoto T, Miyashita Y, Iino M, Kasai H (Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons. Nat Neurosci 4:1086-1092.2001). Matthies H, Ruethrich H, Ott T, Matthies HK, Matthies R (Low frequency perforant path stimulation as a conditioned stimulus demonstrates correlations between long-term synaptic potentiation and learning. Physiol Behav 36:811-821.1986). Mattson MP (Pathways towards and away from Alzheimer's disease. Nature 430:631-639.2004). Mayer ML, MacDermott AB, Westbrook GL, Smith SJ, Barker JL (Agonist- and voltage-gated calcium entry in cultured mouse spinal cord neurons under voltage clamp measured using arsenazo III. J Neurosci 7:3230-3244.1987). Mayer ML, Vyklicky L, Jr., Clements J (Regulation of NMDA receptor desensitization in mouse hippocampal neurons by glycine. Nature 338:425-427.1989). Mayer ML, Westbrook GL, Guthrie PB (Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones. Nature 309:261-263.1984). McBain CJ, Mayer ML (N-methyl-D-aspartic acid receptor structure and function. Physiol Rev 74:723-760.1994). McDermott CM, Hardy MN, Bazan NG, Magee JC (Sleep deprivation-induced alterations in excitatory synaptic transmission in the CA1 region of the rat hippocampus. J Physiol 570:553-565.2006). McHugh TJ, Blum KI, Tsien JZ, Tonegawa S, Wilson MA (Impaired hippocampal representation of space in CA1-specific NMDAR1 knockout mice. Cell 87:1339- 1349.1996). Meddows E, Le Bourdelles B, Grimwood S, Wafford K, Sandhu S, Whiting P, McIlhinney RA (Identification of molecular determinants that are important in the assembly of N-methyl- D-aspartate receptors. J Biol Chem 276:18795-18803.2001). Megias M, Emri Z, Freund TF, Gulyas AI (Total number and distribution of inhibitory and excitatory synapses on hippocampal CA1 pyramidal cells. Neuroscience 102:527- 540.2001). Meldrum B, Garthwaite J (Excitatory amino acid neurotoxicity and neurodegenerative disease. Trends Pharmacol Sci 11:379-387.1990). Migaud M, Charlesworth P, Dempster M, Webster LC, Watabe AM, Makhinson M, He Y, Ramsay MF, Morris RG, Morrison JH, O'Dell TJ, Grant SG (Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein. Nature 396:433-439.1998). Milner B (Disorders of learning and memory after temporal lobe lesions in man. Clin Neurosurg 19:421-446.1972). 177  Mody I, Heinemann U (NMDA receptors of dentate gyrus granule cells participate in synaptic transmission following kindling. Nature 326:701-704.1987). Mohn AR, Gainetdinov RR, Caron MG, Koller BH (Mice with reduced NMDA receptor expression display behaviors related to schizophrenia. Cell 98:427-436.1999). Mohrmann R, Kohr G, Hatt H, Sprengel R, Gottmann K (Deletion of the C-terminal domain of the NR2B subunit alters channel properties and synaptic targeting of N-methyl-D- aspartate receptors in nascent neocortical synapses. J Neurosci Res 68:265-275.2002). Montgomery JM, Pavlidis P, Madison DV (Pair recordings reveal all-silent synaptic connections and the postsynaptic expression of long-term potentiation. Neuron 29:691-701.2001). Monyer H, Burnashev N, Laurie DJ, Sakmann B, Seeburg PH (Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12:529-540.1994). Monyer H, Sprengel R, Schoepfer R, Herb A, Higuchi M, Lomeli H, Burnashev N, Sakmann B, Seeburg PH (Heteromeric NMDA receptors: molecular and functional distinction of subtypes. Science 256:1217-1221.1992). Mori H, Masaki H, Yamakura T, Mishina M (Identification by mutagenesis of a Mg(2+)-block site of the NMDA receptor channel. Nature 358:673-675.1992). Morishita W, Lu W, Smith GB, Nicoll RA, Bear MF, Malenka RC (Activation of NR2B- containing NMDA receptors is not required for NMDA receptor-dependent long-term depression. Neuropharmacology.2006). Morishita W, Lu W, Smith GB, Nicoll RA, Bear MF, Malenka RC (Activation of NR2B- containing NMDA receptors is not required for NMDA receptor-dependent long-term depression. Neuropharmacology 52:71-76.2007). Morris RG, Anderson E, Lynch GS, Baudry M (Selective impairment of learning and blockade of long-term potentiation by an N-methyl-D-aspartate receptor antagonist, AP5. Nature 319:774-776.1986). Mu Y, Otsuka T, Horton AC, Scott DB, Ehlers MD (Activity-dependent mRNA splicing controls ER export and synaptic delivery of NMDA receptors. Neuron 40:581-594.2003). Mulkey RM, Malenka RC (Mechanisms underlying induction of homosynaptic long-term depression in area CA1 of the hippocampus. Neuron 9:967-975.1992). Nadler JV, Martin D, Bustos GA, Burke SP, Bowe MA (Regulation of glutamate and aspartate release from the Schaffer collaterals and other projections of CA3 hippocampal pyramidal cells. Prog Brain Res 83:115-130.1990). Nakagawa T, Futai K, Lashuel HA, Lo I, Okamoto K, Walz T, Hayashi Y, Sheng M (Quaternary structure, protein dynamics, and synaptic function of SAP97 controlled by L27 domain interactions. Neuron 44:453-467.2004). Nakazawa K, McHugh TJ, Wilson MA, Tonegawa S (NMDA receptors, place cells and hippocampal spatial memory. Nat Rev Neurosci 5:361-372.2004). Nakazawa K, Quirk MC, Chitwood RA, Watanabe M, Yeckel MF, Sun LD, Kato A, Carr CA, Johnston D, Wilson MA, Tonegawa S (Requirement for hippocampal CA3 NMDA receptors in associative memory recall. Science 297:211-218.2002). Neyton J, Paoletti P (Relating NMDA receptor function to receptor subunit composition: limitations of the pharmacological approach. J Neurosci 26:1331-1333.2006). 178  Niethammer M, Kim E, Sheng M (Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases. J Neurosci 16:2157-2163.1996). Nowak L, Bregestovski P, Ascher P, Herbet A, Prochiantz A (Magnesium gates glutamate- activated channels in mouse central neurones. Nature 307:462-465.1984). Nusser Z, Lujan R, Laube G, Roberts JD, Molnar E, Somogyi P (Cell type and pathway dependence of synaptic AMPA receptor number and variability in the hippocampus. Neuron 21:545-559.1998). O'Keefe J, Dostrovsky J (The hippocampus as a spatial map. Preliminary evidence from unit activity in the freely-moving rat. Brain Res 34:171-175.1971). Obrenovitch TP, Urenjak J (Is high extracellular glutamate the key to excitotoxicity in traumatic brain injury? J Neurotrauma 14:677-698.1997). Olney JW, Wozniak DF, Farber NB (Excitotoxic neurodegeneration in Alzheimer disease. New hypothesis and new therapeutic strategies. Arch Neurol 54:1234-1240.1997). Olson CR, Freeman RD (Profile of the sensitive period for monocular deprivation in kittens. Exp Brain Res 39:17-21.1980). Overstreet-Wadiche LS, Westbrook GL (Functional maturation of adult-generated granule cells. Hippocampus.2006). Pak DT, Yang S, Rudolph-Correia S, Kim E, Sheng M (Regulation of dendritic spine morphology by SPAR, a PSD-95-associated RapGAP. Neuron 31:289-303.2001). Pandis C, Sotiriou E, Kouvaras E, Asprodini E, Papatheodoropoulos C, Angelatou F (Differential expression of NMDA and AMPA receptor subunits in rat dorsal and ventral hippocampus. Neuroscience 140:163-175.2006). Paoletti P, Ascher P, Neyton J (High-affinity zinc inhibition of NMDA NR1-NR2A receptors. J Neurosci 17:5711-5725.1997). Paoletti P, Perin-Dureau F, Fayyazuddin A, Le Goff A, Callebaut I, Neyton J (Molecular organization of a zinc binding n-terminal modulatory domain in a NMDA receptor subunit. Neuron 28:911-925.2000). Passafaro M, Sala C, Niethammer M, Sheng M (Microtubule binding by CRIPT and its potential role in the synaptic clustering of PSD-95. Nat Neurosci 2:1063-1069.1999). Perez-Otano I, Ehlers MD (Learning from NMDA receptor trafficking: clues to the development and maturation of glutamatergic synapses. Neurosignals 13:175-189.2004). Perin-Dureau F, Rachline J, Neyton J, Paoletti P (Mapping the binding site of the neuroprotectant ifenprodil on NMDA receptors. J Neurosci 22:5955-5965.2002). Petralia RS, Esteban JA, Wang YX, Partridge JG, Zhao HM, Wenthold RJ, Malinow R (Selective acquisition of AMPA receptors over postnatal development suggests a molecular basis for silent synapses. Nat Neurosci 2:31-36.1999). Philpot BD, Espinosa JS, Bear MF (Evidence for altered NMDA receptor function as a basis for metaplasticity in visual cortex. J Neurosci 23:5583-5588.2003). Philpot BD, Sekhar AK, Shouval HZ, Bear MF (Visual experience and deprivation bidirectionally modify the composition and function of NMDA receptors in visual cortex. Neuron 29:157-169.2001). Pickard L, Noel J, Duckworth JK, Fitzjohn SM, Henley JM, Collingridge GL, Molnar E (Transient synaptic activation of NMDA receptors leads to the insertion of native AMPA 179  receptors at hippocampal neuronal plasma membranes. Neuropharmacology 41:700- 713.2001). Prange O, Wong TP, Gerrow K, Wang YT, El-Husseini A (A balance between excitatory and inhibitory synapses is controlled by PSD-95 and neuroligin. Proc Natl Acad Sci U S A 101:13915-13920.2004). Prybylowski K, Chang K, Sans N, Kan L, Vicini S, Wenthold RJ (The synaptic localization of NR2B-containing NMDA receptors is controlled by interactions with PDZ proteins and AP-2. Neuron 47:845-857.2005). Prybylowski K, Wenthold RJ (N-Methyl-D-aspartate receptors: subunit assembly and trafficking to the synapse. J Biol Chem 279:9673-9676.2004). Prybylowski KL, Grossman SD, Wrathall JR, Wolfe BB (Expression of splice variants of the NR1 subunit of the N-methyl-D-aspartate receptor in the normal and injured rat spinal cord. Journal of neurochemistry 76:797-805.2001). Rabacchi S, Bailly Y, Delhaye-Bouchaud N, Mariani J (Involvement of the N-methyl D- aspartate (NMDA) receptor in synapse elimination during cerebellar development. Science 256:1823-1825.1992). Racca C, Stephenson FA, Streit P, Roberts JD, Somogyi P (NMDA receptor content of synapses in stratum radiatum of the hippocampal CA1 area. J Neurosci 20:2512-2522.2000). Redila VA, Christie BR (Exercise-induced changes in dendritic structure and complexity in the adult hippocampal dentate gyrus. Neuroscience 137:1299-1307.2006). Redila VA, Olson AK, Swann SE, Mohades G, Webber AJ, Weinberg J, Christie BR (Hippocampal cell proliferation is reduced following prenatal ethanol exposure but can be rescued with voluntary exercise. Hippocampus.2006). Regalado MP, Villarroel A, Lerma J (Intersubunit cooperativity in the NMDA receptor. Neuron 32:1085-1096.2001). Ren H, Honse Y, Karp BJ, Lipsky RH, Peoples RW (A site in the fourth membrane-associated domain of the N-methyl-D-aspartate receptor regulates desensitization and ion channel gating. J Biol Chem 278:276-283.2003). Richter-Levin G, Canevari L, Bliss TV (Long-term potentiation and glutamate release in the dentate gyrus: links to spatial learning. Behav Brain Res 66:37-40.1995). Roberts L, Greene JR (Post-weaning social isolation of rats leads to a diminution of LTP in the CA1 to subiculum pathway. Brain Res 991:271-273.2003). Roche KW, Standley S, McCallum J, Dune Ly C, Ehlers MD, Wenthold RJ (Molecular determinants of NMDA receptor internalization. Nat Neurosci 4:794-802.2001). Rose GM, Dunwiddie TV (Induction of hippocampal long-term potentiation using physiologically patterned stimulation. Neurosci Lett 69:244-248.1986). Rosenmund C, Feltz A, Westbrook GL (Calcium-dependent inactivation of synaptic NMDA receptors in hippocampal neurons. J Neurophysiol 73:427-430.1995a). Rosenmund C, Feltz A, Westbrook GL (Synaptic NMDA receptor channels have a low open probability. J Neurosci 15:2788-2795.1995b). Rosenmund C, Westbrook GL (Rundown of N-methyl-D-aspartate channels during whole-cell recording in rat hippocampal neurons: role of Ca2+ and ATP [published erratum appears in J Physiol (Lond) 1994 Mar 15;475(3):547-8]. J Physiol (Lond) 470:705-729.1993). 180  Rotenberg A, Mayford M, Hawkins RD, Kandel ER, Muller RU (Mice expressing activated CaMKII lack low frequency LTP and do not form stable place cells in the CA1 region of the hippocampus. Cell 87:1351-1361.1996). Sacchetti B, Lorenzini CA, Baldi E, Bucherelli C, Roberto M, Tassoni G, Brunelli M (Long- lasting hippocampal potentiation and contextual memory consolidation. Eur J Neurosci 13:2291-2298.2001). Sah P, Hestrin S, Nicoll RA (Tonic activation of NMDA receptors by ambient glutamate enhances excitability of neurons. Science 246:815-818.1989). Sakimura K, Kutsuwada T, Ito I, Manabe T, Takayama C, Kushiya E, Yagi T, Aizawa S, Inoue Y, Sugiyama H, et al. (Reduced hippocampal LTP and spatial learning in mice lacking NMDA receptor epsilon 1 subunit. Nature 373:151-155.1995). Sans N, Petralia RS, Wang YX, Blahos J, 2nd, Hell JW, Wenthold RJ (A developmental change in NMDA receptor-associated proteins at hippocampal synapses. J Neurosci 20:1260- 1271.2000). Sassoe-Pognetto M, Ottersen OP (Organization of ionotropic glutamate receptors at dendrodendritic synapses in the rat olfactory bulb. J Neurosci 20:2192-2201.2000). Sather W, Dieudonne S, MacDonald JF, Ascher P (Activation and desensitization of N-methyl- D-aspartate receptors in nucleated outside-out patches from mouse neurones. J Physiol 450:643-672.1992a). Sather W, Dieudonne S, MacDonald JF, Ascher P (Activation and desensitization of N-methyl- D-aspartate receptors in nucleated outside-out patches from mouse neurones. J Physiol 450:643-672.1992b). Schell MJ, Molliver ME, Snyder SH (D-serine, an endogenous synaptic modulator: localization to astrocytes and glutamate-stimulated release. Proc Natl Acad Sci U S A 92:3948- 3952.1995). Schmidt-Hieber C, Jonas P, Bischofberger J (Enhanced synaptic plasticity in newly generated granule cells of the adult hippocampus. Nature 429:184-187.2004). Schneggenburger R (Simultaneous measurement of Ca2+ influx and reversal potentials in recombinant N-methyl-D-aspartate receptor channels. Biophys J 70:2165-2174.1996). Schneggenburger R, Zhou Z, Konnerth A, Neher E (Fractional contribution of calcium to the cation current through glutamate receptor channels. Neuron 11:133-143.1993). Schnell E, Sizemore M, Karimzadegan S, Chen L, Bredt DS, Nicoll RA (Direct interactions between PSD-95 and stargazin control synaptic AMPA receptor number. Proc Natl Acad Sci U S A 99:13902-13907.2002). Schulz PE, Cook EP, Johnston D (Changes in paired-pulse facilitation suggest presynaptic involvement in long-term potentiation. J Neurosci 14:5325-5337.1994). Scimemi A, Fine A, Kullmann DM, Rusakov DA (NR2B-containing receptors mediate cross talk among hippocampal synapses. J Neurosci 24:4767-4777.2004). Sessoms-Sikes S, Honse Y, Lovinger DM, Colbran RJ (CaMKIIalpha enhances the desensitization of NR2B-containing NMDA receptors by an autophosphorylation- dependent mechanism. Mol Cell Neurosci 29:139-147.2005). Sheng M, Pak DT (Ligand-gated ion channel interactions with cytoskeletal and signaling proteins. Annu Rev Physiol 62:755-778.2000). Sheng S (The promise and challenge toward the clinical application of maspin in cancer. Front Biosci 9:2733-2745.2004). 181  Shi SH, Hayashi Y, Petralia RS, Zaman SH, Wenthold RJ, Svoboda K, Malinow R (Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation. Science 284:1811-1816.1999). Shors TJ, Miesegaes G, Beylin A, Zhao M, Rydel T, Gould E (Neurogenesis in the adult is involved in the formation of trace memories. Nature 410:372-376.2001). Single FN, Rozov A, Burnashev N, Zimmermann F, Hanley DF, Forrest D, Curran T, Jensen V, Hvalby O, Sprengel R, Seeburg PH (Dysfunctions in mice by NMDA receptor point mutations NR1(N598Q) and NR1(N598R). J Neurosci 20:2558-2566.2000). Snyder EM, Nong Y, Almeida CG, Paul S, Moran T, Choi EY, Nairn AC, Salter MW, Lombroso PJ, Gouras GK, Greengard P (Regulation of NMDA receptor trafficking by amyloid-beta. Nat Neurosci 8:1051-1058.2005). Song HJ, Stevens CF, Gage FH (Neural stem cells from adult hippocampus develop essential properties of functional CNS neurons. Nat Neurosci 5:438-445.2002). Spargo E, Everall IP, Lantos PL (Neuronal loss in the hippocampus in Huntington's disease: a comparison with HIV infection. J Neurol Neurosurg Psychiatry 56:487-491.1993). Spencer JP, Murphy KP (Bi-directional changes in synaptic plasticity induced at corticostriatal synapses in vitro. Exp Brain Res 135:497-503.2000). Sprengel R, Suchanek B, Amico C, Brusa R, Burnashev N, Rozov A, Hvalby O, Jensen V, Paulsen O, Andersen P, Kim JJ, Thompson RF, Sun W, Webster LC, Grant SG, Eilers J, Konnerth A, Li J, McNamara JO, Seeburg PH (Importance of the intracellular domain of NR2 subunits for NMDA receptor function in vivo. Cell 92:279-289.1998). Standaert DG, Landwehrmeyer GB, Kerner JA, Penney JB, Jr., Young AB (Expression of NMDAR2D glutamate receptor subunit mRNA in neurochemically identified interneurons in the rat neostriatum, neocortex and hippocampus. Brain Res Mol Brain Res 42:89-102.1996). Stanton PK, Heinemann U, Muller W (FM1-43 imaging reveals cGMP-dependent long-term depression of presynaptic transmitter release. J Neurosci 21:RC167.2001). Steigerwald F, Schulz TW, Schenker LT, Kennedy MB, Seeburg PH, Kohr G (C-Terminal truncation of NR2A subunits impairs synaptic but not extrasynaptic localization of NMDA receptors. J Neurosci 20:4573-4581.2000). Stevens CF, Wang Y (Changes in reliability of synaptic function as a mechanism for plasticity. Nature 371:704-707.1994). Stocca G, Vicini S (Increased contribution of NR2A subunit to synaptic NMDA receptors in developing rat cortical neurons. J Physiol 507:13-24.1998). Strack S, Colbran RJ (Autophosphorylation-dependent targeting of calcium/ calmodulin- dependent protein kinase II by the NR2B subunit of the N-methyl- D-aspartate receptor. J Biol Chem 273:20689-20692.1998). Stringer JL, Greenfield LJ, Hackett JT, Guyenet PG (Blockade of long-term potentiation by phencyclidine and sigma opiates in the hippocampus in vivo and in vitro. Brain Res 280:127-138.1983). Stuart GJ, Sakmann B (Active propagation of somatic action potentials into neocortical pyramidal cell dendrites. Nature 367:69-72.1994). Szerb JC (Changes in the relative amounts of aspartate and glutamate released and retained in hippocampal slices during stimulation. J Neurochem 50:219-224.1988). 182  Takumi Y, Bergersen L, Landsend AS, Rinvik E, Ottersen OP (Synaptic arrangement of glutamate receptors. Prog Brain Res 116:105-121.1998). Takumi Y, Ramirez-Leon V, Laake P, Rinvik E, Ottersen OP (Different modes of expression of AMPA and NMDA receptors in hippocampal synapses. Nat Neurosci 2:618-624.1999). Thomas CG, Miller AJ, Westbrook GL (Synaptic and extrasynaptic NMDA receptor NR2 subunits in cultured hippocampal neurons. J Neurophysiol 95:1727-1734.2006). Tingley WG, Ehlers MD, Kameyama K, Doherty C, Ptak JB, Riley CT, Huganir RL (Characterization of protein kinase A and protein kinase C phosphorylation of the N- methyl-D-aspartate receptor NR1 subunit using phosphorylation site-specific antibodies. J Biol Chem 272:5157-5166.1997). Tonegawa S, Tsien JZ, McHugh TJ, Huerta P, Blum KI, Wilson MA (Hippocampal CA1-region- restricted knockout of NMDAR1 gene disrupts synaptic plasticity, place fields, and spatial learning. Cold Spring Harb Symp Quant Biol 61:225-238.1996). Tong G, Jahr CE (Regulation of glycine-insensitive desensitization of the NMDA receptor in outside-out patches. J Neurophysiol 72:754-761.1994). Tong G, Shepherd D, Jahr CE (Synaptic desensitization of NMDA receptors by calcineurin. Science 267:1510-1512.1995). Tovar KR, Westbrook GL (The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro. J Neurosci 19:4180-4188.1999). Townsend M, Yoshii A, Mishina M, Constantine-Paton M (Developmental loss of miniature N- methyl-D-aspartate receptor currents in NR2A knockout mice. Proc Natl Acad Sci U S A 100:1340-1345.2003). Traven HG, Brodin L, Lansner A, Ekeberg O, Wallen P, Grillner S (Computer simulations of NMDA and non-NMDA receptor-mediated synaptic drive: sensory and supraspinal modulation of neurons and small networks. J Neurophysiol 70:695-709.1993). Turrigiano GG, Nelson SB (Homeostatic plasticity in the developing nervous system. Nat Rev Neurosci 5:97-107.2004). Usdin MT, Shelbourne PF, Myers RM, Madison DV (Impaired synaptic plasticity in mice carrying the Huntington's disease mutation. Hum Mol Genet 8:839-846.1999). van Praag H, Christie BR, Sejnowski TJ, Gage FH (Running enhances neurogenesis, learning, and long-term potentiation in mice. Proc Natl Acad Sci U S A 96:13427-13431.1999). van Praag H, Schinder AF, Christie BR, Toni N, Palmer TD, Gage FH (Functional neurogenesis in the adult hippocampus. Nature 415:1030-1034.2002). Vicini S, Wang JF, Li JH, Zhu WJ, Wang YH, Luo JH, Wolfe BB, Grayson DR (Functional and pharmacological differences between recombinant N-methyl-D-aspartate receptors. J Neurophysiol 79:555-566.1998). Villarroel A, Regalado MP, Lerma J (Desensitization of NMDA receptors as a mechanism of neuroprotection. Methods Find Exp Clin Pharmacol 19 Suppl A:51-53.1997). Villarroel A, Regalado MP, Lerma J (Glycine-independent NMDA receptor desensitization: localization of structural determinants. Neuron 20:329-339.1998). Vyklicky L, Jr., Benveniste M, Mayer ML (Modulation of N-methyl-D-aspartic acid receptor desensitization by glycine in mouse cultured hippocampal neurones. J Physiol 428:313- 331.1990). Wagey R, Hu J, Pelech SL, Raymond LA, Krieger C (Modulation of NMDA-mediated excitotoxicity by protein kinase C. J Neurochem 78:715-726.2001). 183  Watanabe M, Inoue Y, Sakimura K, Mishina M (Developmental changes in distribution of NMDA receptor channel subunit mRNAs. Neuroreport 3:1138-1140.1992). Weitlauf C, Honse Y, Auberson YP, Mishina M, Lovinger DM, Winder DG (Activation of NR2A-Containing NMDA Receptors Is Not Obligatory for NMDA Receptor-Dependent Long-Term Potentiation. Journal of Neuroscience 25:8386-8390.2005). Wenzel A, Fritschy JM, Mohler H, Benke D (NMDA receptor heterogeneity during postnatal development of the rat brain: differential expression of the NR2A, NR2B, and NR2C subunit proteins. J Neurochem 68:469-478.1997). Wikstrom MA, Matthews P, Roberts D, Collingridge GL, Bortolotto ZA (Parallel kinase cascades are involved in the induction of LTP at hippocampal CA1 synapses. Neuropharmacology 45:828-836.2003). Winblad B, Poritis N (Memantine in severe dementia: results of the 9M-Best Study (Benefit and efficacy in severely demented patients during treatment with memantine). Int J Geriatr Psychiatry 14:135-146.1999). Wong RK, Prince DA (Afterpotential generation in hippocampal pyramidal cells. J Neurophysiol 45:86-97.1981). Xiong W, Wei H, Xiang X, Cao J, Dong Z, Wang Y, Xu T, Xu L (The effect of acute stress on LTP and LTD induction in the hippocampal CA1 region of anesthetized rats at three different ages. Brain Res 1005:187-192.2004). Xiong ZG, Raouf R, Lu WY, Wang LY, Orser BA, Dudek EM, Browning MD, MacDonald JF (Regulation of N-methyl-D-aspartate receptor function by constitutively active protein kinase C. Mol Pharmacol 54:1055-1063.1998). Xu L, Anwyl R, Rowan MJ (Behavioural stress facilitates the induction of long-term depression in the hippocampus. Nature 387:497-500.1997). Yamada Y, Iwamoto T, Watanabe Y, Sobue K, Inui M (PSD-95 eliminates Src-induced potentiation of NR1/NR2A-subtype NMDA receptor channels and reduces high-affinity zinc inhibition. J Neurochem 81:758-764.2002). Yang CH, Huang CC, Hsu KS (Behavioral stress modifies hippocampal synaptic plasticity through corticosterone-induced sustained extracellular signal-regulated kinase/mitogen- activated protein kinase activation. J Neurosci 24:11029-11034.2004). Yang CH, Huang CC, Hsu KS (Behavioral stress enhances hippocampal CA1 long-term depression through the blockade of the glutamate uptake. J Neurosci 25:4288- 4293.2005). Yang J, Han H, Cao J, Li L, Xu L (Prenatal stress modifies hippocampal synaptic plasticity and spatial learning in young rat offspring. Hippocampus 16:431-436.2006). Yang J, Hou C, Ma N, Liu J, Zhang Y, Zhou J, Xu L, Li L (Enriched environment treatment restores impaired hippocampal synaptic plasticity and cognitive deficits induced by prenatal chronic stress. Neurobiol Learn Mem 87:257-263.2007). Yasuda H, Barth AL, Stellwagen D, Malenka RC (A developmental switch in the signaling cascades for LTP induction. Nat Neurosci 6:15-16.2003). Yoshii A, Sheng MH, Constantine-Paton M (Eye opening induces a rapid dendritic localization of PSD-95 in central visual neurons. Proc Natl Acad Sci U S A 100:1334-1339.2003). Zhang S, Ehlers MD, Bernhardt JP, Su CT, Huganir RL (Calmodulin mediates calcium- dependent inactivation of N-methyl-D- aspartate receptors. Neuron 21:443-453.1998). 184  Zheng F, Erreger K, Low CM, Banke T, Lee CJ, Conn PJ, Traynelis SF (Allosteric interaction between the amino terminal domain and the ligand binding domain of NR2A. Nat Neurosci 4:894-901.2001). Zheng F, Gingrich MB, Traynelis SF, Conn PJ (Tyrosine kinase potentiates NMDA receptor currents by reducing tonic zinc inhibition. Nature neuroscience 1:185-191.1998). Zorumski CF, Thio LL, Clark GD, Clifford DB (Blockade of desensitization augments quisqualate excitotoxicity in hippocampal neurons. Neuron 5:61-66.1990).               185  APPENDIX Intracellular in fusion of NR2B-9C p eptide i ncreases N MDAR d esensitization by unc oupling P SD-95 f rom NMDA re ceptors ex pressed i n mature h ippocampal neurons An interference peptide, NR2B-9C, which disrupts the binding of NMDARs and PSD-95, has been used as another means to test our hypothesis, that a direct interaction of the two is sufficient to regulate desensitization. NR2B-9C, a small peptide resembling the sequence of nine amino acids that form the distal C-terminal region of the NR2B subunits (Lys-Leu-Ser-Ser-Ile-Glu-Ser-Asp-Val), has been used before for the purpose of preventing the receptor to bind PSD-95 (see introduction). In cultured rat hippocampal neurons incubated with Tat-NR2B9c versus a control (Tat-NR2BAA) peptide for 1h before harvesting lysates for immunoprecipiation and western blot analysis, results indicated differential peptide interference with PSD-95 binding to NR2B compared to NR2A (Zhang L and Raymond LA, unpublished data). At low concentrations (200nM), Tat-NR2B9c reduced co-immunoprecipitation of PSD-95 with NR2B by ~50% (compared to treatment with control peptide) without affecting association of PSD-95 with NR2A; higher concentrations (1μM) reduced PSD-95 / NR2A co-IP by ~50% as well (data not shown). For my electrophysiological experiments, a small amount of the stock peptide has been dissolved in the intracellular (recording pipette) solution to a final concentration of 1μM and then allowed to diffuse into the cell through the patch pipette, after the whole-cell configuration has been attained. 186  Currents mediated by NMDARs were elicited and recorded at least 10 minutes after the breaking into the cell, to allow diffusion of the peptide in cellular compartments. Steady-state-to peak ratio was diminished in neurons treated with the NR2B-9C peptide, to a value significantly different from that recorded in control neurons (0.46 ± 0.01, n=4, in treated neurons versus 0.59 ± 0.04, n=4 in control neurons; P<0.05 by unpaired t-test).      Co ntr ol NR 2B -9C 0.0 0.2 0.4 0.6 0.8 * Is s/ Ip A. B. 187  Appendix Figure The effect of NR2B-9C peptide on NMDAR-mediated current desensitization. A. Representative traces of NMDAR currents in mature hippocampal neurons, in control conditions or treated with NR2B-9c (1μM).  Current amplitudes were normalized for comparison of desensitization. B. Treatment with NR2B-9c (1μM) increased the extent of NMDA-evoked current desensitization. Control, n=4; NR2B-9c, n=4, *P<0.05, student t-teast, whereas a lower concentration (200nM) of the NR2B-9c had no significant effect on Iss/Ip (Control, n=14; NR2B9c, n=14). 

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