UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Neural stem cell transplantation : neuroprotection and LTP-induced facilitation of neurogenesis Cho, Taesup 2011

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata


24-ubc_2011_fall_cho_taesup.pdf [ 2.88MB ]
JSON: 24-1.0072116.json
JSON-LD: 24-1.0072116-ld.json
RDF/XML (Pretty): 24-1.0072116-rdf.xml
RDF/JSON: 24-1.0072116-rdf.json
Turtle: 24-1.0072116-turtle.txt
N-Triples: 24-1.0072116-rdf-ntriples.txt
Original Record: 24-1.0072116-source.json
Full Text

Full Text

 Neural stem cell transplantation: Neuroprotection and LTP-induced facilitation of neurogenesis  by  Taesup Cho  B.Sc., Ajou University, 2000 M.Sc., Ajou University, 2002  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  The Faculty of Graduate Studies (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August, 2011  © Taesup Cho, 2011 ii Abstract  Transplantation of neural progenitor cells (NPC) constitutes a putative therapeutic maneuver for use in treatment of neurodegenerative diseases. At present, effects of NPC transplantation in the Alzheimer’s disease (AD) brain are largely unknown and a primary objective of this work is to demonstrate possible efficacy of NPC administration in an AD animal model. The benefits of transplantation could involve a spectrum of effects including replacement of endogenous neurons, conferring neuroprotection with enhancement of neurotrophic factors, and diminishing levels of neurotoxic agents. Additionally, since chronic inflammation is a characteristic property of the AD brain, I considered NPC transplantation could have a particular utility in inhibiting ongoing inflammatory reactivity. Accordingly, intra-hippocampal transplantation of NPC has been examined for efficacy in attenuating inflammatory responses and conferring neuroprotection in the hippocampus. These findings indicate efficacy for NPC transplantation with effects consistent with cellular actions to attenuate inflammatory reactivity. Synaptic plasticity, such as long-term potentiation (LTP), is thought to play a critical role in modification of neuronal circuitry in learning and memory, but the role in neurogenesis is not well known. A critical aspect of my study was to examine potential roles of N-methyl-D-aspartate receptor (NMDAR)-dependent LTP in promoting neurogenesis by facilitating proliferation/survival and neuronal differentiation of endogenous NPCs in the dentate gyrus (DG) and exogenously transplanted neural stem cells (NSCs) in the CA1. I found that LTP induction significantly facilitates proliferation/survival and neuronal differentiation of endogenous NPCs and exogenously transplanted NSCs in the hippocampus. These effects were eliminated by a NMDAR competitive antagonist, CPP. Accordingly, chemical LTP stimulation reproduced enhanced proliferation/survival and neuronal differentiation of NSCs when co-cultured with hippocampal neurons. These effects were eliminated by a NMDAR competitive antagonist, D- APV and inhibited by the tyrosine kinase inhibitor, K252a. ELISA and biotinylation results iii revealed that NMDAR-mediated LTP facilitates the release of a neurotrophic factor, BDNF. The conditioned media from cLTP-induced hippocampal neurons were sufficient to activate the BDNF receptor, TrkB. Overall, my results suggest that NMDAR-dependent LTP plays a critical role in neurogenesis and may contribute to the utility of NSC transplantation as an effective cell therapy for a variety of neurodegenerative diseases.                     iv Preface  This thesis work has been a collaborative effort between me, my supervisor Dr. Yu Tian Wang, and our collaborators Dr. James G. McLarnon, and his previous doctoral student, Dr. Jae K. Ryu. The experiments shown in chapter 2 were designed by Jae K. Ryu, me, Yu Tian Wang, and Dr. James G. McLarnon. Dr. Jae K. Ryu; I carried out all experiments and data analysis. Dr. James G. McLarnon and Dr. Yu Tian Wang drafted and finalized the manuscript. All of the authors have read and approved the final manuscript. The experiments shown in chapter 3 were designed by Dr. Yu Tian Wang and me. I performed all of the experiments. The preparation of many of the materials and techniques was a collaborative effort. The image quantification and statistical analysis shown in Figure 3-5C, and 3-5D as well as NSC transplantation were done by me and Dr. Jae K. Ryu. The biotinylation data shown in Figure 3-5B were done by me and Dr. Changiz Taghibiglou. The ELISA results shown in Figure 3-7A were done by me and Dr. Yuan Ge. All of electrophysiological data shown in chapter 3 were performed by me, Dr. Lidong Liu, Mr. Evans Gary and Dr. Allen W. Chan. All of the viral vectors used in chapter 3 were cloned and produced by myself, Dr. Yuping Li, and Dr. Jie Lu. The retrovirus was generously provided by Dr. Fabio Rossi. All of cultured neurons and NSCs used in this work were prepared by me and Dr. Yuping Li. Some of this thesis work has been published and prepared as manuscripts for publication listed below:  Ryu JK*, Cho T*, Wang YT, and McLarnon JG (2009) Neural progenitor cells attenuate inflammatory reactivity and neuronal loss in an animal model of inflamed AD brain. Journal of Neuroinflammation 6:39. (*co-first authors) Cho T, Ryu JK, Taghibiglou C, Ge Y, Chan AW, Lu J, Liu L, Gary E, McLarnon JG, and Wang YT. v NMDAR-dependent LTP promotes proliferation and neuronal differentiation of NSCs. Submitted. All animal protocols were approved by the UBC Animal Care Ethics Committee while adhering to the guidelines of the Canadian Council on Animal Care. All research or course work involving biological hazards is conducted in accordance with the University of British Columbia Policies and Procedures, Biosafety Practices and Public Health Agency of Canada guidelines.  Animal care certificate number: A08-0807, A08-0207, A06-0356, A05-0206, A03-0313  Biohazard approval certificate number B50080                 vi Table of Contents  Abstract .................................................................................................................................... ii Preface ..................................................................................................................................... iv Table of Contents .................................................................................................................... vi List of Tables ........................................................................................................................... ix List of Figures .......................................................................................................................... x List of Abbreviations .............................................................................................................. xi Acknowledgements .............................................................................................................. xvi Dedication ............................................................................................................................ xvii 1 Introduction ........................................................................................................................... 1 1.1 Neural stem cell and adult neurogenesis ..................................................................... 2  1.1.1 Neuronal transcription factors .................................................................................... 5 1.1.2 Neural stem/progenitor cell markers ........................................................................... 6 1.2 A role of GABA receptors during development ......................................................... 10  1.3 Neural stem cell transplantation in neurodegenerative diseases ............................. 12  1.4 Alzheimer’s disease and microglia ............................................................................. 14  1.4.1 Microglia .................................................................................................................. 15 1.5 Long-term potentiation ................................................................................................ 16  1.5.1 NMDA receptors ...................................................................................................... 18 1.5.2 AMPA receptors ....................................................................................................... 22 1.5.3 Long-term potentiation in the CA3 region ................................................................. 30 1.5.4 Chemical long-term potentiation ............................................................................... 32 1.6 Long-term potentiation and neurogenesis ................................................................. 33  1.7 Brain-derived neurotrophic factor ............................................................................... 36  1.7.1 Brain-derived neurotrophic factor and neurogenesis ................................................ 37 1.7.2 Brain-derived neurotrophic factor and long-term potentiation ................................... 38 1.8 Research hypothesis ................................................................................................... 41  1.9 Summary of research objectives ................................................................................. 42 vii 2 Neural progenitor cells attenuate inflammatory reactivity and neuronal loss in an animal model of inflamed AD brain ...................................................................................... 43 2.1 Introduction .................................................................................................................. 43 2.2 Methods ........................................................................................................................ 45  2.2.1 Neurosphere cultures ............................................................................................... 45 2.2.2 Immunostaining of neurospheres ............................................................................. 45 2.2.3 Stereotaxic injection of fibrillar Aβ1-42 ....................................................................... 46 2.2.4 Transplantation of GFP labeled neural progenitor cells ............................................ 46 2.2.5 Immunohistochemical analysis ................................................................................. 47 2.2.6 Cell-associated immunostaining ............................................................................... 48 2.2.7 Statistical analysis .................................................................................................... 48 2.3 Results .......................................................................................................................... 49  2.3.1 Patterns of distribution and differentiation of transplanted NPC, in vivo .................... 49 2.3.2 Effect of NPC on Aβ1-42-induced inflammatory reactivity .......................................... 50 2.3.3 Effect of neural progenitors on Aβ1-42 -induced neuronal injury ................................ 51 2.4 Discussion .................................................................................................................... 53  3 NMDAR-dependent long-term potentiation promotes proliferation/survival and neuronal differentiation of neural progenitor/stem cells ..................................................... 61 3.1 Introduction .................................................................................................................. 61  3.2 Materials and methods ................................................................................................. 63  3.2.1 Primary cell culture and neural stem cell isolation .................................................... 63 3.2.2 Lentivirus production and infection in vitro ................................................................ 63 3.2.3 Retrovirus preparation and intra-hippocampal injection into the dentate gyrus ......... 64 3.2.4 In vivo field EPSP recordings ................................................................................... 65 3.2.5 Neural stem cell transplantation in the CA1 .............................................................. 66 3.2.6 Chemical LTP induction and conditioned medium stimulation .................................. 66 3.2.7 ELISA assay ............................................................................................................ 67 3.2.8 Antibodies ................................................................................................................ 68 3.2.9 Western blotting ....................................................................................................... 68 3.2.10 Biotinylation of cell surface proteins ...................................................................... 69 3.2.11 Immunocytochemistry and BrdU labeling .............................................................. 70 3.2.12 Immunohistochemistry .......................................................................................... 70 viii 3.2.13 Image quantification and statistical analysis .......................................................... 71 3.3 Results .......................................................................................................................... 73  3.3.1 Induction of LTP increases proliferation/survival of endogenous NPCs ini the subgranular zone of the DG region ....................................................................................... 73 3.3.2 LTP promotes neuronal differentiation of endogenous NPCs in the DG region ........ 74 3.3.3 LTP enhanced neurogenesis of exogenously transplanted NSCs into the hippocampal CA1 region ....................................................................................................... 75 3.3.4 LTP increases neurogenesis of NSCs in cultures in vitro ......................................... 76 3.3.5 BDNF plays a critical role in mediating LTP promoted neurogenesis of NSCs in NSC- neuron co-cultures ................................................................................................................ 79 3.4 Discussion .................................................................................................................... 81  4 Conclusion ..................................................................................................................... 101 References ........................................................................................................................... 106 Appendix A: Publications .................................................................................................... 148                 ix List of Tables  Table 1-1 Representative cell-type specific markers in the brain ......................................... 8                        x List of Figures  Figure 2-1 GFP-labeled NPC in vitro and diffusion of GFP-positive neural progenitor in vivo ................................................................................................................................... 57 Figure 2-2 Expression of markers in injected NPC .............................................................. 58 Figure 2-3 Effects of NPC transplantation on inflammatory reactivity ............................... 59 Figure 2-4 Effects of NPC transplantation on neuronal viability ........................................ 60 Figure 3-1 NMDAR-dependent LTP enhances proliferation of NPCs in the DG ................. 87 Figure 3-2 NMDAR-dependent LTP enhances neuronal differentiation and maturation of NPCs in the DG ................................................................................................................ 89 Figure 3-3 induction of LTP increases neuronal differentiation of exogenous NSCs transplanted into the CA1 region ................................................................................... 91 Figure 3-4 Isolation and characterization of NSCs in cultures ........................................... 93 Figure 3-5 Chemical LTP enhances proliferation/survival and neuronal differentiation in NSC-neuron co-cultures ................................................................................................. 95 Figure 3-6 Conditioned media from cLTP treated neuronal cultures increases neurogenesis of NSCs .................................................................................................... 97 Figure 3-7 LTP promotes neurogenesis of NSCs in cultures at least in part through BDNF- TrkB system ..................................................................................................................... 99       xi List of Abbreviations  ABP: AMPA-binding protein AEBSF: 4-(2-aminoethyl)-benzenesulfonyl-fluoride hydrochloride AD: Alzheimer’s disease AIP: autocamtide-2-related inhibitory peptide AKAP: A-kinase anchor protein ALS: Amyotrophic lateral sclerosis AMPA: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid AMPAR: AMPA receptor AP: anterior posterior APP: amyloid precursor proteins Aβ: amyloid beta BAPTA: 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid BBB: blood-brain barrier BDNF: brain-derived neurotrophic factor β-LRP1: β-low density lipoprotein receptor-related protein1 bFGF: basic fibroblast growth factor bHLH: basic helix-loop-helix BrdU: 5-bromo-2’-deoxyuridine BSA: bovine serum albumin C/EBP: CAAT enhancer-binding protein pathway CA: Cornu Ammonis CaMK: calcium/calmodulin-dependent kinase cAMP: cyclic adenosine monophosphate cLTP: chemical LTP xii CNS: central nervous system CPP: 3-[(±)-2-carboxypiperazin-4-yl]-propyl-1-phosphate CREB: cyclic AMP-response element binding protein DAPI: 4',6-diamidino-2-phenylindole dihydrochloride D-APV: 2-amino-5-phosphonopentanoic acid DBS: deep brain stimulation DCX: doublecortin DG: dentate gyrus DV: dorsoventral ECS: embryonic stem cell EDTA: ethylenediamine tetraacetate EGF: epidermal growth factor EGTA: ethylene glycol tetraacetic acid ELISA: Enzyme-linked immunosorbent assay EPSC: excitatory postsynaptic current EPSP: excitatory postsynaptic potential ER: endoplasmic reticulum ERK: extracellular signal-regulated kinase fEPSP: field excitatory postsynaptic potentials GABA: γ-aminobutyric acid GABAR: GABA receptor GCL: granule cell layer GFAP: glial fibrillary acidic protein GFP: green fluorescent protein GRIP: glutamate receptor interacting protein HBSS: Hank’s balanced salt solution HD: Huntington’s disease xiii HEK: human embryonic kidney HFS: high frequency stimulation HRP: horseradish peroxidase IF: immunefluorescence IL: interleukin KCC2: K+-Cl- co-transporter 2 LFS: low frequency stimulation LIF: leukemia inhibitory growth factor LTD: long-term depression LTP: long-term potentiation MAP2: microtubule associated protein 2 Mash1: achaete scute homologue ash1 MCAO: middle cerebral artery occlusion MCP-1: monocyte chemoattractant protein-1 MEK: MAP kinase kinase mEPSP: miniature EPSP mEPSC: miniature EPSC mGluR: metabotropic glutamate receptor MF: mossy fiber MK-801: (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10-imine maleate ML: medial lateral ML: molecular layer MCP-1: monocyte chemoattractant protein-1 MPP: medial perforant path NeuN: neuronal nuclei NGF: nerve growth factor Ngn: neurogenin xiv NKCC1: K+-2Cl- co-transporter 1 NMDA: N-methyl-D-aspartate NMDAR: NMDA receptor NPC: neural progenitor cell NSC: Neural stem cell NSF: N-ethylmaleimide-sensitive factor NT-3, -4, -5: neurotrophin-3, neurotrophin-4, neurotrophin-5 OB: olfactory bulb PBS: phosphate buffered saline PCNA: proliferating cell nuclear antigen PD: Parkinson's disease PFA: paraformaldehyde PI3K: phosphatidylinositol 3-kinases PICK1: protein interacting with C-kinase 1 PKA: protein kinase A PKC: protein kinase C PLC: phospholipase C PPDA: (2R*,3S*)-1-(phenanthrenyl-2-carbonyl)piperazine-2,3-dicarboxylic acid PPF: paired pulse facilitation PPD: paired pulse facilitaton PSD-95: postsynpaticdensity protein rasGAP: ras GTPase activating protein ras-MAP: ras-mitogen-activated protein RMS: rostra-migratory stream ROS: reactive oxygen species RSK: ribosomal S6 kinase SAP: synapse-associated protein xv SD: Sprague Dawley SDS: sodium dodecyl sulfate SGZ: subgranular zone SNAP: soluble NSF attachment protein SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor STP: short-term potentiation sTPS: strong theta-patterned simulation SVG: subventricular zone TBS: theta-burst stimulation TBST: tris-buffered saline with Tween-20 TeTx: tetanus toxin TM: transmembrane domain TNF-α: tumor necrosis factor-α Trk: tropomysosin receptor kinase VEGF: vascular endothelial growth factor WB: western blotting            xvi Acknowledgements  I would like to thank foremost Dr. Yu Tian Wang for giving me the opportunity to conduct my Ph.D. thesis research in his laboratory and for his supervision throughout the duration of my graduate work. I am grateful to my committee members Dr. James G. McLarnon and Dr. Kurt Haas for their assistance and invaluable insights into this work. A special thanks to the Heart and Stroke foundation of Canada for financial support through a Doctoral Research Award (2005-2008).    I would like to acknowledge my gratitude to the following: Dr. Seung U. Kim, Dr. Churl K. Min, Dr. Byung-Soon Moon, Dr. Tae Woo Kim, Dr. Hyun B. Choi, Dr. Jae K. Ryu, and Mr. Taesik Chae. Thanks to previous and current labmates: Dr. Changiz Taghibiglou, Dr. Lidong Liu, Dr. Jie Lu, Dr. Allen W. Chan, Dr. Tak Pan Wong, Dr. Yuan Ge, Mr. Evans Gary, Mr. Edmund Lo, Mr. Ted W. Lai, Mr. Joon-Hyuk Imm. Dr. Dongchuan Wu, Dr. Clarrisa A. Bradley, Dr. Yushan Wang, Ms. Shu Zhang and Ms. Agnes Kwok. I would also like to thank to Dr. Yuping Li for her technical assistance and Ms. Kimberly Girling, Mr. Ainsley Coquinco, and Dr. Tom Bartlett for the thesis editing. I would like to thank my parents, my brother, Tae Joon Cho, sister, Joo Young Cho, my uncle, Christopher Lim, aunt, Dr. Deok Hye Lee, and my friends whose have supported me throughout my years as a graduate student.       xvii Dedication  I would like to dedicate this thesis to my mother, father, brother, and sister.                        1 1 Introduction  Stem cell transplantation has been considered as a cell replacement therapy. Neural stem cells (NSC) is an example of multipotent stem cells that has been extensively investigated to replace lost neurons as a result of neurodegenerative diseases, such as Alzheimer’s disease (AD), Huntington’s disease (HD), stroke, and Parkinson’s disease (PD) (Lindvall and Kokaia, 2010; Orlacchio et al., 2010). However, stem cell replacement as a clinical therapy has been largely ineffective (Lindvall and Kokaia, 2010; Orlacchio et al., 2010). The use of embryonic stem cells (ESCs) as a cell source raises ethical issues, and requires additional engineering control to produce disease-specific multipotent cell types, such as dopaminergic neurons for treatment of PD (Kim et al., 2002; Singec et al., 2007). On the other hand, multipotent NSCs are thought to have potential to differentiate into either neurons or glia (Reynolds and Weiss, 1992; Gage, 2000; Conti and Cattaneo, 2010). Therefore, NSC transplantation could be an effective therapy for neurodegenerative disorders in the brain. Within the adult mammalian brain, the subventricular zone (SVZ) of the lateral ventricles and  the subgranular zone (SGZ) in the dentate gyrus (DG) contain multipotent NSCs that self-renew, proliferate and give rise to neurons and glia (Reynolds and Weiss, 1992; Richards et al., 1992; McKay, 1997; Gage, 2000; Ma et al., 2009). Within the DG, two types (radial glia-like stem/ type I stem/progenitor cell and nestin-positive type II progenitors) of NSCs can be identified in the SGZ (Fukuda et al., 2003; Garcia et al., 2004; Suh et al., 2007). Ultimately, nestin-positive NSCs become granule cells having axonal projections to the Cornu Ammonis 3 (CA3). Newly formed neurons are thought to play a role in brain function including learning and memory (Zhao et al., 2006; Kee et al., 2007). The dramatic decline in neurogenesis with age may contribute to progressive loss of learning and memory, and cognitive impairments (Bernal and Peterson, 2004; Enwere et al., 2004; Sharpless and DePinho, 2007), which are generally observed in neurodegenerative diseases, such as AD  (Ashe, 2001; Bizon et al., 2004). Thus, manipulation of endogenous neurogenesis 2 and exogenous transplantation of NSCs may be a promising therapeutic target in the diseased and injured brain (Lindvall and Kokaia, 2010). In particular, both neurogenesis and synaptic plasticity occur in the hippocampal DG. However, their relevance to each other is poorly understood. Hippocampal long-term potentiation (LTP) has been shown to have a role in learning and memory (Bliss and Lomo, 1973; Bliss and Collingridge, 1993). A widely studied form of synaptic plasticity is LTP defined as long-lasting enhancement in synaptic strength induced by high frequency stimulation (HFS) (Bliss and Collingridge, 1993; Kandel, 2001). Different types of LTP have been reported in different neural structures, including the hippocampus, cerebral cortex, amygdala and cerebellum (Fox, 2002; Ji et al., 2003). LTP has been most extensively investigated in the hippocampus. Moreover, LTP can be further divided into N-methyl-D- aspartate receptor (NMDAR)-dependent (Collingridge, 1992; Bliss and Collingridge, 1993; Bailey et al., 2000; Malenka and Bear, 2004) and NMDAR-independent (Harris and Cotman, 1986; Zalutsky and Nicoll, 1990; Salin et al., 1996). In the hippocampus, NMDAR-dependent LTP is the most widely characterized form of synaptic plasticity (Kauer et al., 1988a; Muller et al., 1988; Malenka and Bear, 2004). Thus, NMDAR-dependent LTP is the best candidate for hippocampal neurogenesis.  1.1 Neural stem cell and adult neurogenesis     In the brain, neurons and glia are generated during development by multipotent NSCs. Multipotent stem cells give rise to phenotype committed to the tissues from which they are derived. Since 1928, it was believed that neurons could not be regenerated in the adult brain. However, adult neurogenesis in the cerebral cortex of the adult mammalian brain was first suggested in the 1960s (Altman, 1962) followed by the evidence of adult neurogenesis in the DG (Altman, 1963; Altman and Das, 1965) of the hippocampus and the olfactory bulb (OB) via rostra-migratory stream (RMS) (Altman, 1969). Adult neurogenesis studies have shown that 3 neurogenesis actually continues throughout adult life during which cell replacement occurs (Lois et al., 1996; Curtis et al., 2007). The adult mammalian central nervous system (CNS), including human, contains a population of NSCs of the SVZ of the lateral ventricles and the SGZ, a layer beneath the granule cell layer (GCL) of the hippocampal DG (Reynolds and Weiss, 1992; Richards et al., 1992; McKay, 1997; Eriksson et al., 1998; Ma et al., 2009; Conti and Cattaneo, 2010). Adult neurogenesis in the SVZ and SGZ declines with age, observed as a decrease in the production of newly generated neurons (Bernal and Peterson, 2004). Except in these two areas, neurons are not normally generated in the adult brain. NSCs directly isolated from the adult hippocampal SGZ and SVZ have shown confirmed multipotency (Gage, 2000; Ma et al., 2009; Conti and Cattaneo, 2010). The culture efficiency of NSCs was determined by the ability of individual cells to generate multipotent neurospheres grown in suspension (Reynolds and Weiss, 1992; Gritti et al., 1996; Gritti et al., 1999). These NSCs are capable of self-renewal, can differentiate into either neurons or glia (Reynolds and Weiss, 1992; Craig et al., 1999; Kukekov et al., 1999; Nait-Oumesmar et al., 1999; Garcia et al., 2004; Conti and Cattaneo, 2010), and form functional connections with neighboring neurons in the DG and SVZ (Carlen et al., 2002; van Praag et al., 2002; Belluzzi et al., 2003; Kempermann et al., 2004; Toni et al., 2008). Indeed, several thousand multipotent NSCs are generated daily in the SGZ and SGZ (Cameron and McKay, 2001; Rao and Shetty, 2004). Within a few days following their birth, at least half of the daughter cells undergo apoptosis (Dayer et al., 2003; Sun et al., 2004). Newly generated cells in the SVZ migrate through the RMS to the OB where they differentiate into granule cells, interneurons, and periglomerular neurons (Lois and Alvarez-Buylla, 1993, 1994), but the vast majority of these cells die (Morshead and van der Kooy, 1992). In the DG, of the cells that survive, most of newly generated cells are fully mature granule neurons at 4 weeks and survive for several months (Cameron and McKay, 2001). Additionally, newly generated cells migrate into the GCL, differentiate into granule neurons, and form functional integration with host neurons (Cameron et al., 1993; van Praag et al., 2002). Fully differentiated granule neurons generally express neuronal markers, neuronal nuclei (NeuN) and calbindin-D28k (Kuhn et al., 1996), and 4 extend axonal projections to the CA3 (Markakis and Gage, 1999; Cameron and McKay, 2001). Even though several thousand new cells are generated daily, a substantial fraction of newly born neurons die before they mature. Therefore, there is a very small population of proliferating cells in the hippocampus and the effect that those cells exert on host circuitry is likely very small as well. The survival of new neurons is competitively regulated by their own NMDAR-type glutamate receptors during a short, critical period soon after neuronal birth (Tashiro et al., 2006a). Several studies have already reported that newly generated neurons significantly contribute to spatial and temporal memory (Drapeau et al., 2003; Dupret et al., 2007). As well, hippocampus-dependent learning, especially spatial learning can affect adult-generated neuronal survival (Gould et al., 1999; Drapeau et al., 2003). Similarly, exercise has shown to enhance neurogenesis, LTP, and spatial pattern separation indicating that learning is one of the most crucial regulators in hippocampal neurogenesis. Adult neurogenesis may support DG- mediated fine spatial distinctions (van Praag et al., 1999b; Dobrossy et al., 2003; Creer et al., 2010). In a different approach, ablation of neurogenesis in the DG has shown impaired contextual fear conditioning and synaptic plasticity, such as LTP, but not spatial memory (Saxe et al., 2006). However, it is still unclear from this study whether the consequent behavioral effects resulted directly from ablation of neurogenesis or other impairments in the brain. More recent publications have also suggested possible models for how adult hippocampal neurogenesis may affect learning and memory (Deng et al., 2009; Deng et al., 2010). In addition, more hippocampal neurons were detected in adult mice living in an enriched environment as compared to controls in a non-enriched environment (Kempermann et al., 1997). Thus, the fate of neurogenesis and their survival could also be dependent on environmental conditions (Kempermann et al., 1997; van Praag et al., 1999b; Lemaire et al., 2000; Doetsch and Hen, 2005), types of exercise (van Praag et al., 1999b; van Praag et al., 1999a; Farmer, 2004), and various growth factors (Craig et al., 1996). Stem cell research must be performed under well- established controls as there are many environmental factors that can affect neurogenesis.  5 1.1.1 Neuronal transcription factors     Neural stem and progenitor cells in the telencephalon are heterogenous in their proliferative and differentiation potential during development (Lillien, 1998). Thus, a detailed understanding of brain development focusing on the cellular level is necessary to the study of adult neurogenesis, as well as improvement of therapeutic transplantation of NSCs. However, molecular mechanisms underlying the regulation of neurogenesis are poorly understood in the brain. A number of neural basic helix-loop-helix (bHLH) transcription factors, such as neurogenin (Ngn) 1, Ngn2 and NeuroD are expressed in the developing telencephalon (Lee, 1997) while achaete scute homologue ash1 (Mash1) is expressed in the ventral telencephalon (Lo et al., 1991) and affects the generation of neuronal precursor cells in the SVZ (Guillemot and Joyner, 1993). Particularly, Ngn1, Ngn2, Ngn3, NeuroD1 and NeuroD2 are important regulators of neurogenesis (Ma et al., 1996; Sommer et al., 1996; Fode et al., 1998; Scardigli et al., 2001). Ngn1 and 2 have been well studied in the sensory and ventral spinal cord neurons (Ma et al., 1999; Scardigli et al., 2001) whereas Mash1 has been reported as a key regulator for autonomic ganglia, olfactory receptor neurons and noradrenergic neuronal differentiation of the hind brain (Guillemot and Joyner, 1993; Hirsch et al., 1998; Casarosa et al., 1999). Mash1 is also required in early progenitor cells to trigger neuronal differentiation in the olfactory epithelium (Cau et al., 1997). A downstream mediator of Ngn activity, transcription factor NeuroD (Beta2) is expressed in neurons of all brain areas, spinal cord, peripheral ganglia and sensory organs (Lee et al., 1995; Mutoh et al., 1997). NeuroD heterodimerizes with the products with the products of E2A gene and controls the transcription of a variety of genes by binding E- boxes in the promoter region. (Aronheim et al., 1993; Ma et al., 1996). NeuroD-null mice have shown neuronal deficit in the granule layers of both the cerebellum and hippocampus (Miyata et al., 1999), a profound deafness due to the failure of neurotrophic receptors, such as tropomysosin receptor kinase (Trk) B and TrkC, resulting in reduced survival of the inner ear sensory neuron during development (Kim et al., 2001b), and reduction of retinal photoreceptor 6 cells (Pennesi et al., 2003). In the brain, NeuroD and neuronal HLH protein, NEX are necessary for granule cell differentiation in the cerebellum and hippocampal DG (Miyata et al., 1999; Schwab et al., 2000), and can even convert non-neuronal epidermal cells into fully differentiated neurons. Therefore, NeuroD acts as a neuronal differentiation factor in postnatal brain development (Lee et al., 1995; McCormick et al., 1996). Such bHLH transcription factors act as heterodimers, which bind to the consensus sequence CANNTG E-box. E-box is related variant sequence that enhances transcription of downstream genes, resulting in regulation of many biological processes including the determination of neuronal cell fate and differentiation when ectopically expressed during development. However, the normal function of neuronal bHLH proteins still remains unclear. Thus, specific regulatory sequences which enable activity of neuronal bHLH protein during development must be identified to distinguish among many potential targets in the genome as E-box sequences are present in regulatory regions of many non-neuronal target genes and also occur frequently in the genome.  1.1.2 Neural stem/progenitor cell markers     One of major obstacles in studies of adult neurogenesis is the lack of specific marker, defining adult NSCs. As complementary strategies, there are several ways to label dividing cells. Thymidine analogs, such as, IdU, CldU and EdU (Altman and Das, 1965; Kaplan and Hinds, 1977; Barnea and Nottebohm, 1994) and 5-bromo-2’-deoxyuridine (BrdU) (Kuhn et al., 1996; Kempermann et al., 1997; Eriksson et al., 1998; van Praag et al., 1999b) are used to label dividing cells in the adult brain. BrdU is a halogenated pyrimidine and thymidine analog, and is specifically incorporated into DNA during DNA synthesis in the S-Phase. Incorporated BrdU represents a replication of DNA and is transferable to the divided cell (Burns et al., 2006), indicating proliferating cells. It has been widely used to assess the phenotype of BrdU-positive cells by co-labeling with other mature cell markers, such as glial fibrillary acidic protein (GFAP), type III intermediate filament (Tardy et al., 1990) for glia, and either microtubule associated 7 protein 2 (MAP2) (Bernhardt and Matus, 1984) or NeuN (Mullen et al., 1992) for neurons. Higher concentration of 60-600mg/kg BrdU causes toxicity (Taupin, 2007; Ross et al., 2008) and induces neuronal cell death during embryonic and neonatal development (Bannigan, 1985; Nagao et al., 1998). However, the concentration needed for effective labeling is much lower than the toxic dosage. The occurrence of neurogenesis in adult rodent brains has been confirmed using BrdU (Seki and Arai, 1993; Kuhn et al., 1996). In the rat DG, during the hours following BrdU administration, newborn cells express a marker of the cell cycle as the S-phase cells that have incorporated BrdU. As early as 2 days after BrdU injection, newly generated neuronal cells express nestin, a marker of neural progenitor and stem cells (Lendahl et al., 1990). The main advantage of BrdU labeling is that it can be administered to living cells as a cell tracer through a number of routes, intravenously, intraperitoneally, or orally because of its permeability to the blood-brain barrier (BBB). Furthermore, other cell cycle markers, such as Ki67 and proliferating cell nuclear antigen (PCNA) for G1, S- and G2 phase have been used for determining NSC and progenitors. However, application of Ki67 and PCNA is less useful for NSC research compared to BrdU because there is no way to treat and inject them into a living cell or animal. (Kurki et al., 1986; Zacchetti et al., 2003). Although BrdU, Ki67 and PCNA indicate the cell cycle, they are not specific enough for confirming NSCs because BrdU, including other cell cycle markers, was initially developed as an alternative approach for determining the proliferative index of tumors (Hoshino et al., 1989; Struikmans et al., 1997). Moreover, BrdU is a toxic and mutagenic substance that induces several side effects. Therefore, the cell cycle markers should be used together with other alternative markers. These cell-type markers include vimentin, a type III intermediate filament for mitotically active cells, (Liem, 1993), neural progenitors (Eliasson et al., 1999) and radial glial cells (Kinoshita et al., 2005), nestin, (Lendahl et al., 1990), Musashi 1, (Kaneko et al., 2000) and Sox-2 (Graham et al., 2003), which have been widely used for detecting NSCs or neural progenitor cells (NPCs) with other cell-type specific markers, such as β-tubulin III (Tuj-1) (Fanarraga et al., 1999), doublecortin (DCX) (Francis et al., 1999; Gleeson et al., 1999; Jin et al., 2004b; Rao and Shetty, 2004), 8 prox1 (Lavado et al., 2010); an immature neuronal marker, NeuN (Mullen et al., 1992), MAP2 (Bernhardt and Matus, 1984); a mature neuronal marker, and GFAP; a glial cell marker. In addition, ablation of dividing GFAP-expressing cells in the SVZ and SGZ impairs neuroblasts and new neurons in the OB and the hippocampal DG (Garcia et al., 2004). Thus, GFAP could also be recognized as a progenitor marker in the forebrain and hippocampus. Furthermore, CD133, also known as prominin-1 has been reported to be a specific marker for NSC in the cerebellum (Lee et al., 2005), but it has not been confirmed in its specificity in other brain areas. Nonetheless, all listed markers excluding BrdU labeling, are not suitable for living cell regardless of their specificity for NSCs. Even though BrdU is applicable for cultured cells and living animals; its application is much more complicated compared to other markers. For example, BrdU needs additional steps to detect the cell body and it needs to be introduced in multiple injections to be effective. However, the accumulated dosage through multiple injections often causes side effects, such as reducing the size of the cerebellum (Sekerkova et al., 2004). To circumvent these limitations, the retrovirus infection system has been used to label dividing cells in the animal brain (Lewis and Emerman, 1994; van Praag et al., 2002; Tashiro et al., 2006a; Zhao et al., 2006). The retrovirus containing green fluorescent protein (GFP) detects the soma as well as the neurites. Moreover, retrovirus can genetically be modified with genes of interest together with reporter genes, such as GFP and lacZ. As a result, retroviral system allows for the structural and functional study in living NSCs/NPCs. However, retrovirus is unable to cross the BBB, so that infection must be conducted by means of intra-brain injection into the animal brain. Therefore, application of various markers underlying stem cell research must be considered in cell or animal type of experiments.  Table 1-1 Representative cell-type specific markers in the brain  Marker name Cell type Reference CD133 (Prominin-1) NSC; Cell-surface protein (Lee et al., 2005) 9 Marker name Cell type Reference Nestin  Musashi 1 Sox-2: Vimentin      BrdU  PCNA  Ki67  GFAP DCX Prox1 PSA-NCAM    Tuj1  Neural progenitor cell; type IV Intermediate filament structural protein Neural progenitor cell; Neural progenitor and radial glial cell; type III Intermediate filaments, characteristic of primitive neuroectoderm formation     Proliferating cell; during DNA duplication in S- phase Proliferating cell; during DNA repair in G1- (peak), S- and G2/M-phase Proliferating cell; all phases except in G0-phase, not detectable during DNA repair. Astrocyte and type II radial glial cell; Immature neuron Immature neuron Immature neuron; polysialic acid-neural cell adhesion molecule   Immature neuron; β-tubulin III  (Frederiksen et al., 1988) (Kaneko et al., 2000) (Graham et al., 2003) (Houle and Fedoroff, 1983; Liem, 1993) (Eliasson et al., 1999) (Kinoshita et al., 2005)    (Kurki et al., 1986)  (Zacchetti et al., 2003) (Debus et al., 1983) (Tardy et al., 1990) (Francis et al., 1999). (Lavado et al., 2010) (Seki and Arai, 1993; Bonfanti and Theodosis, 1994) (Fanarraga et al., 1999) 10 Marker name Cell type Reference Hu  Noggin  MPB  O4  O1  Calbindin-D28k GAP43 MAP2  Neural tubulin NeuN Neurofilament Tau immature neuronal  Immature neuron; a neuron-specific gene during development Oligodendrocyte; Myelin basic protein, sheath neuronal structure Immature oligodendrocyte  Mature oligodendrocyte  Neuron; Calcium-binding protein Mature neuron; structural protein in axon Mature neuron; Microtubule-associated prote2 in dendrites Mature neuron; structural protein Mature neuron; neuronal nuclei Mature neuron; structural protein in axon Neuron; type of MAP, structural protein in axon (Marusich et al., 1994)     (Sommer and Schachner, 1981 (Sommer and Schachner, 1981) (Kuhn et al., 1996)  (Bernhardt and Matus, 1984)  (Mullen et al., 1992) (Carden et al., 1987)  1.2 A role of GABA receptors during development  The glutamate receptors, permeable to cations are principal excitatory whereas γ- aminobutyric acid (GABA) receptors (GABARs), permeable to anions are primarily inhibitory in the adult brain (Moriyoshi et al., 1991; Kutsuwada et al., 1992; Seeburg, 1993; Farrant et al., 1994; Leinekugel et al., 1995; Leinekugel et al., 1997; Leinekugel et al., 1999; Ganguly et al., 11 2001; Ben-Ari, 2002). A balance between excitation and inhibition is important for regulating brain functions. For instance, blockade of GABARs results in seizures whereas enhancement of GABAergic synapse generate sedative, anticonvulsant and anxiolytic actions. When glutamate receptors are poorly expressed during early development, GABAR expression is highly conserved. As a result, GABAR initially acts as an excitatory neurotransmission. Once a density of glutamatergic and GABAergic synapses is sufficient GABAR shifts to mature inhibitory action as a result of neuronal activity (Obata et al., 1978; Ben-Ari et al., 1989). Accumulating evidence has been provided in different brain regions exhibiting during early development, activation of GABAARs shows depolarization followed by an increase of intracellular Ca2+ concentration (Luhmann and Prince, 1991; Reichling et al., 1994; Wang et al., 1994; Leinekugel et al., 1995; Chen et al., 1996; Owens et al., 1996; Leinekugel et al., 1997; Barker et al., 1998; Leinekugel et al., 1999; Dammerman et al., 2000; Ye, 2000; Eilers et al., 2001; Ganguly et al., 2001; Wang et al., 2001). Another inhibitory glycine receptor in the brain stem and spinal cord has displayed a similar role (Wu et al., 1992; Ehrlich et al., 1999; Kakazu et al., 1999). The developmental shift in the action of GABARs is thought to be a general rule in most vertebrates and this shift from excitatory to inhibitory actions is modulated by activity-dependent mechanisms. This developmental shift is mainly due to higher concentration of intracellular Cl- (Rohrbough and Spitzer, 1996; Ganguly et al., 2001). It has been demonstrated by perforated-patch clamp that Cl- concentration is higher in immature than in adult neurons. Thus, Cl- flows out of neurons when GABAR channels are opened (Barry and Lynch, 1991). This raises the question, “How is higher Cl- concentration maintained during early development?” A role of excitatory GABARs has been studied in two families of transporters, which mainly regulates higher concentration of intracellular Cl-. Na+- K+-2Cl- co-transporter1 (NKCC1)’s expression is upregulated in immature neurons, whereas K+-Cl- co-transporter 2 (KCC2)’s expression predominates in mature neurons. In general, NKCC1 raises intracellular Cl- concentration whereas KCC2, decreases intracellular Cl- concentration (Delpire, 2000). Thus, when GABARs act as an excitatory receptor, intracellular Cl- concentration is higher at the resting membrane potential (Cherubini et al., 1991; 12 Ben-Ari et al., 1994). Later on, GABARs ultimately become inhibitory by the delayed expression of KCC2, leading to a negative shift in the reversal potential for Cl- ions. Further studies will be required to determine the relationship between KCC2 and functional maturation. Furthermore, GABAR- mediated synaptic plasticity during development has been demonstrated in changes in synaptic efficacy. The postsynaptic activation of voltage-dependent Ca2+ channels is sufficient to induce LTP of GABAergic synaptic transmission whereas the induction of LTD requires a GABAAR- mediated membrane depolarization to remove Mg2+, which blocks the pore of NMDARs at resting, indicating that activation of both GABAAR and postsynaptic NMDARs is required. These alterations in synaptic efficacy by a persistent change in the release of GABA have been demonstrated in the neonatal rat hippocampus (Caillard et al., 1999b, a). In addition, a recent study has showed a relationship between GABAR and synaptic plasticity in the CA3 (Sivakumaran et al., 2009). These GABAR-mediated depolarizing potentials affect initial stages of early development up to the first two weeks of postnatal life in the rodent hippocampus (Berninger et al., 1995; Leinekugel et al., 1995; Khalilov et al., 1997; Leinekugel et al., 1997; Leinekugel et al., 1998; Leinekugel et al., 1999; Khazipov et al., 2001; Leinekugel et al., 2002).  1.3 Neural stem cell transplantation in neurodegenerative diseases  Neuronal loss occurs continuously as a natural consequence of aging and as a result of injury or disease throughout life, resulting in functional deterioration and poor repair from injury and disease. The lost neurons and circuitry could be recovered by manipulating endogenous adult neurogenesis or grafting exogenous NSCs/NPCs (Lindvall and Kokaia, 2010; Orlacchio et al., 2010). NSCs are capable of proliferating and providing an unlimited cell source as they are capable of giving rise to brain-specific cell types, such as neurons and glia (Temple, 1989). Evidence has provided that cultured precursor cells maintained for 1 year through multiple passages and transplanted into adult rat hippocampi show differentiated mature neurons and 13 survival for 3 months post-implantation in the GCL without tumor formation (Gage et al., 1995). In addition, transplanted immortalized NPC can differentiate into neurons with typical morphological features and can also generate action potentials and receive functional excitatory and inhibitory synaptic inputs from neighboring cells (Englund et al., 2002). Thus, clinical studies have shown NSCs/NPCs to be a promising cell source to treat disease or injury (Brustle and McKay, 1996; Ogawa et al., 2002). Cell replacement has been clinically applied in PD, HD, AD, Amyotrophic lateral sclerosis (ALS), epilepsy, spinal cord injury, and stroke (Bachoud-Levi et al., 2006; Akesson et al., 2008; Appel et al., 2008; Li et al., 2008b; Baker, 2009; Deda et al., 2009; Orlacchio et al., 2010). However, the clinical results have been largely unsuccessful (Lindvall and Kokaia, 2010). Moreover, the final result of implanted NSCs has been differently resulted in each neurodegenerative disease (Ogawa et al., 2009) and outcomes of the therapy are thereby inconsistent (Dunnett et al., 2001; Dunnett and Rosser, 2007). This is mainly due to difficulties to regenerate the functional neurons because differentiation of NSC in vivo is poorly understood. To circumvent this, NSCs have been transplanted with growth factors, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), or more lineage restricted cells have been grafted (Srivastava et al., 2008; Lindvall and Kokaia, 2010; Orlacchio et al., 2010). Secondly, different types of neurons may be affected in each type of neurodegenerative disorder. Therefore, transplantation with other multipotent stem cells, such as hematopoietic, bone marrow, mesenchymal, umbilical cord blood stem cells has been considered as a rational therapeutic approach (Appel et al., 2008; Biffi et al., 2008; Baker, 2009; Deda et al., 2009; Park and Eve, 2009). Finally, the potential of the brain’s self-repair and NSC transplantation is actually unexplored thus far. Therefore, intensive research, for example, the understanding of the physiology of NSCs and the stimulation of endogenous and transplanted progenitors to increase functional recovery is necessary prior to performing clinical trials in neurodegenerative disorders (Singec et al., 2007; Yu and Silva, 2008).   14 1.4  Alzheimer’s disease and microglia  Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease affecting more than 35 million people worldwide. Most AD cases have shown progressive memory and cognitive impairment with age, leading to severe incapacity and death (Whitehouse et al., 1981; Bartus et al., 1982; Coyle et al., 1983; Brookmeyer et al., 2007). AD is the most common form of dementia among older people. Aging is the greatest risk factor for AD that is characterized by progressive loss of memory, cognitive decline, and inability to carry out daily activities (Ashe, 2001; Selkoe, 2001). AD selectively damages the frontal and temporal lobes, including the hippocampus and amygdala having properties of amyloid plaques, neurofibrillary tangles and neuronal loss (Arnold et al., 1991). Senile plaques and neurofibrillary tangles are the neuropathological hall markers of AD and appear in the hippocampus and other cortex areas (Arnold et al., 1991). Senile plaques are extracellular aggregates of both soluble and insoluble amyloid beta (Aβ) derived from a membrane protein, amyloid precursor proteins (APP) (Masters et al., 1985; Snyder et al., 1994; Selkoe, 2001; Hardy and Selkoe, 2002). Neurofibrillary tangles, an aggregate form of hyperphosphorylation tau (p-tau) protein occupy the neuronal cell body and dendrites. AD is caused by the neuronal cell death that follows the accumulation of insoluble Aβ deposits (neurotoxic hypothesis) (Pike et al., 1993; Selkoe, 2000) and toxic effects of hyperactivated tau which also contribute to impaired neurogenesis in AD (Verret et al., 2007; Zhang et al., 2007; Hong et al., 2010). However, little is known about their exact relationship to the different pathologies (McKee et al., 1991; Terry et al., 1991). Inflammation facilitated by the presence of microglia also plays a role in AD pathogenesis. Evidence has continuously supported the involvement of chronic inflammation in AD. Thus, anti-inflammatory drugs can prevent or slow down the progression of AD (Akiyama et al., 2000).    15 1.4.1 Microglia  Microglia are the immune cells in the CNS that represent approximately 10-20% of all glia in the CNS. Evidence has been provided that microglia originate from monocytes entering the CNS at early stages of embryonic development (Perry et al., 1985; Ling and Wong, 1993), and they shares properties in common with macrophages, such as phagocytosis (McGeer et al., 1988; Kreutzberg, 1996). Microglia have two classic morphologies: ramified (non-activated) or ameboid (activated). The ramified stage is capable of rapidly shifting to the ameboid in response to pathology in the brain (Gehrmann et al., 1995). In general, microglia play a role in immune functions, such as neuroprotection, repair processes, phagocytosis and propagation of the inflammatory response (Banati and Graeber, 1994). A pronounced feature of microglia reactivity is the ability to secrete growth factors, cytokines, neurotoxins and reactive oxygen species (ROS) (Benveniste, 1992; Yates et al., 2000). Chronically activated microglia through pro-inflammatory cytokines and chemokines can affect negatively neurodegenerative diseases. Moreover, these substances lead to recruitment of more microglia to the place where injury occurs (McGeer and McGeer, 1995). These activated microglia cluster around amyloid plaques and release pro-inflammatory cytokines and chemokines that induce neurotoxicity (Benveniste, 1992; Yates et al., 2000; Combs et al., 2001a; Combs et al., 2001b). Thus, microglia play a crucial role in AD. Aβ is able to stimulate microglia through activation of tyrosine kinase cascades (McDonald et al., 1997; Combs et al., 1999) and several receptors have been proposed as the binding sites of Aβ on microglia. This Aβ acts to mediate secretion of pro- inflammatory cytokines, chemokines and ROS (El Khoury et al., 1996; Yan et al., 1996; Husemann et al., 2001; Lue et al., 2001b; Husemann et al., 2002). It is believed that Aβ alone is not toxic to neurons, but instead acts as a chemotactic agent for microglia (Davis et al., 1992). Thus, Aβ serves as a microglial activator to stimulate release of pro-inflammatory cytokines, such as IL-1β, TNF-α, IL-6 and chemokines, resulting in neurotoxicity (Giulian, 1997).  16 1.5 Long-term potentiation  Hebbian theory describes a basic mechanism for synaptic plasticity that arises from coincident pre- and post-synaptic activity. It was first suggested by Donald Hebb in 1949. True LTP was first observed in granule neurons of the DG of the hippocampus following stimulation of the medial perforant path (MPP) from the entorhinal cortex in the anaesthetized rabbit (Bliss and Lomo, 1973). Since then, synaptic plasticity, such as LTP and long-term depression (LTD), the converse process of LTP showing long-lasting decrease in synaptic transmission (Malenka and Nicoll, 1993; Bear and Malenka, 1994; Stevens and Wang, 1994) has been thought to play important roles in learning and memory (Collingridge et al., 1983; Morris et al., 1986; Alkon and Nelson, 1990; Fujii et al., 1991; Dudek and Bear, 1992; Mulkey and Malenka, 1992; Bliss and Collingridge, 1993; Kandel, 1997; Collingridge et al., 2010). Indeed, memories are thought to be encoded by modification of synaptic transmission and long-lasting synaptic enhancement. Thus, this research provides an important key into understanding the cellular and molecular mechanisms by which memories are formed and stored (Bliss and Collingridge, 1993). Much evidence has accumulated related to the role of synaptic plasticity in learning and memory. For instance, synapses are potentiated during inhibitory avoidance learning, but no further potentiation can be induced by HFS, indicating that inhibitory avoidance training occludes LTP (Whitlock et al., 2006). These long lasting memories formed have been attributed to long-lasting changes in synaptic efficacy. As of long lasting forms of memory, the late phases of LTP (L-LTP) appear to require gene transcription and new protein synthesis (Frey et al., 1988; Frey et al., 1996). LTP is one of several phenomena and major cellular mechanisms underlying synaptic plasticity, showing long-lasting enhancement in synaptic transmission between neurons in the brain (Bliss and Collingridge, 1993). It has been intensively studied at excitatory synapses in the Cornu Ammonis 1 (CA1) region of the hippocampus (Teyler and DiScenna, 1987; Gustafsson and Wigstrom, 1988; Kauer et al., 1988b; Nicoll et al., 1988; Madison et al., 1991; Bliss and Collingridge, 1993; Larkman and Jack, 1995). LTP can last from less than few hours in vitro to 17 weeks or months in vivo, depending on the stimulation protocol (Bliss and Gardner-Medwin, 1973; de Jonge and Racine, 1985; Abraham et al., 2002). LTP has been demonstrated at different synapses in rodents (Fox, 2002; Ji et al., 2003) and in primates (Nosten-Bertrand et al., 1996; Urban et al., 1996). Initially, LTP was demonstrated at glutamatergic synapses between MPP and granule neurons in the DG of rabbit hippocampi (Bliss and Lomo, 1973). NMDAR- dependent LTP was first demonstrated in the CA1 region using the competitive antagonist, CPP and non-competitive antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10- imine maleate (MK-801) which blocks the induction of LTP without altering basal synaptic transmission (Collingridge et al., 1983; Abraham and Mason, 1988). Since then, it has been further confirmed that the induction of LTP is primarily NMDAR-dependent in both the CA1 and DG region of the hippocampus (Collingridge, 1992; Bliss and Collingridge, 1993; Bailey et al., 2000; Malenka and Bear, 2004), and NMDARs act as integrators of coincident synaptic signals (Malenka and Nicoll, 1999). NMDARs are required for the induction of LTP in most hippocampal areas (Kauer et al., 1988a; Muller et al., 1988). What are the detailed molecular mechanisms that account for the change in synaptic strength? Presynaptically released glutamate binds to postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) permeable to Na+ and K+, resulting in partial membrane depolarization sufficient to remove Mg2+ ions from postsynaptic NMDARs. Ultimately, this allows Ca2+ influx through NMDARs which results in the consequent rise of intracellular Ca2+. This influx is the critical trigger for LTP (Mayer et al., 1984; Perkel et al., 1993; Chen and Lipton, 1997; Papadia and Hardingham, 2007). Blockade of intracellular Ca2+ by injection of chelators, such as ethylene glycol tetraacetic acid (EGTA) in postsynaptic neurons prevents induction of hippocampal LTP (Lynch et al., 1983). Similarly, directly increasing the amount of postsynaptic Ca2+ by photolysis of caged Ca2+ is sufficient to potentiate synaptic transmission (Malenka et al., 1988; Malenka et al., 1992). Numerous studies have supported postsynaptic modifications that could cause LTP by changes in AMPAR function or number (Kauer et al., 1988b; Malenka et al., 1988; Muller et al., 1988; Davies et al., 1989; Manabe et al., 1992). AMPAR response appears during LTP, but 18 no change in the NMDAR response is seen, supporting postsynaptic modifications by a functional recruitment of AMPARs (Kauer et al., 1988b; Muller et al., 1988). In fact, there has been much controversy regarding whether the changes in synaptic strength occur pre- or postsynaptically. For example, probability of quantal neurotransmitter release from presynaptic areas increases synaptic transmission without showing postsynaptic modification (Malinow and Tsien, 1990; Kullmann and Nicoll, 1992; Isaac et al., 1996). However, non-responsive postsynaptic areas can be explained by a postsynaptic “silence synapse”. Synapses are deemed postsynaptically silent if the postsynaptic membrane contains NMDARs but not AMPARs and thereby normal AMPAR-mediated signaling is not present (Isaac et al., 1995; Durand et al., 1996). Additionally, expression of LTP in area CA1 of the hippocampus is not accompanied by an increase in the probability of release of synaptic vesicles (Hjelmstad et al., 1997). Regardless of pre- and post-synaptic controversy, synaptic modification is primarily mediated by glutamate receptors, such as a NMDARs, AMPARs and Kainate receptors. A subtype of glutamate receptors is also involved in rapid induction, input specificity, persistence, and dependence on correlated pre- and postsynaptic activity. However, LTP is still poorly understood because the molecular and cellular mechanisms of glutamate receptors are very complicated. Therefore, more intense studies of glutamate receptors are required for a better understanding of LTP.  1.5.1 NMDA receptors  Glutamate receptors, such as NMDAR and AMPAR have been extensively studied in relation to LTP. NMDARs and AMPARs play critical roles in excitatory synaptic transmission and in particular, NMDARs are important for the induction of synaptic plasticity in the CNS (Bashir et al., 1991; Berretta et al., 1991; Asztely et al., 1992; Collingridge, 1992; O'Connor et al., 1995; Grosshans et al., 2002). The NMDARs are ligand-gated ionotropic glutamate receptors with allosteric modulation sites for H+, Zn2+ and polyamines, and which have a specific affinity for a 19 selective agonist, NMDA. NMDARs are blocked in a voltage-dependent manner by Mg2+ ions at voltages close to the resting membrane potential. Presynaptic release of glutamate causes Na+ influx through AMPARs in the post synaptic neurons that is sufficient to remove Mg2+ ions by means of membrane depolarization. This results in the opening of an ion channel that is permeable to Na+ and small amounts of Ca2+ influx and K+ efflux (Mayer et al., 1984; Chen and Lipton, 1997; Papadia and Hardingham, 2007). Ca2+ influx via NMDARs contributes to a cascade of intracellular processes that trigger AMPAR trafficking, resulting in LTP and LTD. Structurally, the native NMDAR appears in heterotetrameric assemblies (Laube et al., 1998), typically containing two glycine-binding NR1 subunits, required for a functional NMDAR and two glutamate-binding NR2 subunits (Hollmann and Heinemann, 1994; Premkumar and Auerbach, 1997; Laube et al., 1998; Schorge and Colquhoun, 2003; Papadakis et al., 2004). Several NR1 splicing variations give rise to eight isoforms that can affect electrophysiological and pharmacological channel properties differentially, such as inhibition by H+ or Zn2+ ions or potentiation by polyamines (Zukin and Bennett, 1995). NR2 is classified into four different subunits NR2A, NR2B, NR2C and NR2D and forms the NMDAR in combination with the obligatory subunit NR1 subunit (Moriyoshi et al., 1991; Kutsuwada et al., 1992; Monyer et al., 1994; Behe et al., 1995). The identity of the NR2 subunit is crucial in determining the functional properties of the NMDARs, such as channel high affinity for glutamate, sensitivity to Mg2+, fractional Ca2+ current, and channel conductance and deactivation time. In general, heteromeric NMDARs that consist of NR1/NR2 subunits show different deactivation times (NR2A<NR2C=NR2B<<NR2D; from fast to slow order) (Farrant et al., 1994; Momiyama et al., 1996; Wyllie et al., 1998). In some cases, NR3 (NR3A and NR3B) is able to take the place of NR2, which has a glutamate-binding site. Most native NMDARs do not include NR3 subunits, but studies have shown that NR3A can act as a regulatory NMDAR subunit, expressed primarily during brain development (Das et al., 1998). Addition of NR3A to NR1/NR2 reduces Ca2+ permeability and conductance (Das et al., 1998; Perez-Otano et al., 2001). The subunits are differentially expressed both regionally in the brain and temporally during development. NR2A 20 and NR2B are both present in the brain and the most widely expressed NMDARs are made up of heteromeric NR1/NR2A and NR1/NR2B. In some cases, heterotrimeric NR1/NR2A/NR2B and NR1/NR2A/NR2D receptors exist (Cull-Candy and Leszkiewicz, 2004; Waxman and Lynch, 2005) having differences in their pharmacological and kinetic properties (Tovar and Westbrook, 1999; Cathala et al., 2000; Brickley et al., 2003). Furthermore, opposing roles of NR2A- and NR2B-containing NMDARs in mediating cell survival and cell death have been reported in a middle cerebral artery occlusion (MCAO)-mediated stroke model (Ihrie and Alvarez-Buylla, 2008). NR3A-containing NMDARs have shown neuroprotective effects against excitotoxicity in stroke and neurodegenerative disorders (Nakanishi et al., 2009). This cell death is triggered by extreme Ca2+ influx through the intense NMDAR activation during stroke. Expression of NR3 subunits is regionally and temporally restricted in the brain and expression level of NR3A subunits is very low in the adult cortex and hippocampus (Wong et al., 2002). On the other hand, NR2B-containng NMDARs are greatly expressed during early development and thereby they are mainly observed in immature neurons, and NR2D-contatining NMDARs are highly expressed in the early postnatal brain (Akazawa et al., 1994; Monyer et al., 1994). In contrast, NR2A and NR2C expression levels increase postsynaptically in the brain (Monyer et al., 1994; Liu et al., 2004b; Kohr, 2006). Eventually NR2A subunits outnumber NR2B. NR2C has similar deactivation kinetics to NR2B and is mostly restricted to the cerebellum and expressed sparsely in CA4 hilar cells of the  hippocampus (Waxman and Lynch, 2005). NR2D is expressed in small numbers of cells in select brain regions, and has slow decay kinetics and weak Mg2+ block. In the hippocampus, NR2A and NR2B subunits are mainly expressed at synapses (Tovar and Westbrook, 1999; Janssen et al., 2005; Thomas et al., 2006) whereas NR2D subunits are expressed extrasynaptically (Momiyama et al., 1996; Misra et al., 2000b; Brickley et al., 2003). Physiologically, NR1/NR2D-containing NMDARs have low conductance (Momiyama et al., 1996; Wyllie et al., 1996; Misra et al., 2000b; Misra et al., 2000a; Momiyama, 2000) and extremely slow decay time constants of 4-5 seconds (Lozovaya et al., 2004). This has been confirmed using the NR2D antagonist, (2R*,3S*)-1-(phenanthrenyl-2-carbonyl)piperazine-2,3-dicarboxylic 21 acid (PPDA) and UBP141 (Harney et al., 2008). Extrasynaptic NR2D-containing NMDARs are recruited to synapses during NMDAR-LTP in the DG (Harney et al., 2008). This is important because activity dependent NMDAR trafficking also contributes to synaptic transmission and neuronal excitability (Wenthold et al., 2003; Williams et al., 2007; Stephenson et al., 2008). Furthermore, NMDAR-excitatory postsynaptic currents (EPSCs) are very slow with a large Ca2+ influx that affects Ca2+-dependent intracellular signals and synaptic activity (O'Connor et al., 1995; Harney et al., 2006; Lau and Zukin, 2007). The NR1/NR2 NMDARs can assemble prior to leaving the endoplasmic reticulum (ER). NR1 subunits primarily lead NMDAR trafficking from the ER to membrane (Fukaya et al., 2003) whereas NR2A or NR2B is not able to reach the membrane surface unless co-assembled with NR1 subunits (McIlhinney et al., 1998; Scott et al., 2001; Xia et al., 2001). On the other hand, NR2 subunits are more involved in synaptic localization and stabilization of NMDARs (Wenthold et al., 2003) controlled by interaction between the NR2 C-terminus and PDZ domains (Sheng and Sala, 2001). The interaction of exocyst proteins, synapse-associated protein (SAP)-102 and Sec8 promotes the membrane delivery of NR2B containing NMDARs whereas NR2A binds to postsynaptic density protein (PSD-95) at the synapse (Losi et al., 2003; Sans et al., 2003). The C-terminal domain of NR2A or NR2A/NR2C subunits is important for the induction of LTP in regulating synaptic NMDAR activation and function by enhancing channel open probability in the hippocampus and cerebellum (Steigerwald et al., 2000; Rossi et al., 2002). In addition, NMDARs control bidirectional synaptic plasticity in the hippocampus (Liu et al., 2004a) by regulating postsynaptic AMPARs (Kim et al., 2005). Indeed, NR2A-containing NMDARs are involved in the induction of LTP by increasing the surface expression of the AMPAR subunit GluR1 whereas NR2B- containing NMDARs are involved in the induction of LTD by decreasing surface expression of the AMPAR subunit GluR1GluR1. LTD is primarily triggered by extrasynaptic NR2B-contaiting NMDARs in the hippocampus. Interestingly, de novo LTD requires activation of NR2B-contating NMDAR, whereas LTP and depotentiation require activation of NR2A-contating NMDARs in the adult cortex (Massey et al., 2004). Therefore, intense investigation of the various NMDAR 22 subtypes and individual subunits in different brain areas is required for a better understanding of LTP and LTD.  1.5.2 AMPA receptors  Glutamatergic AMPARs mediate fast excitatory synaptic transmission (Seeburg, 1993; Hollmann and Heinemann, 1994). Activity-dependent trafficking of AMPARs to synapses is a dynamic and powerful mechanism underlying synaptic plasticity in the mammalian CNS (Carroll et al., 1999a; Luscher et al., 1999; Shi et al., 1999; Hayashi et al., 2000; Man et al., 2000; Wang and Linden, 2000; Shi et al., 2001). AMPARs were first reported in 1982 as one of the non- NMDAR channels with a specific affinity for an artificial analogue, AMPA (Honore et al., 1982; Keinanen et al., 1990). AMPAR is a ligand-gated ionotropic glutamate receptor that consists of GluR1, GluR2, GluR3 and GluR4 (also known as GluRA-D or GluA1-4) subunits (Boulter et al., 1990; Wisden and Seeburg, 1993; Hollmann and Heinemann, 1994; Dingledine et al., 1999). Each subunit has a glutamate biding site. Structurally, AMPAR appears in heterotetrameric or homoterameric assemblies (Rosenmund et al., 1998; Gardner et al., 2001). Most AMPARs contain the GluR2 subunit which is predominant and heterotetrameric with GluR1, GluR3 or GluR4 and permeable primarily to monovalent cations, such as Na+ and K+ (Mayer, 2005). Although the exact subunit composition of most AMPARs remains largely unknown, it has been reported that AMPARs made of GluR1 and GluR2 (~80%) or composed of GluR2 and GluR3 (16%) are the major types. Approximately 8% of total AMPAR complexes are homomeric GluR1 in the hippocampus (Wenthold et al., 1996; Lu et al., 2009). AMPAR lacking GluR2 is Ca2+- peameable (Hollmann et al., 1991; Verdoorn et al., 1991; Burnashev et al., 1992; Geiger et al., 1995). Other brain regions including early postnatal hippocampus express GluR4 which forms AMPAR complexes with GluR2 (Zhu et al., 2000). The receptor Ca2+ permeability is governed by not only regulated expression of the GluR2 subunit, but also as a consequence of RNA editing (Hollmann et al., 1991; Liu and Zukin, 2007). Indeed, AMPARs containing GluR2 23 subunits restrict Ca2+ permeability determined by post-transcriptional modification (RNA editing) of the glutamine (Q)/arginine (R) editing site of the GluR2 in the transmembrane domain 2 (TM2) region subunit that only has single amino acid difference from GluR1, GluR3 and GluR4 (Sommer et al., 1991; Dingledine et al., 1992). The arginine has a positive charge that makes it energetically unfavorable for Ca2+ entry through the pore. AMPARs lacking GluR2 subunits that are also permeable Zn2+ have distinctly fast kinetics, which inwardly rectify current-voltage (I-V) relation (Burnashev et al., 1992; Geiger et al., 1995; Swanson et al., 1997). However, most GluR2 subunits are edited to the GluR2 (R) form, indicating that most AMPARs in the CNS are primarily Na+ and K+ permeable (Mayer, 2005). Tetrameric AMPARs assemble as dimers of dimers in the ER and exit to synaptic sites (Greger et al., 2007). Synaptic efficiency is dependent on the number of channels at synapses (Ellgaard and Helenius, 2003; Tichelaar et al., 2004). Each AMPAR subunit has a large extracellular domain, four membrane-associated domains with considerable homology, and different cytoplasmic carboxyl tails. These cytoplasmic tails determine interactions with scaffolding and cytoplasmic proteins. All AMPARs contain PDZ-binding domains, but each binding domain differs (Hollmann et al., 1994; Sheng and Sala, 2001). GluR1, an alternative splice form of GluR2, and GluR4 have longer C-terminus domains and are homologous. In contrast, the predominant splice form of GluR2, GluR3 and an alternative splice form of GluR4 have shorter C-terminus domains and are homologues (Kohler et al., 1994). The GluR4 subunit is mainly expressed during early development while other subunits are expressed in the mature brain (Zhu et al., 2000). The GluR1 subunit interacts with SAP97 through SAP97’s group I PDZ domain (Leonard et al., 1998). The C-terminus contains conserved PDZ binding motif that serves as a binding site for glutamate receptor interacting protein/AMPA-binding protein (GRIP/ABP) and protein interacting with C-kinase 1 (PICK1) as well as a separate site that binds N-ethylmaleimide-sensitive factor (NSF) (Kim and Huganir, 1999; Braithwaite et al., 2000). GluR2, GluR3, and GluR4 bind to PICK1 (Dev et al., 1999; Xia et al., 1999; Greger et al., 2002). PICK1 has a single PDZ domain that interacts with protein kinase Cα (PKCα) and GluR2. On 24 the other hand, GluR2 and GluR3 interacts with GRIP (Dong et al., 1997) and ABP/GRIP2 (Dong et al., 1999a; Dong et al., 1999b) with six to seven PDZ domains as anchors that contribute to synaptic abundance of AMPARs. PICK1 and GRIP/ABP bind to the AMPAR GluR2 and GluR3 subunit C-terminus and LTD is regulated by the interaction between PICK1-GluR2, and disruption of the interaction between GluR2 and GRIP/ABP (Xia et al., 2000; Seidenman et al., 2003; Steinberg et al., 2006). The C-terminus of GluR2, Serine 880 (S880) can be phosphorylated by PKC. This phosphorylated S880 in GluR2 results in disrupting interactions with GRIP/ABP, but not with PICK1 (Matsuda et al., 1999; Chung et al., 2000). Cerebellar LTD requires PKC-regulated interactions between the carboxyl-terminal of GluR2/GluR2 and PDZ binding domain (Xia et al., 1999). Thus, the association of GluR2 with GRIP/ABP is essential for maintaining AMPAR at synapses which can be regulated by PKC and S880 in GluR2 (Matsuda et al., 1999; Chung et al., 2000). Conversely, peptide disruption of the interaction between GluR2/GluR3 and GRIP/ABP increases synaptic current (Daw et al., 2000). Thus, binding of GluR2/GluR3 and GRIP/ABP may stabilize AMPARs in an intracellular pool and block their exocytosis into the synaptic membrane. This may be due to the multifunctional roles of GRIP/ABP proteins that are involved in receptor delivery and stabilization as well as synaptic endocytosis. Overexpression of PICK1 reduces the level of surface expression of GluR2 in cultured neurons (Perez et al., 2001). In contrast, PICK1 expression in the CA1 leads to increases in AMPAR-mediated EPSC amplitude. These effects are blocked by applying inhibitors for PKC or calcium/calmodulin-dependent kinase II (CAMKII) (Terashima et al., 2004). Moreover, postsynaptic intracellular perfusion of GluR2 C-terminal peptides that disrupt GluR2 interaction with PICK1 impairs hippocampal LTD, and LTD in hippocampal slices increases phosphorylation of S880 in GluR2 (Kim et al., 2001a). In addition, phosphorylation of S880 in GluR2 does not affect NMDAR-dependent GluR2 endocytosis, but alters the recycling of GluR2 following activation of NMDARs. Also, neurons lacking PICK1 shows accelerated GluR2 recycling following activation of NMDARs (Lin and Huganir, 2007). These data show that recycling of GluR2 seems to be regulated by phosphorylation of S880 in GluR2 and PICK1. 25 Thus, the interaction of GluR2-PICK1 plays a regulatory role in the expression of LTD in the hippocampus. In addition, another cytosolic protein, NSF, also binds to the C-terminus of GluR2 (Hanson et al., 1997; Fleming et al., 1998). The homohexameric ATPase NSF requires all three domain, and is an essential component of the protein machinery responsible for various membrane fusion processes underlying intracellular protein trafficking and presynaptic exocytosis including intercisternal Golgi protein transport (Rothman, 1994). NSF- dependent membrane fusion needs interactions between NSF and soluble, α-, β- and γ-soluble NSF attachment protein (SNAP) (Sollner et al., 1993). In general, NSF binds to its receptor, vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (v-SNARE) synaptobrevin with the target SNAREs (t-SNARE) syntaxin and SNAP-25 at the presynaptic terminal (Rothman, 1994; Sudhof, 1995; Hay and Scheller, 1997). It has also been reported that NSF regulates AMPAR function as well as synaptic transmission (Nishimune et al., 1998; Song et al., 1998). Rapid continual cycling of receptors requires interactions between the GluR2 subunit and NSF, indicating that GluR2-containing AMPARs participate in receptor trafficking (Nishimune et al., 1998; Osten et al., 1998; Song et al., 1998; Luscher et al., 1999; Luthi et al., 1999). It has been demonstrated that peptide disruption of the interaction between NSF and GluR2 triggers a rapid decrease of the synaptic current, but GluR2-lacking AMPARs are not affected, suggesting that synaptic AMPARs is stabilized by NSF binding to GluR2 (Nishimune et al., 1998; Luscher et al., 1999; Noel et al., 1999; Kim and Lisman, 2001; Shi et al., 2001). These data show that NSF increases the surface expression of GluR2 (Huang et al., 2005). Furthermore, postsynaptic application of α-SNAP increases synaptic transmission and inhibition of either NSF or SNAP decreases LTP (Lledo et al., 1998). Thus, postsynaptic SNAP-NSF- GluR2 interaction plays an important role in the delivery of AMPARs to the membrane surface and stabilization of AMPARs at the synapses (Osten et al., 1998). Although the role of cytosolic proteins in GluR2 is not fully understood, their interactions do play crucial roles in controlling AMPAR trafficking at synapses (Song and Huganir, 2002; Santos et al., 2009). The number of AMPARs at synapses can be regulated in plasticity. AMPAR trafficking has 26 firstly been demonstrated via clathrin-mediated endocytosis followed by increased co- localization with the clathrin adaptor protein, AP2 (Carroll et al., 1999b; Man et al., 2000). Indeed, regulation of the vesicle-mediated plasma membrane trafficking can lead to postsynaptic modifications in the number of AMPARs, which then can lead to changes in synaptic strength (Luscher et al., 1999; Beattie et al., 2000; Lin et al., 2000; Man et al., 2000) and LTP (Carlezon et al., 2000; Lu et al., 2001; Pickard et al., 2001). Rapid AMPAR trafficking from non-synaptic to synaptic areas shows activity-dependent synaptic enhancement during LTP (Shi et al., 1999; Hayashi et al., 2000). However, these subunit-specific molecular mechanisms underlying AMPAR trafficking is still not clear. Importance of the GluR1 subunits in LTP has been confirmed using adult knockout mice lacking GluR1 (Zamanillo et al., 1999). Accumulating evidence has suggested that activation of CaMKII or LTP induces delivery of AMPARs into synapses that require interaction between GluR1 and a PDZ domain protein (Shi et al., 1999; Carlezon et al., 2000; Hayashi et al., 2000). However, AMPARs containing GluR4 are delivered to synapses by spontaneous activity during early development regardless of CaMKII activity (Zhu et al., 2000). It been demonstrated that AMPARs made of the GluR1 and GluR2 subunits requiring interactions between GluR1 and group I PDZ domain proteins are added to synapses during synaptic plasticity. In contrast, AMPARs composed of the GluR2 and GluR3 subunits requiring interaction between GluR2-NSF and GluR2-PDZ II binding domain are continuously added to replace synaptic GluR1-contining AMPARs. This occurs only at synapses that already have AMPARs (Shi et al., 2001). In addition, increased GluR1 and GluR2 subunits are detected following LTP without altering NR1 subunits (Heynen et al., 2000). The destiny of AMPARs is dependent on activation of NMDARs. The AMPAR internalization triggered by NMDAR activation requires Ca2+ influx and protein phosphatases, resulting in protein kinase A (PKA)-dependent AMPAR reinsertion via recycling endosomes. In contrast, activated AMPARs in the absence of NMDAR activity are targeted to late endosome and degraded by the lysosome which is independent of Ca2+ and protein phosphatases (Ehlers, 2000). In addition, a recent publication has reported that ubiquitination of GluR1-containing AMPARs by E3ligase, Nedd4-1 27 is required for receptor internalization and their trafficking to the lysosome in a NMDAR- independent manner (Schwarz et al., 2010). In addition, protein kinase-dependent AMPAR function plays a crucial role in the production of LTP (Madison et al., 1991; Bliss and Collingridge, 1993; Malenka and Nicoll, 1999). The properties of AMPARs, such as conductance, open probability and trafficking can be similarly regulated by phosphorylation. S818, S831, Threonine 840 (T840) and S845 are well known phosphorylation sites in GluR1. S818 is a PKC phosphorylation site that is increased during LTP contributing to synaptic incorporation of AMPARs (Boehm et al., 2006). Postsynaptically localized CaMKII directly controls AMPARs as a result of the phosphorylation of AMPARs in LTP (Miller and Kennedy, 1986; Lisman and Goldring, 1988; Lisman, 1994; Lisman et al., 1997). S831 is phosphorylated by CaMKII during LTP (Barria et al., 1997a; Barria et al., 1997b; Mammen et al., 1997) and this activity drives AMPARs to synapses involved in the interaction between GluR1 and PDZ domain proteins (Hayashi et al., 2000), as well as enhances channel conductance of AMPARs (Benke et al., 1998; Derkach et al., 1999; Derkach, 2003). Moreover, phosphorylation at this site together with S845 is also important for AMPAR incorporation at synapses, suggesting a crucial role of phosphorylation in AMPAR trafficking in an activity-dependent manner (Esteban, 2003). However, mutations on GluR1-S831 do not prevent delivery of the receptor to synapses by active CaMKII (Hayashi et al., 2000), indicating that it may control other targets, such as ras GTPase activating protein (rasGAP) because it is the downstream signal of CaMKII (Chen et al., 1998; Kim et al., 1998). Furthermore, MAP kinase, the rasGAP downstream signal, is activated in response to LTP-inducing HFS in the CA1 area. Thus, MAP kinase may be an important regulator of synaptic plasticity in the CA1 (English and Sweatt, 1996). T840 is not a substrate for protein kinases, such as PKA, PKC and CaMKII. Instead, T840 is a substrate for p70S6 kinase and its phosphorylation is decreased by a LTP induction protocol. Thus, a T840 site may have a role in LTD (Delgado et al., 2007). S845 is a site for both PKA and PKC (Roche et al., 1996). The open probability of AMPAR can be regulated by PKA through phosphorylation of GluR1 S845 (Banke et al., 2000). Moreover, phosphorylation at S845 of GluR1 is also involved 28 in PKA-dependent AMPAR insertion (Esteban, 2003) and reinsertion via recycling endosomes (Ehlers, 2000). Furthermore, dephosphorylation at S845 triggers NMDA-dependent AMPAR internalization (Man et al., 2007). However, AMPARs phosphorylated by exogenous application of cyclic adenosine monophosphate (cAMP) cannot trigger their delivery (Shi et al., 2001). The PKA-scaffolding molecule A-kinase anchor protein (AKAP) is involved because it binds to the GluR1 subunit, which binds to group I PDZ domain, SAP97 (Colledge et al., 2000; Sans et al., 2001). Under basal conditions, AMPAR trafficking is dynamic and continuously occurs at synapses to maintain the steady state levels of AMPARs (Choquet and Triller, 2003; Shepherd and Huganir, 2007; Newpher and Ehlers, 2008; Triller and Choquet, 2008). Rapid changes in the number of AMPARs at postsynaptic sites account for the efficacy of fast synaptic transmission (Shi et al., 2001). These trafficking properties of AMPARs can be regulated by the subunit-specific assembly of receptors (Newpher and Ehlers, 2008). For instance, basal AMPAR-EPSCs in the CA1 pyramidal neurons are thought to arise primarily from GluR2/GluR3 receptors. AMPARs that consist of GluR1/GluR2 or GluR2/GluR4 are inserted during strong synaptic stimulation (Hayashi et al., 2000; Shi et al., 2001). This synaptic AMPAR trafficking has been explained using exocytosis during synaptic plasticity. However, AMPAR exocytosis is not proportionally elevated by an increase of synaptic transmission. Evidence has been provided that overexpression of Stargazin, the AMPAR-associated protein, drastically increases extrasynaptic AMPARs, but it is not sufficient to increase synaptic currents (Schnell et al., 2002). Thus, AMPARs may be inserted to non-synaptic areas, such as extrasynaptic (Yudowski et al., 2007; Lin et al., 2009; Makino and Malinow, 2009; Opazo and Choquet, 2011) and perisynaptic areas (Park et al., 2006; Wang et al., 2008; Kennedy et al., 2010). Evidence has shown that phosphorylation of GluR1 at S845 regulates extrasynaptic AMPAR trafficking for LTP (Oh et al., 2006). Furthermore, AMPARs need to be positioned in exact opposition to the presynaptic sites to receive the released glutamate because they have a low binding affinity with glutamate (Lisman and Raghavachari, 2006). It is only possible if AMPARs are highly mobile at the membrane surface (Borgdorff and Choquet, 2002; Choquet and Triller, 2003; Adesnik et al., 29 2005; Ashby et al., 2006; Bats et al., 2007; Heine et al., 2008). Therefore, LTP could also be explained by lateral movement or diffusion of AMPARs from extrasynaptic sites onto the synaptic membrane sites (Chen et al., 2000; Borgdorff and Choquet, 2002; Yudowski et al., 2007; Newpher and Ehlers, 2008; Makino and Malinow, 2009). Initially, exocytosis was thought to be sufficient to increase the number of AMPARs at synapses (one-step model). However, accumulating evidence has supported the need of additional steps, such as a two-step (Oh et al., 2006) and three-step model (Yudowski et al., 2007; Lin et al., 2009; Makino and Malinow, 2009; Opazo and Choquet, 2011). These two- and three-step models support lateral movement and diffusion with trapping of AMPARs at synapses. In general, the most common LTP is induced by activation of NMDARs and Ca2+-dependent kinase which leads to an increase in synaptic strength through exocytosis of AMPARs. In addition, recent publications have shown that activation of NMDARs and Ca2+-dependent kinase also contribute to AMPAR lateral diffusion (Heynen et al., 2000; Borgdorff and Choquet, 2002; Petrini et al., 2009) including GluR2 lateral movement at postsynaptic sites (Borgdorff and Choquet, 2002). Indeed, activation of NMDARs promotes AMPAR exocytosis mainly to an extra/perisynaptic site and exocytic zone of the plasma membrane, t-SNARE syntaxin 4, followed by lateral diffusion of newly inserted AMPARs to synapses in order to promote LTP (Passafaro et al., 2001; Yudowski et al., 2007; Lin et al., 2009; Kennedy et al., 2010). Finally, a recent study has suggested that during LTP, most AMPARs containing GluR1 are incorporated into synapses by lateral movement followed by GluR1-containing AMPAR exocytosis from intracellular pools to the local extrasynaptic pool as replenishment. Whereas exocytosis of AMPARs shows small contribution to LTP (Makino and Malinow, 2009). These results clarify the role of intracellular and surface AMPARs during synaptic plasticity. However, there is still controversy regarding the non-synaptic site from which AMPARs move into synapses during LTP.   30 1.5.3 Long-term potentiation in the CA3 region  Many excitatory synapses in the CNS show activity-dependent long-term changes in synaptic strength. The most common form of LTP in the hippocampus requires activation of NMDARs that ultimately leads to a postsynaptic increase of AMPARs in synaptic transmission (Bliss and Gardner-Medwin, 1973; Collingridge et al., 1983; Coan and Collingridge, 1985; Bliss and Collingridge, 1993). However, LTP at mossy fiber (MF)/CA3 synapses is fundamentally different from other hippocampal plasticity. The DG provides input from the entorhinal cortex through the MPP to the CA3, named MF/CA3 synapses (Bortolotto et al., 2005). As the major input, this MF/CA3 has several distinct properties in the probability of neurotransmitter release, which affects such activity-dependent presynaptic forms of plasticity as paired pulse facilitation (PPF) and frequency facilitation (Moore et al., 2003). In particular, MF/CA3 synapses have the low probability of basal neurotransmitter release and larger amplitude compared to PPF at associational/commissural synapses (Harris and Cotman, 1986; Zalutsky and Nicoll, 1990; Salin et al., 1996). This heterogeneity in presynaptic function reflects differences in the intrinsic properties of the synaptic terminal and activation of presynaptic neurotransmitter receptors. This low probability of basal neurotransmitter release (low firing rates of granule cells) is maintained by presynaptic A1 adenosine receptors (Moore et al., 2003) and the presynaptic inhibitory action is mediated by the axonal group II metabotropic glutamate receptor (mGluR) at MF/CA3 synapses (Kamiya and Ozawa, 1999). Frequency facilitation has been widely described at MF/CA3 synapses in vitro and in vivo. (Hagena and Manahan-Vaughan). Frequency facilitation comprises unique properties at MF synapse and is not found at any other hippocampal area (Salin et al., 1996; Dobrunz and Stevens, 1999; Toth et al., 2000; Klausnitzer and Manahan- Vaughan, 2008). Another distinguishable difference is the bouton size. At MF terminals, the terminals of axons of granule cells in the DG are functionally separated. Large boutons contact CA3 pyramidal neurons and hilar mossy cells showing large PPF, whereas, small boutons and filopodial extensions contact GABA-containing interneurons in the dentate hilus 31 and stratum lucidum of CA3 areas showing much less frequency-dependent facilitation and paired pulse depression (PPD) (Henze et al., 2000; Toth et al., 2000; Nicoll and Schmitz, 2005). LTP at MF/CA3 synapses is induced by the activation of presynaptic kainate receptors (Monaghan and Cotman, 1982; Harris and Cotman, 1986) in large boutons. Once glutamate is released it can diffuse from synaptic areas back onto the presynaptic GluR5-containing kainate receptors that lead to Ca2+ entry. Ultimately, Ca2+ induces another Ca2+ release from intracell ular stores that, in turn, initiates processes that results in LTP (Bortolotto et al., 1999; Lerma, 2003). Briefly, there are five types of kainate receptor subunits, GluR5, GluR6, GluR7, KA1 and KA2, that can be arranged in different ways to form a tetramer (Dingledine et al., 1999). Structurally, GluR5-7 can form homomers (ex. a receptor composed entirely of GluR5) and heteromers (ex. a receptor composed of both GluR5 and GluR6). However, KA1 and KA2 can only form functional receptors by combining with one of the GluR5-7 subunits (ex. a KA1- containing receptor composed of KA1 together with GluR5-7) (Monaghan and Cotman, 1982; Bettler and Mulle, 1995). Kainate receptors exist at both pre- and postsynaptic sides and regulate transmission at many synapses in a specific manner. However, GluR5-containing kainate receptors predominantly exist at MF/CA3 synapses (Lerma, 2003). In addition, presynaptic L-type or R-type Ca2+channels are an alternative Ca2+ source that can trigger the induction of LTP at MF/CA3 synapses (Breustedt et al., 2003; Dietrich et al., 2003; Lerma, 2003). Therefore, In order to prevent the induction of LTP at MF/CA3 synapses, both kainate receptors and L-type Ca2+ channels have to be blocked (Lauri et al., 2003; Bortolotto et al., 2005). However, LY382884, a specific antagonist for GluR5-containing kainate receptors, has failed to demonstrate any effect on MF LTP reduction in mice lacking the GluR6, but not the GluR5, kainate receptor subunit, suggesting that the GluR6 subunit plays an important role in modulating MF LTP (Contractor et al., 2001; Breustedt and Schmitz, 2004). Similarly, studies using these knockout mice suggest that although kainate receptors are not required, presynaptic kainate receptors facilitate the induction of MF LTP (Schmitz et al., 2003). In spite of numerous conflicting results regarding the kainate receptor subunit, kainate receptors do have 32 roles in the initiation of MF LTP associated with Ca2+ entry into the presynaptic terminal. Although the mechanisms of the LTP induction are less clear, there is unanimous agreement that its production is due to the an increase in presynaptic neurotransmitter release (Xiang et al., 1994; Weisskopf and Nicoll, 1995; Lopez-Garcia et al., 1996; Tong et al., 1996; Reid et al., 2004). What causes enhancement of neurotransmitter release during LTP at MF/CA3 synapses? Several studies have indicated a rise in presynaptic cAMP and Ca2+ during LTP (Huang et al., 1994; Weisskopf et al., 1994; Tong et al., 1996; Reid et al., 2004). The induction of MF LTP is critically dependent on the entry of Ca2+ via voltage-dependent calcium channels into the presynaptic terminal and activation of PKA by cAMP (Castillo et al., 1994; Tong et al., 1996; Breustedt et al., 2003; Dietrich et al., 2003). Its downstream signal, PKA is required for MF/CA3 LTP (Huang et al., 1995). Basic mechanisms of LTP at MF/CA3 synapses have shown NMDAR- independent plasticity. However, associational/commissural synapses at the CA3 show NMDAR-dependent LTP (Zalutsky and Nicoll, 1990; Kakegawa et al., 2004). Previous reports indicate that LTP at associational/commissural synapses, but not MF/CA3 synapses can be inhibited by application of an NMDAR antagonist prior to or during the tetanus (Harris and Cotman, 1986; Zalutsky and Nicoll, 1990). LTP at MF/CA3 synapses still requires more intensive study because of a number of existing controversies.  1.5.4 Chemical long-term potentiation     The typical LTP induction protocol involves tetanus stimulation. Electrical stimulation of neurons or neural pathways for induction is restricted to a localized area. However, global application of chemically induced synaptic plasticity, such as chemical LTP (cLTP) can also be used as a reliable technique. Application of forskolin (an adenylyl cyclase activator) and rolipram (a phosphodiesterase inhibitor) is an effective technique to induce LTP by activation of postsynaptic NMDARs (Yoshihara et al., 2000; Gomes et al., 2004; Kopec et al., 2006; Oh et al., 2006). Presynaptically injected 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid 33 (BAPTA), a Ca2+ chelator cannot block the induction of LTP whereas application of competitive NMDAR antagonist, 2-amino-5-phosphonopentanoic acid (D-APV) in postsynaptic sites can block potentiation. Moreover, chemically induced potentiation by treatment of forskolin and rolipram can persistently increase phosphorylation at S845 to 60% (Oh et al., 2006). Thus, application of forskolin and rolipram is sufficient to elevate postsynaptic intracellular Ca2+ via NMDARs, resulting in activity-dependent AMPAR exocytosis. cAMP application has a similar effect to forskolin-induced potentiation. Inhibition of cAMP-dependent PKA by application of Rp- cAMP, an inhibitor of PKA, blocks forskolin-induced potentiation (Harvey and Collingridge, 1993). In addition, forskolin-induced potentiation is also blocked by autocamtide-2-related inhibitory peptide (AIP), indicating a requirement for activation of CaMKII. Accumulated evidence supports that cLTP-induced potentiation is mediated by AMPAR trafficking (Otmakhov et al., 2004; Kopec et al., 2006; Grey and Burrell, 2008). In addition, the successful induction of cLTP in hippocampal neuronal cultures was first confirmed with a rapid increase in cell surface expression of AMPARs following glycine treatment (Lu et al., 2001; Passafaro et al., 2001; Park et al., 2004). Indeed, postsynaptic NMDARs can be briefly stimulated with NMDAR co-agonist glycine, resulting in inducing LTP accompanied by a rapid insertion of AMPARs and increased clustering of AMPARs at the surface of dendritic membranes. This glycine-facilitated AMPAR insertion is blocked by intracellular tetanus toxin (TeTx), indicating that AMPARs are regulated through SNARE-dependent exocytosis during LTP. Furthermore, glycine-induced LTP by an increase of synaptic AMPARs has been successfully observed in hippocampal slices (Broutman and Baudry, 2001; Thomas-Crusells et al., 2003). Thus, cLTP is a leading experimental model for investigating synaptic plasticity in vitro.  1.6 Long-term potentiation and neurogenesis  In the mammalian CNS, new neurons are continuously observed (Reynolds and Weiss, 1992; Richards et al., 1992; McKay, 1997; Gage, 2000) and they are consistently integrated at a 34 low rate into existing neural circuits in the adult DG of the hippocampus (Altman and Das, 1965; Gage, 2000; van Praag et al., 2002; Jessberger et al., 2007; Taupin, 2007). Adult neurogenesis has been reported as an important factor in learning and memory (Shors et al., 2002; Ming and Song, 2005; Aimone et al., 2006; Christie and Cameron, 2006; Neves et al., 2008; Deng et al., 2009). This is supported by the evidence that newly generated neurons in the DG increase memory capacity (Becker, 2005). Furthermore, voluntary exercise and environmental enrichment lead to an increase of neurogenesis as well as enhancement of learning and LTP in the DG (van Praag et al., 1999b; Farmer, 2004; van Praag et al., 2005). As previously stated, the hippocampus is the primary brain area in which LTP is reliably induced. The most common form of LTP is triggered by activation of NMDARs and Ca2+ -dependent kinase cascades which ultimately lead to an increase in synaptic transmission. NMDAR-dependent LTP in the DG has been the object of intense investigation involving learning and memory in the past 20 years (Bliss and Collingridge, 1993; Malenka and Bear, 2004). For instance, relevance between LTP and learning and memory has been addressed by using inhibitory avoidance in vivo. Inhibitory avoidance is one of the most common animal tests in behavioral memory. It is performed in a two-chambered apparatus with light and dark chambers, the latter being fitted with a device that delivers a foot shock to the rat upon entry. This behavioral paradigm can produce the same LTP as induction of LTP induced by HFS in vitro; the learning-induced synaptic potentiation occludes HFS-induced LTP (Whitlock et al., 2006). In contrast to studies of LTP in learning and memory, relevance between LTP and neurogenesis is poorly elucidated. Initially, the induction of LTP has been proposed to increase neurogenesis, as the efferent activity at MF inputs to the CA3 region is sufficient to increase the number of newly formed granule neurons in the DG (Derrick et al., 2000). More recent studies have reported increased proliferation of NPCs in the DG by the induction of LTP at MPP inputs to the DG (Chun et al., 2006). As well, enhanced neurogenesis and survival in the DG has been accomplished by induction of LTP at MPP to the GCL (Bruel- Jungerman et al., 2006). Furthermore, HFS-induced LTP within a narrow critical period enhances the survival of newly generated neurons (Kitamura et al., 2010). A similar study has 35 been reported that high K+-induced excitation through L-type Ca2+ channel (Cav1.2) and NMDARs on the proliferating precursors is sufficient to increase proliferation without altering neuronal differentiation in isolated NPCs from the hippocampal area (Deisseroth et al., 2004), suggesting that hippocampal activity can influence neurogenesis. In the SGZ of the adult DG, neurogenesis involves a series of differentiation steps from glia-like stem/progenitor (type-1) cells to transiently amplifying NPCs (type-2). Ultimately, type-2 NPCs undergo a change to postmitotic neurons (Fukuda et al., 2003; Garcia et al., 2004; Suh et al., 2007). Type-2 NPCs receive GABAergic excitation input, not glutamatergic input, that increases Ca2+ influx and the expression of neuroD, which promotes activity-dependent neuronal differentiation (Tozuka et al., 2005). In addition, similar results have been reported that excitatory Cl--mediated signaling via glycine receptors during early development can regulate interneuron differentiation accompanied by a concomitant increase in the number of mitotic cells (McDearmid et al., 2006). Glycine receptors have shown a similar role in GABARs stem and spinal cord, mediating Cl-- conducting ligand-gated ion channels that mediate fast synaptic inhibition in the adult CNS and fast synaptic excitation during early development (Ben-Ari et al., 1989; Cherubini et al., 1991; Wu et al., 1992; Wang et al., 1994; Ehrlich et al., 1999; Kakazu et al., 1999). Conversely, during neurogenesis, activation of NMDARs promotes BrdU-positive NPC proliferation and neuronal differentiation (Joo et al., 2007), and competitively regulates survival of new-born neurons along with NMDAR-containing proximity neurons in the DG (Tashiro et al., 2006b). Although a few studies have shown the possible relevance between excitation and neurogenesis, it is still confusing because under pathological conditions associated with AD, enhanced synaptic plasticity does not rescue impairment of neurogenesis and can even lead to decreased survival of newborn neurons (Poirier et al., 2010). However, this paper does show regulation of neurogenesis by synaptic plasticity.   36 1.7 Brain-derived neurotrophic factor  Brain-derived neurotrophic factor (BDNF) from pig brain was discovered in 1982 and was shown to increase survival of cultured embryonic chick sensory dorsal root ganglion neurons (Barde et al., 1982). Since then, neurotrophin-3(NT-3) (Maisonpierre et al., 1990a) and neurotrophin-4/5 (NT-4/5) have also been  discovered as neurotrophic factors (Hallbook et al., 1991). BDNF shares around 50% amino acid identified with NGF, NT-3 and NT-4/5. The mature neurotrophins associate tightly as biologically active homodimers (Chao and Bothwell, 2002). Pro-neurotrophins, such as pro-BDNF, Pro-NGF, pro-NT3 and Pro-NT-4/5 have different binding characteristics and biological activity in comparison with mature neurotrophins (Lee and Chao, 2001; Lee et al., 2001; Chao and Bothwell, 2002). BDNF binds to the receptor tyrosine kinase TrkB (also known as NTRK2) (Patapoutian and Reichardt, 2001). In addition, NT-4/5 and NT-3 also bind to TrkB. Although NT-3 primarily activates the TrkC receptors, it also has low-binding affinity to TrkB (Barbacid, 1994). Once ligand binds to TrkB receptors they form dimer and activate kinases. Subsequently, receptor autophosphorylation on multiple tyrosine residues creates specific binding sties for intracellular target proteins, such as ras-mitogen-activated protein (ras-MAP), phosphatidylinositol 3-kinases (PI3K), phospholipase C (PLC), Shc (SH2- containing sequence) and cyclic AMP-response element binding protein (CREB) (Kaplan and Miller, 2000; Huang and Reichardt, 2001; Patapoutian and Reichardt, 2001; Segal, 2003). Through TrkB receptors, BDNF activates many intracellular signaling, which affects various functions of the nervous system, such as development, synaptic plasticity and neurodegenerative diseases. BDNF promotes protein synthesis by an increase in translation initiation through PI3k-Akt-mTOR (rapamycin) that is required for BDNF-mediated LTP in the hippocampus (Kang and Schuman, 1996; Takei et al., 2001). In fact, BDNF and its receptor, TrkB can be found in most brain regions of the CNS (Conner et al., 1997; Yan et al., 1997). Particularly, the level of BDNF expression is highly conserved in the hippocampus (Conner et al., 1997). 37 1.7.1 Brain-derived neurotrophic factor and neurogenesis  A variety of neurotrophic factors have been reported to influence cell fate decisions, axonal outgrowth, and dendrite pruning of distinct neuronal populations (Chao, 1992; Acheson et al., 1995; Huang and Reichardt, 2001; Abrous et al., 2005). Newly generated cells exposed to BDNF have shown enhanced neuronal maturation, proliferation, differentiation, and survival in rodent models (Kirschenbaum and Goldman, 1995; Minichiello and Klein, 1996; Goldman et al., 1997; Pincus et al., 1998; Zigova et al., 1998; Suh et al., 2009; Pathania et al., 2010). BDNF has also shown enhanced human NSC differentiation and neuronal cell survival (Poo, 2001). BDNF null mice fail to survive postnatally past 3rd weeks, but heterozygous BDNF knock (+/-) survive exhibiting abnormalities in eating behavior and locomotor (Ernfors et al., 1995; Lyons et al., 1999; Kernie et al., 2000). Furthermore, BDNF has been directly shown to promote neurogenesis (Maisonpierre et al., 1990b; Fukumitsu et al., 1998). The administration of exogenous BDNF or adenoviral BDNF that results in overexpression of BDNF into the lateral ventricle of adult rat brains promotes the number of BrdU-positive cells in the adult OB (Zigova et al., 1998; Benraiss et al., 2001), striatum, septum, thalamus and hypothalamus (Pencea et al., 2001). In the absence of BDNF, one of the progeny from each cell division either generates glia or dies in the subependymal layer of the adult mouse brain (Morshead and van der Kooy, 1992). In contrast, inhibition of glial differentiation by overexpression of a soluble bone morphogenetic protein, noggin, has shown BDNF-mediated neuronal differentiation and recruitment of new neurons in the subependymal zone (Chmielnicki et al., 2004). Furthermore, inhibition of endogenous BDNF has shown a decrease in the survival and proliferation of cortical progenitors as well as suppression of Trk receptor signaling pathways, such as PI3K and MAP kinase kinase (MEK)-extracellular signal-regulated kinase (ERK). The signaling mechanisms underlying neurogenesis suggests that the progenitor cell survival is regulated by PI3K whereas MEK is involved in neuronal differentiation without affecting survival or proliferation (Barnabe- Heider and Miller, 2003; Lim et al., 2008). The MAP kinase with ribosomal S6 kinase (RSK) and 38 CAAT enhancer-binding protein pathway (C/EBP) has been already reported to be relevant to neuronal differentiation (Menard et al., 2002). In addition, neurotrophins stimulate neuronal differentiation by altering the expression of various neural bHLH transcription factors. Neuronal differentiation is promoted by Mash1, Math1, or NeuroD, but suppressed by Hes1 and Hes5 (Ito et al., 2003). The association between BDNF levels and hippocampal neurogenesis has been elucidated and it has been shown that prolonged elevation of hippocampal BDNF increases the number of newly generated cells rather than survival of existing NPCs in the DG (Katoh-Semba et al., 2002). In contrast, BDNF contributes to the survival of newly generated neurons in dietary restricted mice (Lee et al., 2002). Although two experiments have shown different results, it has been demonstrated that DG neurogenesis is regulated by BDNF. A recently study has shown that activation of NMDARs increases both proliferation and differentiation of hippocampal NPCs through vascular endothelial growth factor (VEGF) and BDNF (Joo et al., 2007). Both of these neurotrophic factors have been well studied in their effect on stimulating neurogenesis (Benraiss et al., 2001; Pencea et al., 2001; Jin et al., 2002; Zhu et al., 2003).  1.7.2 Brain-derived neurotrophic factor and long-term potentiation  BDNF and two other members of the neurotrophin family, NGF and NT-3 are expressed at comparatively high levels in both hippocampal pyramidal and dentate granule neurons (Gall and Lauterborn, 1992; Drake et al., 1999). A role of BDNF in plasticity of synaptic transmission has been firstly reported in an increase of the frequency of miniature excitatory postsynaptic potential (mEPSP) in developing neuromuscular synapses (Lohof et al., 1993). For example, application of BDNF, but not NGF, for 2-3 hours, sustains enhancement of synaptic strength in the CA1, which can be blocked by applying the tyrosine kinase inhibitor, K252a (Kang and Schuman, 1995). Accumulating evidence has supported the role of BDNF in hippocampal LTP (Korte et al., 1995; Patterson et al., 1996) and in rescuing this impaired basal synaptic transmission (Patterson et al., 1996). In the absence of BDNF, short-term potentiation (STP) is 39 only detected and BDNF knockout animals also show impaired LTP induction which can be restored by exogenous application of BDNF (Korte et al., 1995; Figurov et al., 1996; Patterson et al., 1996). Furthermore, Tetanic stimulation HFS that induces LTP in the hippocampus promotes a BDNF secretion without altering the level of NT-4, as well as enhances the expression of TrkB (Patterson et al., 1992; Castren et al., 1993), but LTD induced by low frequency stimulation (LFS) fails to increase BDNF release (Gartner and Staiger, 2002). Exercise has been shown to increase BDNF expression in the hippocampus (Neeper et al., 1995; Cotman and Berchtold, 2002). The role of BDNF has been proposed in activity-dependent synaptic plasticity regulating synaptic transmission in the hippocampus (Schinder and Poo, 2000). However, BDNF-induced synaptic potentiation has been debated, as it is unsure whether the BDNF release is primarily from the presynaptic area or from the postsynaptic site. TrkB mutant reduces expression in a normal pattern of TrkB throughout the brain, and affects presynaptic function by reducing the ability of tetanic stimulation to induce LTP, but does not affect postsynaptic glutamate receptors (Xu et al., 2000). Further studies have demonstrated that presynaptic BDNF is required for enhancing presynaptic neurotransmitter release as well as activates L-type voltage-gated Ca2+ channels (Tyler et al., 2002; Zakharenko et al., 2003). In contrast, BDNF enhances synaptic transmission through a phosphorylation mechanism (Levine et al., 1995) and activation of NMDARs by enhancing the phosphorylation of NR1 and NR2B subunits in the postsynaptic density (Suen et al., 1997; Black, 1999). In addition, BDNF- mediated LTP has been demonstrated by BDNF-evoked postsynaptic Ca2+ transient, but not in presynaptic sites (Kovalchuk et al., 2002). In fact, both pre- and postsynaptic mechanisms have been intensively studied in the hippocampus (Minichiello, 2009). In general, activity-dependent synaptic enhancement is considered the mechanism underlying learning and memory. Thus, a role of BDNF in activity-dependent synaptic plasticity such as LTP is thought to be involved in learning and memory mechanisms. It has been demonstrated that BDNF is required for spatial learning tasks such as the Morris water maze (Linnarsson et al., 1997). The signaling mechanisms underlying learning and memory have been provided by evidence that in a 40 positively motivated radial arm maze (RMA) test, consolidation of spatial memory is involved in activation of the TrkB-PI3K signaling pathway and protein synthesis by BDNF whereas activation of MAP kinase appears during acquisition of fear memory in a negatively motivated passive avoidance test (Alonso et al., 2002; Mizuno et al., 2003; Yamada and Nabeshima, 2003). Rapid and selective induction of BDNF expression has been also observed during contextual learning (Hall et al., 2000). A recent study has demonstrated that BDNF is essential for persistence of long-term memory through an ERK-dependent manner.                     41 1.8 Research hypothesis  General cell replacement therapy has been studied as a brain repair strategy where the lost circuitry or dysfunctional cells are replaced by endogenously newly-generated cells or exogenously transplanted NSCs/NPCs. However, most of transplanted NSCs/NPCs are not functionally differentiated into neurons, yet still show cognitive improvement. Therefore, transplantation effects rather than cell replacement on brain recovery function and enhancement of neuronal differentiation of transplanted NPCs/NSCs must be investigated. In order to address the role of NSCs/NPCs in the brain, a different approach is required to maximize recovery as well as understand the transplantation effects of NSCs/NPCs. The hypothesis of my proposed research is that exogenously transplanted NPCs are neuroprotective in an animal model of Aβ-induced neurotoxicity by attenuating pro-inflammatory cytokines from the microglia and that NSC/NPC proliferation/survival and neuronal differentiation are enhanced by NMDAR-dependent LTP in the hippocampus. Therefore, NPC transplantation itself is neuroprotective in Aβ-induced neurotoxicity and NMDAR-dependent LTP-induced facilitation of proliferation/survival and neuronal differentiation of both endogenous NPCs and transplanted NSCs could serve as a rational therapeutic intervention in neurodegenerative disorders.         42 1.9 Summary of research objectives  The overall thesis objective is to investigate the neuroprotective effects of transplanted NPCs in an Aβ1-42 peptide-injected inflamed AD brain and to facilitate endogenous neurogenesis as well as increase neuronal survival of exogenously transplanted NSCs by NMDAR-dependent LTP.  The specific objectives are listed below:  1. To investigate neuroprotective effects of NPC transplantation by inhibiting inflammation in Aβ1-42 injected rat hippocampus.  2. To determine the effects of NMDAR-dependent electrical LTP in promoting endogenous proliferation, survival and neuronal differentiation of NSC in the hippocampal DG as well as increasing survival and neuronal differentiation of transplanted NSCs in the hippocampal CA1.  3. To investigate the effects of chemically induced LTP via the NMDAR co-agonist, glycine in mediating proliferation and differentiation of NSCs in vitro.  4. To assess signaling pathways of LTP-induced neurogenesis in activating BDNF-TrkB.  The specific objectives 1 to 4 are presented in thesis chapter 2 to 3.      43 2 Neural progenitor cells attenuate inflammatory reactivity and neuronal loss in an animal model of inflamed AD brain  2.1 Introduction  Alzheimer’s disease (AD) is a chronic neurodegenerative disorder that in advanced stages is characterized by increased levels of amyloid beta (Aβ) peptide deposits, neurofibrillary tangles, abnormalities in neuronal and synaptic function and evidence for ongoing inflammatory reactivity (Akiyama et al., 2000; Eikelenboom et al., 2006). The changes in underlying brain processes are manifest in a marked deterioration in memory and cognition. Numerous risk factors such as aging are associated with, and exacerbate, the loss of function in AD brain (de la Torre, 2002). Importantly, the multiple processes and risk factors contributing to the slow progression of AD pathology compromise therapeutic strategies for treatment of the disease. Transplantation of neural stem cell (NSC) constitutes a putative therapeutic treatment for cell replacement in brain damage due to their intrinsic properties of self-renewal and capability for differentiation into different cell types including neurons. However, evidence also suggests a stem cell therapy may confer neuroprotection by means other than cell replacement including the enhancement of neurotrophic factors (Ostenfeld et al., 2002) or by diminishing levels of putative neurotoxic factors. In the latter case, recent work has indicated efficacy of NPC may involve inhibition of inflammatory factors and responses (Ohtaki et al., 2008; Kim et al., 2009). Overall, beneficial effects of stem cell administration have been reported in a number of animal models including multiple sclerosis (Pluchino et al., 2003), PD (Redmond et al., 2007) and stroke (Lee et al., 2008). A recent study (Blurton-Jones et al., 2009) has provided the first report for use of stem cell therapy in AD with the finding that transplantation improved cognitive 44 performance in transgenic mice by elevation of brain-derived neurotrophic factor (BDNF). Since chronic inflammation is a critical facet of AD brain, we reasoned that transplantation of neural progenitors could serve as a feasible strategy to attenuate ongoing inflammatory reactivity and thereby protect neurons. Furthermore, capacity for neural progenitors to engage in chemotactic activity has recently been reported (Tran et al., 2007; Miller et al., 2008), a necessary requirement for increased mobility in response to inflammatory factors. We have tested this hypothesis by measuring migration of transplanted neural progenitor cell (NPC) and effects of NPC transplantation on inflammatory responses mediated by microglia and astrocytes, levels of the proinflammatory cytokine, tumor necrosis factor-α (TNF-α) and neuronal viability in an animal model of inflamed AD brain. This model uses intra-hippocampal injection of amyloid- beta peptide (Aβ1-42) to induce marked inflammatory reactivity with concomitant neuronal damage in rat brain (Ryu and McLarnon, 2006, 2008).               45 2.2 Methods  2.2.1 Neurosphere cultures  Spheres of NPCs were grown from dissociated telencephalon tissue of 14 days Sprague Dawley (SD) rat embryos in neurobasal medium (GIBCO) containing B27 (GIBCO) supplement with 20 ng/ml basic fibroblast growth factor (bFGF; PeproTech), 10 ng/ml epidermal growth factor (EGF; PeproTech) and 10 ng/ml leukemia inhibitory growth factor (LIF; Chemicon). The procedure of changing culture medium every 3 days results in the formation of neurospheres (Palmer et al., 1997).  2.2.2 Immunostaining of neurospheres  Neurospheres were plated on 12 mm round cover glass (Deckglaser). Spheres were fixed in 4% paraformaldehyde (PFA) for 10 minutes, permeabilized in 0.1% Triton X-100 (Sigma) in phosphate buffered saline (PBS) for 5 minutes, blocked in 5% goat serum in PBS for 1 hour, and incubated at 4˚C overnight with the following primary antibodies: anti-nestin (1:200; Chemicon), anti-vimentin (1:500; Sigma), anti- microtubule associated protein 2 (MAP2, 1:1000; Chemicon), anti-glial fibrillary acidic protein (GFAP) (1:100; Sigma), and anti-green fluorescent protein (GFP, 1:1000; Invitrogen). The spheres were subsequently incubated with anti-mouse and anti-rabbit secondary antibodies conjugated to Alexa Fluor 488 and 555 (IgG, 1:1000; Molecular probes) for 1 hour at room temperature. The spheres were then incubated in 4',6- Diamidino-2-phenylindole dihydrochloride (DAPI, 1:1000; Sigma) for 30 seconds and coverslipped in polyvinyl alcohol mounting medium with DABCO-antifade solution (Sigma). Control immunostaining was performed by omission of the primary antibody. Fluorescent images were obtained from a Leica DMIRE2 microscope using the software OpenLab 3.7.  46 2.2.3 Stereotaxic injection of fibrillar Aβ1-42  All experimental procedures were approved by the University of British Columbia Animal Care Ethics Committee, adhering to guidelines of the Canadian Council on Animal Care. Full- length peptide (Aβ1-42; California Peptide) was prepared as previously described (Franciosi et al., 2006; McLarnon et al., 2006). The compounds were first dissolved in 35% acetonitrile (Sigma) and further diluted to 500 μM with incremental additions of PBS with vortexing. The peptide solution was subsequently incubated at 37°C for 18 hours to promote fibrilization and aggregation and stored at -20°C. Intra-hippocampal injection of Aβ1-42 was performed as previously described (Ryu and McLarnon, 2006, 2008). In brief, male Sprague-Dawley (SD) rats (Charles River) weighing 280-300 g were anesthetized (ketamine/xylazine, intraperitoneal injection (i.p.)) and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA) and received unilateral injection of 2 nM Aβ1-42 at the following coordinates: anteriorposterior (AP): -3.3 mm, mediallateral (ML): -1.6 mm, dorsoventral (DV): -3.6 mm, from bregma (Paxinos and Watson, 2005). Control animals received injection of PBS at these coordinates.  2.2.4 Transplantation of GFP labeled neural progenitor cells  Dissociated NPCs in Hank’s balanced salt solution (HBSS) were transduced with lentiviral vectors carrying an enhanced GFP (pHR’-CMV-GFP). The efficiency of GFP expression levels was quantified in vitro. Approximately 5 x 105 NPCs were seeded and infected with 3-fold higher titer lentivirus (compared with seeded cell density) in 12mm coverslips in vitro. Three days after infection, NPCs were fixed with 4% and placed under a fluorescence microscope for GFP measurement: the results showed approximately 73% of NPCs expressed GFP expression. The transduced NPC-GFP (5 x 104, 3 μl) were then stereotactically transplanted (0.20 μl/min) into the hippocampus. Site of transplantation was chosen close to the peptide injection site at the following coordinates from bregma (AP: -3.3 mm, ML: -1.8 mm, DV: -3.2 mm) as 47 previously described (Ryu et al., 2004). For control cell graft, dead NPC were prepared by repeated cycles of freezing and thawing and used as control graft (Shihabuddin et al., 1995). Transplantation was performed 3 days after PBS and Aβ1-42 injection. Immunosuppressive agents were not used in the transplantation protocols due to the possibility of anti-inflammatory effects of the agents that could complicate immune-modulatory actions of NPC in vivo.  2.2.5 Immunohistochemical analysis  Seven days after NPC transplantation, rats were anesthetised and killed by transcardiac perfusion of saline, followed by 4% PFA. Brains were then removed, post-fixed, cryoprotected, and sectioned into 40 μm throughout the hippocampus (Ryu and McLarnon, 2008). Free-floating sections were processed for immunohistochemistry as described previously (Ryu and McLarnon, 2008). Briefly, sections were permeabilized in 0.2% Triton X-100, blocked with 10% goat serum, and incubated overnight at 4°C with the primary antibodies: anti-GFP (1:1000; Invitrogen), anti- ionized calcium-binding adapter molecule 1 (Iba-1, 1:1000; Wako Chemicals), anti-GFAP (1:1000; Sigma), anti-tumor necrosis factor-alpha (TNF-α, 1:200; Cedarlane Laboratories Ltd), and anti-MAP2 (1:500; Sigma). Sections were incubated with secondary antibodies for 1 hour at room temperature, mounted on Superfrost/Plus microscope slides (Fisher Scientific), and coverslipped. For immunostaining controls, primary antibodies were omitted from the staining procedures. For double immunofluorescence staining, free-floating sections were incubated overnight at 4ºC with primary antibody to GFP (1:1000; Invitrogen) with nestin (1:500; Chemicon), GFAP (1:1000; Sigma), or MAP2 (1:500; Sigma) and incubated for 1 hour with a mixture of Alexa Fluor-conjugated 488 anti-rabbit IgG (1:100; Molecular Probes) and Alexa Fluor 594-conjugated anti-mouse IgG (1:100; Molecular Probes). Three coronal hippocampal sections were used for immunohistochemical analysis. In order to ensure consistency within and between groups, matched hippocampal tissue sections were always processed and all microscopy parameters were kept constant throughout the experiments. Immunofluorescence 48 images were examined under a Zeiss Axioplan 2 fluorescent microscope (Zeiss) using a DVC camera (Diagnostic Instruments) with Northern Eclipse software (Empix Imaging) and analyzed for colocalization of staining using National Institutes of Health Image J.  2.2.6 Cell-associated immunostaining  The extents of microgliosis (Iba-1 marker), astrogliosis (GFAP marker) and TNF-α immunoreactivity induced by intra-hippocampal Aβ1-42 or control PBS injections, were evaluated by measuring the marker pixel intensities from five hippocampal sections (Ryu and McLarnon, 2008; Ryu et al., 2009b). Immunostaining was done over the specific areas of the DG, molecular layer (ML) and granule cell layer (GCL). The immunostained section images were digitized and analyzed using the image analysis program NIH version 1.57 (Wayne Rasband, NIH). The overall neuronal viability was assessed by measuring the immunoreactivity of MAP2 staining in the ML and GCL of the hippocampus. All quantitative analyses were performed in a blinded manner.  2.2.7 Statistical analysis  In each stained section, the hippocampal subregion boundaries were outlined. The GCL region was defined as the superior blade of the dentate gyrus. The ML region was defined as the area between GCL border and the hippocampal fissure. Four fields within the GCL and ML regions were then selected in each section. Intensity of pixels above a predetermined threshold level of staining intensity was measured. All quantitative analyses were performed in a blind manner All data are expressed as means ± SEM. Statistical significance of differences for group comparisons was assessed using analysis of variance followed by Bonferroni’s post hoc test or Student’s t test. Significance was set at p < 0.05. 49 2.3 Results  2.3.1 Patterns of distribution and differentiation of transplanted NPC, in vivo  Initial studies demonstrated that cultured NPC expressed characteristic markers for undifferentiated stem cells including nestin and vimentin; NPC also expressed the astrocytic marker, GFAP but not neuronal MAP2 (data not shown). As shown in Figure 2-1A, successful transduction of lentiviral vector-GFP was demonstrated in cultured NPC prior to cell intra- hippocampal transplantation. Transplantation of GFP-labeled NPC into the DG was carried out 3 days subsequent to intra-hippocampal injections of control PBS or Aβ1-42 (at 2 nM). Immunohistochemical analysis was carried out at 7 days following NPC transplantation (10 days post-Aβ1-42/PBS injection). Representative GFP immunostaining, in the ML and GCL, indicated increased numbers of NPC in the vicinity of Aβ1-42 (right panel, Figure 2-1B), compared with PBS (left panel, Figure 2-1B), injection site. In order to assess dispersion and net migration of NPC from the site of transplantation, immunoreactivity of GFP-positive cells was measured in regions between the sites of peptide/PBS injection and NPC transplantation. The results (Figure 2-1C) demonstrated considerably increased GFP immunoreactivity in these areas in Aβ1-42- injected, relative to PBS-injected, hippocampus. Overall, intensity of fluorescence GFP immunoreactivity was increased by x2.8-fold with peptide, compared with PBS, injection. Undifferentiated NPC exhibit a number of cell-specific properties and markers such as nestin. We examined for expression of characteristic properties of transplanted cells in vivo in peptide- injected hippocampus. Representative staining patterns of GFP with the different cellular markers (nestin, GFAP and MAP2) are presented in Figure 2-2. Results from double- immunostaining analysis demonstrated GFP-labeled cells to express nestin and GFAP (Figure 2-2, upper and middle panels). Overall, we found in excess of 90% of NPC showed expression of both nestin and GFAP. However, no evidence for MAP2 colocalization with GFP was found in Aβ1-42-injected hippocampus (Figure 2-2, lower panels). The lack of MAP2 association with GFP 50 immunoreactivity suggests that with short term transplantation little or no NPC differentiated into neurons.  2.3.2 Effect of NPC on Aβ1-42-induced inflammatory reactivity  The results shown in Fig 1 indicate NPC migration in response to intra-hippocampal Aβ1-42- injection. Since microgliosis and astrogliosis are upregulated after peptide injection, gliosis could be modulated in the presence of NPC grafting. To examine this point, the effects of 7 days NPC transplantation on microglial and astrocyte inflammatory responses and levels of the proinflammatory cytokine, TNF-α were examined in five animal groups; Aβ1-42 or PBS injected rat hippocampus, Aβ1-42 plus NPC, Aβ1-42 plus dead NPC and NPC alone; PBS served as a control for peptide injection and dead NPC were used as a control for NPC. Representative immunostaining for microglia (Iba-1 marker), localized to areas between injection (Aβ1-42 or PBS) sites and NPC transplantation site, is shown for the different experimental groups (10 days post-Aβ1-42/PBS injection) in Figure 2-3A (upper panels). Peptide- injected brain demonstrated a considerably elevated Iba-1 immunoreactivity compared with PBS-injection. NPC transplantation in Aβ1-42-injected animals showed efficacy in reducing extents of Iba-1 immunoreactivity, however, transplantation of dead NPC with peptide was ineffective in reducing microglial proliferative responses. NPC transplantation alone showed a pattern of Iba-1 immunoreactivity similar to PBS control. Quantification of data is presented in Figure 2-3B (upper bar graph). Overall, microgliosis (measured as area density of Iba-1 immunoreactivity in ML/GCL) was increased x4.3-fold in Aβ1-42, relative to PBS, injected brain. Transplantation of NPC in peptide-injected animals significantly reduced microgliosis (by 38%) compared with Aβ1-42-injected animals receiving no transplantation. Levels of Iba-1 immunoreactivity were not significantly altered with application of dead NPC in peptide-injected animals. NPC transplanted animals, in the absence of peptide administration, showed low extents of microgliosis. 51 Representative immunofluorescent staining for GFAP, in the same regions used for analysis of microgliosis, is shown in Figure 2-3A (middle panels) for the different animal groups. Peptide- injected brain exhibited an increased GFAP immunoreactivity relative to PBS control. Interestingly, levels of astrogliosis appeared relatively unchanged in Aβ1-42-injected rats receiving NPC transplantation. GFAP immunoreactivity in animals receiving transplantation alone was similar to marker immunoreactivity with PBS control injection.  Quantification of data (Figure 2-3B, middle bar graph) showed astrogliosis to be significantly increased (x4.6-fold) in peptide, relative to PBS, injected rat brain. Although a small decrease in GFAP immunoreactivity was measured (14%) with NPC transplantation in peptide-injected brain, this effect was not significant. Extents of GFAP immunoreactivity were not significantly different between animals receiving Aβ1-42 and Aβ1-42 + dead NPC or between groups administered PBS injection and ones receiving NPC transplantation alone. Expression of TNF-α was minimal in PBS-injected hippocampus with high expression of the cytokine evident in peptide-injected brain (Figure 2-3A, lower left panels). Transplantation of NPC, but not dead cells, was highly effective in attenuating expression of TNF-α in peptide- injected hippocampus. NPC transplantation alone had no effect to alter levels of the cytokine compared to PBS control. Overall, expression of TNF-α in ML/GCL was increased x6.7-fold in Aβ1-42, compared with PBS, injected hippocampus Figure 2-3B, lower bar graph). Transplantation of NPC into peptide-injected brain significantly reduced levels of the pro- inflammatory cytokine, by 40%, compared with Aβ1-42 injection alone. No significant differences in TNF-α immunoreactivity were measured between peptide and peptide plus dead NPC animal groups or between PBS injected, and NPC transplanted, brain.  2.3.3 Effect of neural progenitors on Aβ1-42 -induced neuronal injury  A critical objective of this work was to determine efficacy of NPC transplantation on neuronal viability. The region of study was the same as for assessment of NPC migration and gliosis, 52 localized to areas between injection and transplantation sites. Representative high magnification patterns of immunostaining for neurons (MAP2 marker) are presented for PBS and Aβ1-42 injected hippocampus (Figure 2-4A). The results indicate a considerable loss of MAP2-positive neurons with Aβ1-42, compared to PBS, injection (Figure 2-4A, left panels). NPC transplantation in peptide-injected animals (Figure 2-4A, second panel from right) was effective in attenuating the loss of neurons. Dead NPC were ineffective when applied in peptide-injected brain (data not shown). The control NPC graft alone (Figure 2-4A, right panel) presented a similar pattern of MAP2 immunoreactivity as found with PBS injection. Overall (n=5 animals/group), MAP2 immunoreactivity in ML and GCL was diminished by 45% in Aβ1-42, relative to PBS, injected animals (Figure 2-4B). Animals receiving NPC transplantation showed a significant (26%) increase in numbers of MAP2-positive neurons compared to peptide-injected animals not receiving NPC treatment. Levels of MAP2 immunoreactivity were not significantly different between Aβ1-42 alone and Aβ1-42 plus dead NPC or between PBS-injected and NPC- transplanted animals.             53 2.4 Discussion  The primary objective of this study was to provide evidence for the potential clinical utility of NPC transplantation in AD brain. The major findings from the work are that NPC transplantation significantly inhibits inflammatory reactivity and provides neuroprotection in the Aβ1-42-injected rat hippocampus. The results constitute the second report of beneficial effects of stem cell treatment in AD; a recent study has demonstrated improvement in cognitive behavior in transgenic animals with effects attributed to increased levels of hippocampal BDNF (Blurton- Jones et al., 2009). As discussed below, our data showing correlation between inflammatory reactivity and neuronal viability support the possibility that NPC actions to attenuate inflammatory responses may have utility in reducing neuronal damage in inflamed AD brain. We found that cultured NPC, isolated from rat brain, exhibited a spectrum of characteristic features of undifferentiated stem cells including expressions of nestin, vimentin and GFAP. Efficient transduction of the cells with GFP was demonstrated for the cultured NPC prior to their in vivo transplantation into rat hippocampus at 3 days following intra-hippocampal injections of PBS control or Aβ1-42. At 7 days following NPC transplantation (10 days after Aβ1-42 or PBS), immunohistochemical analysis showed higher dispersed GFP immunoreactivity between sites of injection and transplantation with peptide, relative to PBS, injection. These results are consistent with Aβ1-42 injection stimulating a migration of NPC from transplantation to injection site. However, over the whole hippocampus we observed no evident differences between GFP immunoreactivity with peptide or PBS injection suggesting that NPC survival was not a factor in our experiments. Double staining in vivo showed prominent immunoreactivity of GFP, colocalized with progenitor cell nestin and GFAP suggesting a NPC phenotype as undifferentiated cells. No evidence for neuronal differentiation of transplanted NPCs was evident (marker MAP2), a result which could reflect the relatively short duration of NPC grafting employed in this work. We conclude migration of NPC from sites of transplantation to sites of injection was 54 enhanced in peptide-injected, compared to PBS-injected, hippocampus. These findings would be consistent with the presence of chemotactic stimulatory signals which increase NPC mobility in peptide-injected brain. The initiating stimulus for induction of increased NPC migration could be due to direct deposition of Aβ1-42 or indirectly due to signals from microglia (see below) that have been activated by peptide. In the latter case, we have documented that injection of Aβ1-42 into the DG is a potent stimulus for induction of microglial chemotactic responses mediated by a specific receptor for vascular endothelial growth factor (VEGF) (Ryu et al., 2009b). Interestingly, recent work has reported stromal cell derived factor-1 and its receptor CXCR4 as modulators of progenitor cell migration in the dentate gyrus (DG) (Tran et al., 2007; Bhattacharyya et al., 2008; Miller et al., 2008). Intra-hippocampal injection of Aβ1-42 was associated with considerable increases in microgliosis and astrogliosis compared with PBS control. Transplantation of NPC after Aβ1-42 injection significantly inhibited microgliosis, but not astrogliosis, in proximity to peptide injection site. Microgliosis was not altered with dead NPC administered to peptide-injected hippocampus and NPC transplantation alone was associated with similar levels of gliosis as for PBS injection. Cytokine levels are enhanced in AD brain (Griffin et al., 1998) and our results showed elevated TNF-α, a pro-inflammatory cytokine with autocrine function in microglia (Benveniste and Benos, 1995), in Aβ1-42-injected hippocampus. Administration of NPC, but not dead progenitor cells, attenuated levels of TNF-α. As discussed below, the effects of NPC grafting to inhibit microgliosis and levels of TNF-α may be correlated. The injection of Aβ1-42 was associated with a loss of neuronal viability, compared with PBS control injection, consistent with previous findings (Franciosi et al., 2006; McLarnon et al., 2006; Ryu and McLarnon, 2008). Importantly, transplantation of NPC, but not dead cells, was effective in diminishing loss of neurons in peptide-injected brain. Although underlying neuroprotective mechanisms are not well understood, this result could be linked with the finding that NPC transplantation was efficacious in attenuating microgliosis with no significant actions to alter astrogliosis. One possibility to account for effects of NPC on microglial responses is that 55 peptide-induced activation of microglia increases their production of chemokines including monocyte chemoattractant protein-1 (MCP-1) (Lue et al., 2001a) and interleukin-8 (IL-8) (Franciosi et al., 2005). In this event preferential migration of NPC to areas exhibiting microglial proliferative responses may follow. Increased migration of stem cells induced by microglia (Aarum et al., 2003), and specifically by the factor MCP-1 (Widera et al., 2004), have been reported, in vitro. Subsequent NPC release of neurotrophic factors (Blurton-Jones et al., 2009) inhibits microglial activation by blocking cell-specific inflammatory factors such as major histocompatibility class II (Neumann et al., 1998). Since Aβ-stimulated microglia are potent producers of TNF-α (Benveniste and Benos, 1995), NPC-mediated effects to decrease microglial activation would be consistent with the diminished levels of the pro-inflammatory cytokine as found following NPC grafting. Indeed, our results demonstrated very similar extents of reductions (about 40%) in microgliosis and levels of TNF-α with NPC transplantation. Previous studies have suggested that pharmacological treatments that inhibit microglial activation can attenuate neuronal damage in animal models of AD (Ryu and McLarnon, 2006; Ryu et al., 2009b). Overall, our findings are consistent with an enhanced migration of NPC in response to signals from peptide-activated microglia with NPC releasing factors which in turn act to inhibit microglial inflammatory reactivity. At present, however, the specific NPC-dependent factors coupled to reduction in inflammatory responses and neuroprotection have not been determined. Our results, together with those reported in (Blurton-Jones et al., 2009), provide a proof of principle that stem cell therapy could be efficacious in AD. A number of questions need to be addressed in AD animal models including the nature of microglial signals which mediate NPC migration and NPC-derived factors which modify microglial activation and inflammatory responses. The production and release of growth factors other than BDNF (Blurton-Jones et al., 2009) by NPC could also contribute to increased neuronal viability and enhanced cognition. Another unresolved question is the possibility that NPC could also directly differentiate into functional neurons in diseased brain suggesting the utility of future work in using longer 56 durations of NPC transplantation in AD animal models.                          57 Figure 2-1 GFP-labeled NPC in vitro and diffusion of GFP-positive neural progenitor in vivo (A) GFP staining of cultured NPC. (B) Representative immunostaining for GFP-positive NPC in molecular (ML) and granule cell (GCL) layers of the DG for PBS and Aβ1-42-injected rat brain.. The site of PBS or Aβ1-42 injection is indicated by a red asterisk; the location of NPC transplantation is indicated by a yellow asterisk. (C) Quantification of GFP fluorescence intensity in region between site of injection (Aβ1-42 or PBS) and site of NPC transplantation (n=4 animals/group, * denotes p < 0.05).   58 Figure 2-2 Expression of markers in injected NPC Representative double staining for GFP association with nestin (upper panel), GFAP (middle panel) and MAP2 (bottom panel), scale bar = 10 µm.                           59 Figure 2-3 Effects of NPC transplantation on inflammatory reactivity (A) Representative staining for microglial responses (Iba-1 marker, upper panels), astroglial responses (GFAP marker, middle panels) and levels of TNF-α (lower panels) in GCL and ML following 10 days intra-hippocampal injections of PBS/Aβ1-42 and 7 days transplantation of NPC/dead NPC. The animal groups (panels, left to right) are for PBS, Aβ1-42, Aβ1-42 + NPC, Aβ1- 42 + dead NPC and NPC alone, scale bar represents 100 µm. (B) Quantification of data for Iba-1 (left bar graph), GFAP (middle bar graph) and TNF-α (right bar graph) for the different animal groups (n=5 animals/group, * denotes p < 0.05 compared with PBS, # denotes p < 0.05 compared with Aβ1-42).  60 Figure 2-4 Effects of NPC transplantation on neuronal viability (A) Representative high magnification of MAP2 immunoreactivity in GCL and ML. Animal groups (panels left to right) are for PBS, Aβ1-42, Aβ1-42 + NPC and NPC alone. (B) The bar graph presents quantification of MAP2 immunoreactivity in GCL/ML (n=5 animal groups, * denotes p < 0.05 vs. PBS, # p < 0.05 vs. Aβ1-42).      61 3 NMDAR-dependent long-term potentiation promotes proliferation/survival and neuronal differentiation of neural progenitor/stem cells  3.1 Introduction  Neuronal loss is a common pathology of a large number of neurodegenerative diseases, and transplantation of neural stem cells (NSCs) to replace the lost neurons is considered a promising potential treatment (Brustle and McKay, 1996; Ogawa et al., 2002). However, the sustained survival, and neuronal differentiation of exogenously transplanted NSCs, as well as their functional integration into host neuronal circuitries remain a major challenge (Brustle and McKay, 1996). Thus, development of clinically relevant and practicable protocols that can promote proliferation/survival, neuronal differentiation and their functional integration into neuronal network in the host brain is urgently required to improve and augment the clinical use of exogenously transplanted NSCs as part of effective therapies to repair neuronal networks following neuronal damage. Evidence accumulated in the last few years has suggested that activation of N-methyl-D- aspartate receptor (NMDAR), a subtype of the glutamate receptors, might be involved in regulating proliferation,  neuronal differentiation and survival of newly generated neurons in the hippocampal dentate gyrus (DG) (Tashiro et al., 2006b; Joo et al., 2007). However, how NMDARs exert these actions remains poorly understood. NMDARs are known to be required to produce certain forms of activity-dependent synaptic plasticity and NMDAR-dependent long- term potentiation (LTP) and long-term depression (LTD) are most-well characterized in the hippocampus. These forms of synaptic plasticity have long been proposed to play critical roles in learning and memory and developmental maturation of neuronal circuits (Bliss and 62 Collingridge, 1993; Malenka and Nicoll, 1999). However, a recent study has suggested a role of NMDAR-dependent LTP in enhancing proliferation and survival of neuronal progenitor cells (NPCs) in the hippocampal DG (Bruel-Jungerman et al., 2006). In addition, evidence accumulated in recent years has also implicated a potential role of NMDARs, and possibly synaptic plasticity, in regulating neuronal survival and death (Lu et al., 2001; Hardingham et al., 2002; Liu et al., 2007; Leveille et al., 2008; Collingridge et al., 2010). Together, these results strongly suggest that NMDARs and consequently, synaptic plasticity may promote neurogenesis by increasing proliferation/survival and neuronal differentiation of NPCs and also possibly transplanted NSCs. In the present study, we therefore set out to investigate potential roles of NMDAR-dependent synaptic plasticity, particularly LTP, in mediating proliferation/survival and neuronal differentiation of endogenous NPCs in the hippocampal DG and of exogenously transplanted NSCs into the hippocampal CA1. In addition, we have also examined the potential mechanisms using a chemical model of LTP in co-cultures of green fluorescent protein (GFP)-labeled NSCs and hippocampal neurons in vitro.               63 3.2 Materials and methods  3.2.1 Primary cell culture and neural stem cell isolation  NSCs were isolated directly from the telencephalon, a known developmental precursor of the cerebrum, at embryonic day 14 (E14) from Sprague Dawley (SD) rats. The dissociated telencephalon cells were cultured in neurobasal media containing B-27 supplement without retinyl acetate (GIBCO) or N2 supplement with 20 ng/ml basic fibroblast growth factor (bFGF; PeproTech), 10 ng/ml epidermal growth factor (EGF; PeproTech) and 10 ng/ml leukemia inhibitory growth factor (LIF; Chemicon). The media were changed every 3 days. This procedure resulted in the formation of neurospheres, an aggregate form of NSCs. In order to generate secondary neurospheres, primary neurospheres were dissociated and re-plated onto 24-well dishes for an additional 10-14 days (Lee et al., 2005). This second isolation and plating were necessary to obtain pure NSCs, as conventional cell sorting was not feasible due to the lack of a specific NSC surface marker. Primary cultured hippocampal neurons were prepared and cultured as previously described (Lu et al., 2001; Peineau et al., 2007). Briefly, hippocampal neurons were prepared from E18 SD rats and grown in neurobasal media (GIBCO) with B-27 supplement (GIBCO) containing retinyl acetate, 0.5 mM glutamaxTM-1. For initial plating, 25 μM L-glutamic acid (GIBCO) was added. The media were replaced every 3 days. 10 μM of 5-Fluoro-5′-deoxyuridine (5-FDU; Sigma-Aldrich) was added to inhibit the growth of glial cells in hippocampal neuron culture.  3.2.2 Lentivirus production and infection in vitro  To produce lentivirus constructs, three different plasmids were transfected into human embryonic kidney (HEK)-293T cells: the transfer plasmid pHR’-CMV- containing enhanced GFP, the packaging plasmid PR8-2 and the envelope plasmid PVSVG. Lentiviral particles were 64 collected from the media once per day for 4 days and centrifuged at 27,500 rpm for 3 hours to concentrate. Lentiviral particles were resuspended in phosphate buffered saline (PBS) and tittered by serial dilution on primary cultured cells from the brain. Titers were 6.36 x 108 TU/ml (Skarsgard et al., 2005). For NSC-neuronal co-culture, 3 days after the viral infection, GFP- labeled NSCs were re-plated with dissociated hippocampal neurons and maintained in the hippocampal culture media.  3.2.3 Retrovirus preparation and intra-hippocampal injection into the dentate gyrus  Retrovirus was generated using a transient transfection approach in HEK-293T cells. Transfection of the retroviral vectors cytomegalovirus immediate early enhancer-chicken β-actin hybrid-GFP (CAG-GFP), viral proteins (CMV-gag/pol) and capsid (CMV-vsvg) into HEK-293T cells was performed using calcium phosphate (Promega). Virus-containing supernatant was harvested at 48 and 96 hours after transfection, and concentrated by two rounds of ultracentrifugation (Tashiro et al., 2006a; Zhao et al., 2006). All other procedures were the same as the lentivirus concentrate. Titers were 9.51 x 108 TU/ml. Three days prior to in vivo field recording, retrovirus was directly infused onto newly generated cells in the DG through the intra- hippocampal injection manner (3.5 mm posterior to bregma, 2.0 mm lateral to midline, ~3.6 mm ventral). Rats were anesthetized with sodium pentobarbitol (65 mg/kg intraperitoneal injection (i.p.); MTC Pharmaceuticals) and placed in a stereotaxic frame (David Kopf instruments). Stereotaxic unilateral injection of retrovirus was performed with a stereotaxic-motorized nano- injector (Stoelting) as previously described (Ryu et al., 2004). The skin was sutured after removing the needle. The animals were allowed to recover and were returned to their cages    65 3.2.4 In vivo field EPSP recordings  Field excitatory postsynaptic potentials (fEPSPs) from the CA1 and DG region of the hippocampus were recorded as previously described (Farmer et al., 2004; Fox et al., 2007; Wong et al., 2007). Five to six-week-old SD rats were anesthetized using sodium pentobarbitol (65 mg/kg, i.p; MTC Pharmaceuticals) and placed into a stereotaxic frame (David Kopf instruments). Supplemental anesthesia was provided hourly at 10% of the initial dose for the duration of the recording. Rectal temperature was maintained at 37 ± 0.5°C during the course of the surgery using a temperature controller (Harvard Instruments). The scalp was opened and separated. Trephine holes were drilled into the skull and bipolar stimulating electrodes (4.0 mm posterior to bregma, 3.0 mm lateral to midline for the CA1 and 7.4 mm posterior to bregma 3.0 mm lateral to midline for the DG) and monopolar recording electrodes (3.5 mm posterior to bregma, 2.0 mm lateral to midline for both the CA1 and DG) were positioned in the CA1 stratum radiatum area or the DG granule cell layer (GCL) region of the hippocampus respectively. Final depths of the electrodes were adjusted to optimize the magnitude of the evoked responses. fEPSPs were adjusted to 60% of maximal response size for testing. Stimulation was generated by an analog-to-digital interface (1322A, Axon Instruments) and a Digital Stimulus Isolation unit (Getting Instruments). Pyramidal or granule neuron responses to the Schaffer collateral stimulation or MPP were recorded by a differential amplifier (P55 A. C. pre-amplifier, Astro-Med Inc.) and analyzed with WinLTP software (WinLTP Ltd.). Responses were evoked by single pulse stimuli and were delivered at 20-s intervals. A stable baseline was recorded for 30 minutes. LTP was induced by applying high frequency stimulation (HFS; 4 trains of 50 pulses at 100 Hz, 15-s inter-train interval) for the CA1 or the strong theta-patterned simulation (sTPS; 4 trains of 10 bursts of 5 pulses at 400 Hz with a 200 ms inter-burst interval, 15-s inter-train interval) (Farmer et al., 2004) for the DG. 3-[(±)-2-carboxypiperazin-4-yl]-propyl-1-phosphate (CPP; 10 mg/kg, 90 minutes before HFS stimulation, Tocris Bioscience) was injected 90 minutes prior to either sTPS or HFS stimulation to block the induction of LTP. 66 3.2.5 Neural stem cell transplantation in the CA1  Two to five hours after CA1 field recording, rats were anesthetized with sodium pentobarbitol (65 mg/kg, i.p; MTC Pharmaceuticals) and replaced into a stereotaxic frame (David Kopf instruments). Stereotaxic unilateral transplantation of GFP-labeled NSCs was performed with a stereotaxic-motorized nano-injector (Stoelting). Injection coordinates for the hippocampus were the same as the recording electrode. GFP-labeled NSCs (5 x 103, 1 μl, 0.20 μl/min) were slowly injected into the CA1 region of the hippocampus using a 10 μl Hamilton syringe fixed to a 26- gauge needle. After injection, the syringe was left in place for an additional 5 minutes and the needle was slowly withdrawn. The skin was sutured after removing the needle. The animals were allowed to recover and were returned to their cages. All animal protocols were approved by the UBC Animal Care Ethics Committee while adhering to the guidelines of the Canadian Council on Animal Care. All efforts were made to minimize animal suffering and to reduce the number of animals used.  3.2.6 Chemical LTP induction and conditioned medium stimulation  Electrical stimulation of neurons for the induction of LTP is typically used. However, these methods are limited to a localized synaptic area. Therefore, a global application of chemical- induced synaptic plasticity, such chemical LTP (cLTP) was used to reliably induce LTP in vitro. NMDAR-dependent cLTP in the culture was induced by brief bath application of high concentration 200 μM glycine, a co-agonist for NMDAR, 200 mM sucrose to stimulate presynaptic release (Bekkers and Stevens, 1995, 1996; Rosenmund and Stevens, 1996), and 5 μM strychnine to block strychnine-sensitive glycine-gated Cl- receptors (Thomson et al., 1989) in the Mg2+-free extracellular solution (ECS, pH 7.35, 140 mM, NaCl, 5.4  mM KCl, 1.3 mM CaCl2, 33mM, 10 mM HEPES 33 mM glucose; 310-320 mOsm). This glycine-induced cLTP protocol has previously been demonstrated to selectively activate synaptic NMDARs (Yoshihara 67 et al., 2000; Gomes et al., 2004; Kopec et al., 2006; Oh et al., 2006), and thereby causes a rapid exocytosis of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) into the synaptic plasma membrane, leading to LTP of AMPAR-mediated excitatory transmission (Hayashi et al., 2000; Liao et al., 2001; Lu et al., 2001; Passafaro et al., 2001; Man et al., 2003; Park et al., 2004; Li et al., 2009). Dissociated NSCs from neurospheres were labeled by GFP- containing lentivirus and then co-cultured with dissociated hippocampal neurons for 8-10 days in vitro. The cultured hippocampal neurons alone or co-cultured NSCs with hippocampal neurons were stimulated by the cLTP protocol for 2 minutes and further incubated for 8 minutes in the same solution omitting sucrose (Lu et al., 2001; Li et al., 2009). Each conditioned medium was collected from non-stimulated, PBS-stimulated, or cLTP- induced cultured hippocampal neurons 1 hour after stimulation, and then used for either enzyme-linked immunosorbent assay (ELISA) analysis of secreted growth factors or stimulation of cultured NSCs alone. Conditioned media were chronically applied every 3rd days for 2 weeks.  3.2.7 ELISA assay  ELISA assays for various growth factors were performed using ELISA kits purchased from Promega (BDNF Emax-, NGF Emax- and NT-3 Emax immunoassay System). Specifically, 96- well plates (Nunc-Immuno Maxisorp) were pre-coated with anti-monoclonal BDNF, anti- polyclonal nerve growth factor (NGF) and neurotrophin-3 (NT-3). They were subsequently incubated with blocking buffer (goat serum) for 1 hour to prevent non-specific binding. Collected conditioned media from non-stimulated, PBS-stimulated and cLTP-stimulated hippocampal neurons at various time points were incubated in the pre-coated 96-well plates with agitation at room temperature. The plates were then incubated with anti-polyclonal BDNF, and anti- monoclonal NGF, and NT-3 overnight at 4°C, followed by horseradish peroxidase (HRP)- conjugated secondary antibodies for another 2.5 hours with agitation at room temperature. Each plate was washed 5 times between each step with tris-buffered saline containing 0.05% Tween- 68 20 (TBST, Sigma-Aldrich). The color reaction was stopped by the addition of 1 N hydrochloric acid (HCl) and the absorbance wavelengths of the samples were read on a quant spectrophotometer (Bio-Tec Instrument Company) at 450nm.  3.2.8 Antibodies  The following anti-mouse primary antibodies were used against 5-bromo-2’-deoxyuridine (BrdU) (1:4 for immunefluorescence (IF); Upstate): GFP (IgG, 1:1000 for IF; Invitrogen), AMPAR subunit, GluR2 (IgG, 1:1000 for western blotting (WB); Chemicon), nestin (IgG, 1:200 and 1:100; Chemicon), neuronal nuclei (NeuN) (IgG1, 1:500 for IF; Chemicon), NMDAR subunit, NR1 (IgG2a, 1:1000 for WB; Chemicon), proliferating cell nuclear antigen (PCNA) (IgG2a, 1.500 for IF; Abcam), tropomysosin receptor kinase B (TrkB) (IgG1, 1:1000 for WB; BD biosciences), and vimentin (IgG, 1:1000 and 1:500; Sigma-Aldrich). Anti-rabbit Primary antibodies were used against β-actin (IgG, 1:2000 for WB; Abcam), doublecortin (DCX) (IgG, 1:1000 for IF; Abcam), γ- aminobutyric acidA receptor-alpha 1 (GABAAR-α1) (IgG, 1:500 for WB; Upstate), glial fibrillary acidic protein (GFAP) (IgG, 1:100 and 1:80; Sigma-Aldrich), GFP (IgG, 1:1000 for IF; Invitrogen), GluR1 (IgG, 1:25 for WB; Calbiochem), β-low density lipoprotein receptor-related protein1 (β- LRP1) (lgG, 1:1000; provided by Dr. Zemin Yao), microtubule associated protein2 (MAP2) (IgG, 1:1000 for IF; Chemicon) and phosphor-Trk (IgG, 1:1000 for WB; Cell Signaling). The order of dilution was WB and IF. Anti-mouse and rabbit secondary Alexa Fluor 488 and 555 (IgG, 1:1000; Molecular probes) antibodies were used for IF, and HRP-linked secondary antibodies were used for WB (1:10,000; GE Healthcare Biosciences).  3.2.9 Western blotting  Western blotting was performed as previously described (Peineau et al., 2007). Cells were washed 3 times with PBS and subsequently harvested with the lysis buffer containing the 69 following protease inhibitors: 300 μM 4-(2-aminoethyl)-benzenesulfonyl-fluoride hydrochloride (AEBSF) (Sigma-Aldrich), 10 μg/ml leupeptin (Bioshop), and 10 μg/ml aprotinin (Bayer). The sample pellet was dissolved in the loading buffer and boiled for 5 minutes. Samples were subjected to sodium dodecyl sulfate (SDS)-PAGE, and the proteins were transferred onto a PVDF membrane (Millipore), blocked with either 5% skim milk or bovine serum albumin, and probed with the relevant antibodies. HRP-conjugated secondary antibodies (GE Healthcare biosciences) were used to develop immunoblots. Blots were developed by using enhanced chemiluminescence detection (GE Healthcare biosciences). Band intensities were quantified using ImageJ software (NIH) and normalized to the quantity of either β-actin, a surface marker of β-LRP1 or TrkB as a loading control. All primary antibodies were diluted in TBST and washes were done with shaking between each step.  3.2.10 Biotinylation of cell surface proteins  Membrane trafficking of AMPAR subunits GluR1 and GluR2 were quantified by surface biotinylation in control or cLTP-stimulated hippocampal neurons as previously described (Peineau et al., 2007). Cultured cells were tagged with 1 mg/ml sulfosuccinimidy1-6- [biotinamido] hexanoate (sulfo-NHS-LC-Biotin; MJS BioLynx Inc.) for 30 minutes. Neurons were rinsed 3 times in cold PBS and then harvested in the lysis buffer with 1mM ethylenediamine tetraacetate (EDTA), 0.5% Triton X-100 and 1% SDS in PBS with three kinds of protease inhibitors (300 μM AEBSF, 10 μg/ml aprotinin, and 10 μg/ml leupeptin). The lysates were subjected to overnight avidin precipitation (280 μg of total protein/60 μl of avidin suspension; Sigma-Aldrich), washed four times and subjected to SDS-PAGE. Western blotting was performed as described above.   70 3.2.11 Immunocytochemistry and BrdU labeling  Immunocytochemistry was performed on 12 mm round cover glasses (Deckglaser). Cultured cells were washed briefly with PBS. They were subsequently fixed in 4% paraformaldehyde (PFA) for 10 minutes and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 5 minutes at room temperature. Cells were then blocked in 5% goat serum in PBS overnight at 4°C or 1 hour at room temperature. Goat-raised secondary antibodies were incubated overnight at 4°C or 1 hour at room temperature. For nuclear staining, cells were incubated with DAPI (1:1000; Sigma-Aldrich) in PBS for 30 seconds prior to mounting on slides in polyvinyl alcohol mounting media with DABCO antifade (Sigma-Aldrich). BrdU was used to detect the NSC proliferation. One day prior to the cLTP induction, 10 μM of BrdU (Sigma-Aldrich) was added to NSCs with or without hippocampal neurons respectively. After the cLTP induction, the NSCs were then fixed in 95% ethanol containing 5% glacial acetic acid and followed by incubation in mouse anti-BrdU (1:4; Upstate) for 1 hour at room temperature. Immunocytochemistry was subsequently performed as described above. Extensive PBS washings were performed between each step.  3.2.12 Immunohistochemistry  Rats were anesthetized with 65 mg/kg of sodium pentobarbital (65 mg/kg, i.p; MTC Pharmaceuticals) and then transcardially perfused with heparinized cold saline and subsequently perfused with 4% PFA in PBS (pH 7.4). Brains were removed from the skull, post- fixed in the same fixative overnight and then dehydrated in 30% sucrose-containing PBS. The brains were then rapidly frozen in powdered dry ice. Coronal sections (30 μm) were cut on a cryostat throughout the hippocampus and stored in the glycerol-cryoprotectant solution. Free- floating sections were processed for single immunohistochemistry as described previously (Ryu et al., 2009b; Ryu et al., 2009a). Briefly, sections were permeabilized by 0.2% Triton X-100 71 (Sigma-Aldrich) in PBS containing 5% bovine serum albumin (BSA) for 30 to 60 minutes. Slices were then incubated overnight at 4°C with primary and secondary antibodies in 1% BSA- containing PBS one after another. Finally, slices were mounted onto Superfrost/Plus microscope slides (Fisher Scientific) with mount media (Sigma-Aldrich). For negative controls, immunohistochemistry was performed omitting incubation of primary antibody.  3.2.13 Image quantification and statistical analysis  To analyze in vivo retroviral injection in the DG and transplanted NSCs in the CA1, GFP- labeled cells throughout the rostral-caudal extent of the transduced region were counted. We performed statistical analysis using at least 5 sections per rat and then entire CA1 or DG images were acquired using a Leica DMIRE2 microscope. Digitized images were then analyzed using OpenLab 3.7. For measurement of proliferation/survival of NPCs in the DG and transplanted NSCs in the CA1, the numbers of GFP-positive cells were counted. To quantify neuronal differentiation of the NPCs in the DG and of transplanted NSCs in the CA1, the total number of GFP-positive cells labeled with MAP2 were counted and expressed as the ration of the percentage change from the vehicle-treated control group. To analyze in vitro culture quantitative analysis, five non-overlapping fields in each coverslip (total 3 coverslips for each condition) were randomly selected and images were acquired using either a Zeiss Axioplan-2 microscope equipped with a DVC camera (Diagnostic Instruments, Sterling Heights, MI) or a Leica DMIRE2 microscope. Digitized images were then analyzed using Northern Eclipse software (Empix Imaging) and OpenLab 3.7. Representative images were adjusted to maximize the signal-to-noise ratio. Relative intensities from each fluorophore channel were adjusted to a 50:50 contribution of signal intensities prior to merging 2 or 3 channel images. For measurement of proliferating NSCs, the numbers of BrdU-positive cells on GFP-labeled NSCs were counted and the percentage of BrdU-labeled cells relative to the total number of GFP-labeled NSCs per field of vision was determined. To quantify neuronal 72 differentiation of NSCs under different treatment procedures, the number of GFP-positive cells labeled with MAP2 were counted and expressed as the ratio of the percentage change from the PBS-treated control group. DAPI (1:1000, Sigma-Aldrich) was applied for the total number of the cells. All quantifications were done in a blinded manner.                          73 3.3 Results  3.3.1 Induction of LTP increases proliferation/survival of endogenous NPCs ini the subgranular zone of the DG region  NPCs in the adult DG are one of the major sources of neuronal precursors in the brain (Reynolds and Weiss, 1992; Richards et al., 1992; McKay, 1997; Gage, 2000). However, only a small fraction of these cells survive and differentiate into mature neurons. To determine if the induction of LTP promotes proliferation and/or survival of these NPCs in the subgranular zone (SGZ) of the DG, we induced NMDAR-mediated LTP in the rat hippocampus. Field excitatory postsynaptic potentials (fEPSPs) evoked by electrical stimulation of the medial perforant path (MPP) at 0.05 Hz for 1 minute were recorded from the DG region and normalized to the averaged slope. LTP was induced by sTPS (4 trains of 10 bursts of 5 pulses at 400 Hz with a 200 ms inter-burst interval, 15-s inter-train interval). As shown in Figure 3-1A, this sTPS stimulation reliably elicited LTP in the DG region. LTP was mediated by NMDARs as it was completely abolished by a pretreatment with the competitive NMDAR antagonist CPP (10 mg/kg, i.p., 90 minutes prior to the induction). Control electrical stimulation (0.05 Hz) administered with either 0.9% saline (i.p., 90 minutes prior to the induction) or CPP injection did not introduce any change in the baseline fEPSPs (Figure 3-1A). One week after electrical field recordings, the animals were sacrificed and the extent of proliferation of NPCs in the SGZ of the DG was assessed with immunohistochemistry with antibody against proliferating cell nuclear antigen (PCNA), a cell cycle marker for proliferation (Kurki et al., 1986). A significant increase in numbers of PCNA-positive NPCs was observed in rats in which LTP was successfully induced (Figure 3-1B, right image) in comparison with saline injected rats (0.05Hz+0.9% saline; Figure 3-1B, left image). The LTP-induced increase in PCNA-positive NPCs was eliminated by the blockade of NMDARs with CPP. Neither 0.9% saline nor CPP injection altered the number of PCNA-positive NPCs (Figure 3-1B). Therefore, our results strongly support that NMDAR- 74 dependent LTP increased the number of endogenous NPCs in the SGZ of the DG.  3.3.2 LTP promotes neuronal differentiation of endogenous NPCs in the DG region  The increased numbers of PCNA-positive NPCs following LTP may result from enhanced proliferation and/or survival of newly generated NPCs. In addition, LTP may also promote neuronal differentiation of these NPCs. Previous studies have well established that NPCs can be specifically labeled by retrovirus GFP vector infection due to their exclusive expression in dividing cells during mitosis (Lewis and Emerman, 1994; van Praag et al., 2002; Tashiro et al., 2006a; Zhao et al., 2006). To determine if LTP promotes the survival and neuronal differentiation of NPCs, we next labeled hippocampal DG NPCs with a GFP-containing retrovirus (CAG-GFP) via intra-hippocampal injection into the DG area and investigated effects of LTP induction on the number of GFP-labeled NPCs and their neuronal differentiation using an experimental protocol illustrated in Figure 3-2A. LTP was induced by sTPS 3 days following virus infection. One week after LTP induction, the survival of NPCs was assessed by counting the total number of GFP- labeled cells and neuronal differentiation was estimated by expression of either doublecortin (DCX), an immature neuronal marker (Francis et al., 1999; Gleeson et al., 1999; Jin et al., 2004b; Rao and Shetty, 2004), or Neuronal Nuclei (NeuN), a mature neuronal marker (Mullen et al., 1992) in GFP-expressing NPCs. As shown in Figure 3-2B and C, the induction of LTP resulted in a significant increase in the number of DCX-positive GFP-NPCs and this increase was not produced by basal, non-LTP-inducing stimulation (0.05Hz+0.9% saline; Figure 3-2B, upper panel), and was prevented by a pretreatment of the rats with the NMDAR competitive antagonist CPP (Figure 3-2C). Thus, LTP appears to promote neuronal differentiation of NPCs into immature neurons. In addition, LTP induction was also associated with a significant increase in the number of NeuN-positive GFP-NPCs (Figure 3-2D and E). In accordance with a general pattern of NPC migration into the granule cell layer (GCL) from the SGZ as they differentiated and matured (Doetsch and Hen, 2005), these fully differentiated, mature GFP- 75 NeuN-positive neurons were primarily located in the inner GCL (Figure 3-2D). Furthermore, LTP-induced neuronal maturation and migration was eliminated by CPP application (Figure 3- 2E). Since neither 0.9% saline nor CPP injection in the absence of LTP induction altered the number of GFP-, DCX- and NeuN-positive cells, the results indicate that LTP promotes neuronal maturation of GFP-NPCs. In addition to the increased NPC neuronal differentiation (Figure 3-2C) and maturation (Figure 3-2E), LTP induction also significantly increased the total number of GFP-labeled cells in a NMDAR-dependent manner (Figure 3-2C and E), suggesting an increased NPC survival. Thus, NMDAR-dependent LTP increases proliferation/survival and neuronal differentiation of endogenous NPCs in the SGZ of the hippocampal DG.  3.3.3 LTP enhanced neurogenesis of exogenously transplanted NSCs into the hippocampal CA1 region  The increased proliferation/survival and neuronal differentiation of endogenous NPCs prompted us next to examine if LTP has similar actions on exogenously transplanted NSCs. To this end, we chose to examine the effects of LTP in NSCs transplanted into the hippocampal CA1 (but not the DG) of anesthetized rats (Figure 3-3A) because this will minimize potential influence on the transplanted NSCs from an endogenous stem cell niche presented in the DG (Kempermann et al., 1997) and because CA1 LTP is the most well-characterized NMDAR- dependent form of synaptic plasticity (Collingridge et al., 2004) and can be reliably induced in vivo (Ge et al., 2010). As shown in Figure 3-3B, LTP of CA1 fEPSPs was reliably induced by a short train of high frequency stimulation (HFS; 4 trains of 50 pulses at 100 Hz, 15-s inter-train interval) of the Schaffer collaterals. LTP was an NMDAR-mediated phenomenon as it was abolished by NMDAR antagonist CPP (10 mg/kg, i.p., 90 minutes prior to the induction). Control stimulation (0.05 Hz) following either 0.9% saline (i.p., 90 minutes prior to the induction) or CPP injection did not introduce any significant alteration in the basal level of fEPSPs (Figure 3-3B). NSCs derived from embryonic cultures were pre-labeled by GFP-containing lentivirus and then 76 transplanted into the CA1 region 2-5 hours after LTP induction. Effects of LTP on proliferation/survival and neuronal differentiation of transplanted NSCs were then examined by quantification of the total number of GFP-positive transplanted cells and percentage of neuronal marker NeuN-positive GFP cells, respectively, within the CA1 region 7 days after transplantation. As shown in Figure 3-3C, the number of GFP-positive transplanted cells was significantly higher in the CA1 region that experienced LTP in comparison with that in the CA1 region stimulated with a non-LTP-inducing control protocol (0.05Hz+0.9% saline). Moreover, injection of CPP (10 mg/kg, i.p.) had no effect on the number of GFP-positive cells (0.05Hz+CPP) on its own, but prevented LTP-induced increase (CPP+HFS). Thus, induction of LTP increases proliferation/survival of the transplanted NSCs. As shown in Figure 3-3D, immunohistochemical staining of mature neuronal marker NeuN also revealed that the induction of CA1 LTP caused a significant increase in the proportion of NeuN-positive transplanted GFP cells in the CA1 region, suggesting an increased neuronal differentiation of exogenously transplanted NSCs. Application of CPP prior to LTP induction abolished LTP-increased neuronal differentiation (CPP+HFS). In contrast, CPP application alone (0.05Hz+CPP) had little effect on the basal neuronal differentiation as it did not alter the proportion of NeuN-positive NSCs in comparison with saline application group (0.05Hz+0.9% saline). Thus, the induction of NMDAR-dependent LTP promotes proliferation/survival and neuronal differentiation of exogenously transplanted NSCs in the CA1 region.  3.3.4 LTP increases neurogenesis of NSCs in cultures in vitro  We next sought to investigate the underlying mechanisms by which LTP promotes proliferation/survival and neuronal differentiation of NSCs using mixed NSC and neuronal cultures (NSC-Neuron co-culture). NSCs were isolated from neurospheres which were derived from E14 rats, then dissociated and re-plated for an additional 10-14 days (Figure 3-4A). Immunocytochemical staining of both neurospheres and dissociated NSCs confirmed that the 77 isolated cells have characteristics of immature NSCs. As shown in Figure 3-4A, the vast majority of the isolated cells (labeled with nuclei fluorescent dye DAPI) expressed type IV intermediate filament protein nestin (Nestin; Green), a protein marker for NPCs (Lendahl et al., 1990), and type III intermediate filament protein vimentin, a protein marker for mitotically active (Liem, 1993), neural progenitors (Eliasson et al., 1999) and radial glia (Kinoshita et al., 2005). Further supporting their identity as immature NSCs, we found that the vast majority of these neurosphere-derived cells did not express MAP2, a protein marker for fully differentiated mature neurons (Figure 3-4A and B; MAP2) (Bernhardt and Matus, 1984). Consistent with recent reports that both NSCs and progenitors can express intermediate filament protein GFAP, a protein marker for radial glia (Wei et al., 2002; Garcia et al., 2004; Ihrie and Alvarez- Buylla, 2008), we also found that these cells expressed GFAP (Figure 3-4A, B). Thus, our immunocytochemical characterization demonstrated that the neurosphere-derived cells isolated in the present study appear as GFAP-positive NSCs that are immature and capable of self- renewal and proliferation. The identity of these cells as NSCs was further confirmed with western blotting of the cellular lysate using antibodies against various cell-type specific markers. Consistent with  the immunocytochemistry results, NSCs express nestin, vimentin and GFAP, but not MAP2 (Figure 3-4B) and this is in great contrast with the adult brain lysates, which expresses mature neuronal MAP2 and glia marker GFAP proteins, but not immature cell markers, nestin and vimentin (Figure 3-4B). To further confirm that these NSCs did not fully differentiate into functional neurons, we also examined their expression of ligand-gated ionotropic glutamate and γ- aminobutyric acid (GABA) receptors (GABARs) that mediate excitatory and inhibitory synaptic transmissions, respectively, at the vast majority of synapses in fully differentiated neurons (Collingridge et al., 2004). As shown in Figure 3-4C, in contrast to the brain lysates, NSCs did not have detectable levels of NMDAR subunit NR1, AMPA (α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid) subtype glutamate receptor subunits GluR1 or GluR2, or GABAA receptor α1 subunits. These results further support the idea that NSCs isolated from 78 neurospheres are immature and contain few fully differentiated functional neurons. Following isolation and characterization of NSCs, we investigated the effect of LTP on proliferation and neuronal differentiation of these NPCs using a well-characterized glycine- based chemical LTP (cLTP) stimulation protocol (for details see method sections) (Lu et al., 2001; Li et al., 2009). In this set of experiments, proliferation of NSCs was evaluated by quantifying the number of cells incorporated with BrdU after cLTP induction. BrdU is a thymidine analog that can be specifically incorporated into the DNA of dividing cells during the S-phase and has been widely used for labeling proliferating cells in studies of neurogenesis (Kuhn and Cooper-Kuhn, 2007; Taupin, 2007). Neuronal differentiation was assayed with immunofluorescent staining of neuronal marker MAP2. As shown in Figure 3-4D and E, a glycine stimulation protocol had no obvious effect on either proliferation or neuronal differentiation in the NSC cultures. This result is not surprising because LTP can only be induced in mature neurons, which have glutamate-mediated excitatory synaptic transmissions. Therefore, we subsequently used NSC-neuron co-cultures to examine LTP effects (Figure 3- 5A). In order to facilitate the identification of NSCs from fully differentiated neurons in the co- cultures, we pre-labeled these NSCs using lentivirus vectors containing GFP 3 days before co- culturing them with dissociated embryonic hippocampal neurons. Both dissociated NSCs and neurospheres were infected by lentivirus-GFP at high efficiency (approximately 73%). Unlike in pure NPC cultures, glycine-based cLTP protocol stimulation reliably induced LTP in NSC-neuron co-cultures. Thus, as shown in Figure 3-5B, surface biotinylation assays revealed that glycine stimulation resulted in a rapid, a significant increase in the amount of both GluR1 and GluR2 subunits of AMPA receptors (AMPARs) on the membrane surface (Lu et al., 2001; Li et al., 2009). An increase in surface AMPARs is not a result of general increases in vesicle membrane fusion, as it was not associated with a detectable change in the amount of membrane protein β- low density lipoprotein receptor-related protein1 (β-LRP1) (Figure 3-5B). Moreover, a glycine- induced increase in cell-surface expression of AMPARs was prevented by blockade of NMDARs with competitive NMDAR antagonist, D-APV (50 μM) (Figure 3-5B). Thus, a glycine- 79 based cLTP stimulation protocol could reliably induce LTP in the NSC-neuron co-cultures. As we expected, cLTP induction was able to significantly increase the number of both BrdU- (Figure 3-5C, upper panel) and MAP2-positive NSCs (Figure 3-5C, bottom panel) and importantly, an increase in both BrdU- and MAP2-labeled NPCs were prevented by D-APV (Figure 3-5D). Neither a PBS treatment as a control nor D-APV alone had any detectable effect on proliferation and neuronal differentiation of the NSCs (Figure 3-5D). Thus, our results suggest that the induction of cLTP with glycine in neurons promotes both proliferation and neuronal differentiation of NSCs co-cultured with neurons.  3.3.5 BDNF plays a critical role in mediating LTP promoted neurogenesis of NSCs in NSC-neuron co-cultures  The requirement of the presence of neurons in the cultures to enable cLTP-increased proliferation and neuronal differentiation of the NSCs suggests that the effects of cLTP are from either a physical contact between NSCs and neurons, or from one or more diffusible factors from the cLTP-stimulated neurons. To differentiate between these two possibilities, we treated the pure NSC cultures with conditioned media from non-, PBS-, or cLTP-stimulated neuronal cultures (Figure 3-6A). As shown in Figure 3-6B, conditioned media from neuronal cultures that experienced LTP induction significantly increased the number of MAP2-positive NSCs in comparison with either conditioned media from either non-stimulated or PBS-stimulated controls. These results strongly suggest that the cLTP effects observed in the NSC-neuron co-cultures may not require the direct physical contact between the two cell types, but indicate that following the induction of cLTP, neurons may secrete some diffusible factors that promote proliferation and neuronal differentiation of the co-cultured NSCs. Several growth factors have previously been shown to promote NSC neuronal differentiation. We therefore next investigated if one or more of these growth factors could be involved in the cLTP-induced proliferation and neuronal differentiation of NSCs. Using ELISA assay, we 80 investigated concentrations of three growth factors previously implicated in stimulating NSC neuronal differentiation (Maisonpierre et al., 1990b; Acheson et al., 1995; Ahmed et al., 1995; Pincus et al., 1998; Zigova et al., 1998; Barnabe-Heider and Miller, 2003), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) in the media obtained from the neuronal cultures at 0, 10, 30, 60 minutes, or 1 day following cLTP induction. We found that the level of BDNF, but not NGF or NT-3, was significantly elevated at 30 and 60 minutes (Figure 3-7A), suggesting BDNF may have a critical role in mediating the effects of cLTP on NSCs. In order to investigate this idea directly, we next treated pure NSC cultures with conditioned neuronal culture media obtained 60 minutes following cLTP induction and determined if it could stimulate TrkB receptors in these NSCs by measuring the level of TrkB receptor tyrosine phosphorylation, an indication of increased TrkB receptor activation by BDNF (Maisonpierre et al., 1990b; Barbacid, 1994; Haniu et al., 1997; Arevalo and Wu, 2006). Western blotting with anti-phopho-TrkB (p-TrkB) antibody revealed an increased level of tyrosine phosphorylation of TrkB receptors expressed on the plasma surface of NSCs and moreover, this conditioned medium-induced increase was blocked by the tyrosine kinase inhibitor, K252a (Tapley et al., 1992) (Figure 3-7B). Finally, in supporting the critical role of BDNF in mediating the effects of cLTP on NPCs in NSC-neuron co-cultures, we found that inhibition of TrkB receptor activation with bath application of K252a prevented cLTP-enhanced the number of GFP-positive and neuronal differentiation of NSCs (Figure 3-7C, D). Thus, the signaling system of BDNF released from neurons and its consequent activation of TrkB receptors on NSCs may, at least in part, be responsible for certain actions of cLTP on NSCs. However, cLTP-induced neuronal differentiation was not completely blocked by treatment with K252a (Figure 3-7B). It therefore appears that in addition to BDNF-TrkB signaling pathway, cLTP-induced neuronal differentiation may also require a physical contact between neurons and NSCs in co-cultures.   81 3.4 Discussion  In the present study, we showed that the induction of NMDAR-mediated LTP at MPP to the DG enhances proliferation/survival, and neuronal differentiation of both endogenous NSCs in the DG and exogenously transplanted NSCs into the CA1 region. These findings strongly suggest that a LTP-inducing protocol may be used to facilitate neurogenesis of these NPCs, thereby facilitating clinical efforts using these cells to repair damaged neurons under certain clinical conditions. Furthermore, using in vitro NSC-neuronal co-cultures, we demonstrated that LTP-induced enhancement of neurogenesis may be, at least in part, mediated through BDNF- TrkB pathways.  The adult mammalian central nervous system (CNS) contains a population of NSCs, which can be isolated from the adult subventricular zone (SVZ) and SGZ of hippocampus to be used for cell replacement research (Reynolds and Weiss, 1992; Richards et al., 1992; McKay, 1997; Gage, 2000). Increasingly, evidence suggests that newly generated neurons in the adult brain are able to integrate into existing neural circuitry receiving functional input (van Praag et al., 2002; Kempermann et al., 2004; Tozuka et al., 2005; Toni et al., 2008) and may compensate for loss of function in injured regions (van Praag et al., 2002; Lledo and Gheusi, 2006). However, because the population of newly-generated cells is extremely low under normal conditions their impact on host circuitry may be minimized in the context of repair under pathological conditions (Lie et al., 2004; Ming and Song, 2005; Arias-Carrion et al., 2007). Indeed, in the absence of an intervention, intrinsic neurogenesis may be limited in its role to compensate for continuous neuronal loss as a natural consequence of aging, injury, and disease throughout life (Fuchs and Gould, 2000; Taupin, 2006). Our results demonstrate that the induction of synaptic plasticity results in a dramatic enhancement of proliferation/survival and differentiation of both endogenous NPCs as well as transplanted NSCs and may have profound implications in the efficacy of potential cell replacement therapies. NMDAR-dependent LTP in learning and 82 memory has been primarily established in the hippocampal region (Collingridge, 1992; Bliss and Collingridge, 1993; Bailey et al., 2000). However, the role of NMDARs or NMDAR-mediated LTP is poorly understood in the context of neurogenesis. The blockade of NMDARs has been suggested to enhance adult neurogenesis (Cameron et al., 1995; Gould et al., 2000). However, most of the previous studies have suggested a role of NMDARs in enhancing hippocampal neurogenesis. NMDAR activation is involved in both proliferation and differentiation (Joo et al., 2007), and survival of new neurons is competitively regulated by NMDARs during a short, critical period soon after neuronal birth (Tashiro et al., 2006b). A similar result has been reported in early developmental interneurons via activation of excitatory chloride-mediated glycine receptors in the spinal network accompanied by a concomitant increase in the number of mitotic cells (McDearmid et al., 2006). A previous study has proposed the possible involvement of LTP in neurogenesis (Derrick et al., 2000). This study has been done in the efferent activity at mossy fiber (MF) inputs to the CA3 region LTP, resulting in an increase of new neurons in the DG. Recent studies also demonstrated enhanced proliferation and survival in the SGZ by induction of LTP at MPP to the DG region (Bruel-Jungerman et al., 2006; Chun et al., 2006). The most recent study has further supported our results that survival of newly generated neurons is enhanced by HFS-induced LTP (Kitamura et al., 2010). However, our study further revealed that the induction of LTP promotes proliferation/survival and neuronal differentiation in both endogenous and exogenous NPCs, and these enhanced effects are through activation of NMDARs. The late finding is consistent with previous studies which suggested a role of NMDARs in hippocampal neurogenesis. NMDAR activation is involved in both proliferation and differentiation (Joo et al., 2007), and survival of new neurons is competitively regulated by NMDARs during a short, critical period soon after neuronal birth (Tashiro et al., 2006b). In contrast, a recent publication has reported that LTP is enhanced during Alzheimer’s disease’s (AD) pathological conditions, but fails to promote neurogenesis (Poirier et al., 2010). In addition, the newly-generated neurons in the DG do not become mature neurons although proliferation is increased in AD (Li 83 et al., 2008a). However, most studies have already shown conflicting results of increased neurogenesis under different pathological conditions including AD (Parent et al., 1997; Parent et al., 2002; Jin et al., 2004a; Jin et al., 2004b; Tattersfield et al., 2004; Parent et al., 2006). For instance, NPC proliferation is enhanced in the DG region during stroke and seizure. This effect is eliminated by the blockade of NMDARs (Parent et al., 1997; Parent et al., 2002; Jessberger et al., 2007). Therefore, pathological condition-induced neurogenesis may also be regulated by NMDARs and/or NMDAR-dependent LTP. In addition to the study of endogenous NPCs, we provided evidence for the first time that NMDAR-dependent LTP enhances proliferation/survival and neuronal differentiation of exogenously transplanted NSCs in the CA1 region. In addition, these results further support the idea that LTP-induced enhancement of proliferation/survival and neuronal differentiation are distinct from effects resulting from the intrinsic stem cell niche of the DG (Kempermann et al., 1997; Brown et al., 2003). However, the results of LTP-regulated proliferation/survival of exogenously transplanted NSCs in the CA1 region were not as dramatic as was observed with endogenous NPCs in the DG area. Technically, a proliferation cycle of in vitro cultured NSCs is much slower at each passage (Ma et al., 2006) and exogenous NSCs were transplanted into the CA1 following HFS. Thus, environmental and intrinsic cell type differences between the CA1 pyramidal neurons and DG granule neurons as well as differing tetanus stimulation paradigms between HFS and sTPS may have contributed to the observed differences in proliferation in these two settings.  To address the underlying mechanisms of LTP-induced enhancement of proliferation/survival and differentiation of NSCs, an in vitro co-culture system was used. Because electrical stimulation of neurons or neural pathways for the induction of LTP is spatially limited to the synaptic area being stimulated, we used a previously characterized cLTP protocol that could reliably stimulate a global area of cells in vitro. Several protocols have been reported to enhance neuronal excitability and induce cLTP. Application of an adenylyl cyclase activator, 84 forskolin and a phosphodiesterase inhibitor, rolipram is an effective technique for the induction of LTP (Yoshihara et al., 2000; Gomes et al., 2004; Kopec et al., 2006; Oh et al., 2006). In addition, high K+ (20mM) is sufficient to increase mEPSCs (Deisseroth et al., 2004). In this study, we used glycine-mediated cLTP to investigate NMDAR-dependent LTP on neurogenesis. Glycine-mediated NMDAR-dependent cLTP was sufficient to insert AMPARs onto the surface membrane in cultured hippocampal neurons as previously described (Lu et al., 2001; Li et al., 2009). Glycine is a NMDAR co-agonist with glutamate and binds to NR1 and NR3 subunit of NMDARs (Wafford et al., 1995; Hirai et al., 1996). High K+-induced enhanced activity-dependent neurogenesis has been reported in isolated adult hippocampal-derived NPCs (Deisseroth et al., 2004). These isolated hippocampal NPCs are already restricted to neuronal progenitors expressing NMDARs (Seaberg and van der Kooy, 2002; Deisseroth et al., 2004). However, NMDARs and AMPARs were not present in isolated NSCs derived from early embryonic telencephalons. Therefore, application of cLTP treatment onto cultured NSCs alone did not show any changes in proliferation/survival and differentiation. We further confirmed the expression level of GABARs because they play an important role in excitatory synapses along with a regulation of co-transporters during development (Cherubini et al., 1991; Ben-Ari et al., 1994). However, GABARs were not found to be present on NSCs at this stage, in which they still exhibited multipotent stem cell properties. A previous co-culture study indicated that cultured NSCs with either neurons or astrocytes promoted cells to adopt a neuronal fate (Song et al., 2002). Thus, we used a co-culture of NSCs and a system of hippocampal neurons to investigate the cues associated with cLTP-induced enhancement of neurogenesis. Consistent with our in vivo results, application of cLTP in co- culture exhibited enhanced proliferation/survival and differentiation of NSCs. Blockade of NMDARs by applying D-APV inhibited not only AMPAR insertion in hippocampal neurons but also proliferation/survival and neuronal differentiation of NSCs. We surmised that cLTP- stimulated neurons may exert their effects on NSCs in the co-culture system via paracrine signaling. We further confirmed paracrine effects using conditioned media from hippocampal 85 neurons following cLTP. Previous work has shown that instantaneous secretion of BDNF is triggered by electrically inducing LTP (Gartner and Staiger, 2002) and activation of NMDARs (Joo et al., 2007). Consistent with previous studies, ELISA and surface biotinylation data revealed that cLTP is sufficient to facilitate BDNF release, but neither NGF nor NT-3 from neurons that is able to activate its receptor, TrkB, on NSCs in vitro. In general, portions of the brain exhibit high levels of many neurotrophic factors (Gall and Lauterborn, 1992) some of which have been demonstrated to regulate proliferation and survival of NPCs during development (Abrous et al., 2005). Furthermore, particularly BDNF binding to TrkB enhances neuronal differentiation in neuronal precursors derived from CNS stem cells (Ahmed et al., 1995; Binder and Scharfman, 2004) and maturation in co-cultured NSCs with astrocytes (Song et al., 2002). However, in this conditioned medium study, neuronal differentiation appeared without altering proliferation. A previous study has explained the environmental importance of transition between proliferation and differentiation of NSCs (Deisseroth et al., 2004). Thus, cLTP-induced conditioned media may be able to enhance proliferation of NSCs under different environmental conditions. We further confirmed cLTP-induced proliferation/survival and neuronal differentiation in co-cultured NSCs using the tyrosine kinase inhibitor, K252a. cLTP-induced proliferation/survival was eliminated by K252a while neuronal differentiation was partially inhibited. Thus, neurotrophins including BDNF and tyrosine kinase receptor, TrkB are required for neurogenesis.  Taken together, we have demonstrated that LTP enhances proliferation/survival and neuronal differentiation of both endogenous NPCs and transplanted NSCs in the hippocampus. In addition, we have shown via in vitro studies based on NSC characterization, a potential role for BDNF-TrkB signaling as an underlying mechanism of this enhancement. The use of in vivo electrical stimulation to promote proliferation/survival and neuronal differentiation of NSCs during development may have profound implications in aiding in the efficacy of   future cell replacement therapies in the context of brain injury aimed at replacing loss of function of 86 existing neural circuitry in the brain. Clinical use of electrical brain stimulation has previously been demonstrated with promising results, from studies involving chronic deep brain stimulation (DBS) at high frequency in human PD patients. (Tronnier et al., 1997b; Tronnier et al., 1997a; Bronstein et al., 2011). However, the mechanisms underlying the action of DBS are poorly understood. Previous studies have proposed that long-term changes of neural plasticity and neural protection may be induced in the network. In contrast, in our study, we show that synaptic plasticity can be produced by well-characterized electrical stimulation protocols, HFS and theta-burst stimulation (TBS) in specific brain regions, to produce LTP and subsequently the promotion of proliferation/survival and differentiation of neural progenitor/stem cells. Thus, we feel with clearly defined stimulus parameters and target sites, that the induction of LTP may have a critical role in promoting improved outcomes of potential cell replacement treatments and therapeutic intervention.                87 Figure 3-1 NMDAR-dependent LTP enhances proliferation of NPCs in the DG A. Evoked-field EPSPs were recorded from the DG of anesthetized rats and LTP was induced by application of sTPS to the MPP. Induction of LTP was abolished by treatment with the competitive NMDAR antagonist, CPP. Treatment with CPP alone or control electrical stimulation alone (0.05 Hz) did not introduce any changes in baseline recording. B. Representative images from coronal sections show LTP enhanced PCNA (red) staining of DAPI (blue)-positive cells in the SGZ compared with controls group (0.05Hz+saline). Boxed areas indicating areas of PCNA staining with enhanced magnification (right panels). Bar graph summary of LTP enhancement of proliferation. Enhanced NPC proliferation, quantified as the number of PCNA-positive NPCs on DAPI-positive cells, by LTP induction was blocked by CPP application. However, CPP application alone did not alter proliferation. All quantification analyses were performed on the basis of electrical field recording and immunohistochemistry results. **p<0.01, n=8, 9, 9, and 8. Statistical analyses were performed with One Way ANOVA. Data are mean ± SEM.             88            89 Figure 3-2 NMDAR-dependent LTP enhances neuronal differentiation and maturation of NPCs in the DG A. Schematic representation of the experimental design. B. The induction of LTP increases the total number of GFP-labeled NPCs, and DCX and GFP double-labeled immature neurons differentiated from NPCs in the DG. Left: Representative images from coronal sections double- stained with antibodies against an immature neuronal marker DCX (red) and GFP (green) in the DG. Examples of double-stained immature neurons in boxed areas are shown in higher magnification in the panels on the right. C. Right: Bar graphs summarizing effects of LTP induction on the total number of GFP-positive cells (top panel), and DCX and GFP double- labeled cells (bottom panel) in the DG. *p<0.05 and **p<0.01; n=8 or 9 in each group. D. The induction of LTP increases the total number of GFP-labeled NPCs, and NeuN and GFP double- labeled differentiated mature neurons in the DG. Left: Representative images from coronal sections double-stained with antibodies against a mature neuronal marker NeuN (red) and GFP (green) in the DG. Examples of double-stained mature neurons in boxed areas are shown in higher magnification in the panels on the right. E. Right: Bar graphs summarizing effects of LTP induction on the total number of GFP-positive cells (top panel), and NeuN and GFP double- labeled cells (bottom panel) in the DG. *p<0.05 and **p<0.01; n=8 or 9 in each group. Statistical analyses were performed with One Way ANOVA. Data are mean ± SEM.     90  91 Figure 3-3 induction of LTP increases neuronal differentiation of exogenous NSCs transplanted into the CA1 region A. Schematic representation of the experimental design. B NMDAR-dependent LTP in the hippocampal CA1 region was reliably induced by HFS of the Schaffer collateral inputs in anesthetized rats. Representative traces of fEPSP taken at time indicated in the graph of fEPSPs on the top. Systemic application of the competitive NMDAR antagonist CPP prevented the induction of LTP (CPP+HFS), without affecting basal level of fEPSP (0.05Hz+CPP). C. Representative images from coronal sections show LTP (LTP) enhanced the total GFP (green)- positive cells in the CA1 compared with a control group (0.05Hz+saline). Bar graph below summarizing data from groups of rats. LTP increased the total numbers of transplanted NSCs. D. Representative images from coronal sections double-stained for GFP and NeuN showing LTP (LTP) increased numbers of NeuN-positive (red) NSCs (green) in the CA1 region, compared with a control group (0.05Hz+saline). Bar graph below summarizing data of LTP enhancement of neuronal differentiation. *p<0.05, n = 7, 7, 10, and 8. Statistical analyses were performed with One Way ANOVA. Data are mean ± SEM.        92  93 Figure 3-4 Isolation and characterization of NSCs in cultures NSCs were isolated and characterized from E14 telencephalon. A. Immunocytochemistry was performed with various cell type-specific markers (green). The representative phase contrast image shows formation of secondary neurospheres and expressing nestin. Triturated cells from neurospheres were double-stained DAPI with antibodies against nestin, vimentin, MAP2, and GFAP. Consistent with immunocytochemistry, western blotting results also show properties of NSCs. Western blotting was performed in different B-27 and N2 media with B. various cell type- specific antibodies against nestin, vimentin, MAP2 and GFAP, and C, various ionotropic receptor antibodies against , NR1, GluR1, GluR2 and GABAAR-α1 subunit in comparison with cell lysates of adult brain tissues. Excitatory and inhibitory receptor subunits were not detected during early stage of NSCs in vitro. D. Representative images from cultured NSCs alone show that a cLTP induction protocol did not alter BrdU (red, right and upper panel) and MAP2 (red, right bottom panel) staining of GFP (green)-positive NSCs compared with control group (PBS, left upper and bottom panels respectively). E. Bar graphs are summary of results illustrating no cLTP effect on proliferation (upper panel, p=0.421, n=5) and neuronal differentiation (p=0.464, n=5) in cultured NSCs alone. All quantification analyses were performed on the basis of immunocytochemical results. Statistical analyses were performed with Student’s t-test. Data are mean ± SEM.          94  95 Figure 3-5 Chemical LTP enhances proliferation/survival and neuronal differentiation in NSC-neuron co-cultures A. Schematic representation of the experimental design for proliferation and differentiation of NSCs co-cultured with hippocampal neurons in vitro. B. Surface biotinylation results show cLTP induction was sufficient to enhance the surface expression of AMPAR subunits GluR1 and GluR2 in cultured hippocampal neurons and that this enhancement could be eliminated by treatment with the competitive NMDAR antagonist, D-APV. Bar graph summary of cLTP enhancement of the surface expression of AMPARs. Enhanced surface expression of AMPARs in hippocampal neurons, quantified as GluR1 and GluR2 expression intensity, by cLTP induction was blocked by D-APV application. ***p<0.001, n=7. C. Representative images showing enhanced BrdU (top panel, red) and MAP2-positive cells (bottom panel, red) were facilitated by cLTP in co-cultured NSCs with hippocampal neurons. D. Bar graph summary of results illustrating enhanced proliferation (left panel, n=5, *p<0.05) and neuronal differentiation (right panel, n=10, **p<0.001) of NSCs by cLTP induction and its elimination by the blockade of NMDAR by D-APV application. D-APV application alone did not alter proliferation or neuronal differentiation of NSCs. The quantification of biotinylation results was standardized with the loading control values with β-LRP1. All quantification analyses were performed on the basis of surface biotinylation and immunocytochemical results. Statistical analyses were performed with One Way ANOVA. Data are mean ± SEM.         96            97 Figure 3-6 Conditioned media from cLTP treated neuronal cultures increases neurogenesis of NSCs A. Schematic representation of the experimental design for conditioned media from cultured hippocampal neurons onto cultured NSCs alone. B. Representative images illustrate cLTP- induced conditioned media enhanced MAP2 (red) staining in cultured NSCs alone compared with control group (non-stimulated and PBS-stimulated media). Bar graph summary of conditioned media enhancement of neuronal differentiation. Enhanced neuronal differentiation of cultured NSCs alone, quantified as the number of MAP2-positive mature neurons of DAPI- positive (blue) cells.  *p<0.05, n=6.                   98            99 Figure 3-7 LTP promotes neurogenesis of NSCs in cultures at least in part through BDNF-TrkB system A, ELISA assay was performed to measure a variety of growth factors from cultured hippocampal neuron media following cLTP induction. cLTP-induced conditioned media were collected at 0, 10, 30, 60 minutes, and 1day following cLTP induction. BDNF release (left panel) was significantly increased at 30 minutes and 1 hour following cLTP induction. *p<0.05, **p<0.01, n=5. The secreted levels of NGF (n=5) and NT-3 (right panel, n=5) were not significantly increased. B, Cultured NSCs were treated with conditioned media. Surface biotinylation results show conditioned media enhanced the expression of phosphorylated tyrosine kinase receptor, p-TrkB and that this enhancement could be eliminated by treatment with the tyrosine kinase inhibitor, K252a. Bar graph summary of activated TrkB by cLTP-induced conditioned media and its inhibition by K252a application. **p<0.01, n=4. The quantification of biotinylation results was standardized with the loading control values with TrkB. C, Representative images illustrate that cLTP enhanced the total GFP (green)-positive cells and MAP2 (red) staining of GFP-positive NSCs co-cultured with hippocampal neurons compared with control group (PBS) and its elimination by K252a application. Bar graph summary of cLTP enhancement of the total GFP-positive cells (left panel) and neuronal differentiation (right panel). Enhanced neuronal differentiation of co-cultured NSCs, quantified as MAP2-positive mature neurons on GFP-positive cells, by cLTP induction was blocked by K252a application.  **p<0.01, n=6.        100            101 4 Conclusion  Cell replacement therapy has been actively investigated as an effective treatment for many neurodegenerative disorders, ranging from acute brain insults such as stroke and brain trauma to chronic neurodegenerative diseases such as AD, HD and PD. Stem cell technology is a rapidly evolving field that will likely significantly impact the replacement therapy for neurodegenerative diseases by providing cell resources. Many pre-clinical animal studies have shown that multipotent NSCs following transplantation can survive and may thereby contribute to the enhanced functional recoveries (Orlacchio et al., 2010). However, despite these numerous positive studies in animal disease models, there remain major challenges that have been limiting the clinical use of NSC transplantation as an effective cell replacement therapy (Lindvall and Kokaia, 2010; Orlacchio et al., 2010). NPCs following transplantation may not properly differentiate into neurons and do not usually survive long enough to form functional connections with host neurons. Moreover, mechanisms by which these transplanted NPCs result in functional recoveries, albeit limited, remain not fully understood. Thus, studies on all these fronts in stem technology are urgently needed to enhance the potential use of NPCs for CNS cell replacement therapies.  Stem cell therapy has generally been considered as a cell replacement therapy to replace damaged neurons in the affected brain area, thereby restoring the function of impaired neuronal circuitry. However, emerging evidence suggests that transplantation of NSCs may also lead to clinically appreciable functional improvements through other mechanisms. To this end, my results presented in Chapter 2 demonstrate that transplantation of NPCs provides a significant neuroprotection, at least in part, via inhibition of inflammatory responses in an animal model of AD. In AD patients, microglia can be activated by Aβ, and the activated microglia may compromise the survival of neurons and/or impair the function of neuronal circuitries likely via 102 increased release of pro-inflammatory cytokines, such as IL-1β, TNF-α, IL-6 (Giulian, 1997). Consistent with these previously reported results, I also found that intra-hippocampal injection of Aβ1-42 markedly increased microgliosis and astrogliosis in the brain area surrounding the injection sites along with an enhanced level of proinflammatory cytokine TNF-α. Importantly, transplantation of NPCs not only attenuated the microglia activation and consequent release of TNF-α, but also reduced the accompanied neuronal loss. It must be noted that any benefit from stem cell therapy, such as obtained here in this AD model, is confounded by a number of complex issues, including multiple factors involved in disease pathology, and the loss of neuronal and synaptic viability in widespread affected brain regions. Nevertheless, our data are noteworthy in demonstrating that neuroprotective efficacy of NSC transplantation in this animal model of AD is likely, at least in part, resulting from the inhibition of  inflammatory activity (McLarnon and Ryu, 2008). Although the effect of NPC transplantation in promoting neuronal viability is modest, it is reasonable to assume that levels of neuroprotection may be further improved by optimizing transplantation protocols, such as earlier transplantation time point and higher doses of NPCs. Overall, our findings, along with recent work using transgenic mice (Blurton-Jones et al., 2009), suggest that NPC transplantation represents a novel and plausible approach that warrants extensive testing as an effective anti-inflammatory and neuroprotective therapy in AD animal models.     While promoting neurogenesis of endogenous NPCs or transplantation of exogenous NSCs/NPCs have generally been considered as promising cell replacement therapies to replace damaged neurons in affected brain areas for many neurodegenerative diseases, their practical use as a clinical therapy for any brain disorder remains disappointing, due at least in part to the short period of existence of these NPCs, and to their failure of neuronal differentiation and functional integration into the host neuronal networks (Brustle and McKay, 1996; Bakshi et al., 2005; Burns et al., 2006). Therefore, clinically relevant protocols that can facilitate proliferation/survival and neuronal differentiation of both endogenous and transplanted 103 NSCs/NPCs are urgently needed to improve the clinical use of NSCs/NPCs as cell replacement therapies. My results presented in chapter 3 provide strong evidence that electrical stimulation with various protocols such as HFS and sTPS can reliably induce NMDAR-mediated LTP; the induction of LTP in turn promotes proliferation/survival and neuronal differentiation of not only endogenous NPCs in the DG area of the hippocampus, but also of exogenous transplanted NSCs into the hippocampal CA1 region in the rat. Given that various devices have been developed for delivering deep brain stimulation (DBS) for clinical use as effective therapies for treatment of a number brain disorders, the LTP protocols demonstrated by my study may have immediate clinical application for increasing proliferation/survival and neuronal differentiation of either endogenous or transplanted NSCs/NPCs, thereby promoting their functional integration into the neuronal network and hence increasing the success rate and efficacy of NSC/NPC replacement therapy. Using a NSC-neuron co-culture, I further demonstrated that the induction of LTP may also be accomplished by a chemical protocol, i.e. by selective activation of synaptic NMDARs with NMDAR co-agonist glycine. Most importantly, I found that similar to the electrically induced LTP, this chemically induced LTP (cLTP) can also promote the differentiation/survival and neuronal differentiation of NSCs. Technically, electrical stimulation to induce LTP requires special stimulation devices and also is limited to the stimulated synapses in the host neurons. However, cLTP is able to stimulate all synapses on neurons in the stimulated regions. In comparison with electrical stimulation protocol, our chemical stimulation protocol may be much easier to use and have higher efficacy in terms of promoting neurogenesis of both endogenous and transplanted NSCs, thereby representing another clinically relevant protocol for cell replacement therapy. Finally, my results from ELISA analysis demonstrate the increased release of BDNF, but not NGF and NT-3 (Figure 3-7A) from hippocampal neurons following LTP induction. This supports the idea that the LTP-induced release of BDNF may be, at least in part, responsible for the facilitated neurogenesis of NSCs/NPCs following LTP, because I found that the LTP-induced neurogenesis could be significantly suppressed by inhibition of TrkB receptor activation. These 104 results are not only in a good agreement with well-characterized effects of BDNF-TrkB system in facilitating LTP induction (Lohof et al., 1993; Kang and Schuman, 1995; Korte et al., 1995; Figurov et al., 1996; Patterson et al., 1996), neuronal survival and neurogenesis (Kirschenbaum and Goldman, 1995; Minichiello and Klein, 1996; Goldman et al., 1997; Pincus et al., 1998; Zigova et al., 1998; Suh et al., 2009; Pathania et al., 2010), but may also suggest that BDNF might have clinical utility in facilitating neurogenesis of both endogenous and transplanted NSCs/NPCs.  The present investigation has raised several important issues which should be addressed in future studies. Firstly, basic mechanisms underlying inhibition of microglial inflammatory responses by transplanted NPCs could be carried out. It would be of interest to determine the role of some diffusible factors from undifferentiated NPCs in inhibiting microglial inflammatory responses and whether they could serve as potential inhibitors of inflammatory responses in AD patients. A recent study has supported this idea that NSCs improve cognition via BDNF in a transgenic model of AD (Blurton-Jones et al., 2009). Secondly, LTP-increased total numbers of NSCs/NPCs could be further investigated to determine whether this effect is from proliferation and/or survival. Proposed studies would include measurements of either survival or cell death signals in NSCs/NPCs following LTP stimulation. Moreover, additional studies could be carried out using specific markers for proliferating cells.    Thirdly, further research should be carried out to determine whether the functional integration of LTP-increased NSCs/NPCs into the neuronal network is effective in ameliorating damaged neuronal circuitry. It would be relevant to complete electrophysiological analysis including measurements of voltage-dependent channels for action potentials and/or EPSCs. Furthermore, the effect of LTP-NSC/NPC therapy on the neuronal network could be compared to the electrophysiological dysfunction in each disease state. Uncontrollable and inappropriate 105 differentiation could result in production of unexpected cells (Barker and Widner, 2004; Chen and Palmer, 2008). Thus, electrophysiological analysis will give better understanding for an appropriate control of NSCs/NPCs.  As advances are made on all of these fronts, more effective and clinically relevant cell replacement based on facilitation of either endogenous or transplanted NSCs/NPC for treating numerous neurodegenerative diseases should be emerge.                    106 References  Aarum J, Sandberg K, Haeberlein SL, Persson MA (2003) Migration and differentiation of neural precursor cells can be directed by microglia. Proc Natl Acad Sci U S A 100:15983-15988. Abraham WC, Mason SE (1988) Effects of the NMDA receptor/channel antagonists CPP and MK801 on hippocampal field potentials and long-term potentiation in anesthetized rats. Brain Res 462:40-46. Abraham WC, Logan B, Greenwood JM, Dragunow M (2002) Induction and experience- dependent consolidation of stable long-term potentiation lasting months in the hippocampus. J Neurosci 22:9626-9634. Abrous DN, Koehl M, Le Moal M (2005) Adult neurogenesis: from precursors to network and physiology. Physiol Rev 85:523-569. Acheson A, Conover JC, Fandl JP, DeChiara TM, Russell M, Thadani A, Squinto SP, Yancopoulos GD, Lindsay RM (1995) A BDNF autocrine loop in adult sensory neurons prevents cell death. Nature 374:450-453. Adesnik H, Nicoll RA, England PM (2005) Photoinactivation of native AMPA receptors reveals their real-time trafficking. Neuron 48:977-985. Ahmed S, Reynolds BA, Weiss S (1995) BDNF enhances the differentiation but not the survival of CNS stem cell-derived neuronal precursors. J Neurosci 15:5765-5778. Aimone JB, Wiles J, Gage FH (2006) Potential role for adult neurogenesis in the encoding of time in new memories. Nat Neurosci 9:723-727. Akazawa C, Shigemoto R, Bessho Y, Nakanishi S, Mizuno N (1994) Differential expression of five N-methyl-D-aspartate receptor subunit mRNAs in the cerebellum of developing and adult rats. J Comp Neurol 347:150-160. Akesson E, Sandelin M, Kanaykina N, Aldskogius H, Kozlova EN (2008) Long-term survival, robust neuronal differentiation, and extensive migration of human forebrain stem/progenitor cells transplanted to the adult rat dorsal root ganglion cavity. Cell Transplant 17:1115-1123. Akiyama H et al. (2000) Inflammation and Alzheimer's disease. Neurobiol Aging 21:383-421. Alkon DL, Nelson TJ (1990) Specificity of molecular changes in neurons involved in memory storage. FASEB J 4:1567-1576. Alonso M, Vianna MR, Depino AM, Mello e Souza T, Pereira P, Szapiro G, Viola H, Pitossi F, Izquierdo I, Medina JH (2002) BDNF-triggered events in the rat hippocampus are required for both short- and long-term memory formation. Hippocampus 12:551-560. Altman J (1962) Are new neurons formed in the brains of adult mammals? Science 135:1127- 1128. Altman J (1963) Autoradiographic investigation of cell proliferation in the brains of rats and cats. 107 Anat Rec 145:573-591. Altman J (1969) Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. J Comp Neurol 137:433-457. Altman J, Das GD (1965) Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J Comp Neurol 124:319-335. Appel SH, Engelhardt JI, Henkel JS, Siklos L, Beers DR, Yen AA, Simpson EP, Luo Y, Carrum G, Heslop HE, Brenner MK, Popat U (2008) Hematopoietic stem cell transplantation in patients with sporadic amyotrophic lateral sclerosis. Neurology 71:1326-1334. Arevalo JC, Wu SH (2006) Neurotrophin signaling: many exciting surprises! Cell Mol Life Sci 63:1523-1537. Arias-Carrion O, Olivares-Bunuelos T, Drucker-Colin R (2007) [Neurogenesis in the adult brain]. Rev Neurol 44:541-550. Arnold SE, Hyman BT, Flory J, Damasio AR, Van Hoesen GW (1991) The topographical and neuroanatomical distribution of neurofibrillary tangles and neuritic plaques in the cerebral cortex of patients with Alzheimer's disease. Cereb Cortex 1:103-116. Aronheim A, Shiran R, Rosen A, Walker MD (1993) The E2A gene product contains two separable and functionally distinct transcription activation domains. Proc Natl Acad Sci U S A 90:8063-8067. Ashby MC, Maier SR, Nishimune A, Henley JM (2006) Lateral diffusion drives constitutive exchange of AMPA receptors at dendritic spines and is regulated by spine morphology. J Neurosci 26:7046-7055. Ashe KH (2001) Learning and memory in transgenic mice modeling Alzheimer's disease. Learn Mem 8:301-308. Asztely F, Wigstrom H, Gustafsson B (1992) The Relative Contribution of NMDA Receptor Channels in the Expression of Long-term Potentiation in the Hippocampal CA1 Region. Eur J Neurosci 4:681-690. Bachoud-Levi AC, Gaura V, Brugieres P, Lefaucheur JP, Boisse MF, Maison P, Baudic S, Ribeiro MJ, Bourdet C, Remy P, Cesaro P, Hantraye P, Peschanski M (2006) Effect of fetal neural transplants in patients with Huntington's disease 6 years after surgery: a long- term follow-up study. Lancet Neurol 5:303-309. Bailey CH, Giustetto M, Huang YY, Hawkins RD, Kandel ER (2000) Is heterosynaptic modulation essential for stabilizing Hebbian plasticity and memory? Nat Rev Neurosci 1:11-20. Baker M (2009) Stem cells: Fast and furious. Nature 458:962-965. Bakshi A, Keck CA, Koshkin VS, LeBold DG, Siman R, Snyder EY, McIntosh TK (2005) Caspase-mediated cell death predominates following engraftment of neural progenitor cells into traumatically injured rat brain. Brain Res 1065:8-19. Banati RB, Graeber MB (1994) Surveillance, intervention and cytotoxicity: is there a protective 108 role of microglia? Dev Neurosci 16:114-127. Banke TG, Bowie D, Lee H, Huganir RL, Schousboe A, Traynelis SF (2000) Control of GluR1 AMPA receptor function by cAMP-dependent protein kinase. J Neurosci 20:89-102. Bannigan JG (1985) The effects of 5-bromodeoxyuridine on fusion of the cranial neural folds in the mouse embryo. Teratology 32:229-239. Barbacid M (1994) The Trk family of neurotrophin receptors. J Neurobiol 25:1386-1403. Barde YA, Edgar D, Thoenen H (1982) Purification of a new neurotrophic factor from mammalian brain. EMBO J 1:549-553. Barker JL, Behar T, Li YX, Liu QY, Ma W, Maric D, Maric I, Schaffner AE, Serafini R, Smith SV, Somogyi R, Vautrin JY, Wen XL, Xian H (1998) GABAergic cells and signals in CNS development. Perspect Dev Neurobiol 5:305-322. Barker RA, Widner H (2004) Immune problems in central nervous system cell therapy. NeuroRx 1:472-481. Barnabe-Heider F, Miller FD (2003) Endogenously produced neurotrophins regulate survival and differentiation of cortical progenitors via distinct signaling pathways. J Neurosci 23:5149-5160. Barnea A, Nottebohm F (1994) Seasonal recruitment of hippocampal neurons in adult free- ranging black-capped chickadees. Proc Natl Acad Sci U S A 91:11217-11221. Barria A, Derkach V, Soderling T (1997a) Identification of the Ca2+/calmodulin-dependent protein kinase II regulatory phosphorylation site in the alpha-amino-3-hydroxyl-5-methyl- 4-isoxazole-propionate-type glutamate receptor. J Biol Chem 272:32727-32730. Barria A, Muller D, Derkach V, Griffith LC, Soderling TR (1997b) Regulatory phosphorylation of AMPA-type glutamate receptors by CaM-KII during long-term potentiation. Science 276:2042-2045. Barry PH, Lynch JW (1991) Liquid junction potentials and small cell effects in patch-clamp analysis. J Membr Biol 121:101-117. Bartus RT, Dean RL, 3rd, Beer B, Lippa AS (1982) The cholinergic hypothesis of geriatric memory dysfunction. Science 217:408-414. Bashir ZI, Alford S, Davies SN, Randall AD, Collingridge GL (1991) Long-term potentiation of NMDA receptor-mediated synaptic transmission in the hippocampus. Nature 349:156- 158. Bats C, Groc L, Choquet D (2007) The interaction between Stargazin and PSD-95 regulates AMPA receptor surface trafficking. Neuron 53:719-734. Bear MF, Malenka RC (1994) Synaptic plasticity: LTP and LTD. Curr Opin Neurobiol 4:389-399. Beattie EC, Carroll RC, Yu X, Morishita W, Yasuda H, von Zastrow M, Malenka RC (2000) Regulation of AMPA receptor endocytosis by a signaling mechanism shared with LTD. Nat Neurosci 3:1291-1300. 109 Becker S (2005) A computational principle for hippocampal learning and neurogenesis. Hippocampus 15:722-738. Behe P, Stern P, Wyllie DJ, Nassar M, Schoepfer R, Colquhoun D (1995) Determination of NMDA NR1 subunit copy number in recombinant NMDA receptors. Proc Biol Sci 262:205-213. Bekkers JM, Stevens CF (1995) Quantal analysis of EPSCs recorded from small numbers of synapses in hippocampal cultures. J Neurophysiol 73:1145-1156. Bekkers JM, Stevens CF (1996) Cable properties of cultured hippocampal neurons determined from sucrose-evoked miniature EPSCs. J Neurophysiol 75:1250-1255. Belluzzi O, Benedusi M, Ackman J, LoTurco JJ (2003) Electrophysiological differentiation of new neurons in the olfactory bulb. J Neurosci 23:10411-10418. Ben-Ari Y (2002) Excitatory actions of gaba during development: the nature of the nurture. Nature Reviews Neuroscience 3:728-739. Ben-Ari Y, Cherubini E, Corradetti R, Gaiarsa JL (1989) Giant synaptic potentials in immature rat CA3 hippocampal neurones. J Physiol 416:303-325. Ben-Ari Y, Tseeb V, Raggozzino D, Khazipov R, Gaiarsa JL (1994) gamma-Aminobutyric acid (GABA): a fast excitatory transmitter which may regulate the development of hippocampal neurones in early postnatal life. Prog Brain Res 102:261-273. Benke TA, Luthi A, Isaac JT, Collingridge GL (1998) Modulation of AMPA receptor unitary conductance by synaptic activity. Nature 393:793-797. Benraiss A, Chmielnicki E, Lerner K, Roh D, Goldman SA (2001) Adenoviral brain-derived neurotrophic factor induces both neostriatal and olfactory neuronal recruitment from endogenous progenitor cells in the adult forebrain. J Neurosci 21:6718-6731. Benveniste EN (1992) Inflammatory cytokines within the central nervous system: sources, function, and mechanism of action. Am J Physiol 263:C1-16. Benveniste EN, Benos DJ (1995) TNF-alpha- and IFN-gamma-mediated signal transduction pathways: effects on glial cell gene expression and function. FASEB J 9:1577-1584. Bernal GM, Peterson DA (2004) Neural stem cells as therapeutic agents for age-related brain repair. Aging Cell 3:345-351. Bernhardt R, Matus A (1984) Light and electron microscopic studies of the distribution of microtubule-associated protein 2 in rat brain: a difference between dendritic and axonal cytoskeletons. J Comp Neurol 226:203-221. Berninger B, Marty S, Zafra F, da Penha Berzaghi M, Thoenen H, Lindholm D (1995) GABAergic stimulation switches from enhancing to repressing BDNF expression in rat hippocampal neurons during maturation in vitro. Development 121:2327-2335. Berretta N, Berton F, Bianchi R, Brunelli M, Capogna M, Francesconi W (1991) Long-term Potentiation of NMDA Receptor-mediated EPSP in Guinea-pig Hippocampal Slices. Eur J Neurosci 3:850-854. 110 Bettler B, Mulle C (1995) Review: neurotransmitter receptors. II. AMPA and kainate receptors. Neuropharmacology 34:123-139. Bhattacharyya BJ, Banisadr G, Jung H, Ren D, Cronshaw DG, Zou Y, Miller RJ (2008) The chemokine stromal cell-derived factor-1 regulates GABAergic inputs to neural progenitors in the postnatal dentate gyrus. J Neurosci 28:6720-6730. Biffi A, Lucchini G, Rovelli A, Sessa M (2008) Metachromatic leukodystrophy: an overview of current and prospective treatments. Bone Marrow Transplant 42 Suppl 2:S2-6. Binder DK, Scharfman HE (2004) Brain-derived neurotrophic factor. Growth Factors 22:123-131. Bizon JL, Lee HJ, Gallagher M (2004) Neurogenesis in a rat model of age-related cognitive decline. Aging Cell 3:227-234. Black IB (1999) Trophic regulation of synaptic plasticity. J Neurobiol 41:108-118. Bliss TV, Gardner-Medwin AR (1973) Long-lasting potentiation of synaptic transmission in the dentate area of the unanaestetized rabbit following stimulation of the perforant path. J Physiol 232:357-374. Bliss TV, Lomo T (1973) 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. Bliss TV, Collingridge GL (1993) A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361:31-39. Blurton-Jones M, Kitazawa M, Martinez-Coria H, Castello NA, Muller FJ, Loring JF, Yamasaki TR, Poon WW, Green KN, LaFerla FM (2009) Neural stem cells improve cognition via BDNF in a transgenic model of Alzheimer disease. Proc Natl Acad Sci U S A 106:13594- 13599. Boehm J, Kang MG, Johnson RC, Esteban J, Huganir RL, Malinow R (2006) Synaptic incorporation of AMPA receptors during LTP is controlled by a PKC phosphorylation site on GluR1. Neuron 51:213-225. Bonfanti L, Theodosis DT (1994) Expression of polysialylated neural cell adhesion molecule by proliferating cells in the subependymal layer of the adult rat, in its rostral extension and in the olfactory bulb. Neuroscience 62:291-305. Borgdorff AJ, Choquet D (2002) Regulation of AMPA receptor lateral movements. Nature 417:649-653. Bortolotto ZA, Nistico R, More JC, Jane DE, Collingridge GL (2005) Kainate receptors and mossy fiber LTP. Neurotoxicology 26:769-777. Bortolotto ZA, Clarke VR, Delany CM, Parry MC, Smolders I, Vignes M, Ho KH, Miu P, Brinton BT, Fantaske R, Ogden A, Gates M, Ornstein PL, Lodge D, Bleakman D, Collingridge GL (1999) Kainate receptors are involved in synaptic plasticity. Nature 402:297-301. Boulter J, Hollmann M, O'Shea-Greenfield A, Hartley M, Deneris E, Maron C, Heinemann S (1990) Molecular cloning and functional expression of glutamate receptor subunit genes. Science 249:1033-1037. 111 Braithwaite SP, Meyer G, Henley JM (2000) Interactions between AMPA receptors and intracellular proteins. Neuropharmacology 39:919-930. Breustedt J, Schmitz D (2004) Assessing the role of GLUK5 and GLUK6 at hippocampal mossy fiber synapses. J Neurosci 24:10093-10098. Breustedt J, Vogt KE, Miller RJ, Nicoll RA, Schmitz D (2003) Alpha1E-containing Ca2+ channels are involved in synaptic plasticity. Proc Natl Acad Sci U S A 100:12450-12455. Brickley SG, Misra C, Mok MH, Mishina M, Cull-Candy SG (2003) NR2B and NR2D subunits coassemble in cerebellar Golgi cells to form a distinct NMDA receptor subtype restricted to extrasynaptic sites. J Neurosci 23:4958-4966. Bronstein JM et al. (2011) Deep brain stimulation for Parkinson disease: an expert consensus and review of key issues. Arch Neurol 68:165. Brookmeyer R, Johnson E, Ziegler-Graham K, Arrighi HM (2007) Forecasting the global burden of Alzheimer's disease. Alzheimers Dement 3:186-191. Broutman G, Baudry M (2001) Involvement of the secretory pathway for AMPA receptors in NMDA-induced potentiation in hippocampus. J Neurosci 21:27-34. Brown J, Cooper-Kuhn CM, Kempermann G, Van Praag H, Winkler J, Gage FH, Kuhn HG (2003) Enriched environment and physical activity stimulate hippocampal but not olfactory bulb neurogenesis. Eur J Neurosci 17:2042-2046. Bruel-Jungerman E, Davis S, Rampon C, Laroche S (2006) Long-term potentiation enhances neurogenesis in the adult dentate gyrus. J Neurosci 26:5888-5893. Brustle O, McKay RD (1996) Neuronal progenitors as tools for cell replacement in the nervous system. Curr Opin Neurobiol 6:688-695. Burnashev N, Monyer H, Seeburg PH, Sakmann B (1992) Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit. Neuron 8:189-198. Burns TC, Ortiz-Gonzalez XR, Gutierrez-Perez M, Keene CD, Sharda R, Demorest ZL, Jiang Y, Nelson-Holte M, Soriano M, Nakagawa Y, Luquin MR, Garcia-Verdugo JM, Prosper F, Low WC, Verfaillie CM (2006) Thymidine analogs are transferred from prelabeled donor to host cells in the central nervous system after transplantation: a word of caution. Stem Cells 24:1121-1127. Caillard O, Ben-Ari Y, Gaiarsa JL (1999a) Long-term potentiation of GABAergic synaptic transmission in neonatal rat hippocampus. J Physiol 518 ( Pt 1):109-119. Caillard O, Ben-Ari Y, Gaiarsa JL (1999b) Mechanisms of induction and expression of long-term depression at GABAergic synapses in the neonatal rat hippocampus. J Neurosci 19:7568-7577. Cameron HA, McKay RD (2001) Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. J Comp Neurol 435:406-417. Cameron HA, McEwen BS, Gould E (1995) Regulation of adult neurogenesis by excitatory input and NMDA receptor activation in the dentate gyrus. J Neurosci 15:4687-4692. 112 Cameron HA, Woolley CS, McEwen BS, Gould E (1993) Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat. Neuroscience 56:337-344. Carden MJ, Trojanowski JQ, Schlaepfer WW, Lee VM (1987) Two-stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation patterns. J Neurosci 7:3489-3504. Carlen M, Cassidy RM, Brismar H, Smith GA, Enquist LW, Frisen J (2002) Functional integration of adult-born neurons. Curr Biol 12:606-608. Carlezon WA, Jr., Haile CN, Coppersmith R, Hayashi Y, Malinow R, Neve RL, Nestler EJ (2000) Distinct sites of opiate reward and aversion within the midbrain identified using a herpes simplex virus vector expressing GluR1. J Neurosci 20:RC62. Carroll RC, Lissin DV, von Zastrow M, Nicoll RA, Malenka RC (1999a) Rapid redistribution of glutamate receptors contributes to long-term depression in hippocampal cultures. Nat Neurosci 2:454-460. Carroll RC, Beattie EC, Xia H, Luscher C, Altschuler Y, Nicoll RA, Malenka RC, von Zastrow M (1999b) Dynamin-dependent endocytosis of ionotropic glutamate receptors. Proc Natl Acad Sci U S A 96:14112-14117. Casarosa S, Fode C, Guillemot F (1999) Mash1 regulates neurogenesis in the ventral telencephalon. Development 126:525-534. Castillo PE, Weisskopf MG, Nicoll RA (1994) The role of Ca2+ channels in hippocampal mossy fiber synaptic transmission and long-term potentiation. Neuron 12:261-269. Castren E, Pitkanen M, Sirvio J, Parsadanian A, Lindholm D, Thoenen H, Riekkinen PJ (1993) The induction of LTP increases BDNF and NGF mRNA but decreases NT-3 mRNA in the dentate gyrus. Neuroreport 4:895-898. Cathala L, Misra C, Cull-Candy S (2000) Developmental profile of the changing properties of NMDA receptors at cerebellar mossy fiber-granule cell synapses. J Neurosci 20:5899- 5905. Cau E, Gradwohl G, Fode C, Guillemot F (1997) Mash1 activates a cascade of bHLH regulators in olfactory neuron progenitors. Development 124:1611-1621. Chao MV (1992) Neurotrophin receptors: a window into neuronal differentiation. Neuron 9:583- 593. Chao MV, Bothwell M (2002) Neurotrophins: to cleave or not to cleave. Neuron 33:9-12. Chen G, Trombley PQ, van den Pol AN (1996) Excitatory actions of GABA in developing rat hypothalamic neurones. J Physiol 494 ( Pt 2):451-464. Chen HJ, Rojas-Soto M, Oguni A, Kennedy MB (1998) A synaptic Ras-GTPase activating protein (p135 SynGAP) inhibited by CaM kinase II. Neuron 20:895-904. Chen HS, Lipton SA (1997) Mechanism of memantine block of NMDA-activated channels in rat retinal ganglion cells: uncompetitive antagonism. J Physiol 499 ( Pt 1):27-46. Chen L, Chetkovich DM, Petralia RS, Sweeney NT, Kawasaki Y, Wenthold RJ, Bredt DS, Nicoll 113 RA (2000) Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms. Nature 408:936-943. Chen Z, Palmer TD (2008) Cellular repair of CNS disorders: an immunological perspective. Hum Mol Genet 17:R84-92. Cherubini E, Gaiarsa JL, Ben-Ari Y (1991) GABA: an excitatory transmitter in early postnatal life. Trends Neurosci 14:515-519. Chmielnicki E, Benraiss A, Economides AN, Goldman SA (2004) Adenovirally expressed noggin and brain-derived neurotrophic factor cooperate to induce new medium spiny neurons from resident progenitor cells in the adult striatal ventricular zone. J Neurosci 24:2133- 2142. Choquet D, Triller A (2003) The role of receptor diffusion in the organization of the postsynaptic membrane. Nature Reviews Neuroscience 4:251-265. Christie BR, Cameron HA (2006) Neurogenesis in the adult hippocampus. Hippocampus 16:199-207. Chun SK, Sun W, Park JJ, Jung MW (2006) Enhanced proliferation of progenitor cells following long-term potentiation induction in the rat dentate gyrus. Neurobiol Learn Mem 86:322- 329. Chung HJ, Xia J, Scannevin RH, Zhang X, Huganir RL (2000) Phosphorylation of the AMPA receptor subunit GluR2 differentially regulates its interaction with PDZ domain- containing proteins. J Neurosci 20:7258-7267. Coan EJ, Collingridge GL (1985) Magnesium ions block an N-methyl-D-aspartate receptor- mediated component of synaptic transmission in rat hippocampus. Neurosci Lett 53:21- 26. Colledge M, Dean RA, Scott GK, Langeberg LK, Huganir RL, Scott JD (2000) Targeting of PKA to glutamate receptors through a MAGUK-AKAP complex. Neuron 27:107-119. Collingridge GL (1992) The Sharpey-Schafer Prize Lecture. The mechanism of induction of NMDA receptor-dependent long-term potentiation in the hippocampus. Exp Physiol 77:771-797. Collingridge GL, Kehl SJ, McLennan H (1983) Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus. J Physiol 334:33-46. Collingridge GL, Isaac JT, Wang YT (2004) Receptor trafficking and synaptic plasticity. Nat Rev Neurosci 5:952-962. Collingridge GL, Peineau S, Howland JG, Wang YT (2010) Long-term depression in the CNS. Nat Rev Neurosci 11:459-473. Combs CK, Bates P, Karlo JC, Landreth GE (2001a) Regulation of beta-amyloid stimulated proinflammatory responses by peroxisome proliferator-activated receptor alpha. Neurochem Int 39:449-457. Combs CK, Karlo JC, Kao SC, Landreth GE (2001b) beta-Amyloid stimulation of microglia and 114 monocytes results in TNFalpha-dependent expression of inducible nitric oxide synthase and neuronal apoptosis. J Neurosci 21:1179-1188. Combs CK, Johnson DE, Cannady SB, Lehman TM, Landreth GE (1999) Identification of microglial signal transduction pathways mediating a neurotoxic response to amyloidogenic fragments of beta-amyloid and prion proteins. J Neurosci 19:928-939. Conner JM, Lauterborn JC, Yan Q, Gall CM, Varon S (1997) Distribution of brain-derived neurotrophic factor (BDNF) protein and mRNA in the normal adult rat CNS: evidence for anterograde axonal transport. J Neurosci 17:2295-2313. Conti L, Cattaneo E (2010) Neural stem cell systems: physiological players or in vitro entities? Nat Rev Neurosci 11:176-187. Contractor A, Swanson G, Heinemann SF (2001) Kainate receptors are involved in short- and long-term plasticity at mossy fiber synapses in the hippocampus. Neuron 29:209-216. Cotman CW, Berchtold NC (2002) Exercise: a behavioral intervention to enhance brain health and plasticity. Trends Neurosci 25:295-301. Coyle JT, Price DL, DeLong MR (1983) Alzheimer's disease: a disorder of cortical cholinergic innervation. Science 219:1184-1190. Craig CG, D'sa R, Morshead CM, Roach A, van der Kooy D (1999) Migrational analysis of the constitutively proliferating subependyma population in adult mouse forebrain. Neuroscience 93:1197-1206. Craig CG, Tropepe V, Morshead CM, Reynolds BA, Weiss S, van der Kooy D (1996) In vivo growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain. J Neurosci 16:2649-2658. Creer DJ, Romberg C, Saksida LM, van Praag H, Bussey TJ (2010) Running enhances spatial pattern separation in mice. Proc Natl Acad Sci U S A 107:2367-2372. Cull-Candy SG, Leszkiewicz DN (2004) Role of distinct NMDA receptor subtypes at central synapses. Sci STKE 2004:re16. Curtis MA, Kam M, Nannmark U, Anderson MF, Axell MZ, Wikkelso C, Holtas S, van Roon-Mom WM, Bjork-Eriksson T, Nordborg C, Frisen J, Dragunow M, Faull RL, Eriksson PS (2007) Human neuroblasts migrate to the olfactory bulb via a lateral ventricular extension. Science 315:1243-1249. Dammerman RS, Flint AC, Noctor S, Kriegstein AR (2000) An excitatory GABAergic plexus in developing neocortical layer 1. J Neurophysiol 84:428-434. Das S, Sasaki YF, Rothe T, Premkumar LS, Takasu M, Crandall JE, Dikkes P, Conner DA, Rayudu PV, Cheung W, Chen HS, Lipton SA, Nakanishi N (1998) Increased NMDA current and spine density in mice lacking the NMDA receptor subunit NR3A. Nature 393:377-381. Davies SN, Lester RA, Reymann KG, Collingridge GL (1989) Temporally distinct pre- and post- synaptic mechanisms maintain long-term potentiation. Nature 338:500-503. Davis JB, McMurray HF, Schubert D (1992) The amyloid beta-protein of Alzheimer's disease is 115 chemotactic for mononuclear phagocytes. Biochem Biophys Res Commun 189:1096- 1100. Daw MI, Chittajallu R, Bortolotto ZA, Dev KK, Duprat F, Henley JM, Collingridge GL, Isaac JT (2000) PDZ proteins interacting with C-terminal GluR2/3 are involved in a PKC- dependent regulation of AMPA receptors at hippocampal synapses. Neuron 28:873-886. Dayer AG, Ford AA, Cleaver KM, Yassaee M, Cameron HA (2003) Short-term and long-term survival of new neurons in the rat dentate gyrus. J Comp Neurol 460:563-572. de Jonge M, Racine RJ (1985) The effects of repeated induction of long-term potentiation in the dentate gyrus. Brain Res 328:181-185. de la Torre JC (2002) Alzheimer's disease: how does it start? J Alzheimers Dis 4:497-512. Debus E, Weber K, Osborn M (1983) Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides. Differentiation 25:193-203. Deda H, Inci MC, Kurekci AE, Sav A, Kayihan K, Ozgun E, Ustunsoy GE, Kocabay S (2009) Treatment of amyotrophic lateral sclerosis patients by autologous bone marrow-derived hematopoietic stem cell transplantation: a 1-year follow-up. Cytotherapy 11:18-25. Deisseroth K, Singla S, Toda H, Monje M, Palmer TD, Malenka RC (2004) Excitation- neurogenesis coupling in adult neural stem/progenitor cells. Neuron 42:535-552. Delgado JY, Coba M, Anderson CN, Thompson KR, Gray EE, Heusner CL, Martin KC, Grant SG, O'Dell TJ (2007) NMDA receptor activation dephosphorylates AMPA receptor glutamate receptor 1 subunits at threonine 840. J Neurosci 27:13210-13221. Delpire E (2000) Cation-Chloride Cotransporters in Neuronal Communication. News Physiol Sci 15:309-312. Deng W, Aimone JB, Gage FH (2010) New neurons and new memories: how does adult hippocampal neurogenesis affect learning and memory? Nat Rev Neurosci 11:339-350. Deng W, Saxe MD, Gallina IS, Gage FH (2009) Adult-born hippocampal dentate granule cells undergoing maturation modulate learning and memory in the brain. J Neurosci 29:13532-13542. Derkach V, Barria A, Soderling TR (1999) 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. Derkach VA (2003) Silence analysis of AMPA receptor mutated at the CaM-kinase II phosphorylation site. Biophys J 84:1701-1708. Derrick BE, York AD, Martinez JL, Jr. (2000) Increased granule cell neurogenesis in the adult dentate gyrus following mossy fiber stimulation sufficient to induce long-term potentiation. Brain Res 857:300-307. Dev KK, Nishimune A, Henley JM, Nakanishi S (1999) The protein kinase C alpha binding protein PICK1 interacts with short but not long form alternative splice variants of AMPA receptor subunits. Neuropharmacology 38:635-644. 116 Dietrich D, Kirschstein T, Kukley M, Pereverzev A, von der Brelie C, Schneider T, Beck H (2003) Functional specialization of presynaptic Cav2.3 Ca2+ channels. Neuron 39:483-496. Dingledine R, Hume RI, Heinemann SF (1992) Structural determinants of barium permeation and rectification in non-NMDA glutamate receptor channels. J Neurosci 12:4080-4087. Dingledine R, Borges K, Bowie D, Traynelis SF (1999) The glutamate receptor ion channels. Pharmacol Rev 51:7-61. Dobrossy MD, Drapeau E, Aurousseau C, Le Moal M, Piazza PV, Abrous DN (2003) Differential effects of learning on neurogenesis: learning increases or decreases the number of newly born cells depending on their birth date. Mol Psychiatry 8:974-982. Dobrunz LE, Stevens CF (1999) Response of hippocampal synapses to natural stimulation patterns. Neuron 22:157-166. Doetsch F, Hen R (2005) Young and excitable: the function of new neurons in the adult mammalian brain. Curr Opin Neurobiol 15:121-128. Dong H, Zhang P, Liao D, Huganir RL (1999a) Characterization, expression, and distribution of GRIP protein. Ann N Y Acad Sci 868:535-540. Dong H, O'Brien RJ, Fung ET, Lanahan AA, Worley PF, Huganir RL (1997) GRIP: a synaptic PDZ domain-containing protein that interacts with AMPA receptors. Nature 386:279-284. Dong H, Zhang P, Song I, Petralia RS, Liao D, Huganir RL (1999b) Characterization of the glutamate receptor-interacting proteins GRIP1 and GRIP2. J Neurosci 19:6930-6941. Drake CT, Milner TA, Patterson SL (1999) Ultrastructural localization of full-length trkB immunoreactivity in rat hippocampus suggests multiple roles in modulating activity- dependent synaptic plasticity. J Neurosci 19:8009-8026. Drapeau E, Mayo W, Aurousseau C, Le Moal M, Piazza PV, Abrous DN (2003) Spatial memory performances of aged rats in the water maze predict levels of hippocampal neurogenesis. Proc Natl Acad Sci U S A 100:14385-14390. Dudek SM, Bear MF (1992) Homosynaptic long-term depression in area CA1 of hippocampus and effects of N-methyl-D-aspartate receptor blockade. Proc Natl Acad Sci U S A 89:4363-4367. Dunnett SB, Rosser AE (2007) Cell transplantation for Huntington's disease Should we continue? Brain Res Bull 72:132-147. Dunnett SB, Bjorklund A, Lindvall O (2001) Cell therapy in Parkinson's disease - stop or go? Nat Rev Neurosci 2:365-369. Dupret D, Fabre A, Dobrossy MD, Panatier A, Rodriguez JJ, Lamarque S, Lemaire V, Oliet SH, Piazza PV, Abrous DN (2007) Spatial learning depends on both the addition and removal of new hippocampal neurons. PLoS Biol 5:e214. Durand GM, Kovalchuk Y, Konnerth A (1996) Long-term potentiation and functional synapse induction in developing hippocampus. Nature 381:71-75. Ehlers MD (2000) Reinsertion or degradation of AMPA receptors determined by activity- 117 dependent endocytic sorting. Neuron 28:511-525. Ehrlich I, Lohrke S, Friauf E (1999) Shift from depolarizing to hyperpolarizing glycine action in rat auditory neurones is due to age-dependent Cl- regulation. J Physiol 520 Pt 1:121- 137. Eikelenboom P, Veerhuis R, Scheper W, Rozemuller AJ, van Gool WA, Hoozemans JJ (2006) The significance of neuroinflammation in understanding Alzheimer's disease. J Neural Transm 113:1685-1695. Eilers J, Plant TD, Marandi N, Konnerth A (2001) GABA-mediated Ca2+ signalling in developing rat cerebellar Purkinje neurones. J Physiol 536:429-437. El Khoury J, Hickman SE, Thomas CA, Cao L, Silverstein SC, Loike JD (1996) Scavenger receptor-mediated adhesion of microglia to beta-amyloid fibrils. Nature 382:716-719. Eliasson C, Sahlgren C, Berthold CH, Stakeberg J, Celis JE, Betsholtz C, Eriksson JE, Pekny M (1999) Intermediate filament protein partnership in astrocytes. J Biol Chem 274:23996- 24006. Ellgaard L, Helenius A (2003) Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol 4:181-191. English JD, Sweatt JD (1996) Activation of p42 mitogen-activated protein kinase in hippocampal long term potentiation. J Biol Chem 271:24329-24332. Englund U, Bjorklund A, Wictorin K, Lindvall O, Kokaia M (2002) Grafted neural stem cells develop into functional pyramidal neurons and integrate into host cortical circuitry. Proc Natl Acad Sci U S A 99:17089-17094. Enwere E, Shingo T, Gregg C, Fujikawa H, Ohta S, Weiss S (2004) Aging results in reduced epidermal growth factor receptor signaling, diminished olfactory neurogenesis, and deficits in fine olfactory discrimination. J Neurosci 24:8354-8365. Eriksson PS, Perfilieva E, Bjork-Eriksson T, Alborn AM, Nordborg C, Peterson DA, Gage FH (1998) Neurogenesis in the adult human hippocampus. Nat Med 4:1313-1317. Ernfors P, Kucera J, Lee KF, Loring J, Jaenisch R (1995) Studies on the physiological role of brain-derived neurotrophic factor and neurotrophin-3 in knockout mice. Int J Dev Biol 39:799-807. Esteban JA (2003) AMPA receptor trafficking: a road map for synaptic plasticity. Mol Interv 3:375-385. Fanarraga ML, Avila J, Zabala JC (1999) Expression of unphosphorylated class III beta-tubulin isotype in neuroepithelial cells demonstrates neuroblast commitment and differentiation. Eur J Neurosci 11:517-527. Farmer J (2004) 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. Farmer J, Zhao X, van Praag H, Wodtke K, Gage FH, Christie BR (2004) 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. 118 Farrant M, Feldmeyer D, Takahashi T, Cull-Candy SG (1994) NMDA-receptor channel diversity in the developing cerebellum. Nature 368:335-339. Figurov A, Pozzo-Miller LD, Olafsson P, Wang T, Lu B (1996) Regulation of synaptic responses to high-frequency stimulation and LTP by neurotrophins in the hippocampus. Nature 381:706-709. Fleming KG, Hohl TM, Yu RC, Muller SA, Wolpensinger B, Engel A, Engelhardt H, Brunger AT, Sollner TH, Hanson PI (1998) A revised model for the oligomeric state of the N- ethylmaleimide-sensitive fusion protein, NSF. J Biol Chem 273:15675-15681. Fode C, Gradwohl G, Morin X, Dierich A, LeMeur M, Goridis C, Guillemot F (1998) The bHLH protein NEUROGENIN 2 is a determination factor for epibranchial placode-derived sensory neurons. Neuron 20:483-494. Fox CJ, Russell K, Titterness AK, Wang YT, Christie BR (2007) Tyrosine phosphorylation of the GluR2 subunit is required for long-term depression of synaptic efficacy in young animals in vivo. Hippocampus 17:600-605. Fox K (2002) Anatomical pathways and molecular mechanisms for plasticity in the barrel cortex. Neuroscience 111:799-814. Franciosi S, Choi HB, Kim SU, McLarnon JG (2005) IL-8 enhancement of amyloid-beta (Abeta 1-42)-induced expression and production of pro-inflammatory cytokines and COX-2 in cultured human microglia. J Neuroimmunol 159:66-74. Franciosi S, Ryu JK, Choi HB, Radov L, Kim SU, McLarnon JG (2006) Broad-spectrum effects of 4-aminopyridine to modulate amyloid beta1-42-induced cell signaling and functional responses in human microglia. J Neurosci 26:11652-11664. Francis F, Koulakoff A, Boucher D, Chafey P, Schaar B, Vinet MC, Friocourt G, McDonnell N, Reiner O, Kahn A, McConnell SK, Berwald-Netter Y, Denoulet P, Chelly J (1999) Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons. Neuron 23:247-256. Frederiksen K, Jat PS, Valtz N, Levy D, McKay R (1988) Immortalization of precursor cells from the mammalian CNS. Neuron 1:439-448. Frey U, Krug M, Reymann KG, Matthies H (1988) Anisomycin, an inhibitor of protein synthesis, blocks late phases of LTP phenomena in the hippocampal CA1 region in vitro. Brain Res 452:57-65. Frey U, Frey S, Schollmeier F, Krug M (1996) Influence of actinomycin D, a RNA synthesis inhibitor, on long-term potentiation in rat hippocampal neurons in vivo and in vitro. J Physiol 490 ( Pt 3):703-711. Fuchs E, Gould E (2000) Mini-review: in vivo neurogenesis in the adult brain: regulation and functional implications. Eur J Neurosci 12:2211-2214. Fujii S, Saito K, Miyakawa H, Ito K, Kato H (1991) Reversal of long-term potentiation (depotentiation) induced by tetanus stimulation of the input to CA1 neurons of guinea pig hippocampal slices. Brain Res 555:112-122. Fukaya M, Kato A, Lovett C, Tonegawa S, Watanabe M (2003) Retention of NMDA receptor 119 NR2 subunits in the lumen of endoplasmic reticulum in targeted NR1 knockout mice. Proc Natl Acad Sci U S A 100:4855-4860. Fukuda S, Kato F, Tozuka Y, Yamaguchi M, Miyamoto Y, Hisatsune T (2003) Two distinct subpopulations of nestin-positive cells in adult mouse dentate gyrus. J Neurosci 23:9357-9366. Fukumitsu H, Furukawa Y, Tsusaka M, Kinukawa H, Nitta A, Nomoto H, Mima T, Furukawa S (1998) Simultaneous expression of brain-derived neurotrophic factor and neurotrophin-3 in Cajal-Retzius, subplate and ventricular progenitor cells during early development stages of the rat cerebral cortex. Neuroscience 84:115-127. Gage FH (2000) Mammalian neural stem cells. Science 287:1433-1438. Gage FH, Coates PW, Palmer TD, Kuhn HG, Fisher LJ, Suhonen JO, Peterson DA, Suhr ST, Ray J (1995) Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc Natl Acad Sci U S A 92:11879-11883. Gall C, Lauterborn J (1992) The dentate gyrus: a model system for studies of neurotrophin regulation. Epilepsy Res Suppl 7:171-185. Ganguly K, Schinder AF, Wong ST, Poo M (2001) GABA itself promotes the developmental switch of neuronal GABAergic responses from excitation to inhibition. Cell 105:521-532. Garcia AD, Doan NB, Imura T, Bush TG, Sofroniew MV (2004) GFAP-expressing progenitors are the principal source of constitutive neurogenesis in adult mouse forebrain. Nat Neurosci 7:1233-1241. Gardner SM, Trussell LO, Oertel D (2001) Correlation of AMPA receptor subunit composition with synaptic input in the mammalian cochlear nuclei. J Neurosci 21:7428-7437. Gartner A, Staiger V (2002) Neurotrophin secretion from hippocampal neurons evoked by long- term-potentiation-inducing electrical stimulation patterns. Proc Natl Acad Sci U S A 99:6386-6391. Ge Y, Dong Z, Bagot RC, Howland JG, Phillips AG, Wong TP, Wang YT (2010) Hippocampal long-term depression is required for the consolidation of spatial memory. Proc Natl Acad Sci U S A 107:16697-16702. Gehrmann J, Matsumoto Y, Kreutzberg GW (1995) Microglia: intrinsic immuneffector cell of the brain. Brain Res Brain Res Rev 20:269-287. Geiger JR, Melcher T, Koh DS, Sakmann B, Seeburg PH, Jonas P, Monyer H (1995) Relative abundance of subunit mRNAs determines gating and Ca2+ permeability of AMPA receptors in principal neurons and interneurons in rat CNS. Neuron 15:193-204. Giulian D (1997) Immune responses and dementia. Ann N Y Acad Sci 835:91-110. Gleeson JG, Lin PT, Flanagan LA, Walsh CA (1999) Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons. Neuron 23:257-271. Goldman SA, Kirschenbaum B, Harrison-Restelli C, Thaler HT (1997) Neuronal precursors of the adult rat subependymal zone persist into senescence, with no decline in spatial extent or response to BDNF. J Neurobiol 32:554-566. 120 Gomes AR, Cunha P, Nuriya M, Faro CJ, Huganir RL, Pires EV, Carvalho AL, Duarte CB (2004) Metabotropic glutamate and dopamine receptors co-regulate AMPA receptor activity through PKA in cultured chick retinal neurones: effect on GluR4 phosphorylation and surface expression. J Neurochem 90:673-682. Gould E, Tanapat P, Rydel T, Hastings N (2000) Regulation of hippocampal neurogenesis in adulthood. Biol Psychiatry 48:715-720. Gould E, Beylin A, Tanapat P, Reeves A, Shors TJ (1999) Learning enhances adult neurogenesis in the hippocampal formation. Nat Neurosci 2:260-265. Graham V, Khudyakov J, Ellis P, Pevny L (2003) SOX2 functions to maintain neural progenitor identity. Neuron 39:749-765. Greger IH, Khatri L, Ziff EB (2002) RNA editing at arg607 controls AMPA receptor exit from the endoplasmic reticulum. Neuron 34:759-772. Greger IH, Ziff EB, Penn AC (2007) Molecular determinants of AMPA receptor subunit assembly. Trends Neurosci 30:407-416. Grey KB, Burrell BD (2008) Forskolin induces NMDA receptor-dependent potentiation at a central synapse in the leech. J Neurophysiol 99:2719-2724. Griffin WS, Sheng JG, Royston MC, Gentleman SM, McKenzie JE, Graham DI, Roberts GW, Mrak RE (1998) Glial-neuronal interactions in Alzheimer's disease: the potential role of a 'cytokine cycle' in disease progression. Brain Pathol 8:65-72. Gritti A, Frolichsthal-Schoeller P, Galli R, Parati EA, Cova L, Pagano SF, Bjornson CR, Vescovi AL (1999) Epidermal and fibroblast growth factors behave as mitogenic regulators for a single multipotent stem cell-like population from the subventricular region of the adult mouse forebrain. J Neurosci 19:3287-3297. Gritti A, Parati EA, Cova L, Frolichsthal P, Galli R, Wanke E, Faravelli L, Morassutti DJ, Roisen F, Nickel DD, Vescovi AL (1996) Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor. J Neurosci 16:1091-1100. Grosshans DR, Clayton DA, Coultrap SJ, Browning MD (2002) LTP leads to rapid surface expression of NMDA but not AMPA receptors in adult rat CA1. Nat Neurosci 5:27-33. Guillemot F, Joyner AL (1993) Dynamic expression of the murine Achaete-Scute homologue Mash-1 in the developing nervous system. Mech Dev 42:171-185. Gustafsson B, Wigstrom H (1988) Physiological mechanisms underlying long-term potentiation. Trends Neurosci 11:156-162. Hagena H, Manahan-Vaughan D Frequency facilitation at mossy fiber-CA3 synapses of freely behaving rats contributes to the induction of persistent LTD via an adenosine-A1 receptor-regulated mechanism. Cereb Cortex 20:1121-1130. Hall J, Thomas KL, Everitt BJ (2000) Rapid and selective induction of BDNF expression in the hippocampus during contextual learning. Nat Neurosci 3:533-535. Hallbook F, Ibanez CF, Persson H (1991) Evolutionary studies of the nerve growth factor family 121 reveal a novel member abundantly expressed in Xenopus ovary. Neuron 6:845-858. Haniu M, Montestruque S, Bures EJ, Talvenheimo J, Toso R, Lewis-Sandy S, Welcher AA, Rohde MF (1997) Interactions between brain-derived neurotrophic factor and the TRKB receptor. Identification of two ligand binding domains in soluble TRKB by affinity separation and chemical cross-linking. J Biol Chem 272:25296-25303. Hanson PI, Roth R, Morisaki H, Jahn R, Heuser JE (1997) Structure and conformational changes in NSF and its membrane receptor complexes visualized by quick-freeze/deep- etch electron microscopy. Cell 90:523-535. Hardingham GE, Fukunaga Y, Bading H (2002) Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nat Neurosci 5:405-414. Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297:353-356. Harney SC, Rowan M, Anwyl R (2006) Long-term depression of NMDA receptor-mediated synaptic transmission is dependent on activation of metabotropic glutamate receptors and is altered to long-term potentiation by low intracellular calcium buffering. J Neurosci 26:1128-1132. Harney SC, Jane DE, Anwyl R (2008) Extrasynaptic NR2D-containing NMDARs are recruited to the synapse during LTP of NMDAR-EPSCs. J Neurosci 28:11685-11694. Harris EW, Cotman CW (1986) Long-term potentiation of guinea pig mossy fiber responses is not blocked by N-methyl D-aspartate antagonists. Neurosci Lett 70:132-137. Harvey J, Collingridge GL (1993) Signal transduction pathways involved in the acute potentiation of NMDA responses by 1S,3R-ACPD in rat hippocampal slices. Br J Pharmacol 109:1085-1090. Hay JC, Scheller RH (1997) SNAREs and NSF in targeted membrane fusion. Curr Opin Cell Biol 9:505-512. Hayashi Y, Shi SH, Esteban JA, Piccini A, Poncer JC, Malinow R (2000) Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction. Science 287:2262-2267. Heine M, Groc L, Frischknecht R, Beique JC, Lounis B, Rumbaugh G, Huganir RL, Cognet L, Choquet D (2008) Surface mobility of postsynaptic AMPARs tunes synaptic transmission. Science 320:201-205. Henze DA, Urban NN, Barrionuevo G (2000) The multifarious hippocampal mossy fiber pathway: a review. Neuroscience 98:407-427. Heynen AJ, Quinlan EM, Bae DC, Bear MF (2000) Bidirectional, activity-dependent regulation of glutamate receptors in the adult hippocampus in vivo. Neuron 28:527-536. Hirai H, Kirsch J, Laube B, Betz H, Kuhse J (1996) The glycine binding site of the N-methyl-D- aspartate receptor subunit NR1: identification of novel determinants of co-agonist potentiation in the extracellular M3-M4 loop region. Proc Natl Acad Sci U S A 93:6031- 6036. 122 Hirsch MR, Tiveron MC, Guillemot F, Brunet JF, Goridis C (1998) Control of noradrenergic differentiation and Phox2a expression by MASH1 in the central and peripheral nervous system. Development 125:599-608. Hjelmstad GO, Nicoll RA, Malenka RC (1997) Synaptic refractory period provides a measure of probability of release in the hippocampus. Neuron 19:1309-1318. Hollmann M, Heinemann S (1994) Cloned glutamate receptors. Annu Rev Neurosci 17:31-108. Hollmann M, Hartley M, Heinemann S (1991) Ca2+ permeability of KA-AMPA--gated glutamate receptor channels depends on subunit composition. Science 252:851-853. Hollmann M, Maron C, Heinemann S (1994) N-glycosylation site tagging suggests a three transmembrane domain topology for the glutamate receptor GluR1. Neuron 13:1331- 1343. Hong XP, Peng CX, Wei W, Tian Q, Liu YH, Yao XQ, Zhang Y, Cao FY, Wang Q, Wang JZ (2010) Essential role of tau phosphorylation in adult hippocampal neurogenesis. Hippocampus 20:1339-1349. Honore T, Lauridsen J, Krogsgaard-Larsen P (1982) The binding of [3H]AMPA, a structural analogue of glutamic acid, to rat brain membranes. J Neurochem 38:173-178. Hoshino T, Nagashima T, Cho KG, Davis RL, Donegan J, Slusarz M, Wilson CB (1989) Variability in the proliferative potential of human gliomas. J Neurooncol 7:137-143. Houle J, Fedoroff S (1983) Temporal relationship between the appearance of vimentin and neural tube development. Brain Res 285:189-195. Huang EJ, Reichardt LF (2001) Neurotrophins: roles in neuronal development and function. Annu Rev Neurosci 24:677-736. Huang Y, Man HY, Sekine-Aizawa Y, Han Y, Juluri K, Luo H, Cheah J, Lowenstein C, Huganir RL, Snyder SH (2005) S-nitrosylation of N-ethylmaleimide sensitive factor mediates surface expression of AMPA receptors. Neuron 46:533-540. Huang YY, Li XC, Kandel ER (1994) cAMP contributes to mossy fiber LTP by initiating both a covalently mediated early phase and macromolecular synthesis-dependent late phase. Cell 79:69-79. Huang YY, Kandel ER, Varshavsky L, Brandon EP, Qi M, Idzerda RL, McKnight GS, Bourtchouladze R (1995) A genetic test of the effects of mutations in PKA on mossy fiber LTP and its relation to spatial and contextual learning. Cell 83:1211-1222. Husemann J, Loike JD, Kodama T, Silverstein SC (2001) Scavenger receptor class B type I (SR-BI) mediates adhesion of neonatal murine microglia to fibrillar beta-amyloid. J Neuroimmunol 114:142-150. Husemann J, Loike JD, Anankov R, Febbraio M, Silverstein SC (2002) Scavenger receptors in neurobiology and neuropathology: their role on microglia and other cells of the nervous system. Glia 40:195-205. Ihrie RA, Alvarez-Buylla A (2008) Cells in the astroglial lineage are neural stem cells. Cell Tissue Res 331:179-191. 123 Isaac JT, Nicoll RA, Malenka RC (1995) Evidence for silent synapses: implications for the expression of LTP. Neuron 15:427-434. Isaac JT, Hjelmstad GO, Nicoll RA, Malenka RC (1996) Long-term potentiation at single fiber inputs to hippocampal CA1 pyramidal cells. Proc Natl Acad Sci U S A 93:8710-8715. Ito H, Nakajima A, Nomoto H, Furukawa S (2003) Neurotrophins facilitate neuronal differentiation of cultured neural stem cells via induction of mRNA expression of basic helix-loop-helix transcription factors Mash1 and Math1. J Neurosci Res 71:648-658. Janssen WG, Vissavajjhala P, Andrews G, Moran T, Hof PR, Morrison JH (2005) Cellular and synaptic distribution of NR2A and NR2B in macaque monkey and rat hippocampus as visualized with subunit-specific monoclonal antibodies. Exp Neurol 191 Suppl 1:S28-44. Jessberger S, Zhao C, Toni N, Clemenson GD, Jr., Li Y, Gage FH (2007) Seizure-associated, aberrant neurogenesis in adult rats characterized with retrovirus-mediated cell labeling. J Neurosci 27:9400-9407. Ji RR, Kohno T, Moore KA, Woolf CJ (2003) Central sensitization and LTP: do pain and memory share similar mechanisms? Trends Neurosci 26:696-705. Jin K, Zhu Y, Sun Y, Mao XO, Xie L, Greenberg DA (2002) Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo. Proc Natl Acad Sci U S A 99:11946- 11950. Jin K, Galvan V, Xie L, Mao XO, Gorostiza OF, Bredesen DE, Greenberg DA (2004a) Enhanced neurogenesis in Alzheimer's disease transgenic (PDGF-APPSw,Ind) mice. Proc Natl Acad Sci U S A 101:13363-13367. Jin K, Peel AL, Mao XO, Xie L, Cottrell BA, Henshall DC, Greenberg DA (2004b) Increased hippocampal neurogenesis in Alzheimer's disease. Proc Natl Acad Sci U S A 101:343- 347. Joo JY, Kim BW, Lee JS, Park JY, Kim S, Yun YJ, Lee SH, Rhim H, Son H (2007) Activation of NMDA receptors increases proliferation and differentiation of hippocampal neural progenitor cells. J Cell Sci 120:1358-1370. Kakazu Y, Akaike N, Komiyama S, Nabekura J (1999) Regulation of intracellular chloride by cotransporters in developing lateral superior olive neurons. J Neurosci 19:2843-2851. Kakegawa W, Tsuzuki K, Yoshida Y, Kameyama K, Ozawa S (2004) Input- and subunit-specific AMPA receptor trafficking underlying long-term potentiation at hippocampal CA3 synapses. Eur J Neurosci 20:101-110. Kamiya H, Ozawa S (1999) Dual mechanism for presynaptic modulation by axonal metabotropic glutamate receptor at the mouse mossy fibre-CA3 synapse. J Physiol 518 ( Pt 2):497- 506. Kandel ER (1997) Genes, synapses, and long-term memory. J Cell Physiol 173:124-125. Kandel ER (2001) The molecular biology of memory storage: a dialogue between genes and synapses. Science 294:1030-1038. Kaneko Y, Sakakibara S, Imai T, Suzuki A, Nakamura Y, Sawamoto K, Ogawa Y, Toyama Y, 124 Miyata T, Okano H (2000) Musashi1: an evolutionally conserved marker for CNS progenitor cells including neural stem cells. Dev Neurosci 22:139-153. Kang H, Schuman EM (1995) Long-lasting neurotrophin-induced enhancement of synaptic transmission in the adult hippocampus. Science 267:1658-1662. Kang H, Schuman EM (1996) A requirement for local protein synthesis in neurotrophin-induced hippocampal synaptic plasticity. Science 273:1402-1406. Kaplan DR, Miller FD (2000) Neurotrophin signal transduction in the nervous system. Curr Opin Neurobiol 10:381-391. Kaplan MS, Hinds JW (1977) Neurogenesis in the adult rat: electron microscopic analysis of light radioautographs. Science 197:1092-1094. Katoh-Semba R, Asano T, Ueda H, Morishita R, Takeuchi IK, Inaguma Y, Kato K (2002) Riluzole enhances expression of brain-derived neurotrophic factor with consequent proliferation of granule precursor cells in the rat hippocampus. FASEB J 16:1328-1330. Kauer JA, Malenka RC, Nicoll RA (1988a) NMDA application potentiates synaptic transmission in the hippocampus. Nature 334:250-252. Kauer JA, Malenka RC, Nicoll RA (1988b) A persistent postsynaptic modification mediates long- term potentiation in the hippocampus. Neuron 1:911-917. Kee N, Teixeira CM, Wang AH, Frankland PW (2007) Preferential incorporation of adult- generated granule cells into spatial memory networks in the dentate gyrus. Nat Neurosci 10:355-362. Keinanen K, Wisden W, Sommer B, Werner P, Herb A, Verdoorn TA, Sakmann B, Seeburg PH (1990) A family of AMPA-selective glutamate receptors. Science 249:556-560. Kempermann G, Kuhn HG, Gage FH (1997) More hippocampal neurons in adult mice living in an enriched environment. Nature 386:493-495. Kempermann G, Wiskott L, Gage FH (2004) Functional significance of adult neurogenesis. Curr Opin Neurobiol 14:186-191. Kennedy MJ, Davison IG, Robinson CG, Ehlers MD (2010) Syntaxin-4 defines a domain for activity-dependent exocytosis in dendritic spines. Cell 141:524-535. Kernie SG, Liebl DJ, Parada LF (2000) BDNF regulates eating behavior and locomotor activity in mice. EMBO J 19:1290-1300. Khalilov I, Esclapez M, Medina I, Aggoun D, Lamsa K, Leinekugel X, Khazipov R, Ben-Ari Y (1997) A novel in vitro preparation: the intact hippocampal formation. Neuron 19:743-749. Khazipov R, Esclapez M, Caillard O, Bernard C, Khalilov I, Tyzio R, Hirsch J, Dzhala V, Berger B, Ben-Ari Y (2001) Early development of neuronal activity in the primate hippocampus in utero. J Neurosci 21:9770-9781. Kim CH, Lisman JE (2001) A labile component of AMPA receptor-mediated synaptic transmission is dependent on microtubule motors, actin, and N-ethylmaleimide-sensitive factor. J Neurosci 21:4188-4194. 125 Kim CH, Chung HJ, Lee HK, Huganir RL (2001a) 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. Kim JH, Huganir RL (1999) Organization and regulation of proteins at synapses. Curr Opin Cell Biol 11:248-254. Kim JH, Liao D, Lau LF, Huganir RL (1998) SynGAP: a synaptic RasGAP that associates with the PSD-95/SAP90 protein family. Neuron 20:683-691. Kim JH, Auerbach JM, Rodriguez-Gomez JA, Velasco I, Gavin D, Lumelsky N, Lee SH, Nguyen J, Sanchez-Pernaute R, Bankiewicz K, McKay R (2002) Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease. Nature 418:50-56. Kim MJ, Dunah AW, Wang YT, Sheng M (2005) Differential roles of NR2A- and NR2B- containing NMDA receptors in Ras-ERK signaling and AMPA receptor trafficking. Neuron 46:745-760. Kim WY, Fritzsch B, Serls A, Bakel LA, Huang EJ, Reichardt LF, Barth DS, Lee JE (2001b) NeuroD-null mice are deaf due to a severe loss of the inner ear sensory neurons during development. Development 128:417-426. Kim YJ, Park HJ, Lee G, Bang OY, Ahn YH, Joe E, Kim HO, Lee PH (2009) Neuroprotective effects of human mesenchymal stem cells on dopaminergic neurons through anti- inflammatory action. Glia 57:13-23. Kinoshita M, Fukaya M, Tojima T, Kojima S, Ando H, Watanabe M, Urano A, Ito E (2005) Retinotectal transmission in the optic tectum of rainbow trout. J Comp Neurol 484:249- 259. Kirschenbaum B, Goldman SA (1995) Brain-derived neurotrophic factor promotes the survival of neurons arising from the adult rat forebrain subependymal zone. Proc Natl Acad Sci U S A 92:210-214. Kitamura T, Saitoh Y, Murayama A, Sugiyama H, Inokuchi K (2010) LTP induction within a narrow critical period of immature stages enhances the survival of newly generated neurons in the adult rat dentate gyrus. Mol Brain 3:13. Klausnitzer J, Manahan-Vaughan D (2008) Frequency facilitation at mossy fiber-CA3 synapses of freely behaving rats is regulated by adenosine A1 receptors. J Neurosci 28:4836-4840. Kohler M, Kornau HC, Seeburg PH (1994) The organization of the gene for the functionally dominant alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit GluR-B. J Biol Chem 269:17367-17370. Kohr G (2006) NMDA receptor function: subunit composition versus spatial distribution. Cell Tissue Res 326:439-446. Kopec CD, Li B, Wei W, Boehm J, Malinow R (2006) Glutamate receptor exocytosis and spine enlargement during chemically induced long-term potentiation. J Neurosci 26:2000-2009. Korte M, Carroll P, Wolf E, Brem G, Thoenen H, Bonhoeffer T (1995) Hippocampal long-term potentiation is impaired in mice lacking brain-derived neurotrophic factor. Proc Natl Acad 126 Sci U S A 92:8856-8860. Kovalchuk Y, Hanse E, Kafitz KW, Konnerth A (2002) Postsynaptic Induction of BDNF-Mediated Long-Term Potentiation. Science 295:1729-1734. Kreutzberg GW (1996) Microglia: a sensor for pathological events in the CNS. Trends Neurosci 19:312-318. Kuhn HG, Dickinson-Anson H, Gage FH (1996) Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J Neurosci 16:2027-2033. Kukekov VG, Laywell ED, Suslov O, Davies K, Scheffler B, Thomas LB, O'Brien TF, Kusakabe M, Steindler DA (1999) Multipotent stem/progenitor cells with similar properties arise from two neurogenic regions of adult human brain. Exp Neurol 156:333-344. Kullmann DM, Nicoll RA (1992) Long-term potentiation is associated with increases in quantal content and quantal amplitude. Nature 357:240-244. Kurki P, Vanderlaan M, Dolbeare F, Gray J, Tan EM (1986) Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the cell cycle. Exp Cell Res 166:209-219. Kutsuwada T, Kashiwabuchi N, Mori H, Sakimura K, Kushiya E, Araki K, Meguro H, Masaki H, Kumanishi T, Arakawa M, et al. (1992) Molecular diversity of the NMDA receptor channel. Nature 358:36-41. Larkman AU, Jack JJ (1995) Synaptic plasticity: hippocampal LTP. Curr Opin Neurobiol 5:324- 334. Lau CG, Zukin RS (2007) NMDA receptor trafficking in synaptic plasticity and neuropsychiatric disorders. Nat Rev Neurosci 8:413-426. Laube B, Kuhse J, Betz H (1998) Evidence for a tetrameric structure of recombinant NMDA receptors. J Neurosci 18:2954-2961. Lauri SE, Bortolotto ZA, Nistico R, Bleakman D, Ornstein PL, Lodge D, Isaac JT, Collingridge GL (2003) A role for Ca2+ stores in kainate receptor-dependent synaptic facilitation and LTP at mossy fiber synapses in the hippocampus. Neuron 39:327-341. Lavado A, Lagutin OV, Chow LM, Baker SJ, Oliver G (2010) Prox1 is required for granule cell maturation and intermediate progenitor maintenance during brain neurogenesis. PLoS Biol 8. Lee A, Kessler JD, Read TA, Kaiser C, Corbeil D, Huttner WB, Johnson JE, Wechsler-Reya RJ (2005) Isolation of neural stem cells from the postnatal cerebellum. Nat Neurosci 8:723- 729. Lee FS, Chao MV (2001) Activation of Trk neurotrophin receptors in the absence of neurotrophins. Proc Natl Acad Sci U S A 98:3555-3560. Lee FS, Kim AH, Khursigara G, Chao MV (2001) The uniqueness of being a neurotrophin receptor. Curr Opin Neurobiol 11:281-286. Lee J, Duan W, Mattson MP (2002) Evidence that brain-derived neurotrophic factor is required for basal neurogenesis and mediates, in part, the enhancement of neurogenesis by 127 dietary restriction in the hippocampus of adult mice. J Neurochem 82:1367-1375. Lee JE (1997) Basic helix-loop-helix genes in neural development. Curr Opin Neurobiol 7:13-20. Lee JE, Hollenberg SM, Snider L, Turner DL, Lipnick N, Weintraub H (1995) Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein. Science 268:836-844. Lee ST, Chu K, Jung KH, Kim SJ, Kim DH, Kang KM, Hong NH, Kim JH, Ban JJ, Park HK, Kim SU, Park CG, Lee SK, Kim M, Roh JK (2008) Anti-inflammatory mechanism of intravascular neural stem cell transplantation in haemorrhagic stroke. Brain 131:616-629. Leinekugel X, Tseeb V, Ben-Ari Y, Bregestovski P (1995) Synaptic GABAA activation induces Ca2+ rise in pyramidal cells and interneurons from rat neonatal hippocampal slices. J Physiol 487 ( Pt 2):319-329. Leinekugel X, Khalilov I, Ben-Ari Y, Khazipov R (1998) Giant depolarizing potentials: the septal pole of the hippocampus paces the activity of the developing intact septohippocampal complex in vitro. J Neurosci 18:6349-6357. Leinekugel X, Medina I, Khalilov I, Ben-Ari Y, Khazipov R (1997) Ca2+ oscillations mediated by the synergistic excitatory actions of GABA(A) and NMDA receptors in the neonatal hippocampus. Neuron 18:243-255. Leinekugel X, Khazipov R, Cannon R, Hirase H, Ben-Ari Y, Buzsaki G (2002) Correlated bursts of activity in the neonatal hippocampus in vivo. Science 296:2049-2052. Leinekugel X, Khalilov I, McLean H, Caillard O, Gaiarsa JL, Ben-Ari Y, Khazipov R (1999) GABA is the principal fast-acting excitatory transmitter in the neonatal brain. Adv Neurol 79:189-201. Lemaire V, Koehl M, Le Moal M, Abrous DN (2000) Prenatal stress produces learning deficits associated with an inhibition of neurogenesis in the hippocampus. Proc Natl Acad Sci U S A 97:11032-11037. Lendahl U, Zimmerman LB, McKay RD (1990) CNS stem cells express a new class of intermediate filament protein. Cell 60:585-595. Leonard AS, Davare MA, Horne MC, Garner CC, Hell JW (1998) SAP97 is associated with the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR1 subunit. J Biol Chem 273:19518-19524. Lerma J (2003) Roles and rules of kainate receptors in synaptic transmission. Nat Rev Neurosci 4:481-495. Leveille F, El Gaamouch F, Gouix E, Lecocq M, Lobner D, Nicole O, Buisson A (2008) Neuronal viability is controlled by a functional relation between synaptic and extrasynaptic NMDA receptors. FASEB J 22:4258-4271. Levine ES, Dreyfus CF, Black IB, Plummer MR (1995) Brain-derived neurotrophic factor rapidly enhances synaptic transmission in hippocampal neurons via postsynaptic tyrosine kinase receptors. Proc Natl Acad Sci U S A 92:8074-8077. Lewis PF, Emerman M (1994) Passage through mitosis is required for oncoretroviruses but not 128 for the human immunodeficiency virus. J Virol 68:510-516. Li B, Yamamori H, Tatebayashi Y, Shafit-Zagardo B, Tanimukai H, Chen S, Iqbal K, Grundke- Iqbal I (2008a) Failure of neuronal maturation in Alzheimer disease dentate gyrus. J Neuropathol Exp Neurol 67:78-84. Li GH, Jackson MF, Orser BA, Macdonald JF (2009) Reciprocal and activity-dependent regulation of surface AMPA and NMDA receptors in cultured neurons. Int J Physiol Pathophysiol Pharmacol 2:47-56. Li JY, Christophersen NS, Hall V, Soulet D, Brundin P (2008b) Critical issues of clinical human embryonic stem cell therapy for brain repair. Trends Neurosci 31:146-153. Liao D, Scannevin RH, Huganir R (2001) Activation of silent synapses by rapid activity- dependent synaptic recruitment of AMPA receptors. J Neurosci 21:6008-6017. Lie DC, Song H, Colamarino SA, Ming GL, Gage FH (2004) Neurogenesis in the adult brain: new strategies for central nervous system diseases. Annu Rev Pharmacol Toxicol 44:399-421. Liem RK (1993) Molecular biology of neuronal intermediate filaments. Curr Opin Cell Biol 5:12- 16. Lillien L (1998) Neural progenitors and stem cells: mechanisms of progenitor heterogeneity. Curr Opin Neurobiol 8:37-44. Lim JY, Park SI, Oh JH, Kim SM, Jeong CH, Jun JA, Lee KS, Oh W, Lee JK, Jeun SS (2008) Brain-derived neurotrophic factor stimulates the neural differentiation of human umbilical cord blood-derived mesenchymal stem cells and survival of differentiated cells through MAPK/ERK and PI3K/Akt-dependent signaling pathways. J Neurosci Res 86:2168-2178. Lin DT, Huganir RL (2007) PICK1 and phosphorylation of the glutamate receptor 2 (GluR2) AMPA receptor subunit regulates GluR2 recycling after NMDA receptor-induced internalization. J Neurosci 27:13903-13908. Lin DT, Makino Y, Sharma K, Hayashi T, Neve R, Takamiya K, Huganir RL (2009) Regulation of AMPA receptor extrasynaptic insertion by 4.1N, phosphorylation and palmitoylation. Nat Neurosci 12:879-887. Lin JW, Ju W, Foster K, Lee SH, Ahmadian G, Wyszynski M, Wang YT, Sheng M (2000) Distinct molecular mechanisms and divergent endocytotic pathways of AMPA receptor internalization. Nat Neurosci 3:1282-1290. Lindvall O, Kokaia Z (2010) Stem cells in human neurodegenerative disorders--time for clinical translation? J Clin Invest 120:29-40. Ling EA, Wong WC (1993) The origin and nature of ramified and amoeboid microglia: a historical review and current concepts. Glia 7:9-18. Linnarsson S, Bjorklund A, Ernfors P (1997) Learning deficit in BDNF mutant mice. Eur J Neurosci 9:2581-2587. Lisman J (1994) The CaM kinase II hypothesis for the storage of synaptic memory. Trends Neurosci 17:406-412. 129 Lisman J, Raghavachari S (2006) A unified model of the presynaptic and postsynaptic changes during LTP at CA1 synapses. Sci STKE 2006:re11. Lisman J, Malenka RC, Nicoll RA, Malinow R (1997) Learning mechanisms: the case for CaM- KII. Science 276:2001-2002. Lisman JE, Goldring MA (1988) Feasibility of long-term storage of graded information by the Ca2+/calmodulin-dependent protein kinase molecules of the postsynaptic density. Proc Natl Acad Sci U S A 85:5320-5324. Liu L, Wong TP, Pozza MF, Lingenhoehl K, Wang Y, Sheng M, Auberson YP, Wang YT (2004a) Role of NMDA receptor subtypes in governing the direction of hippocampal synaptic plasticity. Science 304:1021-1024. Liu SJ, Zukin RS (2007) Ca2+-permeable AMPA receptors in synaptic plasticity and neuronal death. Trends Neurosci 30:126-134. Liu XB, Murray KD, Jones EG (2004b) Switching of NMDA receptor 2A and 2B subunits at thalamic and cortical synapses during early postnatal development. J Neurosci 24:8885- 8895. Liu Y, Wong TP, Aarts M, Rooyakkers A, Liu L, Lai TW, Wu DC, Lu J, Tymianski M, Craig AM, Wang YT (2007) NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo. J Neurosci 27:2846-2857. Lledo PM, Gheusi G (2006) [Adult neurogenesis: from basic research to clinical applications]. Bull Acad Natl Med 190:385-400; discussion 400-382. Lledo PM, Zhang X, Sudhof TC, Malenka RC, Nicoll RA (1998) Postsynaptic membrane fusion and long-term potentiation. Science 279:399-403. Lo LC, Johnson JE, Wuenschell CW, Saito T, Anderson DJ (1991) Mammalian achaete-scute homolog 1 is transiently expressed by spatially restricted subsets of early neuroepithelial and neural crest cells. Genes Dev 5:1524-1537. Lohof AM, Ip NY, Poo MM (1993) Potentiation of developing neuromuscular synapses by the neurotrophins NT-3 and BDNF. Nature 363:350-353. Lois C, Alvarez-Buylla A (1993) Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glia. Proc Natl Acad Sci U S A 90:2074-2077. Lois C, Alvarez-Buylla A (1994) Long-distance neuronal migration in the adult mammalian brain. Science 264:1145-1148. Lois C, Garcia-Verdugo JM, Alvarez-Buylla A (1996) Chain migration of neuronal precursors. Science 271:978-981. Lopez-Garcia JC, Arancio O, Kandel ER, Baranes D (1996) A presynaptic locus for long-term potentiation of elementary synaptic transmission at mossy fiber synapses in culture. Proc Natl Acad Sci U S A 93:4712-4717. Losi G, Prybylowski K, Fu Z, Luo J, Wenthold RJ, Vicini S (2003) PSD-95 regulates NMDA receptors in developing cerebellar granule neurons of the rat. J Physiol 548:21-29. 130 Lozovaya NA, Grebenyuk SE, Tsintsadze T, Feng B, Monaghan DT, Krishtal OA (2004) Extrasynaptic NR2B and NR2D subunits of NMDA receptors shape 'superslow' afterburst EPSC in rat hippocampus. J Physiol 558:451-463. Lu W, Man H, Ju W, Trimble WS, MacDonald JF, Wang YT (2001) Activation of synaptic NMDA receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons. Neuron 29:243-254. Lu W, Shi Y, Jackson AC, Bjorgan K, During MJ, Sprengel R, Seeburg PH, Nicoll RA (2009) Subunit composition of synaptic AMPA receptors revealed by a single-cell genetic approach. Neuron 62:254-268. Lue LF, Walker DG, Rogers J (2001a) Modeling microglial activation in Alzheimer's disease with human postmortem microglial cultures. Neurobiol Aging 22:945-956. Lue LF, Walker DG, Brachova L, Beach TG, Rogers J, Schmidt AM, Stern DM, Yan SD (2001b) Involvement of microglial receptor for advanced glycation endproducts (RAGE) in Alzheimer's disease: identification of a cellular activation mechanism. Exp Neurol 171:29-45. Luhmann HJ, Prince DA (1991) Postnatal maturation of the GABAergic system in rat neocortex. J Neurophysiol 65:247-263. Luscher C, Xia H, Beattie EC, Carroll RC, von Zastrow M, Malenka RC, Nicoll RA (1999) Role of AMPA receptor cycling in synaptic transmission and plasticity. Neuron 24:649-658. Luthi A, Chittajallu R, Duprat F, Palmer MJ, Benke TA, Kidd FL, Henley JM, Isaac JT, Collingridge GL (1999) Hippocampal LTD expression involves a pool of AMPARs regulated by the NSF-GluR2 interaction. Neuron 24:389-399. Lynch G, Larson J, Kelso S, Barrionuevo G, Schottler F (1983) Intracellular injections of EGTA block induction of hippocampal long-term potentiation. Nature 305:719-721. Lyons WE, Mamounas LA, Ricaurte GA, Coppola V, Reid SW, Bora SH, Wihler C, Koliatsos VE, Tessarollo L (1999) Brain-derived neurotrophic factor-deficient mice develop aggressiveness and hyperphagia in conjunction with brain serotonergic abnormalities. Proc Natl Acad Sci U S A 96:15239-15244. Ma BF, Liu XM, Xie XM, Zhang AX, Zhang JQ, Yu WH, Zhang XM, Li SN, Lahn BT, Xiang AP (2006) Slower cycling of nestin-positive cells in neurosphere culture. Neuroreport 17:377-381. Ma DK, Bonaguidi MA, Ming GL, Song H (2009) Adult neural stem cells in the mammalian central nervous system. Cell Res 19:672-682. Ma Q, Kintner C, Anderson DJ (1996) Identification of neurogenin, a vertebrate neuronal determination gene. Cell 87:43-52. Ma Q, Fode C, Guillemot F, Anderson DJ (1999) Neurogenin1 and neurogenin2 control two distinct waves of neurogenesis in developing dorsal root ganglia. Genes Dev 13:1717- 1728. Madison DV, Malenka RC, Nicoll RA (1991) Mechanisms underlying long-term potentiation of synaptic transmission. Annu Rev Neurosci 14:379-397. 131 Maisonpierre PC, Belluscio L, Squinto S, Ip NY, Furth ME, Lindsay RM, Yancopoulos GD (1990a) Neurotrophin-3: a neurotrophic factor related to NGF and BDNF. Science 247:1446-1451. Maisonpierre PC, Belluscio L, Friedman B, Alderson RF, Wiegand SJ, Furth ME, Lindsay RM, Yancopoulos GD (1990b) NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression. Neuron 5:501-509. Makino H, Malinow R (2009) AMPA receptor incorporation into synapses during LTP: the role of lateral movement and exocytosis. Neuron 64:381-390. Malenka RC, Nicoll RA (1993) NMDA-receptor-dependent synaptic plasticity: multiple forms and mechanisms. Trends Neurosci 16:521-527. Malenka RC, Nicoll RA (1999) Long-term potentiation--a decade of progress? Science 285:1870-1874. Malenka RC, Bear MF (2004) LTP and LTD: an embarrassment of riches. Neuron 44:5-21. Malenka RC, Lancaster B, Zucker RS (1992) Temporal limits on the rise in postsynaptic calcium required for the induction of long-term potentiation. Neuron 9:121-128. Malenka RC, Kauer JA, Zucker RS, Nicoll RA (1988) Postsynaptic calcium is sufficient for potentiation of hippocampal synaptic transmission. Science 242:81-84. Malinow R, Tsien RW (1990) Presynaptic enhancement shown by whole-cell recordings of long- term potentiation in hippocampal slices. Nature 346:177-180. Mammen AL, Kameyama K, Roche KW, Huganir RL (1997) Phosphorylation of the alpha- amino-3-hydroxy-5-methylisoxazole4-propionic acid receptor GluR1 subunit by calcium/calmodulin-dependent kinase II. J Biol Chem 272:32528-32533. Man HY, Sekine-Aizawa Y, Huganir RL (2007) Regulation of {alpha}-amino-3-hydroxy-5-methyl- 4-isoxazolepropionic acid receptor trafficking through PKA phosphorylation of the Glu receptor 1 subunit. Proc Natl Acad Sci U S A 104:3579-3584. Man HY, Lin JW, Ju WH, Ahmadian G, Liu L, Becker LE, Sheng M, Wang YT (2000) Regulation of AMPA receptor-mediated synaptic transmission by clathrin-dependent receptor internalization. Neuron 25:649-662. Man HY, Wang Q, Lu WY, Ju W, Ahmadian G, Liu L, D'Souza S, Wong TP, Taghibiglou C, Lu J, Becker LE, Pei L, Liu F, Wymann MP, MacDonald JF, Wang YT (2003) Activation of PI3- kinase is required for AMPA receptor insertion during LTP of mEPSCs in cultured hippocampal neurons. Neuron 38:611-624. Manabe T, Renner P, Nicoll RA (1992) Postsynaptic contribution to long-term potentiation revealed by the analysis of miniature synaptic currents. Nature 355:50-55. Markakis EA, Gage FH (1999) Adult-generated neurons in the dentate gyrus send axonal projections to field CA3 and are surrounded by synaptic vesicles. J Comp Neurol 406:449-460. Marusich MF, Furneaux HM, Henion PD, Weston JA (1994) Hu neuronal proteins are expressed in proliferating neurogenic cells. J Neurobiol 25:143-155. 132 Massey PV, Johnson BE, Moult PR, Auberson YP, Brown MW, Molnar E, Collingridge GL, Bashir ZI (2004) Differential roles of NR2A and NR2B-containing NMDA receptors in cortical long-term potentiation and long-term depression. J Neurosci 24:7821-7828. Masters CL, Simms G, Weinman NA, Multhaup G, McDonald BL, Beyreuther K (1985) Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci U S A 82:4245-4249. Matsuda S, Mikawa S, Hirai H (1999) Phosphorylation of serine-880 in GluR2 by protein kinase C prevents its C terminus from binding with glutamate receptor-interacting protein. J Neurochem 73:1765-1768. Mayer ML (2005) Glutamate receptor ion channels. Curr Opin Neurobiol 15:282-288. Mayer ML, Westbrook GL, Guthrie PB (1984) Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones. Nature 309:261-263. McCormick MB, Tamimi RM, Snider L, Asakura A, Bergstrom D, Tapscott SJ (1996) NeuroD2 and neuroD3: distinct expression patterns and transcriptional activation potentials within the neuroD gene family. Mol Cell Biol 16:5792-5800. McDearmid JR, Liao M, Drapeau P (2006) Glycine receptors regulate interneuron differentiation during spinal network development. Proc Natl Acad Sci U S A 103:9679-9684. McDonald DR, Brunden KR, Landreth GE (1997) Amyloid fibrils activate tyrosine kinase- dependent signaling and superoxide production in microglia. J Neurosci 17:2284-2294. McGeer PL, McGeer EG (1995) The inflammatory response system of brain: implications for therapy of Alzheimer and other neurodegenerative diseases. Brain Res Brain Res Rev 21:195-218. McGeer PL, Itagaki S, Tago H, McGeer EG (1988) Occurrence of HLA-DR reactive microglia in Alzheimer's disease. Ann N Y Acad Sci 540:319-323. McIlhinney RA, Le Bourdelles B, Molnar E, Tricaud N, Streit P, Whiting PJ (1998) Assembly intracellular targeting and cell surface expression of the human N-methyl-D-aspartate receptor subunits NR1a and NR2A in transfected cells. Neuropharmacology 37:1355- 1367. McKay R (1997) Stem cells in the central nervous system. Science 276:66-71. McKee AC, Kosik KS, Kowall NW (1991) Neuritic pathology and dementia in Alzheimer's disease. Ann Neurol 30:156-165. McLarnon JG, Ryu JK (2008) Relevance of abeta1-42 intrahippocampal injection as an animal model of inflamed Alzheimer's disease brain. Curr Alzheimer Res 5:475-480. McLarnon JG, Ryu JK, Walker DG, Choi HB (2006) Upregulated expression of purinergic P2X(7) receptor in Alzheimer disease and amyloid-beta peptide-treated microglia and in peptide-injected rat hippocampus. J Neuropathol Exp Neurol 65:1090-1097. Menard C, Hein P, Paquin A, Savelson A, Yang XM, Lederfein D, Barnabe-Heider F, Mir AA, Sterneck E, Peterson AC, Johnson PF, Vinson C, Miller FD (2002) An essential role for a MEK-C/EBP pathway during growth factor-regulated cortical neurogenesis. Neuron 133 36:597-610. Miller RJ, Banisadr G, Bhattacharyya BJ (2008) CXCR4 signaling in the regulation of stem cell migration and development. J Neuroimmunol 198:31-38. Miller SG, Kennedy MB (1986) Regulation of brain type II Ca2+/calmodulin-dependent protein kinase by autophosphorylation: a Ca2+-triggered molecular switch. Cell 44:861-870. Ming GL, Song H (2005) Adult neurogenesis in the mammalian central nervous system. Annu Rev Neurosci 28:223-250. Minichiello L (2009) TrkB signalling pathways in LTP and learning. Nat Rev Neurosci 10:850- 860. Minichiello L, Klein R (1996) TrkB and TrkC neurotrophin receptors cooperate in promoting survival of hippocampal and cerebellar granule neurons. Genes Dev 10:2849-2858. Misra C, Brickley SG, Wyllie DJ, Cull-Candy SG (2000a) Slow deactivation kinetics of NMDA receptors containing NR1 and NR2D subunits in rat cerebellar Purkinje cells. J Physiol 525 Pt 2:299-305. Misra C, Brickley SG, Farrant M, Cull-Candy SG (2000b) Identification of subunits contributing to synaptic and extrasynaptic NMDA receptors in Golgi cells of the rat cerebellum. J Physiol 524 Pt 1:147-162. Miyata T, Maeda T, Lee JE (1999) NeuroD is required for differentiation of the granule cells in the cerebellum and hippocampus. Genes Dev 13:1647-1652. Mizuno M, Yamada K, Takei N, Tran MH, He J, Nakajima A, Nawa H, Nabeshima T (2003) Phosphatidylinositol 3-kinase: a molecule mediating BDNF-dependent spatial memory formation. Mol Psychiatry 8:217-224. Momiyama A (2000) Distinct synaptic and extrasynaptic NMDA receptors identified in dorsal horn neurones of the adult rat spinal cord. J Physiol 523 Pt 3:621-628. Momiyama A, Feldmeyer D, Cull-Candy SG (1996) Identification of a native low-conductance NMDA channel with reduced sensitivity to Mg2+ in rat central neurones. J Physiol 494 ( Pt 2):479-492. Monaghan DT, Cotman CW (1982) The distribution of [3H]kainic acid binding sites in rat CNS as determined by autoradiography. Brain Res 252:91-100. Monyer H, Burnashev N, Laurie DJ, Sakmann B, Seeburg PH (1994) Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12:529-540. Moore KA, Nicoll RA, Schmitz D (2003) Adenosine gates synaptic plasticity at hippocampal mossy fiber synapses. Proc Natl Acad Sci U S A 100:14397-14402. Moriyoshi K, Masu M, Ishii T, Shigemoto R, Mizuno N, Nakanishi S (1991) Molecular cloning and characterization of the rat NMDA receptor. Nature 354:31-37. Morris RG, Anderson E, Lynch GS, Baudry M (1986) Selective impairment of learning and blockade of long-term potentiation by an N-methyl-D-aspartate receptor antagonist, AP5. 134 Nature 319:774-776. Morshead CM, van der Kooy D (1992) Postmitotic death is the fate of constitutively proliferating cells in the subependymal layer of the adult mouse brain. J Neurosci 12:249-256. Mulkey RM, Malenka RC (1992) Mechanisms underlying induction of homosynaptic long-term depression in area CA1 of the hippocampus. Neuron 9:967-975. Mullen RJ, Buck CR, Smith AM (1992) NeuN, a neuronal specific nuclear protein in vertebrates. Development 116:201-211. Muller D, Joly M, Lynch G (1988) Contributions of quisqualate and NMDA receptors to the induction and expression of LTP. Science 242:1694-1697. Mutoh H, Fung BP, Naya FJ, Tsai MJ, Nishitani J, Leiter AB (1997) The basic helix-loop-helix transcription factor BETA2/NeuroD is expressed in mammalian enteroendocrine cells and activates secretin gene expression. Proc Natl Acad Sci U S A 94:3560-3564. Nagao T, Kuwagata M, Saito Y (1998) Effects of prenatal exposure to 5-fluoro-2'-deoxyuridine on developing central nervous system and reproductive function in male offspring of mice. Teratog Carcinog Mutagen 18:73-92. Nait-Oumesmar B, Decker L, Lachapelle F, Avellana-Adalid V, Bachelin C, Van Evercooren AB (1999) Progenitor cells of the adult mouse subventricular zone proliferate, migrate and differentiate into oligodendrocytes after demyelination. Eur J Neurosci 11:4357-4366. Nakanishi N, Tu S, Shin Y, Cui J, Kurokawa T, Zhang D, Chen HS, Tong G, Lipton SA (2009) Neuroprotection by the NR3A subunit of the NMDA receptor. J Neurosci 29:5260-5265. Neeper SA, Gomez-Pinilla F, Choi J, Cotman C (1995) Exercise and brain neurotrophins. Nature 373:109. Neumann H, Misgeld T, Matsumuro K, Wekerle H (1998) Neurotrophins inhibit major histocompatibility class II inducibility of microglia: involvement of the p75 neurotrophin receptor. Proc Natl Acad Sci U S A 95:5779-5784. Neves G, Cooke SF, Bliss TV (2008) Synaptic plasticity, memory and the hippocampus: a neural network approach to causality. Nat Rev Neurosci 9:65-75. Newpher TM, Ehlers MD (2008) Glutamate receptor dynamics in dendritic microdomains. Neuron 58:472-497. Nicoll RA, Schmitz D (2005) Synaptic plasticity at hippocampal mossy fibre synapses. Nat Rev Neurosci 6:863-876. Nicoll RA, Kauer JA, Malenka RC (1988) The current excitement in long-term potentiation. Neuron 1:97-103. Nishimune A, Isaac JT, Molnar E, Noel J, Nash SR, Tagaya M, Collingridge GL, Nakanishi S, Henley JM (1998) NSF binding to GluR2 regulates synaptic transmission. Neuron 21:87- 97. Noel J, Ralph GS, Pickard L, Williams J, Molnar E, Uney JB, Collingridge GL, Henley JM (1999) Surface expression of AMPA receptors in hippocampal neurons is regulated by an NSF- 135 dependent mechanism. Neuron 23:365-376. Nosten-Bertrand M, Errington ML, Murphy KP, Tokugawa Y, Barboni E, Kozlova E, Michalovich D, Morris RG, Silver J, Stewart CL, Bliss TV, Morris RJ (1996) Normal spatial learning despite regional inhibition of LTP in mice lacking Thy-1. Nature 379:826-829. O'Connor JJ, Rowan MJ, Anwyl R (1995) Tetanically induced LTP involves a similar increase in the AMPA and NMDA receptor components of the excitatory postsynaptic current: investigations of the involvement of mGlu receptors. J Neurosci 15:2013-2020. Obata K, Oide M, Tanaka H (1978) Excitatory and inhibitory actions of GABA and glycine on embryonic chick spinal neurons in culture. Brain Res 144:179-184. Ogawa D, Okada Y, Nakamura M, Kanemura Y, Okano HJ, Matsuzaki Y, Shimazaki T, Ito M, Ikeda E, Tamiya T, Nagao S, Okano H (2009) Evaluation of human fetal neural stem/progenitor cells as a source for cell replacement therapy for neurological disorders: properties and tumorigenicity after long-term in vitro maintenance. J Neurosci Res 87:307-317. Ogawa Y, Sawamoto K, Miyata T, Miyao S, Watanabe M, Nakamura M, Bregman BS, Koike M, Uchiyama Y, Toyama Y, Okano H (2002) Transplantation of in vitro-expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal cord contusion injury in adult rats. J Neurosci Res 69:925-933. Oh MC, Derkach VA, Guire ES, Soderling TR (2006) Extrasynaptic membrane trafficking regulated by GluR1 serine 845 phosphorylation primes AMPA receptors for long-term potentiation. J Biol Chem 281:752-758. Ohtaki H, Ylostalo JH, Foraker JE, Robinson AP, Reger RL, Shioda S, Prockop DJ (2008) Stem/progenitor cells from bone marrow decrease neuronal death in global ischemia by modulation of inflammatory/immune responses. Proc Natl Acad Sci U S A 105:14638- 14643. Opazo P, Choquet D (2011) A three-step model for the synaptic recruitment of AMPA receptors. Mol Cell Neurosci 46:1-8. Orlacchio A, Bernardi G, Martino S (2010) Stem cells: an overview of the current status of therapies for central and peripheral nervous system diseases. Curr Med Chem 17:595- 608. Osten P, Srivastava S, Inman GJ, Vilim FS, Khatri L, Lee LM, States BA, Einheber S, Milner TA, Hanson PI, Ziff EB (1998) The AMPA receptor GluR2 C terminus can mediate a reversible, ATP-dependent interaction with NSF and alpha- and beta-SNAPs. Neuron 21:99-110. Ostenfeld T, Tai YT, Martin P, Deglon N, Aebischer P, Svendsen CN (2002) Neurospheres modified to produce glial cell line-derived neurotrophic factor increase the survival of transplanted dopamine neurons. J Neurosci Res 69:955-965. Otmakhov N, Khibnik L, Otmakhova N, Carpenter S, Riahi S, Asrican B, Lisman J (2004) Forskolin-induced LTP in the CA1 hippocampal region is NMDA receptor dependent. J Neurophysiol 91:1955-1962. Owens DF, Boyce LH, Davis MB, Kriegstein AR (1996) Excitatory GABA responses in 136 embryonic and neonatal cortical slices demonstrated by gramicidin perforated-patch recordings and calcium imaging. J Neurosci 16:6414-6423. Palmer TD, Takahashi J, Gage FH (1997) The adult rat hippocampus contains primordial neural stem cells. Mol Cell Neurosci 8:389-404. Papadakis M, Hawkins LM, Stephenson FA (2004) Appropriate NR1-NR1 disulfide-linked homodimer formation is requisite for efficient expression of functional, cell surface N- methyl-D-aspartate NR1/NR2 receptors. J Biol Chem 279:14703-14712. Papadia S, Hardingham GE (2007) The dichotomy of NMDA receptor signaling. Neuroscientist 13:572-579. Parent JM, Vexler ZS, Gong C, Derugin N, Ferriero DM (2002) Rat forebrain neurogenesis and striatal neuron replacement after focal stroke. Ann Neurol 52:802-813. Parent JM, Elliott RC, Pleasure SJ, Barbaro NM, Lowenstein DH (2006) Aberrant seizure- induced neurogenesis in experimental temporal lobe epilepsy. Ann Neurol 59:81-91. Parent JM, Yu TW, Leibowitz RT, Geschwind DH, Sloviter RS, Lowenstein DH (1997) Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus. J Neurosci 17:3727-3738. Park DH, Eve DJ (2009) Regenerative medicine: advances in new methods and technologies. Med Sci Monit 15:RA233-251. Park M, Penick EC, Edwards JG, Kauer JA, Ehlers MD (2004) Recycling endosomes supply AMPA receptors for LTP. Science 305:1972-1975. Park M, Salgado JM, Ostroff L, Helton TD, Robinson CG, Harris KM, Ehlers MD (2006) Plasticity-induced growth of dendritic spines by exocytic trafficking from recycling endosomes. Neuron 52:817-830. Passafaro M, Piech V, Sheng M (2001) Subunit-specific temporal and spatial patterns of AMPA receptor exocytosis in hippocampal neurons. Nat Neurosci 4:917-926. Patapoutian A, Reichardt LF (2001) Trk receptors: mediators of neurotrophin action. Curr Opin Neurobiol 11:272-280. Pathania M, Yan LD, Bordey A (2010) A symphony of signals conducts early and late stages of adult neurogenesis. Neuropharmacology 58:865-876. Patterson SL, Grover LM, Schwartzkroin PA, Bothwell M (1992) Neurotrophin expression in rat hippocampal slices: a stimulus paradigm inducing LTP in CA1 evokes increases in BDNF and NT-3 mRNAs. Neuron 9:1081-1088. Patterson SL, Abel T, Deuel TA, Martin KC, Rose JC, Kandel ER (1996) Recombinant BDNF rescues deficits in basal synaptic transmission and hippocampal LTP in BDNF knockout mice. Neuron 16:1137-1145. Paxinos G, Watson C (2005) The Rat Brain in Stereotaxic Coordinates. In: Elsevier Academic Press. Peineau S, Taghibiglou C, Bradley C, Wong TP, Liu L, Lu J, Lo E, Wu D, Saule E, Bouschet T, 137 Matthews P, Isaac JT, Bortolotto ZA, Wang YT, Collingridge GL (2007) LTP inhibits LTD in the hippocampus via regulation of GSK3beta. Neuron 53:703-717. Pencea V, Bingaman KD, Wiegand SJ, Luskin MB (2001) Infusion of brain-derived neurotrophic factor into the lateral ventricle of the adult rat leads to new neurons in the parenchyma of the striatum, septum, thalamus, and hypothalamus. J Neurosci 21:6706-6717. Pennesi ME, Cho JH, Yang Z, Wu SH, Zhang J, Wu SM, Tsai MJ (2003) BETA2/NeuroD1 null mice: a new model for transcription factor-dependent photoreceptor degeneration. J Neurosci 23:453-461. Perez-Otano I, Schulteis CT, Contractor A, Lipton SA, Trimmer JS, Sucher NJ, Heinemann SF (2001) Assembly with the NR1 subunit is required for surface expression of NR3A- containing NMDA receptors. J Neurosci 21:1228-1237. Perez JL, Khatri L, Chang C, Srivastava S, Osten P, Ziff EB (2001) PICK1 targets activated protein kinase Calpha to AMPA receptor clusters in spines of hippocampal neurons and reduces surface levels of the AMPA-type glutamate receptor subunit 2. J Neurosci 21:5417-5428. Perkel DJ, Petrozzino JJ, Nicoll RA, Connor JA (1993) The role of Ca2+ entry via synaptically activated NMDA receptors in the induction of long-term potentiation. Neuron 11:817-823. Perry VH, Hume DA, Gordon S (1985) Immunohistochemical localization of macrophages and microglia in the adult and developing mouse brain. Neuroscience 15:313-326. Petrini EM, Lu J, Cognet L, Lounis B, Ehlers MD, Choquet D (2009) Endocytic trafficking and recycling maintain a pool of mobile surface AMPA receptors required for synaptic potentiation. Neuron 63:92-105. Pickard L, Noel J, Duckworth JK, Fitzjohn SM, Henley JM, Collingridge GL, Molnar E (2001) Transient synaptic activation of NMDA receptors leads to the insertion of native AMPA receptors at hippocampal neuronal plasma membranes. Neuropharmacology 41:700- 713. Pike CJ, Burdick D, Walencewicz AJ, Glabe CG, Cotman CW (1993) Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state. J Neurosci 13:1676-1687. Pincus DW, Keyoung HM, Harrison-Restelli C, Goodman RR, Fraser RA, Edgar M, Sakakibara S, Okano H, Nedergaard M, Goldman SA (1998) Fibroblast growth factor-2/brain-derived neurotrophic factor-associated maturation of new neurons generated from adult human subependymal cells. Ann Neurol 43:576-585. Pluchino S, Quattrini A, Brambilla E, Gritti A, Salani G, Dina G, Galli R, Del Carro U, Amadio S, Bergami A, Furlan R, Comi G, Vescovi AL, Martino G (2003) Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature 422:688- 694. Poirier R, Veltman I, Pflimlin MC, Knoflach F, Metzger F (2010) Enhanced dentate gyrus synaptic plasticity but reduced neurogenesis in a mouse model of amyloidosis. Neurobiol Dis. Poo MM (2001) Neurotrophins as synaptic modulators. Nat Rev Neurosci 2:24-32. 138 Premkumar LS, Auerbach A (1997) Stoichiometry of recombinant N-methyl-D-aspartate receptor channels inferred from single-channel current patterns. J Gen Physiol 110:485-502. Rao MS, Shetty AK (2004) Efficacy of doublecortin as a marker to analyse the absolute number and dendritic growth of newly generated neurons in the adult dentate gyrus. Eur J Neurosci 19:234-246. Redmond DE, Jr., Bjugstad KB, Teng YD, Ourednik V, Ourednik J, Wakeman DR, Parsons XH, Gonzalez R, Blanchard BC, Kim SU, Gu Z, Lipton SA, Markakis EA, Roth RH, Elsworth JD, Sladek JR, Jr., Sidman RL, Snyder EY (2007) Behavioral improvement in a primate Parkinson's model is associated with multiple homeostatic effects of human neural stem cells. Proc Natl Acad Sci U S A 104:12175-12180. Reichling DB, Kyrozis A, Wang J, MacDermott AB (1994) Mechanisms of GABA and glycine depolarization-induced calcium transients in rat dorsal horn neurons. J Physiol 476:411- 421. Reid CA, Dixon DB, Takahashi M, Bliss TV, Fine A (2004) Optical quantal analysis indicates that long-term potentiation at single hippocampal mossy fiber synapses is expressed through increased release probability, recruitment of new release sites, and activation of silent synapses. J Neurosci 24:3618-3626. Reynolds BA, Weiss S (1992) Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255:1707-1710. Richards LJ, Kilpatrick TJ, Bartlett PF (1992) De novo generation of neuronal cells from the adult mouse brain. Proc Natl Acad Sci U S A 89:8591-8595. Roche KW, O'Brien RJ, Mammen AL, Bernhardt J, Huganir RL (1996) Characterization of multiple phosphorylation sites on the AMPA receptor GluR1 subunit. Neuron 16:1179- 1188. Rohrbough J, Spitzer NC (1996) Regulation of intracellular Cl- levels by Na(+)-dependent Cl- cotransport distinguishes depolarizing from hyperpolarizing GABAA receptor-mediated responses in spinal neurons. J Neurosci 16:82-91. Rosenmund C, Stevens CF (1996) Definition of the readily releasable pool of vesicles at hippocampal synapses. Neuron 16:1197-1207. Rosenmund C, Stern-Bach Y, Stevens CF (1998) The tetrameric structure of a glutamate receptor channel. Science 280:1596-1599. Ross HH, Levkoff LH, Marshall GP, 2nd, Caldeira M, Steindler DA, Reynolds BA, Laywell ED (2008) Bromodeoxyuridine induces senescence in neural stem and progenitor cells. Stem Cells 26:3218-3227. Rossi P, Sola E, Taglietti V, Borchardt T, Steigerwald F, Utvik JK, Ottersen OP, Kohr G, D'Angelo E (2002) NMDA receptor 2 (NR2) C-terminal control of NR open probability regulates synaptic transmission and plasticity at a cerebellar synapse. J Neurosci 22:9687-9697. Rothman JE (1994) Mechanisms of intracellular protein transport. Nature 372:55-63. Ryu JK, McLarnon JG (2006) Minocycline or iNOS inhibition block 3-nitrotyrosine increases and blood-brain barrier leakiness in amyloid beta-peptide-injected rat hippocampus. Exp 139 Neurol 198:552-557. Ryu JK, McLarnon JG (2008) Thalidomide inhibition of perturbed vasculature and glial-derived tumor necrosis factor-alpha in an animal model of inflamed Alzheimer's disease brain. Neurobiol Dis 29:254-266. Ryu JK, Cho T, Wang YT, McLarnon JG (2009a) Neural progenitor cells attenuate inflammatory reactivity and neuronal loss in an animal model of inflamed AD brain. J Neuroinflammation 6:39. Ryu JK, Cho T, Choi HB, Wang YT, McLarnon JG (2009b) Microglial VEGF receptor response is an integral chemotactic component in Alzheimer's disease pathology. J Neurosci 29:3-13. Ryu JK, Kim J, Cho SJ, Hatori K, Nagai A, Choi HB, Lee MC, McLarnon JG, Kim SU (2004) Proactive transplantation of human neural stem cells prevents degeneration of striatal neurons in a rat model of Huntington disease. Neurobiol Dis 16:68-77. Salin PA, Scanziani M, Malenka RC, Nicoll RA (1996) Distinct short-term plasticity at two excitatory synapses in the hippocampus. Proc Natl Acad Sci U S A 93:13304-13309. Sans N, Racca C, Petralia RS, Wang YX, McCallum J, Wenthold RJ (2001) Synapse- associated protein 97 selectively associates with a subset of AMPA receptors early in their biosynthetic pathway. J Neurosci 21:7506-7516. Sans N, Prybylowski K, Petralia RS, Chang K, Wang YX, Racca C, Vicini S, Wenthold RJ (2003) NMDA receptor trafficking through an interaction between PDZ proteins and the exocyst complex. Nat Cell Biol 5:520-530. Santos SD, Carvalho AL, Caldeira MV, Duarte CB (2009) Regulation of AMPA receptors and synaptic plasticity. Neuroscience 158:105-125. Saxe MD, Battaglia F, Wang JW, Malleret G, David DJ, Monckton JE, Garcia AD, Sofroniew MV, Kandel ER, Santarelli L, Hen R, Drew MR (2006) Ablation of hippocampal neurogenesis impairs contextual fear conditioning and synaptic plasticity in the dentate gyrus. Proc Natl Acad Sci U S A 103:17501-17506. Scardigli R, Schuurmans C, Gradwohl G, Guillemot F (2001) Crossregulation between Neurogenin2 and pathways specifying neuronal identity in the spinal cord. Neuron 31:203-217. Schinder AF, Poo M (2000) The neurotrophin hypothesis for synaptic plasticity. Trends Neurosci 23:639-645. Schmitz D, Mellor J, Breustedt J, Nicoll RA (2003) Presynaptic kainate receptors impart an associative property to hippocampal mossy fiber long-term potentiation. Nat Neurosci 6:1058-1063. Schnell E, Sizemore M, Karimzadegan S, Chen L, Bredt DS, Nicoll RA (2002) Direct interactions between PSD-95 and stargazin control synaptic AMPA receptor number. Proc Natl Acad Sci U S A 99:13902-13907. Schorge S, Colquhoun D (2003) Studies of NMDA receptor function and stoichiometry with truncated and tandem subunits. J Neurosci 23:1151-1158. 140 Schwab MH, Bartholomae A, Heimrich B, Feldmeyer D, Druffel-Augustin S, Goebbels S, Naya FJ, Zhao S, Frotscher M, Tsai MJ, Nave KA (2000) Neuronal basic helix-loop-helix proteins (NEX and BETA2/Neuro D) regulate terminal granule cell differentiation in the hippocampus. J Neurosci 20:3714-3724. Schwarz LA, Hall BJ, Patrick GN (2010) Activity-Dependent Ubiquitination of GluA1 Mediates a Distinct AMPA Receptor Endocytosis and Sorting Pathway. J Neurosci 30:16718-16729. Scott DB, Blanpied TA, Swanson GT, Zhang C, Ehlers MD (2001) An NMDA receptor ER retention signal regulated by phosphorylation and alternative splicing. J Neurosci 21:3063-3072. Seaberg RM, van der Kooy D (2002) Adult rodent neurogenic regions: the ventricular subependyma contains neural stem cells, but the dentate gyrus contains restricted progenitors. J Neurosci 22:1784-1793. Seeburg PH (1993) The TINS/TiPS Lecture. The molecular biology of mammalian glutamate receptor channels. Trends Neurosci 16:359-365. Segal RA (2003) Selectivity in neurotrophin signaling: theme and variations. Annu Rev Neurosci 26:299-330. Seidenman KJ, Steinberg JP, Huganir R, Malinow R (2003) Glutamate receptor subunit 2 Serine 880 phosphorylation modulates synaptic transmission and mediates plasticity in CA1 pyramidal cells. J Neurosci 23:9220-9228. Sekerkova G, Ilijic E, Mugnaini E (2004) Bromodeoxyuridine administered during neurogenesis of the projection neurons causes cerebellar defects in rat. J Comp Neurol 470:221-239. Seki T, Arai Y (1993) Highly polysialylated neural cell adhesion molecule (NCAM-H) is expressed by newly generated granule cells in the dentate gyrus of the adult rat. J Neurosci 13:2351-2358. Selkoe DJ (2000) Toward a comprehensive theory for Alzheimer's disease. Hypothesis: Alzheimer's disease is caused by the cerebral accumulation and cytotoxicity of amyloid beta-protein. Ann N Y Acad Sci 924:17-25. Selkoe DJ (2001) Alzheimer's disease: genes, proteins, and therapy. Physiol Rev 81:741-766. Sharpless NE, DePinho RA (2007) How stem cells age and why this makes us grow old. Nat Rev Mol Cell Biol 8:703-713. Sheng M, Sala C (2001) PDZ domains and the organization of supramolecular complexes. Annu Rev Neurosci 24:1-29. Shepherd JD, Huganir RL (2007) The cell biology of synaptic plasticity: AMPA receptor trafficking. Annu Rev Cell Dev Biol 23:613-643. Shi S, Hayashi Y, Esteban JA, Malinow R (2001) Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons. Cell 105:331-343. Shi SH, Hayashi Y, Petralia RS, Zaman SH, Wenthold RJ, Svoboda K, Malinow R (1999) Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation. Science 284:1811-1816. 141 Shihabuddin LS, Hertz JA, Holets VR, Whittemore SR (1995) The adult CNS retains the potential to direct region-specific differentiation of a transplanted neuronal precursor cell line. J Neurosci 15:6666-6678. Shors TJ, Townsend DA, Zhao M, Kozorovitskiy Y, Gould E (2002) Neurogenesis may relate to some but not all types of hippocampal-dependent learning. Hippocampus 12:578-584. Singec I, Jandial R, Crain A, Nikkhah G, Snyder EY (2007) The leading edge of stem cell therapeutics. Annu Rev Med 58:313-328. Sivakumaran S, Mohajerani MH, Cherubini E (2009) At immature mossy-fiber-CA3 synapses, correlated presynaptic and postsynaptic activity persistently enhances GABA release and network excitability via BDNF and cAMP-dependent PKA. J Neurosci 29:2637-2647. Skarsgard ED, Huang L, Reebye SC, Yeung AY, Jia WW (2005) Lentiviral vector-mediated, in vivo gene transfer to the tracheobronchial tree in fetal rabbits. J Pediatr Surg 40:1817- 1821. Snyder SW, Ladror US, Wade WS, Wang GT, Barrett LW, Matayoshi ED, Huffaker HJ, Krafft GA, Holzman TF (1994) Amyloid-beta aggregation: selective inhibition of aggregation in mixtures of amyloid with different chain lengths. Biophys J 67:1216-1228. Sollner T, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S, Tempst P, Rothman JE (1993) SNAP receptors implicated in vesicle targeting and fusion. Nature 362:318-324. Sommer B, Kohler M, Sprengel R, Seeburg PH (1991) RNA editing in brain controls a determinant of ion flow in glutamate-gated channels. Cell 67:11-19. Sommer I, Schachner M (1981) Monoclonal antibodies (O1 to O4) to oligodendrocyte cell surfaces: an immunocytological study in the central nervous system. Dev Biol 83:311- 327. Sommer L, Ma Q, Anderson DJ (1996) neurogenins, a novel family of atonal-related bHLH transcription factors, are putative mammalian neuronal determination genes that reveal progenitor cell heterogeneity in the developing CNS and PNS. Mol Cell Neurosci 8:221- 241. Song H, Stevens CF, Gage FH (2002) Astroglia induce neurogenesis from adult neural stem cells. Nature 417:39-44. Song I, Huganir RL (2002) Regulation of AMPA receptors during synaptic plasticity. Trends Neurosci 25:578-588. Song I, Kamboj S, Xia J, Dong H, Liao D, Huganir RL (1998) Interaction of the N- ethylmaleimide-sensitive factor with AMPA receptors. Neuron 21:393-400. Srivastava AS, Malhotra R, Sharp J, Berggren T (2008) Potentials of ES cell therapy in neurodegenerative diseases. Curr Pharm Des 14:3873-3879. Steigerwald F, Schulz TW, Schenker LT, Kennedy MB, Seeburg PH, Kohr G (2000) C-Terminal truncation of NR2A subunits impairs synaptic but not extrasynaptic localization of NMDA receptors. J Neurosci 20:4573-4581. 142 Steinberg JP, Takamiya K, Shen Y, Xia J, Rubio ME, Yu S, Jin W, Thomas GM, Linden DJ, Huganir RL (2006) Targeted in vivo mutations of the AMPA receptor subunit GluR2 and its interacting protein PICK1 eliminate cerebellar long-term depression. Neuron 49:845- 860. Stephenson FA, Cousins SL, Kenny AV (2008) Assembly and forward trafficking of NMDA receptors (Review). Mol Membr Biol 25:311-320. Stevens CF, Wang Y (1994) Changes in reliability of synaptic function as a mechanism for plasticity. Nature 371:704-707. Struikmans H, Rutgers DH, Jansen GH, Tulleken CA, van der Tweel I, Battermann JJ (1997) S- phase fraction, 5-bromo-2'-deoxy-uridine labelling index, duration of S-phase, potential doubling time, and DNA index in benign and malignant brain tumors. Radiat Oncol Investig 5:170-179. Sudhof TC (1995) The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature 375:645-653. Suen PC, Wu K, Levine ES, Mount HT, Xu JL, Lin SY, Black IB (1997) Brain-derived neurotrophic factor rapidly enhances phosphorylation of the postsynaptic N-methyl-D- aspartate receptor subunit 1. Proc Natl Acad Sci U S A 94:8191-8195. Suh H, Deng W, Gage FH (2009) Signaling in adult neurogenesis. Annu Rev Cell Dev Biol 25:253-275. Suh H, Consiglio A, Ray J, Sawai T, D'Amour KA, Gage FH (2007) In vivo fate analysis reveals the multipotent and self-renewal capacities of Sox2+ neural stem cells in the adult hippocampus. Cell Stem Cell 1:515-528. Sun W, Winseck A, Vinsant S, Park OH, Kim H, Oppenheim RW (2004) Programmed cell death of adult-generated hippocampal neurons is mediated by the proapoptotic gene Bax. J Neurosci 24:11205-11213. Swanson GT, Kamboj SK, Cull-Candy SG (1997) Single-channel properties of recombinant AMPA receptors depend on RNA editing, splice variation, and subunit composition. J Neurosci 17:58-69. Takei N, Kawamura M, Hara K, Yonezawa K, Nawa H (2001) Brain-derived neurotrophic factor enhances neuronal translation by activating multiple initiation processes: comparison with the effects of insulin. J Biol Chem 276:42818-42825. Tapley P, Lamballe F, Barbacid M (1992) K252a is a selective inhibitor of the tyrosine protein kinase activity of the trk family of oncogenes and neurotrophin receptors. Oncogene 7:371-381. Tardy M, Fages C, Le Prince G, Rolland B, Nunez J (1990) Regulation of the glial fibrillary acidic protein (GFAP) and of its encoding mRNA in the developing brain and in cultured astrocytes. Adv Exp Med Biol 265:41-52. Tashiro A, Zhao C, Gage FH (2006a) Retrovirus-mediated single-cell gene knockout technique in adult newborn neurons in vivo. Nat Protoc 1:3049-3055. Tashiro A, Sandler VM, Toni N, Zhao C, Gage FH (2006b) NMDA-receptor-mediated, cell- 143 specific integration of new neurons in adult dentate gyrus. Nature 442:929-933. Tattersfield AS, Croon RJ, Liu YW, Kells AP, Faull RL, Connor B (2004) Neurogenesis in the striatum of the quinolinic acid lesion model of Huntington's disease. Neuroscience 127:319-332. Taupin P (2006) The therapeutic potential of adult neural stem cells. Curr Opin Mol Ther 8:225- 231. Taupin P (2007) BrdU immunohistochemistry for studying adult neurogenesis: paradigms, pitfalls, limitations, and validation. Brain Res Rev 53:198-214. Temple S (1989) Division and differentiation of isolated CNS blast cells in microculture. Nature 340:471-473. Terashima A, Cotton L, Dev KK, Meyer G, Zaman S, Duprat F, Henley JM, Collingridge GL, Isaac JT (2004) Regulation of synaptic strength and AMPA receptor subunit composition by PICK1. J Neurosci 24:5381-5390. Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R, Hansen LA, Katzman R (1991) Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment. Ann Neurol 30:572-580. Teyler TJ, DiScenna P (1987) Long-term potentiation. Annu Rev Neurosci 10:131-161. Thomas-Crusells J, Vieira A, Saarma M, Rivera C (2003) A novel method for monitoring surface membrane trafficking on hippocampal acute slice preparation. J Neurosci Methods 125:159-166. Thomas CG, Miller AJ, Westbrook GL (2006) Synaptic and extrasynaptic NMDA receptor NR2 subunits in cultured hippocampal neurons. J Neurophysiol 95:1727-1734. Thomson AM, Walker VE, Flynn DM (1989) Glycine enhances NMDA-receptor mediated synaptic potentials in neocortical slices. Nature 338:422-424. Tichelaar W, Safferling M, Keinanen K, Stark H, Madden DR (2004) The Three-dimensional Structure of an Ionotropic Glutamate Receptor Reveals a Dimer-of-dimers Assembly. J Mol Biol 344:435-442. Tong G, Malenka RC, Nicoll RA (1996) Long-term potentiation in cultures of single hippocampal granule cells: a presynaptic form of plasticity. Neuron 16:1147-1157. Toni N, Laplagne DA, Zhao C, Lombardi G, Ribak CE, Gage FH, Schinder AF (2008) Neurons born in the adult dentate gyrus form functional synapses with target cells. Nat Neurosci 11:901-907. Toth K, Suares G, Lawrence JJ, Philips-Tansey E, McBain CJ (2000) Differential mechanisms of transmission at three types of mossy fiber synapse. J Neurosci 20:8279-8289. Tovar KR, Westbrook GL (1999) The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro. J Neurosci 19:4180-4188. Tozuka Y, Fukuda S, Namba T, Seki T, Hisatsune T (2005) GABAergic excitation promotes neuronal differentiation in adult hippocampal progenitor cells. Neuron 47:803-815. 144 Tran PB, Banisadr G, Ren D, Chenn A, Miller RJ (2007) Chemokine receptor expression by neural progenitor cells in neurogenic regions of mouse brain. J Comp Neurol 500:1007- 1033. Triller A, Choquet D (2008) New concepts in synaptic biology derived from single-molecule imaging. Neuron 59:359-374. Tronnier VM, Fogel W, Kronenbuerger M, Steinvorth S (1997a) Pallidal stimulation: an alternative to pallidotomy? J Neurosurg 87:700-705. Tronnier VM, Fogel W, Kronenbuerger M, Krause M, Steinvorth S (1997b) Is the medial globus pallidus a site for stimulation or lesioning in the treatment of Parkinson's disease? Stereotact Funct Neurosurg 69:62-68. Tyler WJ, Perrett SP, Pozzo-Miller LD (2002) The role of neurotrophins in neurotransmitter release. Neuroscientist 8:524-531. Urban NN, Henze DA, Lewis DA, Barrionuevo G (1996) Properties of LTP induction in the CA3 region of the primate hippocampus. Learn Mem 3:86-95. van Praag H, Kempermann G, Gage FH (1999a) Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat Neurosci 2:266-270. van Praag H, Christie BR, Sejnowski TJ, Gage FH (1999b) Running enhances neurogenesis, learning, and long-term potentiation in mice. Proc Natl Acad Sci U S A 96:13427-13431. van Praag H, Shubert T, Zhao C, Gage FH (2005) Exercise enhances learning and hippocampal neurogenesis in aged mice. J Neurosci 25:8680-8685. van Praag H, Schinder AF, Christie BR, Toni N, Palmer TD, Gage FH (2002) Functional neurogenesis in the adult hippocampus. Nature 415:1030-1034. Verdoorn TA, Burnashev N, Monyer H, Seeburg PH, Sakmann B (1991) Structural determinants of ion flow through recombinant glutamate receptor channels. Science 252:1715-1718. Verret L, Jankowsky JL, Xu GM, Borchelt DR, Rampon C (2007) Alzheimer's-type amyloidosis in transgenic mice impairs survival of newborn neurons derived from adult hippocampal neurogenesis. J Neurosci 27:6771-6780. Wafford KA, Kathoria M, Bain CJ, Marshall G, Le Bourdelles B, Kemp JA, Whiting PJ (1995) Identification of amino acids in the N-methyl-D-aspartate receptor NR1 subunit that contribute to the glycine binding site. Mol Pharmacol 47:374-380. Wang J, Reichling DB, Kyrozis A, MacDermott AB (1994) Developmental loss of GABA- and glycine-induced depolarization and Ca2+ transients in embryonic rat dorsal horn neurons in culture. Eur J Neurosci 6:1275-1280. Wang YF, Gao XB, van den Pol AN (2001) Membrane properties underlying patterns of GABA- dependent action potentials in developing mouse hypothalamic neurons. J Neurophysiol 86:1252-1265. Wang YT, Linden DJ (2000) Expression of cerebellar long-term depression requires postsynaptic clathrin-mediated endocytosis. Neuron 25:635-647. 145 Wang Z, Edwards JG, Riley N, Provance DW, Jr., Karcher R, Li XD, Davison IG, Ikebe M, Mercer JA, Kauer JA, Ehlers MD (2008) Myosin Vb mobilizes recycling endosomes and AMPA receptors for postsynaptic plasticity. Cell 135:535-548. Waxman EA, Lynch DR (2005) N-methyl-D-aspartate receptor subtypes: multiple roles in excitotoxicity and neurological disease. Neuroscientist 11:37-49. Wei LC, Shi M, Chen LW, Cao R, Zhang P, Chan YS (2002) Nestin-containing cells express glial fibrillary acidic protein in the proliferative regions of central nervous system of postnatal developing and adult mice. Brain Res Dev Brain Res 139:9-17. Weisskopf MG, Nicoll RA (1995) Presynaptic changes during mossy fibre LTP revealed by NMDA receptor-mediated synaptic responses. Nature 376:256-259. Weisskopf MG, Castillo PE, Zalutsky RA, Nicoll RA (1994) Mediation of hippocampal mossy fiber long-term potentiation by cyclic AMP. Science 265:1878-1882. Wenthold RJ, Petralia RS, Blahos J, II, Niedzielski AS (1996) Evidence for multiple AMPA receptor complexes in hippocampal CA1/CA2 neurons. J Neurosci 16:1982-1989. Wenthold RJ, Prybylowski K, Standley S, Sans N, Petralia RS (2003) Trafficking of NMDA receptors. Annu Rev Pharmacol Toxicol 43:335-358. Whitehouse PJ, Price DL, Clark AW, Coyle JT, DeLong MR (1981) Alzheimer disease: evidence for selective loss of cholinergic neurons in the nucleus basalis. Ann Neurol 10:122-126. Whitlock JR, Heynen AJ, Shuler MG, Bear MF (2006) Learning induces long-term potentiation in the hippocampus. Science 313:1093-1097. Widera D, Holtkamp W, Entschladen F, Niggemann B, Zanker K, Kaltschmidt B, Kaltschmidt C (2004) MCP-1 induces migration of adult neural stem cells. Eur J Cell Biol 83:381-387. Williams JM, Guevremont D, Mason-Parker SE, Luxmanan C, Tate WP, Abraham WC (2007) Differential trafficking of AMPA and NMDA receptors during long-term potentiation in awake adult animals. J Neurosci 27:14171-14178. Wisden W, Seeburg PH (1993) Mammalian ionotropic glutamate receptors. Curr Opin Neurobiol 3:291-298. Wong HK, Liu XB, Matos MF, Chan SF, Perez-Otano I, Boysen M, Cui J, Nakanishi N, Trimmer JS, Jones EG, Lipton SA, Sucher NJ (2002) Temporal and regional expression of NMDA receptor subunit NR3A in the mammalian brain. J Comp Neurol 450:303-317. Wong TP, Howland JG, Robillard JM, Ge Y, Yu W, Titterness AK, Brebner K, Liu L, Weinberg J, Christie BR, Phillips AG, Wang YT (2007) Hippocampal long-term depression mediates acute stress-induced spatial memory retrieval impairment. Proc Natl Acad Sci U S A 104:11471-11476. Wu WL, Ziskind-Conhaim L, Sweet MA (1992) Early development of glycine- and GABA- mediated synapses in rat spinal cord. J Neurosci 12:3935-3945. Wyllie DJ, Behe P, Colquhoun D (1998) Single-channel activations and concentration jumps: comparison of recombinant NR1a/NR2A and NR1a/NR2D NMDA receptors. J Physiol 510 ( Pt 1):1-18. 146 Wyllie DJ, Behe P, Nassar M, Schoepfer R, Colquhoun D (1996) Single-channel currents from recombinant NMDA NR1a/NR2D receptors expressed in Xenopus oocytes. Proc Biol Sci 263:1079-1086. Xia H, Hornby ZD, Malenka RC (2001) An ER retention signal explains differences in surface expression of NMDA and AMPA receptor subunits. Neuropharmacology 41:714-723. Xia J, Zhang X, Staudinger J, Huganir RL (1999) Clustering of AMPA receptors by the synaptic PDZ domain-containing protein PICK1. Neuron 22:179-187. Xia J, Chung HJ, Wihler C, Huganir RL, Linden DJ (2000) Cerebellar long-term depression requires PKC-regulated interactions between GluR2/3 and PDZ domain-containing proteins. Neuron 28:499-510. Xiang Z, Greenwood AC, Kairiss EW, Brown TH (1994) Quantal mechanism of long-term potentiation in hippocampal mossy-fiber synapses. J Neurophysiol 71:2552-2556. Xu B, Gottschalk W, Chow A, Wilson RI, Schnell E, Zang K, Wang D, Nicoll RA, Lu B, Reichardt LF (2000) The role of brain-derived neurotrophic factor receptors in the mature hippocampus: modulation of long-term potentiation through a presynaptic mechanism involving TrkB. J Neurosci 20:6888-6897. Yamada K, Nabeshima T (2003) Brain-derived neurotrophic factor/TrkB signaling in memory processes. J Pharmacol Sci 91:267-270. Yan Q, Radeke MJ, Matheson CR, Talvenheimo J, Welcher AA, Feinstein SC (1997) Immunocytochemical localization of TrkB in the central nervous system of the adult rat. J Comp Neurol 378:135-157. Yan SD, Chen X, Fu J, Chen M, Zhu H, Roher A, Slattery T, Zhao L, Nagashima M, Morser J, Migheli A, Nawroth P, Stern D, Schmidt AM (1996) RAGE and amyloid-beta peptide neurotoxicity in Alzheimer's disease. Nature 382:685-691. Yates SL, Burgess LH, Kocsis-Angle J, Antal JM, Dority MD, Embury PB, Piotrkowski AM, Brunden KR (2000) Amyloid beta and amylin fibrils induce increases in proinflammatory cytokine and chemokine production by THP-1 cells and murine microglia. J Neurochem 74:1017-1025. Ye J (2000) Physiology and pharmacology of native glycine receptors in developing rat ventral tegmental area neurons. Brain Res 862:74-82. Yoshihara M, Suzuki K, Kidokoro Y (2000) Two independent pathways mediated by cAMP and protein kinase A enhance spontaneous transmitter release at Drosophila neuromuscular junctions. J Neurosci 20:8315-8322. Yu D, Silva GA (2008) Stem cell sources and therapeutic approaches for central nervous system and neural retinal disorders. Neurosurg Focus 24:E11. Yudowski GA, Puthenveedu MA, Leonoudakis D, Panicker S, Thorn KS, Beattie EC, von Zastrow M (2007) Real-time imaging of discrete exocytic events mediating surface delivery of AMPA receptors. J Neurosci 27:11112-11121. Zacchetti A, van Garderen E, Teske E, Nederbragt H, Dierendonck JH, Rutteman GR (2003) Validation of the use of proliferation markers in canine neoplastic and non-neoplastic 147 tissues: comparison of KI-67 and proliferating cell nuclear antigen (PCNA) expression versus in vivo bromodeoxyuridine labelling by immunohistochemistry. APMIS 111:430- 438. Zakharenko SS, Patterson SL, Dragatsis I, Zeitlin SO, Siegelbaum SA, Kandel ER, Morozov A (2003) Presynaptic BDNF required for a presynaptic but not postsynaptic component of LTP at hippocampal CA1-CA3 synapses. Neuron 39:975-990. Zalutsky RA, Nicoll RA (1990) Comparison of two forms of long-term potentiation in single hippocampal neurons. Science 248:1619-1624. Zamanillo D, Sprengel R, Hvalby O, Jensen V, Burnashev N, Rozov A, Kaiser KM, Koster HJ, Borchardt T, Worley P, Lubke J, Frotscher M, Kelly PH, Sommer B, Andersen P, Seeburg PH, Sakmann B (1999) Importance of AMPA receptors for hippocampal synaptic plasticity but not for spatial learning. Science 284:1805-1811. Zhang C, McNeil E, Dressler L, Siman R (2007) Long-lasting impairment in hippocampal neurogenesis associated with amyloid deposition in a knock-in mouse model of familial Alzheimer's disease. Exp Neurol 204:77-87. Zhao C, Teng EM, Summers RG, Jr., Ming GL, Gage FH (2006) Distinct morphological stages of dentate granule neuron maturation in the adult mouse hippocampus. J Neurosci 26:3- 11. Zhu JJ, Esteban JA, Hayashi Y, Malinow R (2000) Postnatal synaptic potentiation: delivery of GluR4-containing AMPA receptors by spontaneous activity. Nat Neurosci 3:1098-1106. Zhu Y, Jin K, Mao XO, Greenberg DA (2003) Vascular endothelial growth factor promotes proliferation of cortical neuron precursors by regulating E2F expression. FASEB J 17:186-193. Zigova T, Pencea V, Wiegand SJ, Luskin MB (1998) Intraventricular administration of BDNF increases the number of newly generated neurons in the adult olfactory bulb. Mol Cell Neurosci 11:234-245. Zukin RS, Bennett MV (1995) Alternatively spliced isoforms of the NMDARI receptor subunit. Trends Neurosci 18:306-313.          148 Appendix A: Publications  1. Published papers  Lee M, Cho T, Jantaratnotai N, Wang YT, McGeer, E, and McGeer PL. (2010) Depletion of GSH in glial cells induces neurotoxicity: relevance to aging and degenerative neurological diseases. FASEB J. 7:2533-45.  Yoon YS, Cho T, Yoon SH, Min CK, and Lee C. (2009) N-methyl amine-substituted fluoxetine derivatives: new dopamine transporter inhibitors. Archives of Pharmacal research 12:1663-71.  Ryu JK*, Cho T*, Wang YT, and McLarnon JG. (2009) Neural progenitor cells attenuate inflammatory reactivity and neuronal loss in an animal model of inflamed AD brain. Journal of Neuroinflammation 6:39. (*contributed equally)  Taghibiglou C, Martin HGS, Lai TW, Cho T, Prasad S, Kojic L, Lu J, Liu Y, Lo E, Zhang S, Wu JZZ, Li YP, Wen YH, Imm JH, Cynader MS, and Wang YT. (2009) Role of NMDAR-dependent activation of SREBP1 in excitotoxic and ischemic neuronal injuries Nature Medicine 12:1399- 406.  Ryu JK, Cho T*, Choi HB*, Wang YT, and McLarnon JG. (2009) Microglial VEGF receptor response is an integral chemotactic component in Alzheimer disease pathology. The Journal of Neuroscience 29(1):3-13. (*contributed equally) 2. Papers in preparation  Cho T, Ryu JK, Taghibiglou C, Ge Y, Chan AW, Lu J, Liu L, Gary E, McLarnon JG, and Wang YT. NMDAR-dependent LTP promotes proliferation/survival and neuronal differentiation of NSCs. In 149 preparation.  Ryu JK, Cho T, Wang YT, and McLarnon JG. Pharmacological inhibition of interleukin-8 receptor CXCR2 inhibits inflammatory reactivity and is neuroprotective in an animal model of Alzheimer’s disease. In preparation.  Ge Y, Lu J, Liu L, Wong TP, Wu D, Cho T, Lin S, Kast J, and Wang YT. Specific modulation of homomeric GluR1 receptors by p97 (VCP). In preparation.  Wang Y, Chavko M, Weiss T, Adeeb S, and Cho T. Blast exposure induced neurodegeneration and changes in the expression of cell surface glutamate receptors in the rat brain. In preparation.  3. Abstracts  Lee M, Cho T, Jantaratnotai N, McGeer E, and McGeer PL. (2009) Depletion of GSH of Human Microglia induces neuronal damage: A new model for oxidative stress-induced neurodegeneration in aging and neurological diseases. Society for Neuroscience 39th Meeting, Chicago, USA.  Ryu JK*, Cho T*, Wang YT, and McLarnon JG. (2009) Neural progenitor cells attenuate inflammatory reactivity and neuronal loss in an animal model of inflamed AD brain. The Canadian Association for Neuroscience 3rd Meeting, Vancouver, Canada. (*contributed equally) Cho T, Ryu JK, Taghibiglou C, Evans G, GE Y, McLarnon JG, and Wang YT. (2007) NMDA- dependent LTP promotes differentiation and proliferation of NSCs. Society for Neuroscience 37th Meeting, San Diego, USA.  150 Choi HB, Ryu JK, Cho T, McLarnon JG, Levin LR, Buck J, MacVicar BA. (2007) Raised external [K+] increases cyclic AMP production in astrocytes via bicarbonate dependent activation of soluble adenylyl cyclase. Society for Neuroscience 37th Meeting, San Diego, USA.  Ryu H, Cho T, Park K, Jin B, Min CK. (2004) Migration and differentiation of NSCs implanted into the brain of the animal model of Huntington’s disease. Society for Neuroscience 37th Meeting, San Diego, USA.


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items