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

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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  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, DAPV and inhibited by the tyrosine kinase inhibitor, K252a. ELISA and biotinylation results  ii  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.  iii  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.  iv  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  v  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 1.5  Microglia .................................................................................................................. 15  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 vi  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 Aβ 1-42 ....................................................................... 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 Aβ 1-42 -induced inflammatory reactivity .......................................... 50  2.3.3  Effect of neural progenitors on Aβ 1-42 -induced neuronal injury ................................ 51  2.4 3  Discussion .................................................................................................................... 53 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 vii  3.2.13 3.3  Image quantification and statistical analysis.......................................................... 71  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 4  Discussion .................................................................................................................... 81 Conclusion ..................................................................................................................... 101  References ........................................................................................................................... 106 Appendix A: Publications .................................................................................................... 148  viii  List of Tables  Table 1-1 Representative cell-type specific markers in the brain ......................................... 8  ix  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 BDNFTrkB system ..................................................................................................................... 99  x  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  xi  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 xii  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 xiii  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 xiv  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  xv  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.  xvi  Dedication  I would like to dedicate this thesis to my mother, father, brother, and sister.  xvii  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  1  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-Daspartate 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 2  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 3  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 DGmediated 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 wellestablished controls as there are many environmental factors that can affect neurogenesis.  4  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 Eboxes 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 5  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 6  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), 7  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)  8  Marker name  Cell type  Reference  Nestin  Neural progenitor cell; type IV Intermediate (Frederiksen  et  al.,  filament structural protein  1988)  Musashi 1  Neural progenitor cell;  (Kaneko et al., 2000)  Sox-2:  Neural progenitor and radial glial cell; type III (Graham et al., 2003)  Vimentin  Intermediate filaments, characteristic of primitive (Houle and Fedoroff, neuroectoderm formation  1983; Liem, 1993) (Eliasson et al., 1999) (Kinoshita  et  al.,  2005)  BrdU  Proliferating cell; during DNA duplication in Sphase  PCNA  Proliferating cell; during DNA repair in G1- (Kurki et al., 1986) (peak), S- and G2/M-phase  Ki67  Proliferating cell; all phases except in G0-phase, (Zacchetti  et  al.,  not detectable during DNA repair.  2003)  GFAP  Astrocyte and type II radial glial cell;  (Debus et al., 1983)  DCX  Immature neuron  (Tardy et al., 1990)  Prox1  Immature neuron  (Francis et al., 1999).  PSA-NCAM  Immature neuron; polysialic acid-neural cell (Lavado et al., 2010) adhesion molecule  (Seki and Arai, 1993; Bonfanti  and  Theodosis, 1994) Tuj1  Immature neuron; β-tubulin III  (Fanarraga  et  al.,  1999)  9  Marker name  Cell type  Reference  Hu  immature neuronal  (Marusich  et  al.,  1994) Noggin  Immature neuron; a neuron-specific gene during development  MPB  Oligodendrocyte; Myelin basic protein, sheath neuronal structure  O4  Immature oligodendrocyte  O1  Mature oligodendrocyte  (Sommer  and  Schachner, 1981 Calbindin-D28k  Neuron; Calcium-binding protein  (Sommer  GAP43  Mature neuron; structural protein in axon  Schachner, 1981)  MAP2  Mature neuron; Microtubule-associated prote2 (Kuhn et al., 1996)  and  in dendrites Neural tubulin  Mature neuron; structural protein  (Bernhardt  NeuN  Mature neuron; neuronal nuclei  Matus, 1984)  Neurofilament  Mature neuron; structural protein in axon  Tau  Neuron; type of MAP, structural protein in axon  and  (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 γaminobutyric 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., 10  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 GABA A Rs 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; 11  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, GABARmediated 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 GABA A Rmediated membrane depolarization to remove Mg2+, which blocks the pore of NMDARs at resting, indicating that activation of both GABA A R 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 12  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).  13  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).  14  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 proinflammatory 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).  15  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 16  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). NMDARdependent 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-5,10imine 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 17  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 18  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 19  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 20  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 NR2Bcontaining 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 21  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 nonNMDAR 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 22  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 23  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. 24  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-NSFGluR2 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 25  firstly been demonstrated via clathrin-mediated endocytosis followed by increased colocalization 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 26  is required for receptor internalization and their trafficking to the lysosome in a NMDARindependent 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 27  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., 28  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.  29  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 ManahanVaughan, 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 30  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 KA1containing 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 31  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 NMDARindependent 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 32  (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 RpcAMP, 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 33  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 (BruelJungerman 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 34  been reported that high K+-induced excitation through L-type Ca2+ channel (Ca v 1.2) 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.  35  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 (SH2containing 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). 36  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 (BarnabeHeider and Miller, 2003; Lim et al., 2008). The MAP kinase with ribosomal S6 kinase (RSK) and 37  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 38  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, BDNFmediated 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 39  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.  40  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.  41  1.9 Summary of research objectives  The overall thesis objective is to investigate the neuroprotective effects of transplanted NPCs in an Aβ 1-42 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 Aβ 1-42 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.  42  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 43  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 amyloidbeta peptide (Aβ 1-42 ) to induce marked inflammatory reactivity with concomitant neuronal damage in rat brain (Ryu and McLarnon, 2006, 2008).  44  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',6Diamidino-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.  45  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. Fulllength peptide (Aβ 1-42 ; 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 Aβ 1-42 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 Aβ 1-42 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 46  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 Aβ 1-42 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), antiionized 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 47  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 Aβ 1-42 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. 48  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 intrahippocampal transplantation. Transplantation of GFP-labeled NPC into the DG was carried out 3 days subsequent to intra-hippocampal injections of control PBS or Aβ 1-42 (at 2 nM). Immunohistochemical analysis was carried out at 7 days following NPC transplantation (10 days post-Aβ 1-42 /PBS injection). Representative GFP immunostaining, in the ML and GCL, indicated increased numbers of NPC in the vicinity of Aβ 1-42 (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 Aβ 1-42 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 peptideinjected hippocampus. Representative staining patterns of GFP with the different cellular markers (nestin, GFAP and MAP2) are presented in Figure 2-2. Results from doubleimmunostaining 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 Aβ 1-42 -injected hippocampus (Figure 2-2, lower panels). The lack of MAP2 association with GFP 49  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 Aβ 1-42 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; Aβ 1-42 or PBS injected rat hippocampus, Aβ 1-42 plus NPC, Aβ 1-42 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 (Aβ 1-42 or PBS) sites and NPC transplantation site, is shown for the different experimental groups (10 days post-Aβ 1-42 /PBS injection) in Figure 2-3A (upper panels). Peptideinjected brain demonstrated a considerably elevated Iba-1 immunoreactivity compared with PBS-injection. NPC transplantation in Aβ 1-42 -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 Aβ 1-42, relative to PBS, injected brain. Transplantation of NPC in peptide-injected animals significantly reduced microgliosis (by 38%) compared with Aβ 1-42 -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. 50  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. Peptideinjected brain exhibited an increased GFAP immunoreactivity relative to PBS control. Interestingly, levels of astrogliosis appeared relatively unchanged in Aβ 1-42 -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 Aβ 1-42 and Aβ 1-42 + 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 peptideinjected 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 Aβ 1-42 , compared with PBS, injected hippocampus Figure 2-3B, lower bar graph). Transplantation of NPC into peptide-injected brain significantly reduced levels of the proinflammatory cytokine, by 40%, compared with Aβ 1-42 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, 51  localized to areas between injection and transplantation sites. Representative high magnification patterns of immunostaining for neurons (MAP2 marker) are presented for PBS and Aβ 1-42 injected hippocampus (Figure 2-4A). The results indicate a considerable loss of MAP2-positive neurons with Aβ 1-42 , 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 Aβ 1-42 , 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 Aβ 1-42 alone and Aβ 1-42 plus dead NPC or between PBS-injected and NPCtransplanted animals.  52  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 Aβ 1-42 -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 (BlurtonJones 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 Aβ 1-42 . At 7 days following NPC transplantation (10 days after Aβ 1-42 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 Aβ 1-42 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 53  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 Aβ 1-42 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 Aβ 1-42 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 Aβ 1-42 was associated with considerable increases in microgliosis and astrogliosis compared with PBS control. Transplantation of NPC after Aβ 1-42 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 Aβ 1-42 -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 Aβ 1-42 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 54  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 55  durations of NPC transplantation in AD animal models.  56  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 Aβ 1-42 -injected rat brain.. The site of PBS or Aβ 1-42 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 (Aβ 1-42 or PBS) and site of NPC transplantation (n=4 animals/group, * denotes p < 0.05).  57  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.  58  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/Aβ 1-42 and 7 days transplantation of NPC/dead NPC. The animal groups (panels, left to right) are for PBS, Aβ 1-42 , Aβ 1-42 + NPC, Aβ 142  + 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 Aβ 1-42 ).  59  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, Aβ 1-42 , Aβ 1-42 + 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. Aβ 1-42 ).  60  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-Daspartate 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 longterm 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 61  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.  62  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 63  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, GFPlabeled 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 intrahippocampal 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 nanoinjector (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  64  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. 65  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 26gauge 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 chemicalinduced 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 CaCl 2, 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 66  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 GFPcontaining 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 cLTPinduced 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, 96well plates (Nunc-Immuno Maxisorp) were pre-coated with anti-monoclonal BDNF, antipolyclonal 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 antimonoclonal 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% Tween67  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) (IgG 1 , 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) (IgG 1 , 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), γaminobutyric acid A receptor-alpha 1 (GABA A R-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 (βLRP1) (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 68  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.  69  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, postfixed 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. Freefloating 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 70  (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% BSAcontaining 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, GFPlabeled 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 71  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.  72  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 NMDAR73  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 GFPlabeled 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 GFP74  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 32E). 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 NMDARdependent 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 75  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 76  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 AlvarezBuylla, 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 selfrenewal 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 γaminobutyric 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-4isoxazolepropionic acid) subtype glutamate receptor subunits GluR1 or GluR2, or GABA A receptor α1 subunits. These results further support the idea that NSCs isolated from 77  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 glycinebased 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 35A). In order to facilitate the identification of NSCs from fully differentiated neurons in the cocultures, we pre-labeled these NSCs using lentivirus vectors containing GFP 3 days before coculturing 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 βlow density lipoprotein receptor-related protein1 (β-LRP1) (Figure 3-5B). Moreover, a glycineinduced 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 glycine78  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 79  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.  80  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 BDNFTrkB 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 81  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 82  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, 83  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 coculture 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 cLTPstimulated 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 84  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 85  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.  86  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.  87  88  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 doublestained 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 doublelabeled 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 doublelabeled 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 doublelabeled 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.  89  90  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.  91  92  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 typespecific antibodies against nestin, vimentin, MAP2 and GFAP, and C, various ionotropic receptor antibodies against , NR1, GluR1, GluR2 and GABA A R-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.  93  94  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.  95  96  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 cLTPinduced 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 DAPIpositive (blue) cells. *p<0.05, n=6.  97  98  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.  99  100  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  101  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 Aβ 1-42 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 102  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 103  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 104  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.  105  References  Aarum J, Sandberg K, Haeberlein SL, Persson MA (2003) Migration and differentiation of neural precursor cells can be directed by microglia. 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(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 12:1399406.  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 148  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) NMDAdependent LTP promotes differentiation and proliferation of NSCs. Society for Neuroscience 37th Meeting, San Diego, USA.  149  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.  150  

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