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Modulation of ligand-gated receptors in the central nervous system Wu, Dongchuan 2010

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MODULATION OF LIGAND-GATED RECEPTORS IN THE CENTRAL NERVOUS SYSTEM by  Dongchuan Wu  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)  July, 2010  © Dongchuan Wu, 2010  i  ABSTRACT Basal level neuronal excitability in the mammalian brain is fundamental for physiological brain functions. It is primarily maintained by a fine balance between two types of synaptic transmission: the excitatory transmission mediated by glutamate-gated ion channels including α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate  receptors  (AMPARs)  and  N-methyl-D-aspartic acid receptors (NMDARs), and the inhibitory transmission mediated by chloride channels including γ-Aminobutyric acid receptors (GABAARs) found in the brain and glycine receptors (GlyRs) orginating in the spinal cord and brainstem. Therefore, understanding mechanisms by which these ligand-gated ion channels (LIGCs) are regulated is critical for our understanding of both brain functions and dysfunctions, and is the major focus of this thesis.  In  particular, this research project investigates: 1) how rapid alteration of AMPAR trafficking results in changes in strength of synaptic transmission, with a particular emphasis on its contribution to amygdala long-term potentiation (LTP) and depression (LTD), the two most well-characterized forms of synaptic plasticity, and 2) how excitatory transmitter glutamate modulates functions of the inhibitory GABAA and Gly receptors.  In chaper 2 and 3, we show in lateral amygdale (LA) slices that the induction of LTP requires NR2A-containing NMDAR activation, while the expression of LTP requires AMPARs insertion (sensitive to TeTx or GluR1-derived peptide). On the contrary, the induction of LTD involves activation of NR2B-containing NMDARs and the expression of LTD involves AMPARs endocytosis (sensitive to GluR2-3Y peptide).  ii  The inhibitory receptors GABAARs and GlyRs are respectively activated by binding with their respective transmitters, GABA and glycine. In chapter 4 and 5, we show novel and unexpected findings where glutamate potentiates currents mediated by either GABA ARs or GlyRs in neurons and in HEK cells over-expressing recombinant GABAARs and GlyRs. This potentiation was not dependent on activation of any known ionotropic or metabotropic glutamate receptors. Thus, our results strongly suggest that glutamate can allosterically potentiate the function of GABAARs and GlyRs, thereby blurring the traditional distinction between excitatory and inhibitory transmitters. Such a rapid homeostatic regulatory mechanism may have a significant role in tuning functional balance between synaptic excitation and inhibition in the central nervous system (CNS).  iii  TABLE OF CONTENTS  ABSTRACT ........................................................................................................................................................ ii TABLE OF CONTENTS ...................................................................................................................................iv LIST OF FIGURES ..........................................................................................................................................vii ACKNOWLEDGEMENTS............................................................................................................................ viii CO-AUTHORSHIP STATEMENT ..................................................................................................................ix 1. INTRODUCTION........................................................................................................................................... 1 1.1. General Introduction .................................................................................................................................. 1 1.2. NMDA Receptors....................................................................................................................................... 3 1.2.1. Molecular diversity, composition and structure of NMDA receptors .................................................. 3 1.2.2. NMDARs distribution .......................................................................................................................... 5 1.2.3. NMDAR channel properties ................................................................................................................ 7 1.2.4. NMDAR pharmacology ....................................................................................................................... 9 1.2.5. Modulation of NMDARs.................................................................................................................... 10 1.2.6. NMDARs and synaptic plasticity in the hippocampus ...................................................................... 13 1.2.7. NMDARs and synaptic plasticity in lateral amygdala ...................................................................... 19 1.3. AMPA Receptors ...................................................................................................................................... 22 1.3.1. Molecular diversity, composition and structure of AMPA receptors ................................................. 22 1.3.2. AMPARs distribution ......................................................................................................................... 24 1.3.3. AMPARs channel property ................................................................................................................ 26 1.3.4. Modulation of AMPARs ..................................................................................................................... 27 1.3.4.1. Phosphorylation of AMPARs ...................................................................................................... 27 1.3.4.2. Pamitoylation of AMPARs .......................................................................................................... 30 1.3.5. AMPARs and synaptic plasticity in hippocumpus ............................................................................. 30 1.3.6. AMPARs in synaptic plasticity in amygdala ...................................................................................... 35 1.4. GABAA Receptors ................................................................................................................................... 36 1.4.1. GABAA receptor molecular diversity and composition and structure ............................................... 36 1.4.2. GABAARs distribution ....................................................................................................................... 39 1.4.3. GABARs channel properties.............................................................................................................. 42 1.4.4. GABAARs channel modulation .......................................................................................................... 44 1.4.4.1. Allosteric modulation ................................................................................................................. 45 1.4.4.2. Phosphorylation of GABAARs .................................................................................................... 47 1.4.5. GABAARs in learning memory and disease ....................................................................................... 50 1.5. Glycine Receptors .................................................................................................................................... 54 1.5.1. Molecular diversity, composition, and structure of glycine receptors .............................................. 54 1.5.2. Glycine receptor distribution ............................................................................................................ 56 1.5.3. Glycine receptor channel properties ................................................................................................. 58 1.5.4. Glycine receptor modulation ............................................................................................................. 59 1.5.4.1. Allosteric modulation ................................................................................................................. 59 1.5.4.2. Phosphorylation of glycine receptors......................................................................................... 61 1.5.5. Glycine receptors physiology and pathology .................................................................................... 62 iv  1.6. Rationale, Hypothesis and Objectives...................................................................................................... 64 1.7. References ................................................................................................................................................ 71 2. THE ROLE OF NMDA RECEPTOR SUBTYPES IN GOVERNING THE DIRECTION OF LATERAL AMYGDALA SYNAPTIC PLASTICITY ................................................................................. 119 2.1. Introduction ............................................................................................................................................ 119 2.2. Materials and Methods ........................................................................................................................... 121 2.3. Results .................................................................................................................................................... 123 2.3.1. Differential roles of NR2A- and NR2B-containing NMDA receptors in the LTP and LTD at the thalamic input synapses in the LA............................................................................................................. 123 2.3.2. The selectivity of NVP on NR2A and NR2B-containing NMDAR at the thalamic input synapses in the LA ........................................................................................................................................................ 124 2.4. Discussion .............................................................................................................................................. 125 2.5. References .............................................................................................................................................. 133 3. ROLE OF AMPA RECEPTOR TRAFFICKING IN NMDA RECEPTOR-DEPENDENT SYNAPTIC PLASTICITY IN THE RAT LATERAL AMYGDALA .............................................................................. 138 3.1. Introduction ............................................................................................................................................ 138 3.2. Materials and Methods ........................................................................................................................... 141 3.2.1. Slice electrophysiology .................................................................................................................... 141 3.2.2. Surface biotinylation assay ............................................................................................................. 143 3.2.3. Statistical analysis ........................................................................................................................... 145 3.3. Results .................................................................................................................................................... 145 3.3.1. LTP induced by either HFS or a pairing protocol at thalamic input synapses in the LA is NMDAR-dependent ................................................................................................................................... 145 3.3.2. Expression of LTP in the LA requires postsynaptic vesicle-mediated exocytosis and is associated with increased cell surface expression of AMPARs ................................................................................... 147 3.3.3. LTD induced by LFS and Pairing protocols is NMDAR-dependent at thalamic input synapses in the LA .............................................................................................................................................................. 149 3.3.4. Regulated AMPAR endocytosis is required for expression of LTD in the LA .................................. 150 3.4. Discussion .............................................................................................................................................. 151 3.5. References .............................................................................................................................................. 166 4. ALLOSTERIC POTENTIATION OF GABAAR ECEPTOR CHLORIDE CHANNELS BY GLUTAMATE ................................................................................................................................................. 174 4.1. Introduction ............................................................................................................................................ 174 4.2. Methods.................................................................................................................................................. 175 4.2.1. Neuronal culture.............................................................................................................................. 175 4.2.2. HEK293 cell culture and transfection ............................................................................................. 175 4.2.3. Electrophysiology ............................................................................................................................ 176 4.2.4. Data analysis................................................................................................................................... 177 4.2.5. Chemicals ........................................................................................................................................ 177 4.3. Results .................................................................................................................................................... 178 4.4. Discussion .............................................................................................................................................. 183 4.5. References .............................................................................................................................................. 195 5. ALLOSTERIC POTENTIATION OF GLYCINE RECEPTOR CHLORIDE CHANNELS BY v  GLUTAMATE ................................................................................................................................................. 197 5.1. Introduction ............................................................................................................................................ 197 5.2. Methods and Materials ........................................................................................................................... 198 5.2.1. Neuronal culture.............................................................................................................................. 198 5.2.2. HEK293 cell culture and transfection ............................................................................................. 198 5.2.3. Electrophysiology ............................................................................................................................ 199 5.2.4. Data analysis................................................................................................................................... 201 5.2.5. Chemicals ........................................................................................................................................ 201 5.3. Results .................................................................................................................................................... 201 5.4. Discussion .............................................................................................................................................. 210 5.5. References .............................................................................................................................................. 224 6. DISCUSSION AND FUTURE DIRECTIONS ......................................................................................... 227 6.1. NMDAR Subunits Have Differential Roles in the Induction of LA-LTP and LTD ............................... 227 6.2. AMPAR Trafficking is Critical for the Expression of LTP and LTD in the LA ..................................... 229 6.3. Cross-talk between Excitatory and Inhibitory Synaptic Transmission via Glutamate-Mediated Allasteric Modulation of GABAARs and GlyRs ........................................................................................................... 231 6.4. Future Directions.................................................................................................................................... 233 6.5. References .............................................................................................................................................. 236 APPENDICES ................................................................................................................................................. 238 Appendix A: Publications ............................................................................................................................. 238 Appendix B: UBC Research Ethics Board Certificate of Approval .............................................................. 240  vi  LIST OF FIGURES Figure 1.1. Molecular structure of ionotropic glutamate receptor subunits. ................................................ 69 Figure 1.2. Molecular structure of Cys-loop pentameric ligand-gated ion channel subunits. ...................... 70 Figure 2.1. The role of NR2A and NR2B subunits in LTP and LTD in LA. .............................................. 129 Figure 2.2. NVP-AAM077 (0.4mM) preferentially inhibits NR2A-containing NMDA EPSCs, and partially inhibits NR2B-containing NMDA EPSCs in LA neurons. ........................................................................ 131 Figure 3.1. The induction of LTP at the thalami-amygdale synapses is NMDAR-dependent. A. .............. 158 Figure 3.2. HFS-induced LTP at the thalami-amygdale synapses requires postsynaptic AMPAR insertion.160 Figure 3.3. The induction of LTD at the thalamic pathway in the LA is NMDAR-dependent. ................. 162 Figure 3.4. LTD induced at the thalamic pathway in the LA is mediated by GluR2-subunit dependent endocytosis of postsynaptic AMPARs. ...................................................................................................... 164 Figure 4.1. Glutamate potentiates GABA currents in a dose dependent manner in HEK293 cells expressing rat recombinant GABAARs. ........................................................................................................................... 185 Figure 4.2. Glutamate analogs potentiate GABA current in HEK293 cells expression rat recombinant GABAARs. ................................................................................................................................................ 187 Figure 4.3. CNQX abolished potentiation of glutamate and APV on GABA currents in HEK293 cells expression rat recombinant GABAARs. .................................................................................................... 189 Figure 4.4. APV potentiates GABA current in dose dependent manner in cultured hippocampal neurons.191 Figure 4.5. APV potentiates GABAAR mediated phasic and tonic inhibition in cultured hippocampal neurons. ................................................................................................................................................................... 193 Figure 5.1. APV and NMDA potentiate postsynaptic GlyR-mediated currents via a NMDAR-independent mechanism. ................................................................................................................................................ 214 Figure 5.2. Potentiation of GlyR-mediated currents by glutamate and AMPA in cultured spinal neurons. 217 Figure 5.3. APV increases the open probability of GlyR-mediated single-channel activities in cultured spinal neurons. ...................................................................................................................................................... 219 Figure 5.4. Glutamate-like ligands potentiate glycine currents in HEK293 cells expressing human recombinant GlyRs. ........................................................................................................................................................ 221  vii  ACKNOWLEDGEMENTS First and foremost I offer my sincerest gratitude to my supervisor Dr. Yu Tian Wang, who showed me what the real meaning of the science and has supported me thoughout my thesis with his patience, knowledge and passions. Many thanks to Dr. Shuyan Yu, Dr. Lidong Liu, Dr. Jun Liu for collaborating on some of the studies described in this thesis. I would also like to thank Ms Yuping Li and Dr. Jie Lu for their technical assistance, Ms. Girling, Mr. Axerio and Mr. Ko for critical reading of the thesis draft.  I would like to thank my supervisory committee members Dr. Stan Floresco and Dr. Brian Macvicar for their continuous support and helpful suggestion.  Finally, I would like to thank my mom, my parents in law, my wife Ning and my daughter Ruoxi. This work could never be done without their love and support.  viii  CO-AUTHORSHIP STATEMENT I was involved in the original conception and planning of all of the experiments involved in this thesis. I also participated in all of the experimental procedures, figure preparations and manuscript writings. For chapter 2, Dr. S. Y. Yu assisted with electrophysiology recordings for figure 2.1. For chapter 3, Dr. S. Y. Yu conducted receptor surface biotinylation and assisted with electrophysiology studies in LTD for figure 3.3 and 3.4, and Dr. L. Liu assisted with electrophysiology for figure 3.2. For chapter 4, Dr. J. Liu conducted experiments in cultured neurons, and prepared figure 4.4, 4.5, and 4.6. For chapter 5, Dr. J. Liu conducted electrophysiology experiments in cultured neurons, and prepared figure 5.1, 5.2, and 5.3. The manuscripts for Chapter 2 and Chapter 4 were mainly prepared by me and Dr. Y. T. Wang; the manuscript for Chapter 3 was writtern by Dr. S. Y. Yu and myself, with assistant from Dr. Y. T. Wang and Dr. L. Liu; the manuscript for Chapter 4 was written by Dr. J. Liu and myself, with assistant from Dr. Y. T. Wang.  ix  1. INTRODUCTION 1.1. General Introduction In the mammalian brain, a stable basal level neuronal excitability is critical for normal brain function, and is achieved by a precise balance between excitatory synaptic and inhibitory synaptic transmission. Modulation of efficacy of excitatory and inhibitory synaptic transmission is the basic cellular mechanism in learning and memory. Presynaptic and postsynaptic sites are two key sites for synaptic transmission modulation. This thesis will focus on how synaptic efficacy can be modulated at the postsynaptic level by affecting the function and/or trafficking of excitatory α-amino-3-hydroxyl-5-methyl-4-isoxazole- propionate receptors (AMPARs) and N-methyl-D-aspartic acid receptors (NMDARs) as well as inhibitory γ-Aminobutyric acid receptors (GABAARs) and glycine receptors (GlyRs) in the CNS.  AMPARs mediate fast synaptic transmission at the majority of CNS excitatory synapses, and the changes to the strength of AMPA receptor–mediated synaptic transmission, such as LTP and LTD, are considered a cellular mechanism of learning and memory. The hippocampus and the amygdala are two important regions the brain that are involved in learning and memory. The hippocampus is responsible for spatial memory while the lateral amygdala is involved in fear memory. It is well characterized that AMPARs trafficking is one of the major underlying mechanisms of LTP and LTD in hippocampus. However, it is still unclear whether AMPARs trafficking also mediates expression of the synaptic plasticity in the  1  lateral amygdala .  NMDARs can mediate induction of either LTP or LTD in the hippocampus and amygdala. Recent studies on the hippocampus showed that NR2A-containing NMDARs are required for LTP induction, whereas NR2B-containing NMDARs are required for LTD induction (Liu et al., 2004; Massey et al., 2004). However, the roles of NR2A and NR2B subunits in LTP and LTD in amygdala remain unclear.  In addition to excitatory synaptic modulation, regulation in inhibitory transmission which is mediated mainly by GABAARs and GlyRs is also very important for brain function, especially for maintaining the balance between synaptic excitation and inhibition.  There are three levels of regulation (cross-talks) between excitatory and inhibitory transmission. Those levels of regulation are the network level, the cellular level and the receptor level. Regulation at the network and cellular levels are common and well studied. The receptor level regulation, on the other hand, is rare. The only example for this regulation is that the inhibitory transmitter glycine potentiates NMDAR mediated current via allosteric modulation.  One previous study has reported that glutamate can potentiate GABA AR mediated current via Ca2+ independent pathway, suggesting a potentially new receptor level interaction  2  between excitatory and inhibitory transmission(Stelzer and Wong, 1989). However, whether glutamate can allosterically modulate the function of GABAARs is still not clear. Furthermore, whether such glutamate regulations can also exist on GlyRs remains unknown as well.  In this chapter, I will introduce background information on NMDARs, AMPARs, GABAARs and GlyRs with a particular emphasis on their structures, compositions, distributions, channel properties, modulations, physiological functions, and their roles in diseases.  1.2. NMDA Receptors Unique functional properties and the molecular diversity of the NMDARs determine their role in initiation and regulation of synaptic plasticity (Bliss and Collingridge, 1993). NMDARs are also involved in various pathological processes, such as ischemia, Alzeimer’s disease and other neuro-degenerative diseases (Lau and Zukin, 2007; Kalia et al., 2008).  1.2.1. Molecular diversity, composition and structure of NMDA receptors NMDARs have seven subunits, including NR1, NR2A-D, and NR3A-B. NR1 has eight splicing forms that are generated from a single gene (Dingledine et al., 1999). Of these eight splicing forms, one is in the N-terminus (N1 cassette) and three are in the C-terminus (C1, C2, C2’ cassette based on alternatively spliced exon). Recombinant NR1 (N1, lacking of exon 5)  3  with NR2 is more sensitive to the blockade by Zn2+ and proton, and also to the potentiation by polyamines (Durand et al., 1993; Traynelis et al., 1995). Recombinant NMDARs containing only N1 are more strongly enhanced by PKC agonist phorbol esters than those with C1 or C2 (Durand et al., 1993). In addition to modulating NMDAR channel properties, NR1 splicing variants also affect cell surface expression of NMDARs in HEK cells. Full length of NR1 C-terminus (containing C1 and C2 cassette) but not the short C-terminus (like containing just C1) produces little NR1/NR2 complex surface expression (Smothers and Woodward, 2009). During development, the C1 cassette form presents about half of the total NR1 subunit proteins without developmental changes in the cortex. However, the expression of C2 cassette form decreases and C2’ cassette increases over age (Prybylowski and Wolfe, 2000).  The functional NMDARs are composed of at least two NR1 and two other NR2 and/or NR3 subunits to form diheterotetramers or triheterotetramers (Dingledine et al., 1999; Kopp et al., 2007; Paoletti and Neyton, 2007). NR3 can assemble with NR1 alone in HEK cells, or with both NR1 and NR2 in neurons (Sasaki et al., 2002; Ulbrich and Isacoff, 2008; Cavara et al., 2009). Each NMDAR subunit has a specific expression pattern in the CNS, and different composition of hetero NMDARs show different channel properties.  The NMDAR is a transmembrane protein containing one extracellular N-terminous (including S1 which is adjacent to TM1), one intracellular C-terminus, three transmembrane domains (TM1, TM3, TM4), a cytoplasm-facing re-entrant membrane loop, and an  4  extracellular loop between TM3 and TM4 (S2) (Fig 1.1). S1 and S2 are considered as the agonist binding area. The C-terminus is also the critical site for the interaction with other binding proteins.  1.2.2. NMDARs distribution Binding assay studies with different radioligands 3H-L-Glutamate, 3H-TCP, 3HMK801, or 3HCPP have shown that NMDARs are expressed predominantly in the forebrain but can also be found in other brain areas (Monaghan et al., 1983; Maragos et al., 1988; Monaghan et al., 1989; Subramaniam and McGonigle, 1991; Ozawa et al., 1998). In the whole brain, the highest expression level for NMDARs was found in the CA1 area of the hippocampus.  Studies with in situ hybridization histochemistry have shown different distribution of NMDARs subunits (Moriyoshi et al., 1991; Kutsuwada et al., 1992; Monyer et al., 1992; Watanabe et al., 1992; Monyer et al., 1994; Mori and Mishina, 1995; Ozawa et al., 1998). The NR1 mRNA can be detected throughout the brain. However, mRNA of the four NR2 subunits exhibit distinct distribution patterns. The NR2A mRNA can be detected widely in the whole brain with highest expression in the cerebral cortex, hippocampus and cerebellum. The NR2B mRNA is mainly distributed in the forebrain, and with high level expression in the olfactory bulb, septum, caudate-putamen, thalamus, hippocampus and cerebral cortex. The NR2C mRNA is distributed predominantly in the granule cell layer of cerebellum, and exhibits weaker expression in the olfactory bulb and thalamus. NR2D mRNA can be detected in the  5  thalamus, brain stem and olfactory bulb. Moreover, the NR2C and NR2D mRNA can also be detected in interneurons within the hippocampus (Mori and Mishina, 1995). NR3A mRNAs are widely expressed in the brain (Ciabarra et al., 1995; Sucher et al., 1995; Sun et al., 1998), whereas NR3B mRNAs are distributed in the ventral horn of spinal cord, the facial and trigeminal nuclei of the brain stem (Chatterton et al., 2002).  In the rodent brain, expression patterns of the NR2 subunits are regulated during development (Watanabe et al., 1992, 1993a, b; Akazawa et al., 1994; Monyer et al., 1994; Watanabe et al., 1994a, b). NR2B and NR2D mRNAs are first expressed prenatally, whereas NR2A and NR2C mRNA are first observed around birth. NR2B mRNAs are distributed widely, whereas NR2D mRNAs are selectively expressed in the middle brain structure. During the first two weeks after birth, the NR2A mRNAs express ubiquitously in the brain and NR2C mRNAs start to express in the cerebellum. Interestingly, NR2B mRNAs almost disappear and are replaced by NR2C mRNAs in the cerebellar granule cells on embryonic day 7. NR3A mRNAs showed the highest level of expression within the late prenatal and early postnatal periods (Sun et al., 1998).  Immunohistochemical studies have shown that the distribution pattern of NR1 in rat brain is consistent with its mRNA distribution by using specific antibodies to NR1 C-terminus (Petralia et al., 1994a). Using selective antibodies to NR2A/NR2B C-terminus, Petralia et al. found that distribution patterns of NR2A/2B are similar to NR1 except in the cerebellum  6  (Petralia et al., 1994b). The ultrastructural examination in cerebral cortex, hippocampus and cerebellar cortex have shown that NR1 and NR2A/2B are located in postsynaptic densities and associated dendrites (Petralia et al., 1994b; Petralia et al., 1994a). NR3B protein is most prominent in motor neurons in the spinal cord and brainstem (Chatterton et al., 2002).  1.2.3. NMDAR channel properties Voltage-dependent Mg2+ blockade and Ca2+ permeability are the two characteristic features of NMDARs (Mayer et al., 1984; Mayer and Westbrook, 1987; Ozawa et al., 1998; Dingledine et al., 1999). NMDARs are permeable to Na+ and K+ with a low selectivity, and are highly permeable to Ca2+. At physiological concentration, Mg2+ blocks NMDAR at resting membrance potential (around -70 mV) and this blockade can be gradually removed by depolarization of cell membrance. NR1/NR2A and NR1/NR2B composition of NMDARs is more sensitive to Mg2+ blockade, compared with NR1/NR2C and NR1/NR2D. In the presence < 1mM of Mg2+, the largest NMDA response can be achieved at -25mV NR1/NR2A and NR1/NR2B whereas the maximam NMDA response can be acheieved around -45mV in NR1/NR2C and NR1/NR2D (Burnashev et al., 1992; Kutsuwada et al., 1992). Mg2+ blockade and Ca2+ permeability are both dependent upon an asparagine (N) site located in the re-entrant pore loop. This site corresponds to the Q/R (glutamine-arginine) site in the M2 segment of AMPARs, which is responsible for the Ca2+ permeability of AMPARs. Replacing asparagine with glutamine at this site of NR1 reduces both Ca2+ permeability and Mg2+ blockade. The replacement of asparagine with arginine, a more positively charged amino acid, almost  7  completely abolishes Ca2+ permeability and Mg2+ blockade (Burnashev et al., 1992; Sakurada et al., 1993). The homologus site of NR2 (NR2A/NR2B) also plays a crucial role in the Mg2+ blockade. Replacing the asparagine with glutamine at this site of NR2 leads to a reduction of Mg2+ blockade and causes permeability to Mg2+ (Burnashev et al., 1992; Monyer et al., 1992).  Desensitization, activation and deactivation kinetics are much slower in NMDARs than in AMPARs (Ozawa et al., 1998). The time constant of desensitization of NR1/NR2A is much faster than that of NR1/NR2B (Vicini et al., 1998). The deactivation time constants also differ remarkably among different NR2 subunit compositions. With Mg2+ free solution, the deactivation time constants in response to 1 ms pulse of 1 mM glutamate from fastest to the slowest are as follows: NR2A < NR2B = NR2C << NR2D (Vicini et al., 1998).  The single channel conductance of native and recombinant NMDARs also depends on subunit composition. NR2A- or NR2B-containing receptors display a high conductance of ~50 pS. NR2C- or NR2D-containing receptors show a lower conductance of ~35 pS and ~18 pS respectively (Farrant et al., 1994; Momiyama et al., 1996; Wyllie et al., 1998)  Combination of NR3A in NR1/NR2 receptors reduces channel conductance, Ca2+ permeability and surface expression (Das et al., 1998; Perez-Otano et al., 2001). In NR3A knockout mice, NMDA current is enhanced and spine density is increased in early postnatal cerebrocortical neurons. More interestingly, NR1/NR3A or NR1/NR3B receptors can only be  8  activated by glycine, but not glutamate nor NMDA. Furthermore, they do not share similiar characteristics of NMDARs like Ca2+ permeability, Mg2+ blockade and influence by NMDAR agonist or antagonist. This makes NR1/NR3A and NR1/NR3B more like glycine-activated cation channel receptors (Chatterton et al., 2002).  1.2.4. NMDAR pharmacology The first finding that glycine enhances NMDARs-mediated current was discovered by Johnson and Ascher. Then Kleckner and Dingledine demonstrated that glycine is a co-agonist of NMDARs (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988). Now we know that most of native NMDARs (NR1/NR2 composition) has a glycine binding site on NR1 subunits and a glutamate binding site on NR2 subunits (Cull-Candy and Leszkiewicz, 2004; Furukawa et al., 2005; Paoletti and Neyton, 2007). NR3 subunits are more similar to NR1 subunits which only have a glycine binding site (Yao and Mayer, 2006).  Antagonists of NMDARs can be divided into several groups based on their action sites involved in channel function (Ozawa et al., 1998; Paoletti and Neyton, 2007). The first group are competitive inhibitors acting on the glutamate binding site of NR2 subunits which are AP-5, CPP, NVP-AAM077, PPDA and PMPA. As for the second set of antagonists, these act on the glycine binding site of NR1. The well known inhibitors for this group are 7-CKA, 5,7-DCKA and CGP 61594. The third type of inhibitors are called open-channel blockers, which act on the channel pore and block other ions from migrating through the channel pore.  9  The corresponding antagonists for this category are MK-801, PCP, ketamine, memantine, argiotoxine-636 and spermine. Finally, the fourth class of antagonists act on unique sites on the NR2 N-terminus, and these are ifenprodil and Ro 25-6981 (Paoletti and Neyton, 2007).  Antagonists that can be used to discriminate between receptor subtypes still remain limited. Ifenprodil and its derivatives Ro 25-6981 and CP 101,606 display high selectivity to NR2B-containing receptors (Williams, 1993). Ifenprodil binds to some N-terminal leucine/isoleucine/valine-binding (LIVBP) protein like domain of NR2B. In this domain, several residues (both hydrophilic and hydrophobic) were found to govern ifenprodil inhibition (Perin-Dureau et al., 2002). Ro 25-6981, which also acts on N-terminal LIVBP-like domain of NR2B, has less off target effect and higher affinity to NR2B than ifenprodil (Fischer et al., 1997; Malherbe et al., 2003; Keiser et al., 2009). The newly developed antagonist NVP-AAM077 is considered an NR2A selective antagonist. However, its selectivity for NR2A over NR2B, which was previously reported to be >100-fold (Auberson et al., 2002; Liu et al., 2004), had been reported merely about 10-fold (Berberich et al., 2005; Frizelle et al., 2006; Neyton and Paoletti, 2006).  1.2.5. Modulation of NMDARs Modulation of NMDARs also leads to two consequences, including changes of channel properties and changes of surface expression of receptors. These two consequences can be achieved extracellularly or intracellularly, by allosteric modulation, phosphorylation or other  10  modulations.  Phosphorylation of NMDARs can be controlled by protein kinases and  protein phosphotases. Studies have shown that several specific sites can be phosphorylated by PKA, PKC, CaMKII, or tyrosine kinases, but until present, there is no direct evidence to show whether phosphorylation of these sites is responsible for any changes in channel function.  Threonine879, serine890 and serine896 on the C1 cassette of NR1 C-terminus can be phosphorylated by PKC (Tingley et al., 1993; Ehlers et al., 1995; Tingley et al., 1997). Serine890 phosphorylation disperses surface associated NR1 clusters in fibroblasts. Interestingly, serine897 neighboring to PKC site (serine896) can be phosphorylated by PKA, but phosphorylation of these two sites has no effect on the subcellular distribution of NMDARs (Tingley et al., 1997). Interestingly, recombinant homomeric NR1 lacking the C1 cassette can still be potentiated by the PKC activator PMA (Durand et al., 1993; Sigel et al., 1994), suggesting two possibilities: (1) PKC phosphorylates other unknown sites of NMDARs; and (2) PKC phosphorylates other proteins that interact with NMDARs to regulate the channel property indirectly. In addition, presence of N-terminus cassette is more sensitive to the potentiation effect of PMA, raising another possibility that extracellular phosphorylation is also important for channel regulation (Durand et al., 1993). NR1/NR2A or NR1/NR2B, but not NR1/NR2C or NR1/NR2D recombinant can be potentiated by PKC activator, indicating potential phosphorylation sites exist in the NR2A/2B C-terminus (Mori et al., 1993). Later studies have shown that PKC phosphorylates Ser1303, Ser1323 on the C-terminus of NR2B (Liao et al., 2001). Mutation of these two sites can dramatically reduce the potentiation effect of  11  PKC on recombinant NR1/NR2B (Liao et al., 2001). In addition to the phosphorylation by PKC, Ser1303 can also be phosphorylated by CaMKII (Omkumar et al., 1996), indicating that this phosphorylation site may be involved in synaptic plasticity. Even though PKC cannot potentiate NR1/NR2C (Mori et al., 1993), NR2C can still be phosphorylated at Serine1244 near the extreme C-terminus by either PKC or PKA. Phosphorylation of this site can accelerate both the activation and decay rate without affecting surface expression (Chen et al., 2006).  Activation of muscarinic acetylcholine receptors (Markram and Segal, 1990, 1992; Marino et al., 1998),  opioid receptors (Chen and Huang, 1991), phosphoinositol-coupled metabotropic glutamate receptors (Aniksztejn et al., 1992) and the protease-activated receptor PAR1(Gingrich et al., 2000) all potentiate neuronal or recombinant NMDARs, probably via PKC-mediated pathways. Activation of  adrenergic receptors potentiates NMDAR activities by inhibiting calcineurin (Raman et al., 1996). NMDARs activities can be inhibited by the serine and threonine phosphatases 1, 2A or 2B (calcineurin) in acutely isolated dentate gyrus granule cells or cultured hippocampal neurons (Wang et al., 1994a; Omkumar et al., 1996).  Wang and Salter first demonstrated that neuronal NMDAR activities can be controlled by a balance between tyrosine phosphorylation and dephosphorylation (Wang and Salter, 1994). Inhibiting protein tyrosine kinases (PTKs) or introducing protein tyrosine phosphatases (PTPs) reduces NMDARs currents (Wang et al., 1996). Introducing exogenous Src (one Src family of PTKs) or inhibiting PTPs increases NMDARs currents (Wang and Salter, 1994).  12  Furthermore, other studies have shown Src or Fyn (one of Src family of PTKs) potentiates recombinant NR1/NR2A mediated currents in HEK cells or Xenopus oocytes, but not recombinant NR1 combined with other NR2 subunits (Chen and Leonard, 1996; Kohr and Seeburg, 1996). Another study has shown that the potentiation effect of Src is due to its release of tonic inhibition of zinc on NR1/NR2A receptors, and that three tyrosines at C-terminus of NR2A (Y1105, Y1267, Y1387) are required for this effect on Src (Zheng et al., 1998). A later study has shown that Y1252, Y1336 and Y1472 on the C-terminus of NR2B can be phosphorylated by Fyn, and that Y1472 is the main phosphorylation site on recombinant NR2B (Nakazawa et al., 2001). In the native system, however, the potentiation effect of Src on NMDARs is poorly understood. Src potentiates native NMDAR currents in neurons but not via the removal of the zinc inhibition (Xiong et al., 1999), a mechanism of which remains to be determined.  PTPs can regulate NMDARs function by two ways. PTP potentiates NMDAR currents via activation of Src in neurons (Lei et al., 2002). The STEP (striatal enriched tyrosine phosphatase) family, which is a brain specific and non-receptor type of PTPs, inhibits NMDAR activities by competing with the Src family (Pelkey et al., 2002).  1.2.6. NMDARs and synaptic plasticity in the hippocampus Synaptic plasticity can be defined as “the ability to change the strength of synaptic transmission” and is believed to be involved in learning and memory processes. Different forms of synaptic plasticity including LTP (increased synaptic strength) and LTD (decreased  13  synaptic strength) exist in the hippocampus. LTP, first described by Bliss and Lomo in the dentate gyrus (Bliss and Lomo, 1973), has three basic properties: cooperativity, associativity and input-specificity. They fit the Hebb’s prediction “a form of synaptic plasticity driven by temporal association of pre- and post-synaptic activities. They represent a cellular model of learning and memory” and make it an ideal synaptic mechanism for memory storage. LTP can be induced by tetanus stimulation (a train of 100 stimuli at 100Hz) or more modest stimulation-theta burst stimulation (mimicking the firing patterns occurred in the hippocampus during learning)(Otto et al., 1991) at the pathway of interest in hippocampus(Bliss and Collingridge, 1993).  The induction of LTP by tetanic stimulation can be prevented by a variety of NMDAR antagonists including AP5 (acting at glutamate binding site), MK-801(acting in the channel pore), and 7-chlorokynurenic acid (acting at the allosteric glycine site) in hippocampal slices or in vivo(Collingridge et al., 1983; Harris et al., 1984; Morris et al., 1986; Coan et al., 1987; Bashir et al., 1990), suggesting that NMDARs activation is essential for induction of this process. Two properties of NMDARs, voltage dependent Mg2+ blockade and Ca2+ permeability, determine their role in basal transmission and initiation of LTP (Mayer et al., 1984; Malenka and Nicoll, 1993). NMDARs cannot be activated when cell membrane potential is close to -70mV due to voltage dependent Mg2+ blockade. NMDARs antagonist have little effect on basal transmission in hippocampus, indicating NMDARs is not necessary for basal transmission. On the other hand, after tetanic stimulation, massive glutamate released from  14  presynaptic site persistently activates AMPARs and causes strong depolarization of the cell. The Mg2+ blockade on NMDARs is then relieved, which leads to further Ca2+ influx through NMDARs and increases the intracellular Ca2+ concentration, which is essential for LTP induction. Intracellular application of Ca2+ chelator EGTA can abolish the induction of NMDAR dependent-LTP (Lynch et al., 1983), which further confirms the role of NMDARs in LTP. In the following study, Morris (Morris et al., 1986) found that AP5 blocks LTP in dentate gyrus of hippocampus and also impairs spatial learning in rats, which is dependent upon hippocampus (Morris et al., 1982). The correlation between NMDAR antagonist blockade of LTP and spatial learning and memory suggests that LTP might be the underlying cellular events of learning and memory. This study provided the first correlative evidence suggesting that synaptic plasticity is involved in learning and memory. Later, by using genetic approaches Tonegawa group found that conditioned knockout of NR1 in CA1 or CA3 region can abolish the NMDARs-dependent LTP induction in CA1 or CA3 region of the hippocampus. Moreover, the mutant animals show impairment on associative spatial memory, strongly indicating NMDARs are very important in both LTP induction and spatial learning and memory (Tsien et al., 1996; Nakazawa et al., 2002).  However, NMDARs are not only sufficient for LTP but also for long-term depression (LTD) (Bashir and Collingridge, 1992; Christie and Abraham, 1992; Dudek and Bear, 1992; Mulkey and Malenka, 1992). Those results make the correlation between LTP and its role in spatial learning unclear.  LTD can be induced by low frequency and longer time stimulation  15  (1Hz, 15mins). Compared with LTP induction, Malenka et al. proposed a “calcium theory” in LTP and LTD induction. In this theory, LTP induction needs transient and very high concentration of Ca2+ whereas LTD induction needs long lasting and lower concentration of Ca2+. Pairing protocol for LTP and LTD induction supports this “calcium theory”, where holding cell membrane potential at 0 mV (mimicing strong depolarization), 2 Hz stimulation for 50 seconds can induce LTP, whereas holding membrane potential at -45 mV (mimicing weak depolarization) can induce LTD(Brebner et al., 2005; Yu et al., 2008). But if one considers distinct channel properties of different composition of NMDARs, we can speculate that different NR2 subunit containing NMDARs may confer distinct roles in LTP and LTD induction. As I mentioned before, NR2A and NR2B subunit predominantly exist in hippocampus in the adult animal. The time constant of desensitization of NR1/NR2A is much faster than that of NR1/NR2B (Vicini et al., 1998), indicating NR2B containing NMDARs can open for longer time which is required for LTD induction. In supporting this hypothesis, several studies have used genetic approaches to selectively knock out the NR2A subunit or delete NR2A subunit carboxyl tail resulting in an impairment of hippocampal CA1 LTP(Sakimura et al., 1995; Sprengel et al., 1998; Berberich et al., 2005; Berberich et al., 2007). On the other hand, knocking-out NR2B subunit produces almost intact hippocampal CA1 LTP(von Engelhardt et al., 2008). The different roles of NMDAR subtypes in determining the direction  of  hippocampal  synaptic  plasticity were  further  supported  by several  pharmacological studies both in vitro slices(Liu et al., 2004; Massey et al., 2004), in vivo anesthetized animals(Fox et al., 2006) and even in freely moving animals (unpublished data).  16  Liu et al. found that preferential inhibition of NR2A-containing NMDARs prevents the induction of LTP without affecting LTD production. In an opposite way; inhibition of NR2B-containing NMDARs prevents the induction of LTD without affecting LTP production (Liu et al., 2004). Fox et al. reported the similar results in anesthetized animals, which further confirmed different roles of NR2 subtypes in LTP and LTD induction (Fox et al., 2006).  However, results contradictory to these pharmacological studies have also been reported by others (Hendricson et al., 2002; Morishita et al., 2007). Reasons underlying these conflicting results, while remaining to be determined, may include the specificity of subunit-preferential antagonists (Berberich et al., 2005; Berberich et al., 2007), developmental stages(Bartlett et al., 2007), and even differences of the slice preparations (Bartlett and Wang, 2009). In particular, some of the controversies have been attributed to various concentrations of NVP being used in these studies.  In one of our previous studies (Liu et al., 2004), we had  found that NVP has a about 100 times more selectivity towards recombinant human NR1/NR2A NMDARs expressed in Oocytes and at a concentration of 0.4 μM, it can fully block NR1/NR2A receptors with little effect on NR1/NR2B receptors.  However, one of the  recent studies using rat NR1/NR2A and NR1/NR2B recombinant receptors over-expressed in HEK cells reported that NVP has only less than 10 times the selectivity towards NR2A over NR2B. It is noted that the selectivity can only be maintained at concentrations of 0.1 μM. Its selectivity is essentially lost at a concentration of 0.4 μM (Berberich et al., 2005). Differences in concentrations for selectivity of NVP have also been observed for native NMDARs in slice  17  preparations.  Thus, while Berberich et al. found that NR2A selectivity towards NVP may  only be achieved at a concentration below 0.1 μM, Wu et al. revealed that a full blockade of NR2A containing NMDA receptors by NVP could not be achieved at a concentration of 0.1 μM, but required a concentration of 0.4 μM in acute cortical brain slices. Importantly, they found that at this higher concentration, NVP in fact has little effect on blocking NR2B-containing NMDARs(Wu et al., 2007). These controversies highlight the importance in determining the subunit specificity of the pharmacological agents under certain types of experimental conditions. Some other controversies may be attributed to different slice preparations, even though the underlying mechanism is not clear. LTD induction can be blocked by NR2B containing NMDARs antagonist Ro25-6981 in coronal hippocampal slices but not in sagital or transverse hippocampal slices(Yang et al., 2005; Morishita et al., 2007: Bartlett and Wang, 2009). Beyond these controversies, the effect of Ro25-6981 on LTD in anesthetized or on freely moving animals is very consistent (Fox et al., 2006; Duffy et al., 2008)(unpublished data), while intraperitoneal application of NVP (1.2mg/kg) always blocks tetanic stimulation (100Hz) induced LTP in anesthesized or on freely moving animals(Fox et al., 2006)(unpublished data). Regardless of the subunit selectivity of NVP and Ro25-6981 on native or recombinant NMDARs, the fact that they can selectively and respectively prevent LTP and LTD validates their utility to distinguish the role of LTP and LTD in hippocampus-dependent learning and memory.  18  1.2.7. NMDARs and synaptic plasticity in lateral amygdala The lateral amygdala (LA) is the key locus for synaptic changes that underpins the long-term memory of conditioned auditory fears. In this Pavlovian fear conditioning paradigm, a neutral auditory tone (conditions stimulus, CS) is paired with electric shock (unconditioned stimulus, US)(Sah et al., 2003) and once the association between the CS and US is formed, the CS alone can then elicit a conditioned fear response. The LA receives sensory information from both the auditory thalamus and cortex, and serves as the first stage of processing of auditory inputs to the amygdala (LeDoux, 2000; Blair et al., 2001). CS and US inputs converge onto specific cells in the LA, and the association of CS and US leads to long-lasting alterations in synaptic efficiency (McKernan and Shinnick-Gallagher, 1997; Repa et al., 2001). Depending on the stimulating protocols, stimulation pathway and the recording conditions used, bidirectional alterations of synaptic efficacy, long-term potentiation (LTP) and long-term depression (LTD) at these LA synapses have previously been reported (Rogan and LeDoux, 1995; Rogan et al., 1997; Heinbockel and Pape, 2000; Kaschel et al., 2004). Recent studies have provide some correlative evidence suggesting an important role for LA LTP in mediating the association between CS and US, and hence the formation and storage of the learned fear in the LA (Rogan and LeDoux, 1995; Rogan et al., 1997). LA LTD is suggested to be mediating the extinction of the learned fear response (Dalton et al., 2008).  As metioned above, auditory inputs to LA come from auditory thalamus and auditory cortex and either of these pathways can mediate conditioned auditory fear memory (Romanski  19  and LeDoux, 1992; LeDoux, 2000). The projection from auditory cortex may be involved with a more complicated auditory stimulus pattern(Jarrell et al., 1987), whereas thalamic pathway may be involved with initiation of the plastic changes in the amygdala(Quirk et al., 1995; Quirk et al., 1997; Morris et al., 1999). In thalamic pathway, different stimulation protocol can induce different factor mediated LTP. Pairing-induced LTP is abolished by L type voltage gated calcium channel (L-VGCC) blockers and with Ca2+ chelator BAPTA but not by NMDAR antagonist APV(Weisskopf et al., 1999). 30Hz tetanus stimulation can produce pure NMDAR dependent LTP (Bauer et al., 2002). Lee et al. observed that pre-induction but not post-induction application of APV can abolish late phase LTP induction by delivering 5 trains of tetanus stimulatinon at thalamus pathway. This late phase LTP can also be reduced by L-VGCC blocker (Lee et al., 2002a). Behavior studies showed intra-amygdala application of APV but not L-VGCC blocker can prevent acquisition of fear conditioning, indicating NMDAR dependent LTP in LA may play a more important role in fear memory formation (Bauer et al., 2002). In cortical pathway, Huang and Kandel showed that APV partially blocked LTP induced by delivering tetanus stimulation (100Hz) at cortical pathway (Huang and Kandel, 1998). Moreover, Humeau et al. found that simultaneous activation of converging cortical and thalamic afferents specifically induced associative, presynaptic NMDAR dependent LTP at the cortical, but not in thalamic inputs (Humeau et al., 2003). Taken together, NMDAR dependent LTP may play a critical role in fear memory formation and induction of LTP is required presynaptic NMDAR activity in cortical pathway and postsynaptic NMDAR activation in thalamic pathway. Furthermore, some studies reported that NR2B containing  20  NMDAR antagonist ifenprodil can block thalamic LTP induction (Bauer et al., 2002; Miwa et al., 2008), indicating that NR2B containing NMDAR may take more of an important role in LTP induction in LA than in the hippocampus. However, it is important to note that ifenprodil actually displays a higher affinity for 1 adrenoceptors than it does for NR2B receptors(Chenard et al., 1991) and also has affinity for serotonergic(Chenard et al., 1991; McCool and Lovinger, 1995) and sigma opioid receptors(Karbon et al., 1990). In this regard, it is highly probable that the effects of ifenprodil on LTP in the LA may be mediated by other receptors in addition to NMDA receptors.  Surprisingly, there are few studies that focus on LTD in LA. Different slice preparation can also affect LTD induction in LA. Heinbockel and Pape reported that LTD can be induced in thalamic, but not in the cortical pathway by theta frequency stimulation with coronal amygdala slices. More interesting, this LTD is mediated by postsynaptic group II metabotropic glutamate receptors(Heinbockel and Pape, 2000). However, Kaschel showed that low frequency stimulation on cortical pathway can induce NMDAR dependent LTD with horizontal amygdala slices (Kaschel et al., 2004). In horizontal slices, cortical input contain afferents from perirhinal and entorinal cortex(von Bohlen und Halbach and Albrecht, 2002), whereas in the coronal slice cortical input only contain afferences derived from sensory cortical areas(Doron and Ledoux, 1999). The different input fiber may play a role in LTD induction in LA, but the role of NMDARs in LTD induction still needs further investigation.  21  1.3. AMPA Receptors AMPARs mediate fast synaptic transmission at a vast majority of excitatory synapses in the CNS. AMPARs are also thought to have a critical role in mediating many forms of synaptic plasticity that are believed to represent the cellular mechanisms underlying learning and memory, addiction, neural degenerative process in amnesia and other CNS related diseases.  1.3.1. Molecular diversity, composition and structure of AMPA receptors AMPARs contain four different subtypes: GluR1, GluR2, GluR3 and GluR4, which are generated from different genes. All four AMPAR subunits contain two alternatively spliced forms; flip and flop. Flip forms are highly expressed before birth in rats, whereas flop forms are poorly expressed before birth and are up-regulated to the same level of flip forms in adult rat. There is a difference of desensitization kinetics between flip and flop forms and the difference can be attenuated to same extent by cyclothiazide and PEPA, respectively. GluR2 and GluR4 also contain C-terminal splice variants, which exhibits either a long or a short C-terminus. In addition to alternative splicing, GluR(2-4) RNA can be post-transcriptionaly modified by RNA editing. GluR(2-4) subunits are edited at the R/G site, which is located just before the flip/flop exons. Substitution of glycine for arginine in GluR3 and GluR4 reduces and accelerates the recovery from desensitization (Lomeli et al., 1994). GluR2 also contains a Q/R editing site. Substitution of arginine for glutamine cause low calcium permeability (Hume, 1991), low single-channel conductance (Swanson et al., 1997), and an approximately linear  22  current-voltage relationship (Hume et al., 1991). Removing 25% of GluR2 Q/R editing will cause epilepsy and early death in mice (Brusa et al., 1995).  The subunit composition of functional AMPA is still largely unknown. A tetrameric structure is the most accepted model for AMPARs. AMPARs can form either homomeric or heteromeric complexes. In vertebrate system, there are calcium permeable, GluR2R-lacking and calcium impermeable GluR2R-containing AMPARs. Both forms of AMPARs can exist in a single neuron in the hippocampus. One well-known form of GluR2R-lacking AMPARs is homozygous GluR1, which plays a very important role in global ischemia-induced cell death or AMPARs mediated synaptic plasticity in either the hippocampus or amygdala. GluR2-containing hetermeric AMPARs are the major form that exists in the CNS and can be formed by GluR2 plus either GluR1 or GluR3 in hippocampal pyramidal neurons.  The AMPAR is a transmembrane protein containing one extracellular N-terminous (including S1 which is adjacent to TM1), one intracellular C-terminus, three transmembrane domains (TM1, TM3, TM4), a cytoplasm-facing re-entrant membrane loop, and an extracellular loop between TM3 and TM4 (S2) (Fig 1.1). S1 and S2 are considered as the agonist binding area. The C-terminus is also the critical site for the interaction with other binding proteins.  23  1.3.2. AMPARs distribution  AMPARs are distributed ubiquitously throughout the whole CNS. The regional distribution was examined by [3H] AMPA binding assay. High level binding was found in the hippocampus and superficial layer of the cerebral cortex. In the deeper layer of cerebral cortex and in the caudate-putamen, the binding level is intermediate. In the diencephalon, midbrain and brain stem, the binding level is lower. The lowest binding level was found in the cerebellum (Monaghan et al., 1984: olsen et al., 1987).  Systematic studies by using in situ hybridization histochemistry revealed mRNA expression patterns of GluR(1-4) in adult rat brains (Keinanen et al., 1990; Sommer et al., 1990; Monyer et al., 1991). The GluR1 mRNA is abundantly expressed in the pyramidal cell layer and dentate gyrus of hippocampus, and is lower expressed in layers 3 and 4 in the cerebral cortex. In the cerebellum, GluR1 mRNA can be found in Purkinje cells but not in granule cells and it is also expressed in Bergmann glial cell as well as GluR4. The GluR2 mRNA is highly expressed throughout hippocampus, cerebral cortex and cerebellum. The GluR3 mRNA has the same expression pattern as GluR1 except in Bergmann glial cells where GluR3 does not express. The GluR4 mRNA is much less abundant than that of GluR(1-3) mRNAs. GluR4 mRNA is highly expressed in layers 3 and 4 of the cerebral cortex and in granule cells of the cerebellum.  The specific antibodies to AMPARs subunits provide strong tools for study more detailed distribution patterns of different AMPARs subunits in CNS. The general distribution 24  patterns in different brain area detected by specific antibodies are similar to the studies with in situ hybridization histochemistry and previous binding experiments. All AMPAR subunits are distributed abundantly and differentially in neuronal cell soma and processes in the cerebral cortex, basal ganglia, limbic system, thalamus, cerebellum and brainstem (Petralia and Wenthold, 1992; Martin et al., 1993). The studies with GluR1, GluR2 and GluR3 specific antibodies showed some populations of interneurons of the cerebral cortex and hippocampus were lacking of GluR2 staining but stained with GluR1or GluR3 (Petralia et al., 1997; Moga et al., 2002). Combined with other studies, populations of principal neurons (including projection neurons and interneurons) in the brainstem and spinal cord show lack of GluR2 expression but high expression of GluR3 and/or GluR4 (Petralia and Wenthold, 1992; Tachibana et al., 1994; Petralia et al., 1997).  Ultrastructural studies have reported subcellular distribution of these AMPARs in the cerebral cortex, hippocampus and amygdala. Different GluR1, GluR2, GluR2/3 and GluR4 staining was restricted mainly to postsynaptic densities and adjacent dendritoplasma, and to neuron cell body cytoplasm. There are no convincing evidence of obvious staining at presynaptic terminals, and only limited evidence of glial staining, such as Bergmann glia that express GluR1 and GluR4 staining (Petralia and Wenthold, 1992; Martin et al., 1993; Petralia et al., 1997). Those studies provide strong evidence for a major postsynaptic location of AMPARs.  25  1.3.3. AMPARs channel property  As mentioned previously, AMPARs can be divided into two groups in CNS, those containing GluR2 subunit and those that lack GluR2 subunit. The two groups of AMPARs showed different ion selectivity and rectification properties. GluR2-containing AMPARs are permeable to Na+ and K+, but almost impermeable to Ca2+, and also show strong outward rectification. In contrast, GluR2 lacking AMPARs are permeable to Na+, K+, and Ca2+, and show strong inward rectification. All studies showed that GluR2 governs the Ca2+ permeability and outward rectification by a positively charged amino acid arginine at the Q/R editing site which is located in the M2 segment. Mutation of arginine to glutamine can totally switch GluR2-containing AMPARs from Ca2+–impermeable, outward rectification forms into Ca2+-permeable, inward rectification forms like GluR2-lacking AMPARs. The inward rectification is due to the permeation of intracellular polyamines in the channel pore of GluR2-lacking receptors at positive potentials (Bowie and Mayer, 1995; Donevan and Rogawski, 1995; Isa et al., 1995; Kamboj et al., 1995; Koh et al., 1995), but the mechanism of the outward rectification of GluR2 containing AMPARs is less clear. In addition GluR2-lacking AMPARs also display another two functional properties: they are easily blocked by external polyamines (Brackley et al., 1993; Herlitze et al., 1993), and show a higher single channel conductance (Swanson et al., 1997). Desensitization kinetics of AMPARs is varied among different subunit compositions or the two splicing alternative forms (flip and flop). Except for homomeric GluR1, the AMPARs assembled with the flop form  26  show a faster desensitization time constant than those with the flip form (Colquhoun et al., 1992; Mosbacher et al., 1994).  1.3.4. Modulation of AMPARs The modulation of functional AMPARs normally causes two consequences: changes in AMPAR channel properties and changes in the number of AMPARs expressed on cell surface. These two consequences can be achieved by modulations such as phosphorylation, palmitoylation, glycosylation, etc.  1.3.4.1. Phosphorylation of AMPARs The phosphorylation sites of AMPARs are mainly located at the C-terminus. Various phosphokinases can phosphorylate special sites at the C-terminus of AMPARs and modulate the channel function. There are three phosphorylation sites at the GluR1 C-terminus. PKA can phosphorylate GluR1 at Ser845 to increase channel open probability. Interestingly, the phosphorylation of Ser845 is not seen in tissue after long-term potentiation (LTP) induction for one hour, but is seen in de-depression tissue. This indicates that phosphorylation of this site is not necessary for normal GluR1 insertion, but instead, it might contribute to the surface reinsertion of GluR1 (Lee et al., 2000). Mutation of Ser845 to Alanine on GluR1 eliminates PKA-induced increase of channel open probability (Banke et al., 2000). However this mutation only reduces LTP but does not block GluR1 insertion (Lee et al., 2003). These studies indicate that PKA plays an 27  important role in regulating GluR1 channel property and, at least in part, in regulating GluR1 surface expression. CaMKII phosphorylates GluR1 at Ser831. The phosphorylation of this site can be detected after LTP induction (Lee et al., 2000), but mutation of Ser831 to Alanine does not affect CaMKII-induced GluR1 insertion (Hayashi et al., 2000). This indicates that Ser831 phosphorylation is not required for LTP induction. Similar to PKA, phosphorylation of GluR1 by CaMKII increases the channel conductance (Derkach et al., 1999). PKC phosphorylates Ser818, causing incorporation of GluR1 into synapses (Boehm et al., 2006; Lin et al., 2009).  Two sites of serine can be phosphorylated in the GluR2 C-terminus, Serine880 and Serine863, which can be phosphorylated by PKC (Matsuda et al., 1999; McDonald et al., 2001). Phosphorylation of Serine880 has been detected after LTD induction. Phosphorylation of Serine880 accelerates AMPAR endocytosis by disrupting the interaction between GluR2 and glutamate receptor-interacting protein 1 (GRIP1), in a PKC independent way (Kim et al., 2001) . Studies using combination of GluR2 knockout and overexpression of Lysine882 mutation of GluR2 (a mutation lacking PKC activation on Serine880 without disruption the interaction between GluR2 and GRIP1) have found that phosphorylation of Serine880 on GluR2 is required for AMPARs endocytosis in Cerebellum (Chung et al., 2003). Other studies have also shown that Serine880 phosphorylation is required for AMPARs endocytosis in CA1 neuron or dorsal horn neurons (Seidenman et al., 2003; Park et al., 2009). Until now, very few studies have shown the detection of Serine863 phosphorylation on GluR2 in neurons.  28  Besides the serine phosphorylation sites, there are three potential tyrosine phosphorylation sites on GluR2 C-terminus, Tyrosine869, 873, and 876 (Ahmadian et al., 2004). Blocking tyrosine phosphorylation on these sites abolishes GluR2-mediated endocytosis of AMPARs (Ahmadian et al., 2004). Furthermore, Hayashi and Huganir have shown that only phosphorylation of Tyrosine876 by Src family tyrosine kinase is responsible of AMPAR endocytosis in neurons (Hayashi and Huganir, 2004). More recently, Thomas et al showed that Jun N-terminal kinases (JNKs) phosphorylate the long C-terminus splicing variants of GluR2 at Threonine912. Dephosphorylation of Thr912 is correlated with AMPAR endocytosis, and rephosphorylation of Thr912 by JNKs is responsible for AMPAR reinsertion (Thomas et al., 2008).  The phosphorylation of GluR3 subunits has not been reported.  There are two sites can be phosphorylated in GluR4 C-terminus. PKA, PKC and CaMK2 can phosphorylate Ser842 in vitro, whereas only PKA and PKC phosphorylate Ser842 in GluR4 in vivo (Carvalho et al., 1999; Correia et al., 2003; Gomes et al., 2007). Phosphorylation of Ser842 of GluR4 is required for disrupting the interaction with a retention molecule and increasing GluR4 expression on cell membrane (Esteban et al., 2003). However, PKC binding to GluR4 C-terminus is necessary for GluR4 surface expression (Gomes et al., 2007). Theronine830 can only be phosphorylated by PKC in vitro (Carvalho et al., 1999) and has been detected in vivo up untill recently.  29  1.3.4.2. Pamitoylation of AMPARs Protein palmitoylation, which is a liable and reversible fatty acylation, can regulate cellular location of proteins. Two cystenines of GluR1-4 can be palmitorylated (Hayashi et al., 2005), one site in the transmembrane domain TM2 and the other in the C-terminus region. TM2 palmitoylation results in accumulation of AMPARs in the Golgi apparatus and reduced cell surface AMPARs expression. C-terminus palmitoylation of GluR1 at C811 or GluR2 at C836 enhances activity-dependent AMPARs endocytosis. GluR1 palmitoylation-induced AMPARs endocytosis is due to the disruption of the interaction with cytoskeletal protein 4.1N (Hayashi et al., 2005) . Furthermore, depalmitoylation of GluR1 can cause GluR1 phosphorylation at 816 or 818 by PKC, increasing GluR1 insertion (Lin et al., 2009).  1.3.5. AMPARs and synaptic plasticity in hippocumpus AMPARs mediate fast synaptic transmission in most excitatory synapses in CNS(Dingledine et al., 1999). Plastic changes to the strength of AMPA receptor–mediated synaptic transmission are the final outcome of either short term or long term synaptic plasticity which includes short-term potentiation (STP), short-term depression (STD), LTP and LTD(Malenka and Nicoll, 1993). Different synaptic plasticity can be triggered by activation NMDARs, metabotropic glutamate receptor and other receptors (Malenka and Bear, 2004).  Both presynaptic and postsynaptic mechanism can be responsible for plastic changes to the AMPAR-mediated synaptic transmission. For instance, mossy fiber LTP is a  30  cAMP-dependent presynaptic form of plasticity whereas CA1 LTP is a NMDAR-dependent postsynaptic form of plasticity (Malenka and Bear, 2004). Here, I will focous on postsynaptic modulation on AMPARs. Postsynaptic modulation on AMPARs can also be achieved by alterations of AMPAR channel properties and alterations of number of AMPARs on synapses. These two modulations may occur separately or together. The “silent synapses in LTP and LTD expression” hypothesis (Kullmann, 1994; Isaac et al., 1995; Liao et al., 1995) suggests that AMPARs trafficking may be involved in LTP and LTD expression. A silent synapse contains functional NMDARs, but lacks functional AMPARs, and it becomes activated during the induction of LTP by recruitment of AMPARs. In contrast, an active synapse may be silenced during LTD by the loss of functional AMPARs. Recently, accumulating evidence indicate that exocytosis (or insertion) and endocytosis (or internalization) number of surface AMPARs expression is the underlying mechanism of LTP and LTD expression, respectively. Moreover, GluR1 and GluR2 may play different role in LTP and LTD expression.  Clathrin-dependent AMPARs endocytosis is involved in LTD expression (Carroll et al., 1999; Luscher et al., 1999; Beattie et al., 2000; Man et al., 2000). There exists two distinct types of clathrin-dependent AMPARs endocytosis, Constitutive and Regulated, classified according to their underlying mechanism and functional significance. Consititutive pathway is not involved in reduction of the number of AMPARs on the cell surface, which may be due to the existence of a constitutive AMPAR exocytosis pathway to counteract the constitutive endocytosis (Man et al., 2000; Liang and Huganir, 2001). This Constitutive endocytosis is  31  controlled by a short stretch of seven amino acids (EFCYKSR) in the most juxtamemberance region of the C terminus of GluR2, which is 100% conserved among all AMPAR GluR subunits. However, stimulating the regulated pathway with insulin or NMDA leads to a rapid reduction in the number of postsynaptic AMPARs and LTD expression (Beattie et al., 2000; Man et al., 2000). This Regulated AMPARs endocytosis is controled by C terminal domain of GluR2 subunit but not GluR1(Man et al., 2000). Insulin can simulate homo GluR2 AMPARs endocytosis but not homo GluR1 AMPARs. After switching the C-terminus of GluR2 on GluR1 subunit, insulin can also induce GluR1Glu2C AMPARs endocytosis (Man et al., 2000). A cluster of tyrosines close to the end of GluR2 C-terminus are required for Regulated AMPARs endocytosis and interfere peptide designed to interfere tyrosine phosphorylation in this cluster can  block  insulin  induced  AMPARs  endocytosis  and  LFS  induced  LTD  in  hippocampus(Ahmadian et al., 2004). Furthermore, Hayashi and Huganir have shown that only phosphorylation of Tyrosine876 is required for AMPAR endocytosis in neurons (Hayashi and Huganir, 2004). In addition to this tyrosine-rich sequence of GluR2, there are still other domains or sequences that are critical for the regulated AMPARs endocytosis. One sequence binds with N-ethylmaleimide-sensitive factor (NSF), an ATPase that is involved in membrane fusion. And it was found in the GluR2 C-terminus from positions 844 to 853 amino acid which just follows the conserved domain for Consititutive endocytosis. Inhibiting the GluR2-NSF interaction reduces the surface expression of AMPARs (Luthi et al., 1999; Noel et al., 1999) and occludes the generation of LTD (Luscher et al., 1999; Luthi et al., 1999). The dissociation of NSF from this domain may allow AP2 to bind to GluR2 and initiate clathrin-dependent  32  endocytosis (Nishimune et al., 1998; Lee et al., 2002b). Another domain is the PDZ domain (S880KVI) in the extreme GluR2 C-terminus and interacts with PDZ domain-containing proteins glutamate receptor-interacting protein (GRIP), AMPAR binding protein (ABP) and protein interacting with C-kinase (PICK1). Both ABP and GRIP probably anchor AMPARs at both synaptic and intracellular locations (Dong et al., 1997; Daw et al., 2000; Osten et al., 2000). PICK1 may be involved in AMPARs endocytosis and inhibition of PICK1-GluR2 binding blocks LTD in cerebellum (Xia et al., 2000). PICK1-GluR2 binding may phosphorylate GluR2 on serine 880 and thereby inhibit the binding of AMPARs to ABP/GRIP and then probably induce AMPARs endocytosis (Matsuda et al., 1999; Chung et al., 2000).  Native AMPARs are thought to be heteromeric protein complexes assembled from combination of GluR subunits 1-4(Hollmann and Heinemann, 1994). It has been suggested that native AMPARs in rat hippocampus largely contain GluR1/2 subunits (Lu et al., 2009). The GluR1 subunit plays a more important role in AMPARs exocytosis and LTP expression in hippocampus (Shi et al., 1999; Collingridge et al., 2004). LTP induction is impaired in GluR1 knockout mice but is intact in GluR2 knockout mice (Jia et al., 1996; Zamanillo et al., 1999). This deficency in LTP can be rescured by genetically expressing GluR1 (Mack et al., 2001). Moreover, GluR1 C-terminal fragment can block AMPARs exocytosis and LTP expression (Shi et al., 2001), indicating GluR1 C-terminus is critical in these processes. After phosphorylation by CaMKII, the PDZ-domain-containing protein synapses-associated protein 97 (SAP97) can bind to PDZ domain of GluR1 C-terminus and deliver GluR1 into spines  33  (Mauceri et al., 2004). Ras p42/44 MAPK pathway activation is also involved in GluR1 and CaMKII dependent insertion of AMPARs (Zhu et al., 2002). Beside GluR1 insertion on the cell surface, phosphorylation of serine 831 at GluR1 C-terminus can directly enhance GluR1 channel opening(Derkach et al., 1999), which is observed after LTP induction in hippocampus(Benke et al., 1998). These results indicate that both alterations in AMPARs channel properties and surface expression are involved in LTP expression.  In addition to SAP97, PSD-95 and stargazin also play a role in AMPARs surface expression(Bredt and Nicoll, 2003). PSD-95 and stargazin can interact with each other through stargazin C-terminal PDZ domain (Schnell et al., 2002). Overexpression of PSD-95 causes a dramatic increase in synaptic AMPARs in either cultured hippocampal neurons or slices (El-Husseini et al., 2000; Schnell et al., 2002). On the other hand, overexpression of stargazin causes a remarkable increase of total surface AMPARs without altering synaptic AMPARs (Schnell et al., 2002). Moreover, overexpression of a stargazin mutant lacking its C-terminal PDZ domain increases AMPARs surface expression but reduces synaptic AMPARs (Schnell et al., 2002). These results suggest stargazin recruits AMPARs to plasma membrane, whereas an interaction between PSD-95 and stargazin recruits AMPARs from extrasynaptic sites to synapses during synaptic plasticity. Finally, overexpression of PSD-95 selectively enhances AMPAR mediated synaptic transmission and occludes LTP (Stein et al., 2003), indicating an interaction of stargazin and PSD-95 may play a role in LTP expression. Stargazin and other stagazin related transmembrane AMPAR regulatory proteins (TARPs) regulate AMPARs  34  surface expression without affecting AMPAR subunit selectivity(Bredt and Nicoll, 2003). How the TARP dependent regulation of AMPARs synaptic expression and GluR1 subunit specific modulation together regulate AMPARs exocytosis during LTP is not clear. One speculation is TARP dependent regulation may determine the availability of AMPARs for surface expression and synaptic incorporation, whereas GluR1 subunit dependent regulation determines the number of AMPARs that will incorporate into synapses.  1.3.6. AMPARs in synaptic plasticity in amygdala Although how AMPARs undergo the LTP and LTD is quite clear in hippocampus, the detailed mechanisms of LTP and LTD in lateral amygdala is not extensively investigated. As I metioned before, GluR1/2 or GluR2/3 complex is the main form of native AMPARs in amygdala. C-tail of GluR1 can inhibit AMPAR insertion and LTP induction in cultured hippocampal neurons. Viral infection of GluR1 C-tail into LA can block both LTP induction in LA and fear memory formation (Rumpel et al., 2005), indicating GluR1 insertion may be involved in these processes. The studies reported that fear conditioning can induce increased surface expression of both GluR1 and GluR2 in amygdala, further confirming that native AMPARs insertion also oocurs in fear memory formation (Yeh et al., 2006; Kim et al., 2007; Yu et al., 2008). The question remained is whether native GluR1/2 insertion is involved in fear memory formation? Kim et al reported fear conditioning can induce increased GluR1/2 surface expression in LA and extinction reverses the enhanced GluR1/2 surface expression to basal level, indicating AMPARs endocytosis may be involved in extinction. Moreover, our study  35  showed that LTD can be abolished by application of GluR2 interfering peptide which can block GluR2 endocytosis. Other studies also showed that this peptide can block extinction of fear condition without affecting acquisition(Kim et al., 2007; Dalton et al., 2008), indicating that GluR2 dependent endocytosis and LTD is required for fear memory extinction.  1.4. GABAA Receptors Gamma-aminobutyric acid (GABA) receptors are divided into ionotrophic GABA A receptors (GABAARs) and metobotrophic GABABRs. GABAARs belong to Cys-loop pentameric ligand-gated ion channel (LGIC) superfamily, which includes cation permeable channels such as nicotinic acetylcholine receptors, serotonin type 3 receptors and zinc receptors, and anion permeable channels such as glycine receptors, invertebrate glutamate and histidine receptors (Olsen and Sieghart, 2008, 2009). Each GABAAR subunit consists of a long extracellular N-terminal domain, followed by four transmembrane domain (TM1-TM4), and ends with a short extracellular C-terminal region (Fig. 1.2) (Olsen and Sieghart, 2008). The channel pore is composed of the M2 and parts of the M1 region (Corringer et al., 2000; Sine and Engel, 2006). Modulation of GABAARs functions is relevant to both phasic and tonic inhibition in CNS (Semyanov et al., 2004).  1.4.1. GABAA receptor molecular diversity and composition and structure Mammalian GABAARs include 19 subunits, 1-6, 1-3, 1-3, , ,, , 1-3 encoded from 19 distinct genes (Simon et al., 2004). A distinct group of GABAARs, which were also 36  termed GABACRs, are insensitive to bicuculline and bacolfen, and can be formed by either homo- or hetero-oligomeric 1-3 subunits (Olsen and Sieghart, 2008). However, it was recommended that the term GABAC should be replaced by the term ρ-containing GABAA receptors by the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, because  subunit-containing receptors are structurally and functionally similar to the other GABAARs and thus should be considered as a minor subspecies of GABAA receptors (Barnard et al., 1998).  Only a few of GABAARs subunits have splice variants. The 2 subunits have two splicing variants;2L and 2S. 2L variant contains an eight-amino-acid sequence in the large intracellular loop, which includes a consensus protein kinase C phosphorylation site that is missing in 2S. The 2 variants show similar distribution patterns in brain, but the relative expression level is differently dependent on neuron types and brain regions (Gutierrez et al., 1994). The transgenic mice lacking 2L showed prolonged sleeping time in response to benzodiazepine agonists (Quinlan et al., 2000), indicating that this eight-amino-acid sequence is involved in the regulation of benzodiazepine sensitivity. 6 subunit has two splice variants, 6 and the short form 6s, but the latter does not form functional channels (Korpi et al., 1994). Simon et al reported that 2,4-5, 2-3, 2-3, , and 1 all have splicing variants in human genome (Simon et al., 2004). To date, however, none of these variants have been demonstrated to be present within functional receptors.  37  The composition of GABAARs is tremendously diverse, largely due to the wide variety of different subunits available for receptor formation. Evidence is greatly in favor of heteropentameric receptors containing two copies of a single , two copies of a single , and one copy of another subunit, such as , , or (Sieghart and Sperk, 2002; Olsen and Sieghart, 2008). However, other homomeric or heteropentameric compositions have also been reported, like homomeric3, homomeric , or heteromeric and compositions, in overexpression systems or probably in native receptors.  Homomeric 3 can form GABA insensitive,  spontaneous opening chloride channels which can be enhanced by pentobabitone and be inhibited by picrotoxin in either transfected Xenopus oocytes or mammalian cells (Connolly et al., 1996; Wooltorton et al., 1997; Taylor et al., 1999). Homomeric 1or 2 can produce GABAARs which are insensitive to bicuculline blockade or bacolfen activation in overexpressed HEK293 cells (Cutting et al., 1991; Qian and Dowling, 1993; Wang et al., 1994b; Wang et al., 1995b; Enz and Cutting, 1999). / is the minimal requirement for the composition of functional GABAARs (Malherbe et al., 1990b; Sigel et al., 1990), which merely lack benzodiazepine sensitivity compared with major native GABAARs (Levitan et al., 1988). The  composition has the full properties of native receptors (Sigel et al., 1990). Futhermore, the   2 composition comprises above 90% of the native GABAARs, and the 122composition comprises about 60% of GABAARs in CNS (Mohler, 2006). Different compositions of GABAARs show distinct distribution and pharmacological properties in CNS.  38  1.4.2. GABAARs distribution GABAARs are distributed ubiquitously throughout the whole CNS. The regional distribution was studied by using [3H] muscimol or [3H] GABA binding assay (Palacios et al., 1981; Bowery et al., 1987). Binding levels vary between different brain area and nuclei. Highest binding levels are found in frontal cortex, the granule cell layer of the cerebellum, the olfactory bulb, and the thalamic medial geniculate. The molecular layer of hippocampus and the external (I–IV) layers of the cortex also showed high levels of binding. Moderate binding levels are observed in the basal ganglia. Low levels are detected in the pons, medulla and brainstem (Palacios et al., 1981; Bowery et al., 1987).  Systematic studies using in situ hybridization revealed mRNA expression patterns of α1-6, β1-3,γ1-3 and  subunits in adult rat brains (Khrestchatisky et al., 1989; Lolait et al., 1989; Shivers et al., 1989; Hironaka et al., 1990; Kato, 1990; Luddens et al., 1990; Malherbe et al., 1990b, a; Seeburg et al., 1990; Ymer et al., 1990; Zhang et al., 1990; Khrestchatisky et al., 1991; MacLennan et al., 1991; Persohn et al., 1991; Wisden et al., 1991; Wisden et al., 1992). mRNAs of different subunits display distinct distribution patterns in brain. 6 mRNAs are restricted to the cerebellum. In cortex, 1 mRNAs express with higher level in layer 2,3,5,6 than layer 4. In contrast, 2 mRNAs are predominantly expressed in layer2. 3 mRNAs are predominant in layer 5 with lower level expression in other layers. 4 mRNAs appear to be highest in layer2 and 3. 5 mRNAs are present with low level in all layers. 1 mRNAs are rare in cortex. 2/3 mRNAs are expressed in the same pattern of lamination, with  39  higher levels in layer 2,3,5,6 than layer 4. 2 mRNAs show high expression level in all layers with the same expression pattern as 2/3. 1 and 3 mRNAs are equally present in all layers and 1 expression level is weaker than 3. mRNAs show modest expression level in layer 2 and low expression level in other layers.  In hippocampus, 2 transcripts are distributed abundantly in CA1, CA3 and dentate gyrus cell layer. 1 and 4 mRNAs are also expressed in all sectors. 3 mRNAs are mainly present in granule cells of dentate gyrus. 5 mRNAs are strongly expressed in CA1 and CA3 region and expressed at low level in the dentate gyrus. 3 mRNAs are present throughout hippocampus with high expression levels. 2 mRNAs appear to be less abundant than 1 and 3. 1 mRNAs are expressed with high level in all brain regions. 2 mRNAs are abundant in hippocampus whereas 1 mRNAs are rare.  mRNAs are mainly restricted to dentate gyrus. In the caudate nucleus and the nucleus accumbens the predominant  subunit mRNAs are 2 and 3 mRNAs are the major  subunits.  mRNAs show low expression.  mRNAs are moderately expressed in the caudate nucleus and the nucleus accumbens. In amygdala area, 2 are the prevalent  subunit mRNAs, and three  subunit mRNAs are expressed from low to moderate levels. 1-2 is the major  subunit mRNAs in amygdala. No  mRNAs can be detected in this region. In most of thalamus, the major GABAAR subunit mRNAs are 1, 4,2.  subunit mRNAs that can be detected at low levels.  mRNAs are expressed in some of the nucleus in thalamus.  40  In cerebellum, distinct groups of cells show different expression patterns. 1, 2 mRNAs are predominant in stellate/basket cells, and 2 mRNAs are detected at very low levels. In purkinje cells, 1, 2, 3,2 are the only existing GABAARs mRNAs. Granule cells show 1, 6, 2,3,2 and  mRNAs expression. ε and  mRNAs are strongly expressed in locus coeruleus (LC) and are also expressed in hypothalamus, but not in cortex and hippocampus in rats (Sinkkonen et al., 2000).  mRNAs are expressed in lung, thymus, and prostate and particularly abundant in the uterus, but have never been detected in human brain tissues (Hedblom and Kirkness, 1997). 1 mRNAs were restricted to retina, whereas 2 and 3 mRNAs are present in retina and most brain areas (Enz and Cutting, 1999; Alakuijala et al., 2005).  During development, different GABAAR subunits present specific temporal and  regional expression patterns (Laurie et al., 1992). 1, 6 and  mRNAs appear after birth and increase markedly with age. The 2 and 2 mRNAs appear in the embryo and their expression increase postnatally. In contrast, 3 mRNA is abundant in the embryo and decline after birth. 2, 4, 5, 1, 3, 1 and 3 mRNAs expression reaches to peak level around birth (Laurie et al., 1992).  Immunohistochemistry studies showed excellent correlation with studies on the mRNA distribution of GABAAR subunits (Fritschy and Mohler, 1995; Pirker et al., 2000). Ultrastructure studies showed GABAAR subunits not only exist in synapses also in extrasynapses (Somogyi et al., 1989; Nusser et al., 1998). The different subunit compositions could contribute to distinct synaptic or extrasynaptic distribution of GABA ARs. In cerebellar  41  granule cells, 6/2/3/ compositions are located at extrasynaptic area, whereas 1/2/3/2, 6/2/3/2 and 16/2/3/2 compositions are located at synapses (Nusser et al., 1995; Nusser et al., 1998). In hippocampus, 1-containing GABAARs are located in most inhibitory postsynaptic domains of primary neurons. In contrast, 2 containing GABAARs are located only in a subset of synapses on the dendrites, but in most synapses on axon initial segments (Nusser et al., 1996).  1.4.3. GABARs channel properties GABAARs are chloride channels, which are also permeable to other anions. Native GABAARs have a permeability sequence of SCN>NO3>I>Br>Cl>HCO3>F (Bormann et al., 1987; Fatima-Shad and Barry, 1993). The permeability to anions can be determined by an intracellular domain close to M2 segment of  subunit in 2/3/3 recombinate GABAARs. Mutation of four amino acids (ASAA) to three amino acids (SG-E) in the 3 but not 2 or 2 subunits results in conversion to cationic selectivity without affecting other pharmacological properties of GABAARs (Jensen et al., 2002). Permeability to different anions is directly related to the function of GABAARs. As we mentioned above, GABAARs are the main inhibitory receptors in CNS. Under most conditions, at inhibitory synaptic or extrasynaptic sites, activiation of GABAARs causes inhibititory effects, namely hyperpolarization of the cell. This depends on the Cl- equilibrium potential being close to or negative to the resting membrane potential, which is maintained by the Cl--extruding K+-Cl- cotransporter (KCC2) activity that regulates chloride distribution across the cell membrane (Payne et al., 2003).  42  However, when the intracellular Cl- concentration increases and Cl- equilibrium potential moves positive to the resting potential, activation of GABAARs induces excitatory effects by depolarizing the cell (Rivera et al., 2005). This occurs, for instance, in GABAAR-containing synapses during the early developmental period, where the lack of KCC2 and enriched expression of Na+-K+-coupled co-transporters (NKCC1) causes intracellular Cl- accumulation (Farrant and Nusser, 2005).  Subunit composition is a major factor that determines the pharmacological properties of GABAARs. In the overexpression system, incorporation of 2S subunits into 12 receptors enhances the extent of desensitization and accelerates deactivation of GABAARs (Boileau et al., 2003; Boileau et al., 2005).  subunits can dramatically reduce GABAARs desensitization (Brown et al., 2002).  In cultured neurons, the  composition which is the major form of  GABAARs in synapses shows a fast desensitization, whereas the  composition which is the major form in extrasynapses shows a slow desensitization (Farrant and Nusser, 2005). Enhanced GABA sensitivity is also observed in containing GABAARs compared with -containing GABAARs (Brown et al., 2002; Farrant and Nusser, 2005). Different  subunits can also influence the extent of desensitization of GABAARs. In hte compostion, 1-incorporated 3receptors have slower desensitization and faster deactivation properties than 6containing 3receptors (Bianchi et al., 2002). However, in the  composition, 1-incorporated 22receptors have faster desensitization and faster deactivation properties than 6-containing 22receptors (Tia et al., 1996).  43  Native GABAARs exhibit three single-channel conductances of 12, 17-20 and 27-30 pS in response to GABA, the largest of which accounts for approximately 95% of all events (Bormann et al., 1987; Macdonald et al., 1989; Mortensen and Smart, 2006). Studies in recombinant receptors indicate that the single-channel conductance depends on their subunit composition.  receptors have a main single-channel conductance of 11~15 pS, whereas  or receptors have a main single-channel conductance of 25~32 pS (Verdoorn et al., 1990; Angelotti and Macdonald, 1993; Fisher and Macdonald, 1997; Brickley et al., 1999; Mortensen and Smart, 2006), indicating that the  composition may exist in native system. Subunit composition of GABAAR affects not only the single-channel conductance but also other single-channel parameters. In the 13 (with either 2 or ) composition, -incorporating receptors show extension in both open time and the duration of bursts of channel openings compared with  containing receptors (Fisher and Macdonald, 1997). In the 42 (with either 2 or ) composition, 422 receptors contain single-channel clusters, but 42 receptors do not exhibit these clusters (Akk et al., 2004).  1.4.4. GABAARs channel modulation GABAARs can be modulated either extracellularlly or intracellularly. Numerous psychoactive drugs, which include benzodiazepines, sedative and anesthetic barbiturates and steroids, general anesthetics, some convulsants and some chemicals, take effect via extracellularlly activation or allosteric modulation of GABAARs. Intracellular modulation can also affect channel properties of GABAARs. For instance, insulin can phosphorylate  44  intracellular part of GABAARs and triggers insertion of GABAARs into the plasma membrane (Wan et al., 1997b). Therefore, modulation of GABAARs produces two consequences, which are changes of the channel properties and changes of receptor numbers on cell surface.  1.4.4.1. Allosteric modulation GABAARs contain a number of different allosteric binding sites, which are located in the N-terminus GABAARs subunits. These allosteric sites are the targets of various small molecules, including benzodiazepines (BZ), barbiturates and steroids.  Tranquilizer drugs of the BZ type bind to GABAARs at the interface between the  and  subunits. The  composition of GABAARs is not sensitive to BZ. Site-directed mutagenesis studies provide the sophisticated insight into the molecular structure of BZ binding site. In 2 subunit, six residues M57, Y58, F77, A79, T81 and M130 are involved in the potentiation effect of BZ (Buhr et al., 1997; Buhr and Sigel, 1997; Wingrove et al., 1997; Sigel et al., 1998; Kucken et al., 2000); andA79 and T81 line the BZ binding pocket (Teissere and Czajkowski, 2001). In the  subunit, a histidine residue at a conserved position (1-H101, 2-H101, 3-H126, or 5-H105) is a critical site for BZ binding. Replacement of this histidine residue with an arginine residue can abolish diazepam sensitivity of 1-, 2-, 3-, or 5- containing receptors (Wieland et al., 1992; Benson et al., 1998).  Barbiturates enhance the binding of GABA and increase GABAARs mediated  45  currents. At low concentrations (<10 μM, within the sedative therapeutic range of 0.5–3 μg/ml), barbiturates act at a positive allosteric site on the GABAAR to increase its response to GABA. Barbiturates cause a leftward shift in the dose-response curve without changing the maximal response to GABA. At higher concentrations (>100 μM), barbiturates act as agonists that directly activate GABAARs (Nicoll and Wojtowicz, 1980; Feng et al., 2004; Muroi et al., 2009; Fisher and Fisher, 2010). At milimolar concentrations, barbiturates inhibit GABA responses (Schwartz et al., 1986; Peters et al., 1988; Thompson et al., 1996). The binding sites of barbiturates on GABAARs are distinct from GABA binding sites (Amin and Weiss, 1993).  subunits are considered as the major location for these binding sites, because homo-oligomeric  receptors directly bind to barbiturates. Studies have shown that several residues in  subunit are involved in the potentiation effect of barbiturates on GABAARs, including threonine 262 and serine 290 in 1 (Birnir et al., 1997; Dalziel et al., 1999; Pistis et al., 1999), glycine 219 in 2 (Carlson et al., 2000; Chang et al., 2003), asparagine 265 and 289 in 3 (Pistis et al., 1999; Cestari et al., 2000). Moreover, some residues can affect barbiturate gating on recombinant GABAARs, like asparagine 265 in 3 (Cestari et al., 2000) and threonine 69 in 6 (Drafts and Fisher, 2006).  Neurosteroids include metabolites of progesterone, metabolites of deoxycorticosterone and the synthetic steroid alphaxalone (Corpechot et al., 1981; Corpechot et al., 1983; Cottrell et al., 1987; Harrison et al., 1987; Majewska, 1992; Kokate et al., 1994). They all enhance GABA-stimulated  36  Cl-fluxes and binding of GABA agonist (Gee, 1988). At low  46  concentrations (nM), steroids increase the open probability of GABAARs without affecting the single channel conductance. At high concentrations (μM), these steroids cause direct activation of GABAARs, which is sufficient to inhibit excitatory transmission. The effect of steroids on GABAARs is not via the barbiturate or benzodiazepine binding site, but the effect instead acts on the steroid binding site. Some residues in the  subunit have been confirmed to be critical for the potentiating effects of steroid. Mutation of glutamine 241 in 1 subunits or the same residue in other isoforms of  subunits abolishes effects of steroid on recombinant  or  receptors (Hosie et al., 2006; Hosie et al., 2007; Akk et al., 2008; Hosie et al., 2009). In addition, serine 240, asparagine407 and tyrosine410 in α1 subunits are also involved in the potentiation effect of steroids (Li et al., 2007; Akk et al., 2008). The proposed common interaction sites that mediate this process include both the 1 glutamine241 acting as an H-bond  acceptor  to  3-hydroxyl  group  of  the  steroid  A  ring  and  the  α1  asparagine407/tyrosine410 interacting with the ketone group in the side chain on the D ring of steroids (Hosie et al., 2007).  1.4.4.2. Phosphorylation of GABAARs The phosphorylation sites of GABAARs are located at the intracellular loop (TM3-TM4) of  or  subunits (Brandon et al., 2002). In  subunits, a conserved serine residue, serine 409 in β1/3 or serine 410 in β2, can be phosphorylated by PKA, PKC, PKG, CaMKII, and Akt in vitro (Moss et al., 1992a; McDonald and Moss, 1994, 1997; Wang et al., 2003). In 2 subunits, serine 327 can be phosphorylated by PKC and CaMK2 in both short (2S) and  47  long (2L) isoforms. In addition, the 2L isoform, with an additional 8 amino acid sequence between TM3 and TM4, can also be phosphorylated at Ser 343 by PKC and CaMKII (Moss et al., 1992a; McDonald and Moss, 1994, 1997). In 11 or 112 recombinant heterologus systems, PKA activation inhibits GABA responses and increases 1 phosphorylation. Mutation of serine 409 in 1 subunit abolishes the effect of PKA, indicating that phosphorylation of serine 409 is required for the inhibition effect of PKA. In cultured neurons, PKA activation can also inhibit native GABAARs responses (Moss et al., 1992b). In contrast, PKA activation enhances 132 recombinant GABAARs responses, along with the phosphorylation of serine 408 and 409 in 3. Mutation of serine 409 with or without serine 408 abolishes the enhancement of PKA. Interestingly, mutation of serine 408 alone shows inhibition of PKA-induced activation, mimicing the effect of PKA on β1-containing receptors (McDonald et al., 1998). The distinctive effects of PKA on recombinant GABAARs with different subunits explains at least partially the contradictory effects of PKA on native GABA ARs observed in different brain regions. For instance, PKA inhibits GABA currents in cultured superior cervical ganglia, spinal cord neurons, cerebellar granule cells, hippocampal pyramidal cells, and olfactory bulb granule cells (Porter et al., 1990; Moss et al., 1992b; Robello et al., 1993; Poisbeau et al., 1999; Flores-Hernandez et al., 2000), whereas it potentiates GABA currents in hippocampal dentate granule neurons, cerebellar purkinje neurons, and olfactory bulb granule cells (Kano and Konnerth, 1992; Kano et al., 1992; Nusser et al., 1999).  In heterologous systems of  or  recombinant GABAARs, PKC inhibits  48  GABAARs function, and causes phosphorylation of serine409 in β1, serine410 in β2, serine327 in γ2S/L, and serine 343 in γ2L(Krishek et al., 1994; Moss and Smart, 1996). Mutation of serine 409 in β1, 327 and 343 in γ2L can totally abolish the effect of PKC on 112L recombinant receptors (Krishek et al., 1994), indicating that the serine phosphorylation is responsible for the effect of PKC on GABAARs. The effects of PKC activation on native GABAARs function are also controversal. PKC activation decreases GABAARs activities, and blockade of PKC increases GABAAR functions in cultured cortical neurons (Brandon et al., 2000). In contrast, PKC increases GABAARs mediated mIPSCs amplitude in dentate gyrus granule cells in hippocampal slices (Poisbeau et al., 1999). The underlying mechanism of these differences remains unclear. Constitutively activated CaMKII can enhance GABAAR mediated currents in cultured rat spinal cord neurons (Wang et al., 1995a). In addition, CaMKII is essential for the rebound potentiation of GABAARs function in cerebellar Purkinje neurons (Kano et al., 1996). This effect can be mimiced by intracellular application of PKA or cyclic AMP analogues (Kano and Konnerth, 1992). Akt phosphorylates serine410 in β2 both in vitro and in vivo, and enhances GABAAR function via an increase in surface GABAAR expression (Wang et al., 2003).  Tyrosine phosphorylation enhances the GABAAR function in recombinant 122 receptors, cultured superior cervical ganglion neurons, and spinal cord neurons (Moss et al., 1995; Wan et al., 1997a; Wan et al., 1997b). Tyrosine phosphorylation sites exist in 1, 2, 3 and 2 subunits (Moss et al., 1995; Wan et al., 1997a; Brandon et al., 2001). Mutations of  49  tyrosine 365/367 on 2L but not tyrosine 384/386 on 1 subunit abolish the Src induced enhancement of GABAAR function in recombinant 122L receptors (Moss et al., 1995). Interestingly, Src enhances GABAAR mediated current in recombinant 12 receptors (Wan et al., 1997a), indicating that 2 is not always required for the Src effect on GABAARs.  1.4.5. GABAARs in learning memory and disease GABAARs mediate both fast synaptic inhibition and slow tonic extrasynaptic inhibition in CNS. GABAARs play a critical role in controlling neuron excitability, which is the basal requirement in learning and memory and other physiological functions. Changes in GABA AR functions cause various diseases like epilepsy and autism, which are due to an imbalance of the excitatory and inhibitory systems.  The role of GABA-releasing interneurons in rhythm oscillation of the neuronal network indirectly reflects the role of GABAARs in brain physiological function. The activity patterns of GABAergic interneurons can shape the dynamics of neural networks. Synchronous firings of principal neurons and interneurons generate different patterns of rhythmic activities, including theta (4-12Hz), gamma (30-100Hz), and fast (>200Hz) oscillations, which are all involved in many functions of CNS. Gamma oscillation occurs during spatial navigation, memory task and visual perception (Buzsaki and Draguhn, 2004), indicating that GABAAR activities may play a very important role in these processes.  50  Genetic manipulation of different subunits of GABAARs reveals important roles of GABAARs in learning and memory. The α5 subunit is expressed mainly in the hippocampus. 5  subunit  knockout  mice  display  a  significantly  improved  performance  in  hippocampal-dependent spatial learning tasks and show no changes in anxiety responses and non-hippocampal-dependent task performance (Collinson et al., 2002). Enhanced paired pulse facilitation, which is an index of presynaptic change, may be related to the observed enhancement in learning and memory. Interestingly, there is no change in LTP in α5 / mice. The enhanced spatial performance can be mimicked by application of a partial inverse agonist acting on 5 GABAARs in wild rats (Chambers et al., 2004), which is consistent with the results from α5 / mice. Furthermore, in point mutated α5 (H105R) mice that lack the benzodiazepine site, trace fear conditioning (hippocampus dependent) but not classic fear conditioning (amygdala dependent) is enhanced (Crestani et al., 2002), strongly indicating that α5-containing GABAARs play an important role in learning and memory. 3 knockout/ mice display a cleft palate, and 90% of the mice died shortly after birth (Homanics et al., 1997). The surviving 3 knockout/ mice display features common to the Angelman syndrome in humans, including poor learning and memory, poor motor coordination, repetitive stereotypical behavior and EEG abnomalities (DeLorey et al., 1998). The 3 subunit is the only currently known  subunit that is expressed in the reticular nucleus of the thalamus. GABA-mediated inhibition is abolished in reticular nucleus in 3 knockout mice. The consequent result is the dramatically enhanced oscillation synchrony, which indicates that β3-containing GABAARs act as “desynchronizers” responsible for recurrent inhibitory  51  connections in the reticular nucleus (Huntsman et al., 1999). Almost all 2 / mice die in the first few days after birth (Gunther et al., 1995). 2 +/ heterozygous mice can survive and display an enhanced aversive memory in trace fear conditioning (Crestani et al., 1999). Combined with reduced inhibitory synaptic clusters in hippocampus, these results suggest that GABAARs play an important role in inhibitory synapse formation and aversive learning and memory (Crestani et al., 1999). Interestingly, 2, 3, 2 and  subunits knockout mice display no differences in learning and memory-related tasks or activities (Rudolph and Mohler, 2004). These results may be caused by developmental compensation of the missing GABA AR subunits.  The knowledge about GABAARs in diseases is mainly from the drug treatments of diseases, and from genetic analysis of patients and follow-up genetic manipulations in animals. Abolishing or reducing GABAAR function results in enhanced neuronal excitability, which in turn causes neurological disorders, such as epilepsy, schizophrenia and autism. Epilepsy, a common chronicneruological disorder characterized by recurrent seizures, can be caused by genetic abolition of β3 subunit of GABAARs. 3 knockout-/- mice show features similar to the Angelman syndrome in humans, including seizure, EEG abnomalities and other abnormal behaviors.  3 knockout mice display sensorimotor gating deficits and hyperdopaminergic phenotypes that are common symptoms in schizophrenia (Yee et al., 2005). The deficit can be  52  completely normalized by treatment with antipsychotic D2-receptor antagonist haloperidol, which is a common treatment in schizophrenia patients (Yee et al., 2005). The cognitive deficits in schizophrenia are associated with a deficit in EEG (electroencephalogram)  rhythm activity and maybe with dysconnectivity (abnormal functional integration of brain processes), indicating a potential deficit in GABAergic control. In addition to the standard antipsychotic therapy, treatment of MK0777, an α2/α3-GABAAR modulator, not only enhances the level of attention and working memory, but also restores  oscillation in a cognitive task (Lewis et al., 2008).  Autism is related to rearrangements or mutations of neuroligin genes in human. In neuroligin 3 (Arg451Cys) mutant mice, impaired social interactions occur along with enhanced GABAAR mediated inhibition (Tabuchi et al., 2007). The similar phenomenon can be observed in neuroligin 2 overexpression mice (Hines et al., 2008). These results strongly indicate enhanced GABAAR containing synapses may involve in autism.  A large group of antiepileptic drugs act via mechanisms of directly or indirectly enhancing GABAAR activities. For instance, Clorazepate, a benzodiazepine derivative, acts at the benzodiazepine binding site of GABAARs, and Valproic acid reverses the transamination process to maintain extracellular GABA levels (French and Faught, 2009). Furthermore, a large scale of drugs for psychotic diseases, insomnia, and anxiety act on GABAARs. According to these data mentioned above, GABAARs are crucial for CNS inhibition and probably also for  53  these neuronal disorders, however, the underlying mechanisms still need further investigation.  1.5. Glycine Receptors Glycine receptors (GlyRs) are anion permeable LGICs that belong to Cys-loop pentameric ligand-gated ion channel superfamily(Lynch, 2004). GlyRs primarily mediate fast synaptic inhibition in spinal cord and brain stem.  1.5.1. Molecular diversity, composition, and structure of glycine receptors There are five subunits of GlyRs, including 1-4) and  subunit (Grenningloh et al., 1987; Grenningloh et al., 1990a; Kuhse et al., 1990a; Akagi et al., 1991; Kuhse et al., 1991; Matzenbach et al., 1994). Among these subunits, 4 subunit exists in mouse, chick and zebrasfish, but not in rat or human (Matzenbach et al., 1994; Harvey et al., 2000; Imboden et al., 2001; Devignot et al., 2003). The primary structure of different  subunits displays 80-90% amino acid sequence identity, and the  subunit shares about ~47% sequence similarity with the 1 subunit (Grenningloh et al., 1990a; Lynch, 2004).  he major splicing variants exist in  subunits. 1 subunit has two isoforms,1 and ins1, in rats. ins1 contains additional eight amino acids that inserts into the large intracellular loop of 1 (Malosio et al., 1991b). On the other hand, 2 subunit has three splice variants in rats. Alternative splicing generates two splice variants, 2A and 2B (Kuhse et al., 1991). In 2B, an isoleueine is found at position 58 and an alanine at position 59, instead of valine and  54  threonine in 2A. Another splice variant is termed as 2* which incorporate a glutamic acid at the 167 position instead of glycine in 2A and is insensitive to strychnine (Kuhse et al., 1990b). Two splice variants in human, termed 3L and 3K, have been identified (Nikolic et al., 1998). Compared with 3L in rat and 3 in human, the 3K variant lacks the coding sequence for 15 amino acids in the large intracellular loop. 3K recombinant receptors show faster desensitization than 3L recombinant receptors in HEK cells (Nikolic et al., 1998). 4 and  subunit variants have not been reported.  GlyR subunit assembly is not clear. An important piece of evidence in favour of heteropentameric receptors was revealed by using crosslinked purified GlyR subunits from rat spinal cord (Langosch et al., 1988), which shows that the majority of crosslink products have five times molecular weight of the mean individual subunit. Trimeric homomeric α1 GlyRs may exist, which was proved by using both laser scattering and single particle electron microscopic analyses (Xue et al., 2001). This result is not contradictory with previous reports where they showed that small crosslink products might be dimers, trimers or tetramers (Langosch et al., 1988). These studies indicate that pentameric composition is probably not the only composition form of GlyRs. Each of  subunits can form homomeric GlyRs, whereas  subunit must accompany one or two types of  subunits to form heterometric GlyRs in heterologous expression systems (Schmieden et al., 1989; Grenningloh et al., 1990b; Kuhse et al., 1990a; Schmieden et al., 1992; Bormann et al., 1993). Native GlyRs have the similar properties as  recombinant receptors in adult rat (Bormann et al., 1993), indicating  is the  55  major form of native composition of GlyRs. Homomeric 2 GlyRs exist in embryonic rat (Becker et al., 1988; Aguayo et al., 2004; McDearmid et al., 2006).  Similar to other cys-loop LGICs, each subunit of GlyRs includes a large N-terminal extracellular domain that contains the agonist binding sites and the conserved cysteine loop, four -helical transmembrane domains (TM1-4), a large intracellular loop (between TM3 and TM4 that contains modulation and interaction sites with cytoplasmic factors), and a small extracellular C-terminus (Lynch, 2004; Hernandes and Troncone, 2009).  1.5.2. Glycine receptor distribution Regional distribution of GlyRs was examined by using H3 strychnine binding assay in the rat and human tissue (Zarbin et al., 1981; Probst et al., 1986). High level binding was found in the spinal cord and medulla. Low level binding can be observed in the pons, thalamus and hypothalamus. No binding was detected in higher brain regions. In the spinal cord, the binding distribution is diverse throughout the gray matter. By contrast, the binding distribution is concentrated to discrete nuclei in brain stem.  Systematic studies by using in situ hybridization revealed mRNA expression patterns of 1-3) and  subunits in rat brains (Fujita et al., 1991; Malosio et al., 1991a; Sato et al., 1991; Watanabe and Akagi, 1995). In adult rat, GlyR 1 subunit mRNAs are expressed at high level in spinal cord, and are also expressed in a few brain areas. 2 mRNAs are observed in  56  several brain regions including layer VI of the cerebral cortex and hippocampus. 3 subunit mRNAs are expressed at low levels in cerebellum, olfactory bulb and hippocampus. In contrast, high amounts of  subunit mRNAs are widely distributed throughout spinal cord and brain. In prenatal rat, 1 subunit mRNAs are first detected at embryonic day 15 and expressed at very low level. 2 mRNAs are distributed widely and abundantly in most of the CNS. However, 2 mRNAs decline dramatically within most of regions of CNS. 3 mRNAs are presented rarely in prenatal period and increase to highest level around 3 weeks after birth.  subunit mRNAs are expressed at low level prenatally but exist throughout the CNS.  Immunohistochemistry studies show similar distribution patterns as strychnine autoradiography studies (Araki et al., 1988; Basbaum, 1988; Murakami et al., 1988; van den Pol and Gorcs, 1988). However, immunolabeling is found in the cerebellum and olfactory bulb, which is not detected by strychnine binding assay. The controversy among these results may be due to low strychnine binding ability of some types of GlyRs or unspecific binding of GlyRs antibody.  Untrastructural studies reveal that GlyRs are accumulated at postsynaptic regions, indicating a functional role of GlyRs in fast synaptic inhibition (Triller et al., 1985; Altschuler et al., 1986; Triller et al., 1987; van den Pol and Gorcs, 1988). In addition, the colocalization of GlyRs and GABAARs was observed at same postsynaptic area in the spinal cord and cerebellum (Bohlhalter et al., 1994; Todd et al., 1996; Dumoulin et al., 2001; Geiman et al.,  57  2002).  1.5.3. Glycine receptor channel properties GlyRs are chloride channels that are also permeable to other anions. Native GlyRs have a permeability sequence of SCN>NO3>I>Br>Cl>HCO3>F (Bormann et al., 1987; Fatima-Shad and Barry, 1993). Mutation of proline250 and alanine251 can reduce anion permeability and produce cation permeability in 1 recombinant GlyRs (Keramidas et al., 2002; Moorhouse et al., 2002).  The desensitization of homomeric 1 recombinants can be influenced by the density of surface GlyRs, gephyrin binding, or modulation of intracellular domains (Legendre et al., 2002). Higher density of GlyRs is related to faster desensitization of GlyRs. Gephyrin binding enhances GlyR desensitization. Human 3K GlyRs, which lacks a 15-amino acid segment in the large intracellular domain between TM3 and TM4, has faster desensitization than 3L recombinant GlyRs (Nikolic et al., 1998). Mutations at Trp243, Ile244, Pro250, and Ala251 in the TM1-TM2 loop of 1 GlyRs enhance the desensitization rate (Lynch et al., 1997; Saul et al., 1999; Lynch, 2004). I244N and P250T mutations of 1 have been reported in humans with startle diseases (Lynch et al., 1997; Saul et al., 1999).  GlyRs display multiple states of single channel conductance (Bormann et al., 1987; Bormann et al., 1993). The conductance range is from 12ps to 110ps and contains seven stages  58  in both recombinant and native GlyRs (Rajendra et al., 1997). Native GlyRs exhibit two main conductance states of 45pS and 30pS, or exhibit one main conductance of 45pS (Hamill et al., 1983; Bormann et al., 1987; Smith et al., 1989; Takahashi and Momiyama, 1991; Twyman and Macdonald, 1991; Takahashi et al., 1992). In recombinant rat GlyRs, the main conductance of homomeric α3 is 110pS, homomeric α1or α2 is 90pS, and heteromeric α1/2/3β is around 50pS (Takahashi et al., 1992; Bormann et al., 1993). The relative open probabilities of the main conductance states do not vary with agonist concentrations (Twyman and Macdonald, 1991; Beato et al., 2002).  1.5.4. Glycine receptor modulation Modulation of GlyRs also can be divided into two types, allosteric modulation and phosphorylation. Allosteric modulation is involved in fast and short-term modulation via extracellular or intracellular sites of GlyRs. Phosphorylation modulation will cause either short- or long-term changes of GlyRs, including changes of channel properties and number of receptors on cell surface.  1.5.4.1. Allosteric modulation Zinc can modulate GlyRs through potentiation or inhibition. Zinc increases native or recombinant GlyR-mediated currents at low concentrations (5 M) by increasing open probability (Laube et al., 1995; Laube et al., 2000). Mutation of aspartic acid at position 80 of the α1 subunit to alanine or glycine can disrupt zinc potentiation on glycine currents (Laube et  59  al., 2000). Mutation of intracellular methionine at position 246 to alanine can also abolish zinc potentiation on glycine currents (Lynch et al., 1998). Zinc inhibits native or recombinant GlyR-mediated currents at high concentrations (>100 M) by decreasing open probability (Laube et al., 1995; Laube et al., 2000). Threonine 112 determines the zinc inhibition on GlyR-mediated currents (Laube et al., 2000). Zinc inhibition can also be abolished by pretreatment with diethylpyrocarbonate, a histidine-specific modifying agent, or mutations of either histidine 101 or 109 on 1 subunits (Harvey et al., 1999).  Different neurosteroids have different effects on GlyRs. Progesterone, its precursor pregnenalone (PREG) and pregnenolone sulfate (PREGS) all inhibit glycine currents in cultured chicken or rat spinal cord neurons (Wu et al., 1990; Wu et al., 1997; Fodor et al., 2006). In heterologous system, PREGS and dehydroepiandrosterone sulfate (DHEAS) inhibit all of the examined 1, 2, 4, 1, 2 recombinant GlyRs (Maksay et al., 2001). α homo GlyRs is more sensitive than -containing GlyRs on DHEAS inhibition. PREG enhances homomeric α1 GlyR-mediated currents but has no effect on 1 or homomeric α2 GlyRs. However, progesterone inhibits homo α2 GlyRs mediated currents while exerting no effect on α1 containing GlyRs (Maksay et al., 2001). This study indicates that the effect of different steroids on α2 and 1 GlyRs are critical for neuronal development. The molecular determinants of neurosteriod action on GlyRs have not been determined.  G protein activation can potentiate GlyR-mediated currents via non-canonical  60  pathways of intracellular modulation (secondary messenger system). Overexpression of distinct G protein  subunits, as well as the  subunit complexes scavenger peptide ct-GRK2, significantly reduces the effect of G-protein activation on GlyRs, indicating that this effect is mediated by  subunits. With an inside-out patch configuration,  subunits alone increase GlyRs open probability, strongly suggesting  subunits may have direct interaction with intracellular domain of GlyRs (Yevenes et al., 2003).  1.5.4.2. Phosphorylation of glycine receptors PKA dramatically increases the GlyR-mediated currents by increasing the probability of the channel openings in neurons isolated from rat spinal trigeminal nucleus (Song and Huang, 1990). However, PKA also decreases GlyR-mediated response in neurons isolated from substantia nigra and ventromedial hypothalamus (Agopyan et al., 1993; Inomata et al., 1993). The potential phosphorylation site by PKA in 1ins is the first amino acid residue (serine) of the eight-amino acid insertion, and in  it is found at position 363 threonine in the TM3-TM4 domain. However, it is still unclear whether these sites can be directly phosphorylated, and if so, whether the phosphorylation changes the GlyRs functions.  In some studies, PKC decreases GlyR-mediated currents (Vaello et al., 1994; Nishizaki and Ikeuchi, 1995), but has no effect on GlyRs in similar heterologus systems (Mascia et al., 1998; Gentet and Clements, 2002). In rat hippocampal neurons, PKC dramatically increases GlyR-mediated currents (Schonrock and Bormann, 1995). The reason for these contradictory  61  results is not clear. 1 subunit contains a PKC phosphorylation consensus sequence at serine 391 in the TM4 domain (Ruiz-Gomez et al., 1991). In vitro assay also proves that  subunit can be phosphorylated by PKC (Vaello et al., 1994), indicating that PKC phosphorylation may affect GlyR functions. However, there is no direct evidence showing that serine 391 phosphorylation is responsible for changes in GlyR function.  Src enhances GlyRs mediated currents, whereas lavendustin A, a protein tyrosine kinase inhibitor, inhibits GlyRs mediated currents in hippocampal and spinal cord neurons (Caraiscos et al., 2002). Similar effects are mimicked in 1 recombinant but not in homomeric 1 GlyRs, indicating that  subunit may contain the phosphorylation site for Src (Caraiscos et al., 2002). Mutation of tyrosine413 in the large intracellular loop of the  subunit abolishes the effect of several tyrosine phosphorylation modulators (Caraiscos et al., 2002), strongly indicating that phosphorylation of tyrosine 413 of  subunit is responsible for the effect of Src on GlyRs.  1.5.5. Glycine receptors physiology and pathology Until now, no evidence has demonstrated that GlyRs play important roles in learning and memory. However, GlyRs are involved in many physiological and pathological processes, including locomotion, breathing rhythm control, retina development, inflammatory pain sensitization and startle diseases.  62  Two inhibition pathways mediated by GlyRs, namely reciprocal Ia inhibition and recurrent inhibition, are involved in stretching reflex. Ia inhibition relax the antagonistic muscles and coordinate the agonistic muscles by Ia interneuron-induced reciprocal action on  motoneurons. Recurrent inhibition reduces the fluctuation resulting from the Ia inhibition to localize the stretching reflex (Windhorst, 2007; Hernandes and Troncone, 2009). Respiratory rhythm is generated by respiratory neurons (Bonham, 1995; Feldman and Del Negro, 2006). Every respiratory neuron receives postsynaptic inhibition wave during silent periods. This inhibition wave is partly mediated by activation of GlyRs. The inhibition wave not only prevents respiratory neuron spiking during the silent period but also fine-tunes its spiking frequency during the active period (Bonham, 1995).  It is known that 2-containing GlyRs express abundantly in the retina. Taurine, an endogenous GlyRs agonist, potentiates the expression of rod photoreceptors, and this potentiation is inhibited by the GlyR antagonist strychnine. Overexpression of 2 GlyRs reduces proliferation of retinal progenitor cells and increases the production of rod photoreceptors. Acute knockout of 2 GlyRs reduces the production of rod photoreceptors, indicating that 2 GlyRs is required for the expression of rod photoreceptors in retina (Young and Cepko, 2004).  Symptoms of strychnine poisoning include not only disinhibition of motor neurons but also enhanced pain perception (Betz and Laube, 2006), indicating that GlyRs may regulate  63  pain perception. In 3 GlyRs knockout mice, prostaglandin E2 (PGE2) induces inhibition of GlyRs mediated currents and reduces pain sensitization, providing a new drug target for analgesia (Harvey et al., 2004).  Human startle disease is a neurological disorder characterized by an exaggerated response to unexpected stimuli (Rajendra and Schofield, 1995). Startle disease is caused by heritage mutations on GlyRs, which lead to reduced GlyR-mediated currents (Lynch, 2004).  1.6. Rationale, Hypothesis and Objectives Modulation of LGICs is directly related to synaptic plasticity which has long been proposed as a primary mechanism of learning and memory processes in CNS. Glutamate receptors, particularly the AMPA and NMDA receptors, are the major LGICs that mediate synaptic transmission at the vast majority of excitatory synapses in the mammalian brain. It is well accepted that AMPARs and NMDARs have critical roles in mediating expression and induction of LTP and LTD, respectively. However, the exact mechanism by which these receptors, particularly with regard to the exact roles of their different receptor subunits, contribute to LTP and LTD remains hotly debated. Nonetheless, recent advancements in our understanding of the detailed molecular mechanisms underlying the expression of LTP and LTD in the hippocampus have led to the development of some reagents that can distinguish between the induction and expression of LTP or LTD. The availability of these reagents has allowed investigators to begin to address the roles of hippocampal LTP or LTD in some learning and memory-related behaviours. By contrast,  64  though the fear conditioning paradigm mediated by lateral amygdala (LA) has been considered as a prominent model for studying learning and memory, the detailed mechanisms underlying either LTP or LTD in the LA region, in comparison with their counterparts in the hippocampus, are poorly understood. Here, we hypothesize that NMDA and AMPA receptor subunits may have differential roles in mediating the induction and expression of amygdala LTP and LTD. Specifically, we propose that NR2A and NR2B may be differentially required for the induction of LTP and LTD, whereas GluR1-dependent insertion and GluR2-dependent endocytosis of AMPARs may be respectively required for the expression of LTP and LTD.  As summarized in Chapter 2 and 3, these hypotheses were primarily tested using amygdala slices acutely prepared from rats (18-24 days). In chapter 2, to differentiate the roles of NR2A- and NR2B-containing NMDARs in the induction of LTP and LTD, NVP was used, which is the only available pharmacological tool to preferentially antagonize NR2A and Ro 25-6981, the highly selective NR2B antagonist. Because the selectivity of NVP on NR2A and NR2B is hotly debated, the selectivity of NVP on NR2A and NR2B was first tested under our experimental conditions by comparing NVP-sensitive component in the presence and absence of Ro 25-6981 to block the NR2B component. Having established the required specificity for both NVP and Ro 25-6981, the slices were treated with NVP or Ro 25-6981 prior to LTP or LTD induction to test the different roles of NR2A and NR2B. The results strongly suggest that NR2A- and NR2B-containing NMDARs may have preferential role in mediating induction of amygdala LTP and LTD respectively. NVP and Ro 25-6981 can  65  respectively inhibit LTP and LTD and may therefore be a useful tool for probing roles of LTP and LTD in amygdala-dependent learning and memory related behaviours.  Blockade of NMDARs also inhibit downstream of NMDARs. The best tool for dissociating roles of LTP and/or LTD in learning and memory should be those targeting the final step of LTP and LTD, i.e. the expression of LTP and LTD. In chapter 3, we confirmed AMPARs insertion and internalization were respectively involved in the expression of LTP and LTD by using a combination of electrophysiological and biochemical measurements of cell surface expression of AMPARs following the induction of LTP or LTD. GluR1 and GluR2 was then tested on whether they were differentially required for AMPAR insertion (LTP) and internalization (LTD) by using selective GluR1 and GluR2 C-terminal derived interfering peptides. Our results not only confirm the utility of GluR23Y peptide as a specific amygdala LTD inhibitor, but also provide the first evidence for the specificity of GluR1 peptide as novel inhibitor for amygdala LTP. Together with results presented in chapter 2 and 3, we provided evidence that suggests NVP and GluR1 peptide can be used as mechanistically distinct inhibitors for LTP, whereas Ro 25-6981 and GluR23Y peptide are specific inhibitors for LTD. The availability of these tools may allow us to dissociate the roles of LTP and LTD in fear learning and memory and amygdala related emotional disorders like anxiety with unprecedent specificity.  GABAARs and GlyRs are the two major inhibitory LGICs that mediate fast inhibitory  66  transmission at a vast majority of inhibitory synapses in the brain and spinal cord respectively. The classic inhibitory transmitter GABA and glycine respectively bind and activate GABA ARs and GlyRs. However, the observation by Ascher that glycine enhances NMDARs mediated excitatory effect blurs the boundary between inhibitory and excitatory transmitters (Johnson and Ascher, 1987) and suggests the potential existence of cross-talk between inhibitory transmitters and excitatory LGICs. Here, we hypothesize that there may also exist a similar cross-talk between excitatory transmitters and inhibitory LGICs.  Therefore, in the  present study, we set to investigate if glutamate can modulate functions of GABAAR and/or GlyRs, and if so, whether this modulation is mediated by a mechanism of direct binding to these receptors very similar to the glycine modulation of NMDARs. These were tested in Chapter 4 (GABAAR) and 5 (GlyRs) using electrophysiological recordings in neurons and in HEK cells transiently expressing recombinant receptors.  GABAARs mediate the tonic and phasic inhibition in the CNS. In chapter 4, we first investigated whether glutamate can potentiate GABAARs mediated currents with recombinant α1β2 or α1β2γ2 GABAARs in HEK cells. The results were consistent with our hypothesis that glutamate allosterically potentiates GABAAR-gated currents.  We then extended these  observations to hippocampal neurons maintained in primary culture, and discovered a similar potentiation of GABAAR-gated currents.  Moreover, we found the glutamate potentiation  affected both GABAAR-mediated tonic and phasic inhibition.  67  GlyRs mediate the majority of inhibition transmission in spinal cord, in chapter 5, we first tested whether glutamate has a modulating effect on GlyRs in spinal neurons maintained in primary culture using whole-cell recordings. We then determined potential mechanisms underlying the glutamate modulation using outside-out and cell attached configurations. These results allow us to conclude that glutamate can directly potentiate function of GlyRs without involvement of any diffusible second messenger system. This was further confirmed by the demonstration of similar glutamate-induced potentiation currents through recombinant GlyRs transiently expressed in HEK cells.  Thus, our results presented in the Chapter 4 and 5 provide strong evidence for a positive cross-talk between excitatory glutamate and inhibitory GABA/glycine transmitter systems.  These results not only blur the traditional distinction between excitatory and  inhibitory transmitters, but may also suggest a novel and efficient mode of homeostatic regulation of neuronal excitability via a reciprocal allosteric enhancement of each other’s receptor function. 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Zheng F, Gingrich MB, Traynelis SF, Conn PJ (1998) Tyrosine kinase potentiates NMDA receptor currents by reducing tonic zinc inhibition. Nat Neurosci 1:185-191.  Zhu JJ, Qin Y, Zhao M, Van Aelst L, Malinow R (2002) Ras and Rap control AMPA receptor trafficking during synaptic plasticity. Cell 110:443-455.  118  2. THE ROLE OF NMDA RECEPTOR SUBTYPES IN GOVERNING THE DIRECTION OF LATERAL AMYGDALA SYNAPTIC PLASTICITY1 2.1. Introduction Fear conditioning is a classical form of associative learning and can be formed by simple pairing of a neutral auditory tone (conditioned stimulus, CS) with electric shock (unconditioned stimulus, US)(Sah et al., 2003; Dityatev and Bolshakov, 2005). After acquisition, it can last for days, months or even years. In the Pavlovian fear conditioning paradigm, once the association between the CS and US is formed, the CS alone can then elicit a conditioned fear response (Pitkanen et al., 1997; McDonald, 1998; Doron and Ledoux, 1999; Blair et al., 2001). This process and outcome is very similar to long-term potentiation (LTP) of synaptic transmission, in which weak stimuli can gain potentiated response after one second to two minutes high frequency stimulation (Bliss and Collingridge, 1993). LTP also can be induced in the lateral amygdala, LA (Rogan and LeDoux, 1995; Rogan et al., 1997; Blair et al., 2001), a key loci for fear conditioning formation. Moreover, CS alone can elicit potentiation of responses (in the LA) after US pairing with CS in an anesthetized rat (Rogan et al., 1997). NMDARs antagonist APV, which blocks LTP in the hippocampus and other brain areas can prevent acquisition of fear conditioning (Kim et al., 1991; Nicoll and Malenka, 1999), strongly indicating that LTP plays an important role in the acquisition of fear conditioning. However, LTP and fear conditioning can be two parallel events. APV also blocks long-term depression  1  A version of this chapter will be submitted for publication. Wu, D.C., Yu S.Y. and Wang Y.T.. The role of  NMDA receptor subtypes in governing the direction of lateral amygdala synaptic plasticity. 119  (LTD)(Malenka and Nicoll, 1993), an opposite form of synaptic plasticity from LTP, making the role of LTP in fear conditioning unclear.  NMDARs contain NR1, NR2A, NR2B, NR2C and NR2D subunits(Dingledine et al., 1999). The major form of NMDARs in the hippocampus and amygdala are NR1/NR2A or NR1/NR2B. Recent studies show that NR2A and NR2B subunits play differential roles in LTP and LTD induction (Liu et al., 2004; Massey et al., 2004).  Blockade of NR2A-containing  NMDARs using NR2A preferential NMDAR antagonist NVP can abolish LTP induction in the hippocampus. Conversely, blockade of NR2B-containing NMDARs using an NR2B selective antagonist Ro25-6981 can abolish LTD induction in the hippocampus (Liu et al., 2004). Similar results are observed in anesthetized rats (Fox et al., 2006) as well as in freely moving rats (unpublished data). However, the relatively small window of specificity of NVP in differentiating NR2A and NR2B-containing NMDARs has challenged the utility of this antagonist as a specific LTD inhibitor.  On the LTD side, besides the specific NR2B  antagonist Ro25-6981, the interference peptide (Tat-GluR23Y) is able to block even the final step of LTD. Administration of these two LTD inhibitors can impair reversal learning and block the extinction of a learned fear response without affecting it’s acquisition(Dalton et al., 2008; Duffy et al., 2008)(unpublished data). In contrast, lacking a specific inhibitor of LTP has significantly hindered progress of further investigation on determining the role of LTP in learning and memory behaviors.  120  The lateral amygdala (LA) is the key locus for synaptic changes that underpins the long-term memory of conditioned auditory fears (McKernan and Shinnick-Gallagher, 1997; Blair et al., 2001). Here we aim to test whether NVP and Ro25-6981 can dissociate LTP and LTD in LA, with an aim of providing new tools for future efforts in dissociating the roles of LTP and LTD in fear conditioning acquisition.  2.2. Materials and Methods Male Sprague Dawley rats (17-24 days old) were placed under deep anesthesia and decapitated. The brain was rapidly removed and placed in ice-cold slicing solution containing (in mM): 87 NaCl, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, 25 glucose and 75 sucrose that was bubbled continuously with carbogen (95% O2 and 5% CO2) to adjust pH to 7.4. Coronal slices of 400 µm thickness containing the amygdala were produced using a vibrating blade microtome and recovered in an incubation chamber with carbogenated artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 2.5 KCl, 1.0 MgCl 2, 2.0 CaCl2, 1.25 NaH2PO4, 26 NaHCO3 and 25 glucose for 30 min at 34C, and were then returned to room temperature for at least 30 min before recording. All experiments were carried out at room temperature (22-24°C).  A single slice was transferred to a recording chamber perfused with carbogenated  121  ACSF at a flow rate of 1.5 - 2.5 ml/min and held beneath a platinum wire. Whole–cell patch clamp recordings were performed using the “blind” method from neurons in the dorsal part of the LA. Immediately after obtaining the whole cell configuration, current clamp was used to identify the firing pattern of the cells. Recording pipettes were filled with pipette solution containing (in mM): 122.5 Cs-gluconate, 17.5 CsCl, 2 MgCl2, 10 HEPES, 0.5 EGTA, 4 K-ATP and 5 QX-314, with pH adjusted to 7.2 by CsOH, (290-300 mOsm).  To isolate  NMDA-mediated component of EPSCs, perfusing solution was replaced by Mg 2+ free ACSF containing AMPA antagonist NBQX (5μM ), and bicuculline methiodide (10μM ). Once stable EPSCs were obtained, Ro25-6981 (3μM) and NVP-AAM007 (0.4 μM) were applied sequentially to assess the NR2B- and NR2A-components of EPSCs. The resistance of electrodes is typically 4-8 MΩ.  In LTP and LTD experiments, the membrane potential was held at -70 mV. Excitatory postsynaptic currents (EPSCs) were evoked by stimulating the auditory thalamic synaptic inputs (Weisskopf et al., 1999; Bauer et al., 2002; Yu et al., 2008) via a constant current pulse (0.05 mS) delivered through a tungsten bipolar electrode and recorded through a MultiClamp 700B amplifier (Axon Instruments). In addition, 10 μM bicuculline methiodide was included in the ACSF. Synaptic responses were evoked at 0.05 Hz except during the induction of LTP and LTD. After obtaining a stable EPSC baseline, either LTP or LTD was induced by applying either 200 pulses at 2Hz while depolarizing the cell to -5 mV (LTP), or 480 pulses at 1 Hz while holding the cell at -50 mV (LTD)(Yu et al., 2008).  The stimulation intensity of 122  induction was the same as that used during baseline recording. The induction of LTP and LTD were performed within 10 min after the establishment of a whole cell configuration to avoid washout of intracellular contents. Each cell was characterized immediately after obtaining the whole cell configuration.  The drugs used in all electrophysiological experiments were made up in stock solution and diluted 1000 times into the perfusion ACSF on the day of recording. NBQX, and the selective  NMDA  NR2B  antangonist  (+/-)-(R*,S*)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidine  propanol  (Ro25-6981) were obtained form Tocris Biosciences ( Ellisville, Missouri). The selective NMDA NR2A antagonist NVP-AAM007 (NVP) was obtained from Novartis Pharma AG, Base (Switzerland). All other chemicals were obtained from Sigma (St. Louis, MO).  2.3. Results 2.3.1. Differential roles of NR2A- and NR2B-containing NMDA receptors in the LTP and LTD at the thalamic input synapses in the LA As we have recently reported, we induced NMDA receptor-dependent LTP and LTD in the pyramidal neurons of LA with standard pairing protocols (Yu et al., 2008). As shown in Fig. 1A, stimulation of the auditory thalamic pathway to the LA with a train of 200 stimuli at 2 Hz while the postsynaptic neuron was held at -5 mV induced robust LTP in the LA (150.1 ±  123  18% of control EPSC amplitude 35 min after the paring protocol), whereas stimulation of the same pathway with a train of 480 pulses at 1Hz while holding the cell at -50mV resulted in stable LTD (the amplitude of EPSCs being 60.7 ± 9 % of the control levels 35 min after LTD induction; Fig. 1B). Both LTP and LTD are NMDA receptor-dependent(Yu et al., 2008). To test the role of different NR2 subunits in LTP and LTD induction in LA, we bath-applied the NR2A selective antagonist NVP (0.4 M) or the NR2B specific antagonist Ro25-6981 (3 M) prior to and during the LTP or LTD induction. 0.4 M of NVP prevented LTP induction (93.9 ± 8% of the control EPSCs 35 min after the paring protocol; Fig. 1A) while having little effect on the LTD induction (61.8 ± 19% of the control 35 min after LTD induction; Fig. 1D).  In contrast,  application of the NR2B selective antagonist Ro25-6981 (3M) failed to affect LTP induction (153.2 ± 25% of the control EPSCs 40 min after the induction stimulation; Fig. 1B), but prevented LTD induction the normalized EPSC amplitude was 90.1 ± 6% of the control 35 min after LTD induction; Fig. 1C). These results indicate that NR2A- and NR2B-containing NMDARs may be differentially required for the production of LTP and LTD.  2.3.2. The selectivity of NVP on NR2A and NR2B-containing NMDAR at the thalamic input synapses in the LA While Ro25-6981 is considered an NR 2B selective antagonist, that can selectively block the NR2B component of the EPSC (Fischer et al., 1997), the selectivity of NVP for NR2A has been more controversial (Frizelle et al., 2006). We therefore next examined their respective selectivity under our experimental conditions by comparing components blocked by 124  Ro25-6981 in the absence and presence of NVP. We found that bath application of 0.4 M NVP inhibited total NMDA EPSCs by 64.3 ± 12% (Fig. 2A and B), suggesting that NMDA component EPSCs at these synapses may be predominantly mediated by NR2A containing NMDARs. In comparison, application of 3 M of Ro25-6981 alone reduced 44.6 ± 22 % of total NMDA EPSCs (Fig 2A and B).  However, in the presence of NVP,  Ro25-6981  sensitive component was reduced to 29.58 ± 12.60%, suggesting a weak antagonizing action of NVP towards NR2B-containing receptors.  However, since NVP, unlike Ro25-6981, has no  effect on the induction of LTD, the small contaminate blockade of NR2B containing receptors may not affect the utility of NVP as a LTP inhibitor. Thus, under our experimental conditions, NVP and Ro25-6981 appear to respectively inhibit the induction of LTP and LTD.  2.4. Discussion The primary finding of this study is that NR2A and NR2B receptor activation results in bi-direction of synaptic plasticity, LTP and LTD in lateral amygdala. We report that blockade of NR2A receptor activation impairs LTP in amygdala slices without affecting LTD. Conversely, blockade of NR2B receptors disrupts LTD in the LA without impairing LTP.  Dissociation between NMDA receptor subunit activation and the direction of synaptic plasticity in the LA is in keeping with some(Liu et al., 2004; Massey et al., 2004; Woo et al., 2005; Yang et al., 2005; Izumi et al., 2006), but different from other (Hendricson et al., 2002; Morishita et al., 2007), previous observations from other brain regions. Many factors, 125  including area and developmental stage differences of the slice preparations and concentrations of the drugs used, can potentially contribute these apparent controversies. In particular, some of the controversies have been attributed to various concentrations of NVP used in these studies. In one of our previous studies (Liu et al., 2004), we found that NVP has about 100 times selectivity towards recombinant human NR1NR2A NMDARs expressed in Oocytes, and at 0.4 M concentrations, it fully blocked NR1/NR2A receptors with little effect on NR1/NR2B receptors.  However, a recent study using rat NR1/NR2A and  NR1/NR2B recombinant receptors over-expressed in HEK cells reported that NVP has less than 10 times selectivity of NR2A over NR2B.  They similarly report that the selectivity can  only be maintained at a concentration of 0.1 M and that selectivity is essentially lost at the concentration of 0.4 M (Berberich et al., 2005).  Differences in concentrations for selectivity  of NVP have also been observed for native NMDARs in slice preparations. Thus, while Berberich et al found that NR2A selectivity for NVP may only be achieved at the concentration below 0.1 M, Wu et al revealed that a full blockade of R2A containing NMDA receptors by NVP could not be achieved at a concentration of 0.1 M, but require a concentration of NVP at 0.4 M in acute cortical brain slices.  Importantly, they found that at  this higher concentration, NVP in fact has little effect on blocking NR2B-containing NMDARs (Wu et al., 2007).  These controversies highlight the importance of determining the subunit  specificity of the pharmacological agents under the experimental conditions.  However, given  the fact that Ro25-6981 has no effect on the induction of LTP, blockade of LTP by NVP observed in the present study may support the notion that activation of NR2A-containing  126  receptors may be preferentially required for the LTP induction at these synapses under present experimental conditions.  On the other hand, prevention of LTD by Ro25-9681 strongly  argues for an essential role of NR2B-containing receptors in LTD induction. The failure to affect LTD production by NVP, despite its partial blockade of NR2B-containing receptors, suggests not only that the induction of LTD does not require NR2A-containing receptors, but also that the small contaminant blockade of NR2B by NVP observed in the present study is not sufficient to affect LTD induction.  Together, the differential blockade of LTP and LTD by  NVP and Ro25-9681 suggests that these two antagonists may have certain utilities in probing roles of LA LTP and LTD in fear learning.  There is another controversy on the role of NR2B in LTP induction in LA slices. Miwa et al have recently reported that ifenprodil, another NR2B antagonist, can abolish LTP induction in LA slices (Miwa et al., 2008). However, it is important to note that ifenprodil actually displays a higher affinity for 1 adrenoceptors than it does for NR2B receptors(Chenard et al., 1991) and also has affinity for serotonergic (Chenard et al., 1991; McCool and Lovinger, 1995) and sigma opioid receptors (Karbon et al., 1990). In this regard, it is highly probable that the effects of ifenprodil on LTP in the LA may be mediated by other receptors in addition to NMDA receptors.  In addition, Higgins et al. have observed ifenprodil  to have behavioural effects which directly contrast with the more selective NR2B antagonists Ro25-6981(Higgins et al., 2005). The NMDA NR2B antagonist used in the present study, Ro25-6981, is an activity dependent and highly selective NR2B receptor antagonist (Fischer et  127  al., 1997; Mutel et al., 1998; Lynch et al., 2001) that is 32 times more selective for NR2B receptors than for the 1 adrenoceptors (Pinard et al., 2001). The use of this more selective compound had no effect on the induction of LTP in amygdala slices.  In summary, our findings provide a useful tool to dissociate LTP and LTD in amygdala and will accelerate the investigation of the role of LTP and LTD in amygdala related disorder like anxiety etc in vivo.  128  Figure 2.1. The role of NR2A and NR2B subunits in LTP and LTD in LA. Top: NR2A receptor activation is required for the induction of LTP at the thalamic pathway in the LA. A) LTP in LA neurons was reliably induced by pairing presynaptic stimulation (2 Hz, 200 pulses) with postsynaptic depolarization to -5 mV. Bath application of NVP-AAM077 (0.4 M) prevented LTP induction (n=8 for each group).  B) Bath application of Ro25-6981  (3mM) did not prevent LTP induction (n = 8 and 5, respectively for control and Ro25-6981 groups) The amplitude of individual EPSCs was normalized to the averaged amplitude of EPSCs during the 5 min baseline recordings just before LTP induction.  Representative traces  on the top of panel A and B are averaged EPSCs from three consecutive responses taken before (1) and 35 min after (2) LTP induction.  Bottom:  NR2B receptor activation is necessary for the induction of LTP at the thalamic  pathway in the LA. C) LTD in LA neurons was reliably induced by pairing presynaptic stimulation (1 Hz, 480 pulses) with postsynaptic depolarization to -50 mV. Bath application of Ro25-6981 (3 M) prevented LTD induction (n = 9 and 7, respectively for control and Ro25-6981 groups). D) Bath application of NVP-AAM077 (0.4 M) failed to prevent LTD induction. Representative traces on the top of panel A and B are averaged EPSCs from three consecutive responses taken before (1) and 35 min after (2) LTP induction.  129  130  Figure 2.2. NVP-AAM077 (0.4 µM) preferentially inhibits NR2A-containing NMDA EPSCs, and partially inhibits NR2B-containing NMDA EPSCs in LA neurons. A) Representative traces of sequential pharmacologically isolated NMDA EPSCs.  (Top)  Representative traces were taken before (black line), after NVP-AMM077 (0.4 µM: blue line), and after combined NVP-AMM077 (0.4 µM) and Ro25-6981 (3 µM) application (green line). (Bottom) Representative traces were taken before (black line), after Ro25-6981 (3 M: orange line), and after combined Ro25-6981 (3 µM) and NVP-AMM077 (0.4 µM) application (green line). The bar graph in (B) summarizes the data of sequential application of Ro25-6981 (3 M) and NVP-AMM077 (0.4 µM). NVP-AMM077 application first inhibited 64.29 ± 12.26% of total NMDA EPSCs, subsequent Ro25-6981 application inhibited 29.58 ± 12.60 of total NMDA EPSCs.  Ro25-6981 application first inhibited 44.55 ± 21.94% of total NMDA  EPSCs, the following NVP-AMM077 application inhibited 52.38 ± 25.62 of total NMDA EPSCs. NVP-AMM077 (0.4mM: hatched) also partially blocked NR2B containing NMDA EPSCs.  131  A control 25 pA  Ro25-6981 3 M  NVP AAM077 0.4 M NVP AAM077 0.4 M + Ro25-6981 3 M  50 pA  100 ms  100 ms  B  100  3  M of Ro25-6981 0.4  M of NVP-AAM077  Percentage of total NMDA current  90 80  NVP first  Ro first  70 60  50 40 30  20 10 0  N  VP  se  i ns  ti v  e  c  r ur  R  o  en  se  t  ve it i s n  r cu  re  nt  R  o  ns se  ve it i  N  c  VP  r ur  se  en  n  t  ti si  ve  c  r ur  en  t  132  2.5. 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Morishita W, Lu W, Smith GB, Nicoll RA, Bear MF, Malenka RC (2007) Activation of NR2B-containing NMDA  135  receptors is not required for NMDA receptor-dependent long-term depression. Neuropharmacology 52:71-76.  Mutel V, Buchy D, Klingelschmidt A, Messer J, Bleuel Z, Kemp JA, Richards JG (1998) In vitro binding properties in rat brain of [3H]Ro 25-6981, a potent and selective antagonist of NMDA receptors containing NR2B subunits. J Neurochem 70:2147-2155.  Nicoll RA, Malenka RC (1999) Expression mechanisms underlying NMDA receptor-dependent long-term potentiation. Ann N Y Acad Sci 868:515-525.  Pinard E, Alanine A, Bourson A, Buttelmann B, Gill R, Heitz M, Jaeschke G, Mutel V, Trube G, Wyler R (2001) Discovery of (R)-1-[2-hydroxy-3-(4-hydroxy-phenyl)-propyl]-4-(4-methyl-benzyl)-piperidi n-4-ol: a novel NR1/2B subtype selective NMDA receptor antagonist. Bioorg Med Chem Lett 11:2173-2176.  Pitkanen A, Savander V, LeDoux JE (1997) Organization of intra-amygdaloid circuitries in the rat: an emerging framework for understanding functions of the amygdala. Trends Neurosci 20:517-523.  Rogan MT, LeDoux JE (1995) LTP is accompanied by commensurate enhancement of auditory-evoked responses in a fear conditioning circuit. Neuron 15:127-136.  Rogan MT, Staubli UV, LeDoux JE (1997) Fear conditioning induces associative long-term potentiation in the amygdala. Nature 390:604-607.  Sah P, Faber ES, Lopez De Armentia M, Power J (2003) The amygdaloid complex: anatomy and physiology. Physiol Rev 83:803-834.  Weisskopf  MG,  Bauer  EP,  LeDoux  JE  (1999)  L-type  voltage-gated  calcium  channels  mediate  NMDA-independent associative long-term potentiation at thalamic input synapses to the amygdala. 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Introduction The lateral amygdala (LA) is the key locus for synaptic changes that underpins the long-term memory of conditioned auditory fears. In this Pavlovian fear conditioning paradigm, a neutral auditory tone (conditions stimulus, CS) is paired with electric shock (unconditioned stimulus, US)(Sah and Lopez De Armentia, 2003; Dityatev and Bolshakov, 2005) and once the association between the CS and US is formed, the CS alone can then elicit a conditioned fear response. The LA receives sensory information from both the auditory thalamus and cortex, and serves as the first stage of processing of auditory inputs to the amygdala (Pitkanen et al., 1997; McDonald, 1998; Doron and Ledoux, 1999; Blair et al., 2001). CS and US inputs converge onto specific cells in the LA, and the association of CS and US leads to long-lasting alterations in synaptic efficiency (McKernan and Shinnick-Gallagher, 1997; Repa et al., 2001). Depending on the stimulating protocols and the recording conditions used, bidirectional alterations of synaptic efficacy, long-term potentiation (LTP) and long-term depression (LTD) at these LA synapses have previously been reported (Rogan and LeDoux, 1995; Rogan et al., 1997; Heinbockel and Pape, 2000; Blair et al., 2001; Kaschel et al., 2004). Recent studies 2  A version of this chapter has been published. Yu S.Y., Wu D.C., Liu L., Ge Y., and Wang Y.T. (2008) Role of  AMPA receptor trafficking in NMDA receptor-dependent synaptic plasticity in the rat lateral amygdala. Journal of Neurochemistry. 106(2):889-99. 138  have provide some correlative evidence suggesting an important role for LA LTP in mediating the association between CS and US, and hence the formation and storage of the learned fear in the LA (Rogan and LeDoux, 1995; Rogan et al., 1997; Blair et al., 2001; Humeau et al., 2007). LA LTD is suggested to be mediating the extinction of the learned fear (Dalton et al., 2008). However, specific and causative roles of either LTP or LTD in various aspects of this fear conditioning paradigm remains largely uncharacterized due in part, to the lack of good specific inhibitors for the expression of LTP or LTD and a more detailed molecular understanding of the pathways activated by LTP or LTD stimuli.  Both LTP and LTD are most well-characterized at the CA1 synapses in the hippocampus. NMDA and AMPA subtypes of glutamate receptors may have distinct roles in the production of hippocampal CA1 LTP and LTD (Bliss and Collingridge, 1993; Malenka and Nicoll, 1999; Malenka and Bear, 2004). Activation of postsynaptic NMDARs is thought to be required for the induction of both LTP and LTD, whereas modification of AMPAR-mediated responses is crucial for the expression of LTP and LTD. How AMPAR-mediated responses are regulated during the expression of LTP and LTD remains hotly debated, likely involving alteration of both presynaptic release and postsynaptic responsiveness. Nonetheless, evidence accumulated in recent years strongly supports a rapid change in the number of postsynaptic AMPARs after LTP and LTD induction. Thus, alterations to AMPAR trafficking may be a major mechanism underlying the modification of postsynaptic responsiveness. While facilitated AMPAR insertion (exocytosis) into postsynaptic membrane may be at least in part responsible for the  139  expression of LTP, the stimulated clathrin-dependent endocytosis (internalization) of AMPARs from the postsynaptic membrane is the required last step in the expression of LTD (Luscher et al., 1999; Man et al., 2000b; Lu et al., 2001; Malinow and Malenka, 2002; Collingridge et al., 2004; Adesnik et al., 2005; Chowdhury et al., 2006; Toyoda et al., 2007).  These alterations to AMPAR trafficking may not be limited to synaptic plasticity at the hippocampal CA1 region, but are suggested to be common mechanisms shared by various forms of synaptic plasticity observed in the other brain areas (Wang and Linden, 2000; Brebner et al., 2005; Kakegawa and Yuzaki, 2005; Sun et al., 2005; Crozier et al., 2007). These recent advancements in our understanding of the detailed molecular mechanisms underlying the expression of LTP and LTD in the hippocampus have lead to the development of some reagents that can interfere specifically with either facilitated insertion or internalization of AMPARs, and thus, as specific inhibitors for the expression of LTP or LTD. The availability of these reagents has allowed investigators to begin to address the roles of hippocampal LTP or LTD in some learning and memory-related behaviours (Wong et al., 2007).  Though the fear conditioning paradigm mediated by LA has been considered a prominent model for studying learning and memory, the detailed mechanisms underlying either LTP or LTD in the LA region, in comparison with their counterparts in the hippocampus, are poorly understood. Several studies have shown that the induction of LA LTP requires activation of  140  NMDARs, L-type voltage-gated calcium channels (VGCCs), or group I metabotropic glutamate receptors depending on experimental conditions (Huang and Kandel, 1998; Weisskopf et al., 1999; Rodrigues et al., 2001; Bauer et al., 2002), LTD induction is dependent on either NMDA or group II metabotropic glutamate receptors (Heinbockel and Pape, 2000; Kaschel et al., 2004).  In contrast, our understanding of how the expression of either LTP or LTD is mediated in the LA has lagged behind. One such study suggests a role of increased AMPAR insertion in LA LTP (Rumpel et al., 2005). However, the involvement of facilitated endocytosis of AMPARs in the expression of LA LTD has not previously been investigated. In the present work, we have explored the possible involvement of rapid alterations in postsynaptic AMPAR trafficking in mediating the expression of LTP and LTD in the LA and provided evidence suggesting that, like hippocampal CA1 LTP and LTD, the NMDAR-mediated alteration of intracellular trafficking and plasma membrane surface expression of AMPARs is also an essential step in the expression of LTP and LTD in the LA area under these conditions.  3.2. Materials and Methods 3.2.1. Slice electrophysiology Male Sprague Dawley rats (16-26 days old) were under deep anesthesia and decapitated. The brain was rapidly removed and placed in ice-cold slicing solution containing (in mM): 87 NaCl, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, 25 glucose and  141  75 sucrose that was bubbled continuously with carbogen (95% O2 and 5% CO2) to adjust pH to 7.4. Coronal slices of 400 µm thickness containing the amygdala were produced using a vibrating blade microtome and recovered in an incubation chamber with carbogenated artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 2.5 KCl, 1.0 MgCl2, 2.0 CaCl2, 1.25 NaH2PO4, 26 NaHCO3 and 25 glucose for 30 min at 34℃, and were then returned to room temperature for at least 30 min before recording. All experiments were carried out at room temperature. A single slice was transferred to a recording chamber perfused with carbogenated ACSF at a flow rate of 1.5 - 2.5 ml/min and held beneath a platinum wire. Whole–cell patch clamp recordings were performed using the “blind” method from neurons in the dorsal part of the LA. Recording pipettes were filled with pipette solution containing (in mM): 122.5 Cs-gluconate, 17.5 CsCl, 2 MgCl2, 10 HEPES, 0.5 EGTA, 4 K-ATP and 5 QX-314, with pH adjusted to 7.2 by CsOH, (290-300 mOsm). The resistance of electrodes is typically 4-8 MΩ. Imediately after obtaining the whole cell configuration, current clamp was used to identify the firing pattern of the cells.  After cell characterization, the membrane potential was held at -70 mV. Excitatory postsynaptic currents (EPSCs) were evoked by stimulating the auditory thalamic synaptic inputs (Weisskopf et al., 1999) via a constant current pulse (0.05 mS) delivered through a tungsten bipolar electrode and recorded through a MultiClamp 700B amplifier (Axon Instruments). In addition, 10 μM bicuculline methiodide was included in the ACSF. Synaptic responses were evoked at 0.05 Hz except during the induction of LTP and LTD. After  142  obtaining a stable EPSC baseline, LTP was induced by either high frequency stimulation (HFS) (delivering three episodes of 100 pulses at 100 Hz in 20 second intervals while freely holding the cell) or with a paired protocol (200 pulses at 2Hz while depolarizing the cell to -5 mV). LTD was induced by either low frequency stimulation (LFS), 900 pulses at 1 Hz while freely holding the cell, or using a paired protocol by delivering 480 pulses at 1 Hz while holding the cell at -50 mV. The stimulation intensity of induction was the same as that used during baseline recording. The induction of LTP and LTD were performed within 10 min after the establishment of a whole cell configuration to avoid washout of intracellular contents.  The drugs used in all experiments were made up in stock solution and diluted 1000 times into the perfusion ACSF on the day of recording. All chemicals, including D-APV and bicuculline, were obtained from Sigma (St. Louis, MO). TeTx was a gift from Dr. William S. Trimble (Department of Biochemistry, University of Toronto).  3.2.2. Surface biotinylation assay Surface biotinylation assays were performed in acute amygdala slices obtained as described above. Briefly, after being recovered in an incubation chamber with carbogenated ACSF for 1 h, slices were transferred to a recording chamber perfused with ACSF at a flow rate of 1.5 - 2.5 ml/min. LTP or LTD were induced by HFS or LFS protocols (the same as electrophysiological recordings) respectively. After another 20 min of incubation in ACSF gassed with carbogen, a small region of the slice surrounding the recording electrode within  143  the LA was dissected and transferred to a well in a 6 well plate and was incubated on ice for 1 h in carbogenated ACSF containing 100 μM NHS-SS-biotin (Sigma, St. Louis, MO). Collected tissue was then washed three times for 5 min with ice-cold ACSF containing 10 mM glycine (Sigma, St. Louis, MO), and was immediately homogenized in 800 μl ice-cold lysis buffer (20 mM Tris-HCl (pH7.5); 1% TX-100; 50 mM NaCl; 1 mM EDTA; 0.1% SDS and a cocktail of protease inhibitors (Roche, Laval, QC). After incubation on ice for 30 min, the homogenate was centrifuged at 14,000 rpm at 4℃ for 10 min. Supernatants were collected. The biotinylated proteins from 300 μg of total protein in the lysate were precipitated with 60μl of Ultra-link immobilized Streptavidin beads (pierce, Rockford, IL), diluted with the addition of 800 μl ACSF, on a rotator overnight at 4℃. Precipitates were collected by centrifuging at 6,000 r.p.m. for 1 min, washed by ACSF three times, and then boiled for 5 min in 28 μl 1x sample buffer. Western blots using 30 μg tissue lysates were used as controls for the total protein.  Proteins eluted from the beads and total lysates were subjected to a 10% SDS-PAGE gel electrophoresis and were transferred to a PVDF membrane. The membrane was blocked by 5% milk for 1 h at room temperature, immunoblotted, sequentially stripped and re-probed on the same blot by using anti-GluR1 (lab raised rabbit polyclonal antibody against C-terminal816-889,, 1:2500) , anti-GluR2 (Chemicon, 1:1000), anti-β-tubulin (Sigma, 1:1000) and anti-transferring receptor (TfR) (Zymed, 1:1000) antibodies. Blots were developed using enhanced chemiluminescence detection (Amersham, Piscataway, VJ) and imaged with the  144  BioRad gel imaging system. Protein band densities were quantified with Image-J software (NIH), using identically sized rectangular objects that could accommodate all bands of the protein and expressed as a percentage of the control.  3.2.3. Statistical analysis The data were presented as mean ± SEM. Statistical analyses were performed using non-paired Student’s t-test. Critical value for statistical significance was set at p < 0.05.  3.3. Results 3.3.1. LTP induced by either HFS or a pairing protocol at thalamic input synapses in the LA is NMDAR-dependent Pyramidal neurons in the LA receives sensory information from both the auditory thalamus and cortex (Pitkanen et al., 1997; McDonald, 1998; Doron and Ledoux, 1999; Blair et al., 2001) and in both pathways, various forms of LTP have been characterized using protocols that largely differ from the ones commonly used in the CA1 of hippocampus (Huang and Kandel, 1998; Weisskopf et al., 1999; Bauer et al., 2002; Lee et al., 2002; Tsvetkov et al., 2002).  In the present study, we have focused our study on NMDAR-dependant LTP at  auditory thalamic synapses onto the pyramidal neurons in the LA region of amygdala brain slices. Neurons in the LA region were recorded with whole-cell recording configurations and the pyramidal-like neurons were characterized according to the firing pattern of the cells under current clamp right after breakthrough. All recorded cells included in the present work showed  145  various degrees of spike frequency adaptation and had relatively broad action potentials (Fig. 1B), typical characteristics for excitatory pyramidal-like neurons in the amygdala (Rainnie et al., 1991; Pare et al., 1995). EPSCs were then recorded under voltage-clamp conditions by electrical stimulation of auditory thalamic inputs as shown in Fig. 3.1A.  We first attempted  to elicit LTP at this input pathway with standard protocols used to induce NMDAR-dependent homosynaptic CA1 LTP in hippocampal slices (Malenka and Nicoll, 1999; Liu et al., 2004). We found that a single train of high frequency stimulation (100 Hz for 1 s) was not able to reliably induce LTP in amygdala (data not shown), but multiple trains of tetanus (three or more trains of 100 Hz for 1 s with an interval of 20 s) could reliably induce LTP in brain slices prepared within three hours (47.56 ± 17.23% increase in the amplitude of EPSCs 40 min after LTP induction; n=6; Fig. 3.1C).  Like hippocampal CA1 LTP (Bliss and Collingridge, 1993; Malenka and Nicoll, 1999), this LA LTP was also NMDAR-dependent. Thus, bath application of NMDAR antagonist D-APV (50 M) prior to, and during, the induction period had little effect on the basal EPSCs, but did prevent the induction of LTP (107.18 ± 9.33% of the control EPSCs 40 min after the induction stimulation; Fig. 3.1C). We next tried to induce LA LTP with a pairing protocol, another standard method used to induce NMDAR-dependent LTP in hippocampal CA1 synapses (Zhu et al., 2002; Liu et al., 2004).  In these experiments, the auditory thalamic  pathway to the LA was stimulated with a train of 200 stimuli at 2 Hz while the postsynaptic neuron was held at -5 mV. As shown in Fig. 3.1D, this pairing protocol was also capable of  146  inducing robust LTP in the LA (81.73 ± 26.95% increases in EPSC amplitude 40 min after LTP induction). This pairing protocol-induced LTP was also NMDAR-dependent as it was prevented by APV (Fig. 3.1D). Thus, the induction of LA LTP at thalamic synapses onto LA pyramidal neurons induced with either HSF or with a pairing protocol, under these experimental conditions, requires activation of NMDARs.  3.3.2. Expression of LTP in the LA requires postsynaptic vesicle-mediated exocytosis and is associated with increased cell surface expression of AMPARs Evidence accumulated in recent years strongly support the hypothesis that the postsynaptic expression of hippocampal CA1 LTP may be in part mediated by a rapid increase in the number of AMPA receptors expressed on the membrane surface (Malenka, 2003; Collingridge et al., 2004), due to  facilitated membrane-fusion of AMPAR-containing  vesicles with the postsynaptic plasma membrane (Shi et al., 1999; Hayashi et al., 2000; Lu et al., 2001; Shi et al., 2001; Rumpel et al., 2005). To test the involvement of a similar membrane fusion-dependent step in the expression of LA LTP, we applied enzymatically active, non-membrane-permeant recombinant light chain of the Clostridium tetanus neurotoxin (TeTx) into postsynaptic neurons via the recording pipette. This toxin blocks SNARE-mediated membrane fusion by selectively cleaving VAMP (Rothman, 1994; Lin and Sheng, 1998) and has previously been shown to prevent Ca2+-evoked dendritic exocytosis of postsynaptic vesicles containing new AMPARs (Maletic-Savatic et al., 1998) and LTP (Lu et al., 2001) in cultured neurons. Application of TeTx (200 nM) prevented LTP expression (95.64 ± 6.43% of  147  the control EPSC amplitude 40 min after the induction of LTP), but did not effect the basal EPSC response (Fig. 3.2A). In contrast, the control (boiled inactive TeTx; 200 nM) failed to prevent the expression of LTP (157.45 ± 10.96% of control EPSCs, n = 8) (Fig. 3.2B). These results suggest that a SNARE-mediated membrane fusion-dependent event, likely exocytosis of AMPARs, also plays an important role in the LTP expression in the thalamic input synapses of the LA.  Recent studies reported that the GluR1 subunit dominantly contributes to the expression of hippocampal LTP (Sheng and Hyoung Lee, 2003; Boehm et al., 2006). To test the involvement of GluR1 containing APMAR insertion in Amygdala LTP, we further applied TAT-GluR1wt peptides (YGRKKRRQRRRQS831INEAIRTST840LPRNS845G) which match the sequence of GluR1 C-terminal residue 830-848 into postsynaptic neurons via the recording pipette (200g/ml). This peptide blocked the expression of LTP (103.16 ± 8.93% of baseline, n = 6), but a non-related control peptide (YGRKKRRQRRRVYKYGGYNE) failed to do so (169.62 ± 21.78% of baseline, n = 6) (Fig. 3.2C).  To confirm an increased exocytosis of AMPARs during the expression of NMDAR-dependant LA LTP, we measured the changes in the levels of surface AMPARs in control and HFS stimulated (LTP-expressed) LA slice preparations using surface biotinylation. Quantification of the average density of individual bands obtained after immunoblotting indicated a dramatic increase in surface GluR1 expression (40.2 ± 10.1%, n = 6, p < 0.01) and  148  a small, but significant increase in GluR2 surface expression (14.8 ± 6.8%, n = 6, p < 0.05) 20 min after HFS (Fig. 3.2E). In contrast, the level of transferrin receptors (TfR; used as a surface protein loading control) expressed on the cell-surface was not notably altered. Probing for the intracellular proteins β-tubulin in each experiment demonstrated that the biotinylation was specific for surface proteins. Together, the TeTx and biotinylation results suggest that this LA LTP involves a rapid increase of AMPAR surface expression, which may contribute to the expression of LTP at these synapses.  3.3.3. LTD induced by LFS and Pairing protocols is NMDAR-dependent at thalamic input synapses in the LA In comparison with LTP, LTD has not been extensively studied in the LA region (Heinbockel and Pape, 2000; Kaschel et al., 2004). We, therefore, carried out a series of experiments to characterize LTD at the same auditory thalamic synapses onto the LA pyramidal neurons described for LTP above.  Following the establishment of stable baseline  EPSCs, induction of LA LTD was attempted using two LTD-induction protocols commonly used to induce hippocampal CA1 LTD in the slice preparation: low frequency stimuli (LFS, 900 pulses at 1 Hz) and pairing protocols (480 pulses at 1Hz while holding the cell at -50mV; (Mulkey and Malenka, 1992; Dudek and Bear, 1993; Zhu et al., 2002; Liu et al., 2004). As shown in Fig. 3.3, both LFS and the pairing protocols reliably induced LTD under our experimental conditions, decreasing the amplitude of EPSCs respectively to 58.07±6.11% and 60.21±4.44 % of the control levels 40 min post LTD induction. Bath application of D-APV  149  prior to, and during, the induction period prevented the induction of LTD elicited by either protocols, indicating that like LA LTP, the induction of LTD at these synapses, under these experimental conditions , is also dependent on the activation of NMDARs.  3.3.4. Regulated AMPAR endocytosis is required for expression of LTD in the LA Previous studies have provided strong evidence suggesting that the expression of various forms of LTD in various brain areas is mediated by a common mechanism that involves a rapid reduction in the number of postsynaptic AMPARs as a result of a facilitation of GluR2-subunit dependent clathrin-mediated endocytosis of AMPARs (Luscher et al., 1999; Man et al., 2000b; Wang and Linden, 2000; Bredt and Nicoll, 2003; Brebner et al., 2005). However, whether a similar mechanism is also involved in the expression of LA LTD remains unknown. To investigate this possibility, we made use of a synthetic peptide (GluR23Y – derived from the sequence in the carboxyl tail between 869YKEGYNVYG877). This peptide has previously been shown to prevent the regulated, but not constitutive AMPAR endocytosis (Ahmadian et al., 2004) and thereby block the expression of LTD, without affecting either the basal synaptic transmission or the expression of LTP in various brain areas (Ahmadian et al., 2004; Wang et al., 2004; Brebner et al., 2005). Postsynaptic application of the GluR23Y peptide (100 μg/ml, in the intracellular recording solution), while not affecting basal synaptic transmission (data not shown), completely abolished the expression of LA LTD (the normalized EPSC amplitude was 104.78 ± 6.51% of the control 40 min after LTD induction; Fig. 3.4A). The control inactive peptide GluR23A, whose tyrosine residues were replaced with  150  alanines (AKEGANVAG) failed to affect LTD (61.16 ± 6.52% of the control level 40 min after LTD induction; Fig. 3.4B). These results suggest that regulated AMPAR endocytosis is required for the LA LTD characterized here.  Consistent with a critical role of AMPAR endocytosis in the expression of LA LTD, biochemical analysis of the surface expression of AMPAR subunits likewise showed that the induction of LTD with a LFS stimulation produced a significant reduction of surface GluR2 (28.4 ± 6.3 %), and, to a lesser extent, GluR1 (11.9 ± 3.5 %) subunits (Fig 3.4D). The results further confirmed that, like its counterparts in many other brain areas, the expression of LTD in the LA region is mediated by a rapid and persistent reduction of the number of postsynaptic AMPARs as a result of facilitated GluR2-dependent, clathrin-mediated AMPAR endocytosis.  3.4. Discussion The lateral amygdala is one of the key loci, which underlies the long-term memory of auditory fear conditioning; a well accepted model to understand the role of synaptic plasticity in learning and memory. However, the molecular mechanisms for the synaptic efficiency changes during LTP and LTD in this region are still largely unknown. In the present study, we have provided strong evidence that supports 1) the induction of both LA LTP and LTD requires activation of NMDARs using standard HFS or pair-pulse protocols; 2) the expression of this NMDAR-dependent LA LTP is associated with a rapid increase in plasma membrane surface expression of AMPARs likely as a result of facilitated AMPAR exocytosis; and 3) the  151  expression of NMDAR-dependent LA LTD is mediated by the reduced number of postsynaptic AMPARs due to facilitated GluR2-dependent AMPAR endocytosis.  While it is well established that the induction of hippocampal CA1 LTP or LTD requires activation of postsynaptic NMDARs (Bliss and Collingridge, 1993; Malenka and Nicoll, 1999), the requirement of such NMDAR activation for the induction of LA LTP or LTD remains controversial.  Several studies have previously shown that the induction of LA  LTP requires either activation of NMDARs, or L-type voltage-gated calcium channels (VGCCs) or group I metabotropic glutamate receptors (Huang and Kandel, 1998; Weisskopf et al., 1999; Rodrigues et al., 2001; Bauer et al., 2002). Similarly, the induction of LTD in the LA has also been previously shown to be either NMDAR dependent or independent (Heinbockel and Pape, 2000; Kaschel et al., 2004).  These results strongly suggest that the  induction of LTP or LTD in LA may be mediated by multiple mechanisms. Which mechanism is activated may depend heavily upon experimental conditions such as the age of animals, recording conditions and the induction protocols used (Rodrigues et al., 2004). In the present work we were able to demonstrate that the protocols commonly used to induce NMDAR-dependent plastic changes in hippocampal CA1 synapses also reliably to induced LTP or LTD in the LA.  Moreover, both the LTP and LTD induced here could be prevented  by the NMDAR antagonist D-APV. Thus, similar to CA1 synapses in the hippocampus, the glutamatergic synapses in the LA also have the capacity to express classical NMDAR-mediated LTP and LTD with these well-defined induction protocols. Our results are  152  consistent with previous studies showing that NMDARs contribute to synaptic transmission in the LA (Mahanty and Sah, 1999; Weisskopf and LeDoux, 1999) and NMDAR-dependent synaptic plasticity occurs at thalamic input synapses to the LA (Bauer et al., 2002; Lee et al., 2002).  Further we have shown that alteration to AMPAR trafficking is strongly implicated in the expression of these synaptic alterations. Although mechanisms by which the activation of postsynaptic NMDARs and subsequent Ca2+ influx during the induction lead to the long-lasting expression of either LTP or LTD remains unclear, rapid changes in the number of postsynaptic AMPARs as a result of altered AMPAR insertion to or internalization from the postsynaptic membrane surface has been proposed to be a prominent mechanism for the expression of LTP and LTD, respectively, in many brain regions (Bredt and Nicoll, 2003; Malenka, 2003; Collingridge et al., 2004). The alteration of AMPARs during LTP or LTD has been most extensively studied in the hippocampus. Many studies have provided strong evidence supporting that AMPA receptors undergo constitutive recycling between the postsynaptic membrane and the intracellular compartment via SNARE-complex mediated vesicle exocytosis and clathrin-mediated endocytosis. Activation of NMDARs during the induction can in turn trigger cascades that cause specific alteration of postsynaptic insertion or endocytosis of AMPARs, thereby leading to a persistent increase or decrease in the number of AMPARs on the postsynaptic membrane surface during the expression of LTP or LTD (Luscher et al., 1999; Man et al., 2000a; Lu et al., 2001; Malinow and Malenka, 2002; Sheng  153  and Kim, 2002; Bredt and Nicoll, 2003; Malenka, 2003; Sheng and Hyoung Lee, 2003; Collingridge et al., 2004).  Consistent with a critical role of enhanced insertion of AMPARs in mediating the expression of LTP in the LA, our surface expression assays revealed a significant increase in the surface levels of GluR1 in amygdala slices 20 min post LTP  stimulation. The  requirement for facilitated insertion of AMPARs in the expression of LA LTP, was demonstrated by the ability of active TeTX to fully block LTP expression by specific disruption of SNARE-mediated membrane fusion dependent exocytosis in the postsynaptic neurons.  These results provide further evidence supporting the hypothesis that a rapid  increase in the number of postsynaptic functional AMPARs, as a result of specific enhancement of vesicle-mediated AMPAR insertion, may also be a critical mechanism responsible for the expression of LA LTP. The greater increase in GluR1, versus GluR2 AMPAR subunits, suggests that similar to hippocampal CA1 LTP (Sheng and Hyoung Lee, 2003; Boehm et al., 2006), the facilitated AMPAR insertion during LTP expression is mediated by a putative homomeric GluR1-dependent mechanism, at least during the early stage of LTP expression. The important role of GluR1 in LA LTP expression is also in a agreement with a recent study reporting that the GluR1 subunit dominantly contributes to amygdala LTP and fear conditioning (Humeau et al., 2007).  In keeping with hippocampal and other brain region synaptic alterations for  154  NMDAR-dependant LTD, biotinylation assays demonstrated that LA LTD also resulted in a significant reduction in the cell surface expression of AMPARs (Beattie et al., 2000; Lin et al., 2000; Luscher et al., 2000; Man et al., 2000b; Turrigiano, 2000; Wang and Linden, 2000; Carroll et al., 2001).  However, the reduction in the amount of GluR2 subunit, following LTD  induction, was much more dramatic than that in GluR1. These results strongly imply an important role for the GluR2 subunit in mediating the facilitated AMPAR endocytosis during the expression of LA LTD. Based on our previous study (Man et al., 2000b), we have proposed that there are two distinct clathrin-mediated endocytotic pathways (constitutive and regulated pathways) involved in the removal of AMPARs from the plasma membrane surface. The constitutive endocytosis pathway may be a common mechanism shared by all GluRs, and plays an important role in keeping a constant number of AMPARs on the cell surface. In contrast, the regulated pathway is GluR2 specific, and responsible for the rapid and long-term reduction of AMPARs on the cell surface. Consistent with this idea, subsequent studies from several laboratories have identified a number of unique sequences present in the carboxyl intracellular tail of GluR2 that are critical for GluR2-dependent AMPAR endocytosis. These studies have lead to the development of several interference peptides that, when delivered into postsynaptic neurons, can specifically block the regulated AMPAR endocytosis without affecting the constitutive endocytosis, thereby preventing the expression of LTD in many brain areas (for review see (Wang, 2008). Here, using one of these such peptides, the GluR23Y peptide, we were able to demonstrate that, similar to mechanisms involved in the expression of LTD in many other brain areas, the expression of LA LTD also involves a GluR2-dependent  155  facilitation of AMPAR endocytosis.  Synaptic plasticity in the LA has been proposed as a prominent cellular mechanism responsible for certain forms of learning and memory encoded by neurons in this area (Rogan and LeDoux, 1995; Rogan et al., 1997; Blair et al., 2001; Dityatev and Bolshakov, 2005; Humeau et al., 2007; Sigurdsson et al., 2007).  Consistent with this idea, evidence  accumulated in recent years has provided much support for a critical role of NMDARs in fear-conditioning releated learning and memory (Miserendino et al., 1990; Kim et al., 1991; Kim et al., 1992; Fanselow and Kim, 1994; Lee and Kim, 1998; Lee et al., 2001; Langton et al., 2007). However, because NMDARs participate in mediating synaptic transmission and is also required for both LTP and LTD, these previous studies provided limited clues about the exact roles of LTP or LTD in this learning paradigm. Recently, the availability of molecularly specific reagents such as the GluR3Y peptide used in this study, has allowed investigators to begin to address the detailed roles of LTP or LTD in some learning and memory-related behaviours (Wong et al., 2007; Dalton et al., 2008; Duffy et al., 2008; Wang, 2008).  In the present study, we have provided further evidence indicating that the induction and expression of LTP and LTD in the LA may share some common molecular mechanisms for LTP/LTD with other brain regions. These include the requirement of NMDARs in the induction and alteration of AMPAR trafficking in the expression. Thus, agents developed to specifically interfere with the induction or expression of either NMDAR dependant LTP or  156  LTD may likely be useful tools in probing the role of LA LTP/LTD in LA-related learning and memory.  It can be expected that studies such as the present work, work aimed at further  advancing our understanding of the molecular mechanisms underlying LA LTP and LTD, will have a significant impact on our efforts to discern the respective roles of LTP and LTD in some learning and memory-related behaviors mediated by the LA and in other brain structures.  157  Figure  3.1.  The  induction  of  LTP  at  the  thalami-amygdale  synapses  is  NMDAR-dependent. A. Photograph showing the positions of both stimulating and recording electrodes in an amygdala slice (LA: lateral amygdala; CeA: central amygdala).  EPSCs evoked by electrical  stimulation of auditory thalamic inputs were recorded under whole-cell voltage-clamp mode at a holding potential of -70 mV. B. LA excitatory pyramidal-like neurons under current-clamp mode exhibits typical spike frequency adaptation and relatively broad action potentials in response to a depolarizing current pulse.  C.  High frequency stimulation induced LTP (HFS,  3 trains of 100Hz for 1 s with an interval of 20 s) in the LA neuron requires activation of NMDARs. LA Neurons were recorded under voltage-clamp mode at a holding membrane potential of -70mV throughout recordings except for the HFS period during which the recording was switched to current-clamp mode. The ability of HFS to induce LTP was prevented by bath application of NMDAR antagonist D-APV (50 μM; n = 6 for each group). D. LTP in LA neurons were also reliably induced by pairing presynaptic stimulation (2 Hz, 200 pulses) with postsynaptic depolarization to -5 mV and the LTP was also abolished by D-APV (n = 8 for each group).  The amplitude of individual EPSCs was normalized to the  averaged amplitude of EPSCs during the 5 min baseline recordings just before the LTP induction.  Representative traces on the top of panel C and D are averaged EPSCs from ten  consecutive responses taken before (1) and 40 min after (2) LTP induction.  158  159  Figure 3.2. HFS-induced LTP at the thalami-amygdale synapses requires postsynaptic AMPAR insertion. A and B. Postsynaptic application of active TeTx (200 nM, n = 7), but not heat-inactivated TeTx (n = 8), prevented the expression of LTP, suggesting the involvement of a SNARE-dependent postsynaptic membrane fusion event (exocytosis). C. Postsynaptic application of GluR1 C-terminal peptide (200g/ml, n=6), but not non-related control peptide (200g/ml, n=6) blocked the expression of LTP, confirming the involvement of GluR1 containing receptor insertion during LTP expression. D. Bar graph summarizing data obtained at 40 min after LTP induction depicted in A, B and C. E. HFS-induced LTP is associated with a specific increase in cell surface expression of AMPARs. Surface biotinylation assays were performed in control slices or slices 20 min after LTP induction (by HFS) and the level of cell surface (biotinylated) AMPARs were then quantified by sequential probing the same membrane of GluR1 and GluR2 on western blots (Left). Reprobing the same membrane for transferrin receptors (TfRs) and β-tubulin (-Tub) served for controls respectively for sample loadings and specific surface protein labeling. Pooled data from six individual experiements are summarized in the histogram on the right.  Note: the asterisk (*) represents statistically  significance differences from control values (p < 0.05).  160  161  Figure 3.3. The induction of LTD at the thalamic pathway in the LA is NMDAR-dependent. Representative traces on the top of each panel are averaged EPSCs from ten consecutive responses taken before (1) and 40 min after (2) LTD induction. A and B. Bath application of D-APV prevented LTD produced either by LFS (900 pulses at 1 Hz; n = 6 for each group; A) or by pairing protocol (1Hz, 480 pulse stimuli while holding the cell at -50mV; n = 7 and 8, respectively for control and APV groups; B).  162  163  Figure 3.4. LTD induced at the thalamic pathway in the LA is mediated by GluR2-subunit dependent endocytosis of postsynaptic AMPARs.  A and B. Postsynaptic  application of GluR23Y peptide (100 μg/ml; n = 6; A), but not the control GluR23A peptide (100 μg/ml; n = 5; B), prevented the expression of LTD. C. 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Introduction In the central nervous system (CNS), excitatory transmitters activate excitatory receptors to depolarize the target cell and to increase the probability of firing an action potential; whereas inhibitory transmitters normally exert the opposite action by activating inhibitory receptors, to hyperpolarize the cell and to reduce neuronal firing.  Reciprocal  interactions between excitatory and inhibitory systems have been well documented, ranging from cellular to network levels. For instance, activation of metabotropic excitatory amino acid receptors can modulate inhibitory synaptic transmission via G-protein signaling (Patenaude et al., 2003; Puyal et al., 2003). The excitatory and inhibitory elements in a neuronal network can interact with each other via a feedback or recurrent inhibitory circuit (Windhorst, 2007). At the single-molecule level, however, it is rare for an inhibitory transmitter to directly act on an excitatory receptor, or vice versa. One exception to this is that the inhibitory transmitter glycine can directly bind to NMDA receptors, and thereby functions as a co-agonist of the receptor (Kleckner and Dingledine, 1988). It is however unclear whether the direct interaction between excitatory and inhibitory systems at the transmitter-receptor level is a general phenomenon. GABAA receptors mediate the chief inhibitory neurotransmission in the CNS.  3  A version of this chapter will be submitted for publication. Wu D.C., Liu J., and Wang Y.T. Allosteric  potentiation of GABAA receptor chloride channels by glutamate. 174  Also, previous studies have shown that glutamate can potentiate GABA-induced currents in neurons although the underlying mechanism remains unknown(Stelzer and Wong, 1989). Here we attempted to test whether glutamate can modulate function of GABAA receptors by an allosteric interaction with the GABAA receptor itself.  4.2. Methods 4.2.1. Neuronal culture Cultured hippocampal neurons were prepared from the brains of D18 fetal Wister rats. Tissues were digested with a 0.25% trypsin solution (Invitrogen) for 25 min at 37 °C, and then mechanically dissociated using a fire-polished Pasteur pipette. Next, the cell suspension was centrifuged at 2500 ×g for 50 s and the cell pellets resuspended in DMEM containing 10% Fetal Bovine Serum (FBS; Sigma-Aldrich). Cells were seeded on poly-D-lysine-coated 24-well coverslips at a density of 2.5 ×105 cells/well. Cultures were maintained in a humidified incubator with 5% CO2 at 37 °C. After 24 hrs, plating medium was changed to Neurobasal medium supplemented with B-27 supplement and L-glutamine and the media changed twice weekly thereafter. Cultured neurons were used for electrophysiological recordings 10-14 days after plating.  4.2.2. HEK293 cell culture and transfection HEK293 cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS. Cells were grown to 20% confluence and transiently transfected using Lipofectamine 2000 175  (Invitrogen) according to the manufacturer's protocols. Cells were transfected with a pBK-CMV NB-200 expression vector containing a rat recombinant GABAAR α1, 2 and 2 or a combination of vectors of α1 and β2 subunits. The transfection ratio with α1/2/2 and 1/ plasmid was 2:2:1 and 1:1. pcDNA3-GFP was also co-transfected along with GABAAR subunits as a transfected marker, in order to facilitate the visualization of the transfected cells during electrophysiological experiments. 15-20 hours following transfection, cells were re-plated on glass coverslips and cultured for an additional 15-24 hrs before whole-cell patch-clamp recordings.  4.2.3. Electrophysiology  Whole cell patch-clamp recordings: Whole-cell recordings were performed under voltage-clamp mode using an Axopatch 200B or 1D patch-clamp amplifier (Molecular Devices). Whole-cell currents were recorded at a holding potential of -60 mV unless indicated elsewhere, and signals were filtered at 2 kHz, digitized at 10 kHz (Digidata 1322A). Recording pipettes (3-5 MΩ) were filled with the intracellular solution that contained (mM): CsCl 140, HEPES 10, Mg-ATP 4, QX-314 5, pH 7.20; osmolarity, 290-295 mOsm. BAPTA (10 mM) was added in the intracellular solution (otherwise specified). The coverslips were continuously superfused with the extracellular solution containing (mM): NaCl 140, KCl 5.4, HEPES 10, MgCl2 1.0, CaCl2 1.3, glucose 20, pH 7.4; osmolarity, 305-315 mOsm. For recording of GABA currents in HEK293 cells, fast perfusion of GABA and/or glutamate analogues were accomplished with a computer-controlled multibarrel fast perfusion system (Warner 176  Instruments). To evoke GABA mediated currents, a glass pipette filled with GABA (100 µM in the extracellular solution) was placed near neurons under recording. Pressure pulses (20-60 ms) were delivered to eject GABA via a Picospritzer at 30 s or 60 s intervals. For recordings of mIPSCs and GABA evoked postsynaptic currents in cultured neurons, strychnine (10 µM), CNQX (10 µM) and TTX (0.5 µM) were added in the extracellular solution to block glycine receptor currents and minimize the activation of ionotropic glutamate receptors and voltage-gated sodium channels, respectively. All experiments were performed at room temperature.  4.2.4. Data analysis  Values are expressed as mean ± SEM (n = number of experiments). The two-tailed Student’s test was used for statistical analysis and P values less than 0.05 were considered statistically significant. Dose–response curves were created by fitting data to Hill equation: I = Imax/(1+EC50/[A]n), where I is the current, Imax is  the maximum current, [A] is a given  concentration of agonist, n is the Hill coefficient.  4.2.5. Chemicals N-methyl-D-aspartate  (NMDA),  D-2-amino-5-phosphonovaleric  α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate  (AMPA),  kinate  acid  (APV),  acid  (KA),  6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and MK-801 were purchased from Torcis (Ellisville, Missouri, US). Gluamate, gamma-Aminobutyric acid (GABA) and strychnine were  177  purchased from Sigma-Aldrich. Bicuculline methobromide was purchased from Alexis Biochemicals.  4.3. Results GABAA receptors are pentameric protein complexes with various subunit combinations. The 2 is the minimally required subunit combination for the formation of a functional GABAA receptor, whereas the 2 subunit combination is most abundantly expressed in native receptors in the brain (Olsen and Sieghart, 2008). Therefore, we first tested whether glutamate can modulate GABAA currents in HEK293 cells transiently expressing recombinant rat GABAA, and 2 subunits. Whole-cell voltage-clamp recording were performed on the transfected cells with a chloride-based pipette solution at a holding potential of -60mV. Fast perfusion of GABA (1M, 4s) induced an inward current, confirming the expression of functional GABAARs in these cells following the transient transfection (Fig 4.1 A and B. Application of 1 mM glutamate alone did not induce any detectable current (Fig 4.1 A and B), indicating that there are no functional glutamate-sensitive ion channels in HEK cells, and GABAARs cannot be activated directly by glutamate. Pretreatment with 1 mM of glutamate dramatically enhanced 1 M GABA-induced current (331.2 ± 4.5% of control, n=6, P<0.001) compared with GABA alone, suggesting that glutamate acts as either a potent allosteric modulator or a co-agonist to GABAA receptors. 100 M of bicuculline completely abolished the glutamate-potentiated current (0.015±0.014% of control GABA current, n=6, p<0.05), confirming that the increased current by glutamate is directly mediated by GABA A receptors,  178  but not by any other ion channels. Glutamate enhanced 1m GABA induced currents with an EC50 180 M and with a lowest effective dose (20% potentiation) of about 30 M (figure 4.1C). 100 M glutamate shifted the dose-response curve of GABA-induced current toward lower concentrations, and decreased the EC50 of GABA from 13.2 M to 5.3 M (Figure 4.1 D). The effect of glutamate is more potent on the currents induced with lower concentrations of GABA (fig 4.1 D). In contrast, when GABA currents were induced by higher concentrations of GABA, such as 100 mM, which induces almost the maximal response, the potentiation effect of glutamate is much smaller (Figure 4.1D).  Next we sought to determine whether other glutamate analogues also exhibit a similar regulatory effect on GABAA currents. 100 M of NMDA, kainate, AMPA enhanced GABA (1 M) currents to 171.7%, 188.7%, and 165.9% of control, respectively (Figure 4.2 A) in the 1 transfected cells. This suggests that a common feature in the structure of glutamate analogues may be important for their allosteric modulation of GABAA receptors. We next tested whether CNQX and APV, which are the competitive non-NMDA and NMDA receptor antagonists respectively, also display enhancing effect on GABA currents. CNQX did not enhance GABA induced current at normal working concentration 10 M (105.6±6.5 of control, P>0.05, N=4, Figure 4.2 D) and even at very high concentration 1mM (93.5±1.3 % of control, P>0.05, n=5, Figure 4.2 D). In contrast, 100 M of APV interestingly exhibited a similar effect as glutamate analogues, as it increased the GABA current to 173.3±3.8% of control (P<0.001, n=10, Figure 4.2C). This is possibly because APV acts at the same site as glutamate analogues  179  on NMDA receptors, so that APV may allosterically modulate GABAARs in a similar way as other glutamate analogues. We then tested the effect of non-competitive NMDAR antagonist MK-801 on GABA currents, because MK-801 blocks NMDAR by binding to the channel pore region, but not the agonist binding site, we suspected that it may not act like a glutamate anologue.  Indeed, we found that 10 M of MK-801 failed to exhibit any effect on GABA  currents (96.9±5.8% of control, n=5, p>0.05, Figure 4.2 C). Then the question is raised whether the inability of CNQX to enhance GABA currents is due to a lack of direct binding to the glutamate binding site on the GABAAR. We found that 1 mM of CNQX blocked the potentiation effect of glutamate (107.3±0.8% of control with glutamate along, n=5, p<0.01 compared) or APV (111.1±5.6% of control with APV along, n=5, p<0.01) on GABA induced current (Figure4.3 A and B), indicating that both glutamate and APV may share a same acting site on GABAARs, which can be competitively blocked by CNQX. In addition, these results suggest that APV can be used as a substitute for glutamate to examine the potentiation effects on GABAARs in neurons, because APV can exert same potentiation effect of glutamate, but without activation of mGluR and other iontrophic glutamate receptors.  Next we investigated whether the 2subunit combination of GABAARs also showed a similar response to glutamate analogues. In the 1 transfected HEK cells, GABA-induced current (1 M) was enhanced by 100 M of glutamate, NMDA, kainate, AMPA, and APV to 356.0+46.0%, 267.6+20.5%, 388.3+61.0%, 339.2+38.0%, and 254.9+33.0%, respectively (Figure 4.2 B). Taken together, our data in HEK cells strongly  180  indicate glutamate can positively modulate GABAARs by directly acting on GABAAR itself, rather than via other indirect pathways involving any known glutamate receptors. At least the composition of 1 and 2 subunits of GABAARs receptor is sufficient for this effect, whereas  subunit is not necessary for this effect.  The composition of native GABAARs is diverse in the CNS, even in a given brain area. 1 subunit is predominant in synaptic sites and 6 subunit is predominated in extrasynaptic sites. Therefore, we investigated whether glutamate can enhance GABA induced currents in cultured hippocampal neurons. To exclude the interference from activation of metabotropic and ionotropic glutamate receptors, we used APV, instead of glutamate, as the glutamate-like ligand to examine its effect on neuronal GABA currents. APV reversibly enhanced GABA induced currents in a dose-dependent manner in neurons, with an EC50 of 184±23 M (Figure 4.4), confirming that, like what we observed in HEK recombinant expression systems, glutamate-like ligands can potentiate function of native GABAARs.  This modulation can be  achieved through either allosteric regulation or changing chloride driving force by regulating chloride transporters. To exclude the possibility that the effect of APV is due to an alteration of chloride transporter activity, we tested GABA currents under various holding voltage conditions with or without APV (Figure 4.4). The reversal potential of GABA current was 0 mV and was not be affected by APV, implying that the effect of APV on neuronal GABA currents is not due to a change in the chloride concentration gradient between intracellular and extracellular solutions. Taken together, our results in neurons strongly suggest that glutamate,  181  the major excitatory transmitter can act as fast, allosteric inhibitory receptor modulator in CNS.  The action of GABA on GABAARs can produce two types of inhibition on neuronal excitability, phasic inhibition and tonic inhibition. The phasic inhibition is mediated by activation of postsynaptic GABAARs, and can be studied by recording miniature inhibitory postsynaptic currents (mIPSCs). Application of 200 M of APV enhanced mIPSC amplitude (control: 35.5±2.3pA, AVP application: 43.3±2.5pA P<0.001, n=8, Figure 4.5) without changing the frequency (0.73±0.16, APV application: 0.72±0.12 P>0.05, n=7), suggesting this effect is through postsynaptic modulation but not changes in presynaptic GABA release. The fast on and off property of this regulation also is consistent with the rapid allosteric modulation mode.  The tonic inhibition by GABA is mainly mediated by activation of extrasynaptic GABAARs, which contains subunits (Semyanov et al., 2004; Belelli et al., 2009). Extrasynaptic GABAARs are very sensitive to low concentrations of ambient GABA. As shown in Fig. 4.5, bath application of 200 M of APV produced an upward shift in the baseline currents (22.05 ±2.78 pA after APV, compared with 12.15 ±0.82 pA in control, p<0.01, n=6, Figure 4.5). These upward shifts represented APV-enhanced tonic GABA currents, as it was blocked by 10 M of bicuculline (n=6, Figure 4.5).  The enhancement of tonic GABA  currents is more than 80%, indicating that extrasynaptic GABAARs are more sensitive to APV  182  than synaptic GABAARs, or the concentration of GABA is lower at extrasynaptic sites. Taken together; our results on GABA phasic and tonic inhibition suggest that the effect of glutamate on GABAARs maybe play an important role in controlling basic neuron excitability.  4.4. Discussion Our study provided a novel mechanism of the transmitter-receptor level interaction between excitatory and inhibitory systems. The potentiation effect of glutamate on GABA mediated current was demonstrated on HEK293 cells transiently transfected with heterogeneous GABAARs. Given that HEK293 cells are a simple system that do not contain any glutamate-activated receptors, our results strongly suggest that glutamate potentiates GABAARs mediated current most likely via an allosteric modulation but not through activation of any known classic glutamate receptors. The observation that glutamate potentiation can be mimicked by most ionotropic glutamate receptor ligands suggests that this putative glutamate-binding site on GABAARs is pharmacologically less stringent than other glutamate binding sites on iontropic glutamate receptors. Also, high doses of CNQX did not enhance GABA currents and can block the potentiation effect of both glutamate and APV. This strongly indicates glutamate and APV act at same site on GABAARs. Furthermore, APV potentiates GABAA receptor mediated mIPSCs and tonic GABA current in neurons, providing putative effect of glutamate on synaptic or non-synaptic GABA systems, highlighting the physiological relevance of the modulation.  183  It is interesting to note that glutamate has also been previously reported to inhibit GABA-induced response (Stelzer et al., 1987).  Thus, glutamate can potentiate or inhibit  GABA induced-current under different conditions. The inhibition of GABA induced current is due to the high elevation of intracellular Ca2+ by activation of glutamate receptors (Connor et al., 1988). In order to eliminate this calcium-dependent inhibition, Stelzer and Wong previously studied glutamate potentiation of GABA-induced currents after buffering intracellular Ca2+. However, the involvement of glutamate receptors in this study cannot be ruled out without the inhibition of glutamate receptors. Our observation that glutamate potentiates GABA current in HEK cells strongly suggest that glutamate can enhance GABA current by a mechanism independent of glutamate receptors and this is further supported by the fact that similar potentiation can be observed with a number of glutamate ligands including APV that act on different glutamate receptors.  It still remains unclear how glutamate reaches GABAA receptors. Glutamate can be released from glutamatergic terminals of neurons or from astrocytes. Synaptically released glutamate can spill over to adjacent GABA synapses or extrasynapses and enhance GABAAR-mediated inhibition as feedback. Glutamate released from astrocytes can also reach both adjacent synaptic and extrasynaptic sites to enhance both phasic and tonic inhibition. Besides these two pathways, there is also evidence suggesting that glutamate can be co-released with GABA in GABAergic terminals in the auditory cortex (Gillespie et al., 2005), which strongly supports that glutamate may affect GABAAR mediated fast synaptic inhibition.  184  Figure 4.1. Glutamate potentiates GABA currents in a dose dependent manner in HEK293 cells expressing rat recombinant GABAARs. HEK cells were transiently transfected with rat 1/2/2 hetermetric GABAARs. Whole-cell voltage-clamp recordings were performed with a Cl- based intracellular recording solution at a holding potential of -60mV. A and B. Glutamate potentiates bicuculline-sensitive GABAARs-mediated chloride currents. Representative traces were current induced by fast perfusion of glutamate through a computer-driven multi-barrel fast perfusion system. Fast perfusion of glutamate 1 mM (Glu) alone produced no detectable currents, but potentiated currents induced by GABA 1 M (331.2 ± 4.5% of control GABA current, n=5, P<0.01). Bicuculline totally abolished the potentiated current, which confirmed that glutamate potentiated GABAA-mediated chloride current, but not other unknown cation channel mediated cation currents. C. The dose response-curve of glutamate on 1um GABA induced current with EC50=180 M. D. 100 M glutamate left-shifted the GABA dose response curve, and reduced the EC50 from 13.2 M to 5.3 M. The maximum potentiation effect can be achieved when GABA concentration is lower. At saturate concentration of GABA, 100 m glutamate did not potentiate on GABA currents.  185  186  Figure 4.2. Glutamate analogs potentiate GABA current in HEK293 cells expression rat recombinant GABAARs. HEK cells were transiently transfected with rat 1/2 (B) or 1/2/2 (A,C,D) hetermetric GABAARs.  A and B. Normalized GABA current amplitude  graphs summarizing effects of glutamate and its various analog ligands on currents induced by GAB A in HEK293 cells overexpression 1/2/2 (A) and 1/2 (B) heteromeric GABAARs. The amplitudes of peak currents were normalized to that of currents induced by GABA alone and the number of cells in each of the group is indicated on top of each bar. C. Competitive NMDARs antagonist APV 100 M potentiated GABA current to 173.3±3.8% of control (P<0.001, n=10), whereas non-competitive MK-801 10 M of MK-801 failed to exhibit any effect on GABA currents (96.9±5.8% of control, n=5, p>0.05). D. Competitive non-NMDARs antagonist CNQX failed to exhibit any effect on GABA current at normal working concentration 10 M (105.6±6.5 of control, P>0.05, N=4) and at very high concentration 1 mM (93.5±1.3 % of control P>0.05, n=5). Representative traces were currents induced by GABA (1 M) with or without 100 M APV, 10 M MK-801, 10 m CNQX and 1 mM CNQX.  187  188  Figure 4.3. CNQX abolished potentiation of glutamate and APV on GABA currents in HEK293 cells expression rat recombinant GABAARs. HEK cells were transiently transfected with rat 1/2/2 heterometric GABAARs. A. CNQX 1 mM reduced potentiation of glutamate 100 M on 1 M GABA induced current from 197.9±13.2% of control to 107.3±0.7% of control (n=5, P<0.01). Representative traces are currents induced by GABA (1 M) or GABA (1 M) plus CNQX (1 mM) with (red line) or without (black line) 100 M glutamate. B. CNQX 1mM reduced potentiation of APV 100uM on 1uM GABA induced current from 173.3±3.8% of control to 111.1±5.6% of control (n=5, P<0.01). Representative traces are currents induced by GABA (1 M) or GABA (1 M) plus CNQX (1 mM) with (red line) or without (black line) 100 M APV.  189  190  Figure 4.4. APV potentiates GABA current in dose dependent manner in cultured hippocampal neurons. Whole-cell patch-clamping of cultured hippocampal neurons were performed with CsCl-based pipette solution at a holding potential of -60mV. GABA-mediated currents were evoked repetitively at an interval of 30s by pressure ejection of GABA (100 M) in the presence of CNQX 10 M in the extracellular solution and all other drugs were applied through bath perfusion. A. APV reversibly potentiates GABA-induced current. Representative current traces produced by with a pulse of GABA (100 M) were obtained under control (Control), 2mins after bath application of APV (200 M) and 2min after washing off APV (Washout). B. APV potentiates GABA current in a dose-dependent manner. Dose-response curve was constructed for GABA (100 M) induced currents with various APV concentration (n=6) and fitted with the Hill equation that yields a Hill coefficient of 1.6 and EC 50 of 184.4 M. C. APV potentiation of GABA currents is not associated with alteration of the reversal potential. Currents were induced by pressure injection of GABA (100 M) without (top traces, control) (superimposed current traces on the top) or with APV (200 M; superimposed traces at the bottom, APV) at various holding potentials from -80 to 60, with a step interval of 20 mV. Current-Voltage (I-V) curves on the right were constructed by measuring the peak amplitude of the currents.  191  192  Figure 4.5. APV potentiates GABAAR mediated phasic and tonic inhibition in cultured hippocampal neurons. Whole-cell patch-clamping of cultured hippocampal neurons were performed with CsCl-based pipette solution at a holding potential of -60mV. Phasic inhibition (mIPSC) and tonic inhibition (bicuculline sensitive holding currents) were recorded in the presence of TTX 0.5µM, CNQX 10µM, MK-801 10µM. A. APV potentiates the amplitude but not the frequency of bicuculline-sensitive GABAAR-mediated mIPSCs.  200µM APV  potentiate the amplitude of mIPSCs (from control: 35.5±2.3 pA to APV: 43.3±2.5 pA, P<0.001, n=8) but not frequency (from control: 0.73±0.16 to APV: 0.72±0.12, P>0.05, n=8). Representative current traces were obtained in absence of APV, in presence of APV and in presence both APV and bicuculline. B. APV potentiates the amplitude of bicuculline-sensitive GABAAR-mediated  holding  currents.  200µM  APV  potentiate  the  amplitude  of  bicuculline-sensitive GABAAR-mediated holding currents (from control: 12.5±0.82 pA to APV: 22.1±2.8 pA, P<0.01, n=6). Representative current traces were obtained from holding currents in presence of bicuculline 10µM or both APV 200µM and bicuculline 10µM.  193  194  4.5. References Belelli D, Harrison NL, Maguire J, Macdonald RL, Walker MC, Cope DW (2009) Extrasynaptic GABAA receptors: form, pharmacology, and function. J Neurosci 29:12757-12763.  Connor JA, Wadman WJ, Hockberger PE, Wong RK (1988) Sustained dendritic gradients of Ca2+ induced by excitatory amino acids in CA1 hippocampal neurons. Science 240:649-653.  Gillespie DC, Kim G, Kandler K (2005) Inhibitory synapses in the developing auditory system are glutamatergic. Nat Neurosci 8:332-338.  Kleckner NW, Dingledine R (1988) Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes. Science 241:835-837.  Olsen RW, Sieghart W (2008) International Union of Pharmacology. LXX. Subtypes of gamma-aminobutyric acid(A) receptors: classification on the basis of subunit composition, pharmacology, and function. Update. Pharmacol Rev 60:243-260.  Patenaude C, Chapman CA, Bertrand S, Congar P, Lacaille JC (2003) GABAB receptor- and metabotropic glutamate receptor-dependent cooperative long-term potentiation of rat hippocampal GABAA synaptic transmission. J Physiol 553:155-167.  Puyal J, Grassi S, Dieni C, Frondaroli A, Dememes D, Raymond J, Pettorossi VE (2003) Developmental shift from long-term depression to long-term potentiation in the rat medial vestibular nuclei: role of group I metabotropic glutamate receptors. J Physiol 553:427-443.  Semyanov A, Walker MC, Kullmann DM, Silver RA (2004) Tonically active GABA A receptors: modulating gain and maintaining the tone. Trends Neurosci 27:262-269.  Stelzer A, Wong RK (1989) GABAA responses in hippocampal neurons are potentiated by glutamate. Nature 337:170-173.  195  Stelzer A, Slater NT, ten Bruggencate G (1987) Activation of NMDA receptors blocks GABAergic inhibition in an in vitro model of epilepsy. Nature 326:698-701.  Windhorst U (2007) Muscle proprioceptive feedback and spinal networks. Brain Res Bull 73:155-202.  196  5.  ALLOSTERIC  POTENTIATION  OF  GLYCINE  RECEPTOR  CHLORIDE CHANNELS BY GLUTAMATE4  5.1. Introduction Neuronal excitability is fundamental to neuronal function, and is primarily controlled by a fine balance between synaptic excitation and inhibition.  In the central nervous system (CNS), the  synaptic excitation is chiefly mediated by the excitatory transmitter glutamate acting on ionotropic glutamate receptor-gated cationic channels.  While in the brain, the synaptic  inhibition is primarily mediated by the inhibitory transmitter -aminobutyric acid (GABA) acting at A type of GABA receptor-gated chloride channel, in the mammalian brainstem and spinal cord, glycine is principally recognized for its role as the primary neurotransmitter mediating fast synaptic inhibition via activating glycine receptor (GlyR)-gated chloride channel(Lynch, 2004; Betz and Laube, 2006).  However, glycine can also contribute to  excitatory transmission by serving as an allosteric modulator for the NMDA subtype glutamate receptor (NMDAR)(Johnson and Ascher, 1987; Kleckner and Dingledine, 1988).  In the  present study, we have unexpectedly discovered that the excitatory transmitter can exert a similar allosteric effect on GlyRs.  Along with the previously demonstrated glycine  potentiation of excitatory NMDA receptors(Johnson and Ascher), our results not only blur the traditional distinction between an excitatory and an inhibitory transmitter system, but also lead  4  A version of this chapter has been submitted for publication. Liu J., Wu D.C., and Wang Y.T. Allosteric  potentiation of glycine receptor chloride channels by glutamate. 197  us to propose a new model of functional cross-talk between the two classical fast transmitters, via the reciprocal allosteric enhancement of each other’s receptor function. This reciprocal modulation between the two classic excitatory and inhibitory transmitter systems could act as a novel, and rapid homeostatic control mechanism for neuronal excitability.  5.2. Methods and Materials 5.2.1. Neuronal culture Cultured spinal cord neurons were prepared from the lumbar spinal cords of D14-16 fetal Wister rats. Tissues were digested with a 0.25% trypsin solution (Invitrogen) for 25 min at 37 °C, and then mechanically dissociated using a fire-polished Pasteur pipette. Next, the cell suspension was centrifuged at 2500 ×g for 50 s and the cell pellets resuspended in DMEM containing 10% Fetal Bovine Serum (FBS; Sigma-Aldrich). Cells were seeded on poly-D-lysine-coated 24-well coverslips at a density of 2.5 ×105 cells/well. Cultures were maintained in a humidified incubator with 5% CO2 at 37 °C. After 24 hrs, plating medium was changed to Neurobasal medium supplemented with B-27 supplement and L-glutamine and the media changed twice weekly thereafter. Cultured neurons were used for electrophysiological recordings 10-14 days after plating.  5.2.2. HEK293 cell culture and transfection  HEK293 cells were cultured in MEM (Invitrogen) supplemented with 10% FBS. Cells  198  were grown to 20% confluence and transiently transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocols. Cells were transfected with a pBK-CMV NB-200 expression vector containing a human recombinant GlyR α1 or a combination of vectors of α1 and β subunits(Caraiscos et al., 2007). To enhance the formation of functional 1 and  heteromeric GlyRs, 1 and  plasmids were transfected at a 1:40 ratio(Burzomato et al., 2003). pcDNA3-GFP was also cotransfected along with GlyR subunits as a transfected marker, in order to facilitate the visualization of the transfected cells during electrophysiological experiments. 15-20 hours following transfection, cells were re-plated on glass coverslips and cultured for an additional 15-24 hrs before whole-cell patch-clamp recordings.  5.2.3. Electrophysiology  Whole cell patch-clamp recordings: Whole-cell recordings were performed under voltage-clamp mode using an Axopatch 200B or 1D patch-clamp amplifier (Molecular Devices). Whole-cell currents were recorded at a holding potential of -60 mV unless indicated elsewhere, and signals were filtered at 2 kHz, digitized at 10 kHz (Digidata 1322A). Recording pipettes (3-5 MΩ) were filled with the intracellular solution that contained (mM): CsCl 140, HEPES 10, Mg-ATP 4, QX-314 5, pH 7.20; osmolarity, 290-295 mOsm. BAPTA (10 mM) was added in the intracellular solution (otherwise specified). The coverslips were continuously superfused with the extracellular solution containing (mM): NaCl 140, KCl 5.4, HEPES 10, MgCl2 1.0, CaCl2 1.3, glucose 20, pH 7.4; osmolarity, 305-315 mOsm. For recordings of mIPSCs and glycine evoked postsynaptic currents in cultured neurons, bicuculline (10 µM), 199  CNQX (10 µM) and TTX (0.5 µM) were added in the extracellular solution to block GABAA receptor currents and minimize the activation of ionotropic glutamate receptors and voltage-gated sodium channels, respectively. To evoke glycine mediated currents, a glass pipette filled with glycine (100 µM in the Ca2+-free extracellular solution) was placed near neurons under recording. Pressure pulses (20-60 ms) were delivered to eject glycine via a Picospritzer at 30 s or 60 s intervals. For recording of glycine currents in HEK293 cells, fast perfusion of glycine and/or glutamate ligands were accomplished with a computer-controlled multibarrel fast perfusion system (Warner Instruments). For experiments of bath application of glutamate, AMPA and NMDA, 5 mM EGTA was added in and calcium was removed from the extracellular solution unless specified elsewhere. All experiments were performed at room temperature.  Single channel analysis: Single-channel currents were recorded in on-cell attached or outside-out patch configurations with the use of thick-wall glass pipettes whose tips were fire polished and coated with Sylgard. The electrodes had resistances of 6-10 MΩ. The pipettes were filled with the extracellular solution for on-cell attached patches or with the intracellular solution for outside-out patches. Signals were filtered at 1 kHz, sampled at 10 kHz and analyzed off-line with Clampfit 9.0 software. An idealized recording of the durations and amplitudes of detectable events of the single-channel data was generated using 50% threshold crossing criteria. Events with a duration less than 300 µs were ignored. Single channel activities were expressed as the product of the number of channels × the open probability (P o); i.e. NPo = ∑[(open time × number of channels open) ∕ total time of record]. In on-cell attached 200  configuration, only patches with one active channel activity was included for data analysis.  5.2.4. Data analysis  Values are expressed as mean ± SEM (n = number of experiments). One-way ANOVA or the two-tailed Student’s test was used for statistical analysis and P values less than 0.05 were considered statistically significant. Dose–response curves were created by fitting data to Hill equation: I = Imax/(1+EC50/[A]n), where I is the current, Imax is  the maximum current, [A]  is a given concentration of agonist, n is Hill coefficient.  5.2.5. Chemicals  N-methyl-D-aspartate propionate  (AMPA),  (NMDA),  α-amino-3-hydroxyl-5-methyl-4-isoxazole-  D-2-amino-5-phosphonovaleric  7-nitroquinoxaline-2,3-dione (CNQX),  acid  (APV),  6-cyano-  Quisqualic acid (QA), kinate acid (KA), MK-801,  YM-298198 and MPEP were purchased from Torcis (Ellisville, Missouri, US). Gluamate and strychnine were purchased from Sigma-Aldrich. Bicuculline methobromide was purchased from Alexis Biochemicals.  5.3. Results We performed whole-cell patch-clamp recordings of spinal dorsal horn neurons maintained in primary cultures. Miniature postsynaptic events in were initially recorded at a holding potential of –60 mV in the presence of voltage-channel blocker TTX (0.5 M) and  201  GABAA receptor antagonist bicuculline (10 M). To isolate the GlyR-mediated miniature inhibitory postsynaptic currents (mIPSCs) from glutamate receptor-mediated miniature excitatory  postsynaptic  currents  (mEPSCs),  we  sequentially  applied  α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) subtype glutamate receptor (AMPAR) antagonist CNQX (10 µM) and N-methyl-D-aspartate subtype glutamate receptor (NMDAR) antagonist APV (50 M) in the extracelluar perfusion solution. As expected, CNQX almost abolished events with fast rising kinetics, likely mEPSCs (Fig. 1Ab).  Surprisingly, bath  perfusion of APV (100 µM) resulted in profound increase in the amplitudes of remained miniature synaptic events (Fig.1Ac).  As these events were recorded in the presence of  AMPAR (CNQX), NMDAR (APV), and GABAA receptor (bicuculline) blockers, these synaptic events were likely mIPSCs mediated by GlyRs.  Indeed, they were blocked by  adding GlyR blocker strychnine (1 µM; Fig. 1Ad) suggesting a significant enhancement of GlyR-mediated mIPSCs by APV.  This potentiation was reversible because mIPSCs  recovered to the control level after washing out APV (Fig. 1Ae). As shown in Fig. 1Af and g, APV increased amplitude of GlyR-mediated mIPSCs without affecting their frequency; the amplitude being increased to 137.4 ± 10.1% of the control (P < 0.05, n = 7), while frequency being remained largely unchanged (0.56 ± 0.19 Hz in presence of APV vs 0.53 ± 0.21 Hz in the absence of APV (P > 0.05; n=7).  We next examined if the increased mIPSC amplitude  by APV was associated with an alteration in the kinetic properties of mIPSCs, by comparing their rising and decay time courses (Fig. 1Ah). There was no significant difference in the 10-90% rising time (control: 1.28 ± 0.2 ms vs APV: 1.26 ± 0.17 ms) and the decay time  202  constant (τD), which fit with a one-time expotential decay function (dotted lines in Fig. 1Ah; control: 13.3 ± 2.5 ms vs APV: 14.7 ± 3.0 ms). reversibly potentiates GlyR-mediated mIPSCs.  These results demonstrate that APV  Moreover, the selective enhancement of  amplitude of mIPSCs, without altering their frequency and kinetics, strongly suggest that this potentiation is most likely mediated by APV acting via a postsynaptic mechanism.  To further confirm the postsynaptic locus of the modulation, we next investigated the effect of APV on GlyR-gated currents evoked by pressure ejection of glycine from a micropipette near the neurons under recordings in the presence of TTX, CNXQ and Bicucullin. The glycine-induced currents under these conditions were blocked by bath application of 1 µM strychnine, confirming the identity of strychnine-sensitive, GlyR-mediated Cl- currents (data not shown). Consistent with a postsynaptic modulation, bath application of APV (50 µM) caused significant increase in glycine induced currents (138.1 ± 12.2 % of the control, P < 0.01, n = 6; Fig. 1B).  However, the potentiated glycine currents by APV may be due to the blockade of an ambient glutamate activation of NMDAR-mediated inhibition of GlyR function.  If it is the  case, we would expect that direct activation of NMDARs by bath application of NMDA should inhibit the GlyR-mediated currents.  To our surprise, application of NMDA (50 µM) in the  absence of extracellular Mg2+ produced even a greater potentiation of glycine current to 180.2 ± 13.3 % of the control (n = 6, P < 0.01, Fig. 1C).  203  The qualitatively similar potentiation of glycine currents by both APV and NMDA strongly suggests that the potentiation is not dependent on activation of NMDA receptors. This conjecture was further tested after blocking NMDAR channels with MK-801, a use-dependent and non-competitive NMDAR antagonist(Wong et al.). To fully blockade of NMDAR channels, MK-801 (10 µM) was first co-applied with NMDA (50 µM) in the Mg2+-free extracellular solution. As shown in Fig. 1D, NMDA-induced currents were progressively and completely inhibited by MK-801 (Fig. 1Da). The MK-801 blockade of NMDA channels was irreversible, as after MK-801 washout, a subsequent application of NMDA (50 M) failed to produce any observable current (Fig. 1Db). As illustrated in Fig. 1Dc, after the blocking of NMDAR channels by MK801, NMDA-induced potentiation of glycine-induced currents largely remained (147.7 ± 14.6 % of the control, n = 5, P < 0.01). These results support the notion that the potentiation does not require the opening of NMDAR channels.  However, it remains possible that the potentiation is mediated by the binding of NMDA or APV to the glutamate-binding site on the NMDARs without activation of the receptor channels. We hypothesized that if the potentiation requires NMDA occupation of the ligand-binding sites of NMDARs in the absence of channel activation, APV should occlude the NMDA’s potentiation by competing the same ligand-binding site. As shown in Fig. 1E, bath application of APV (50 µM) alone increased the glycine-induced current to 134.2 ± 11% of the control (n = 7, P < 0.01, Fig. 1F). However, in contrast to our expectation, APV failed to  204  occlude NMDA-induced potentiation of glycine currents. In fact, NMDA (50 µM) in the presence of APV resulted in a greater enhancement of the glycine current (176 ± 7.8 %, n = 6, P < 0.001, Fig. 1E). The potentiation of glycine currents caused by NMDA+APV was significantly different from APV alone (P < 0.01, n = 5, Fig. 1G). These results strongly suggest that NMDA and APV may potentiate GlyR mediated currents, not by acting on the NMDAR glutamate binding site, but by a previously undescribed novel glutamate-binding site.  These observations prompted us to then test whether the potentiation can be mimicked by other known glutamate ligands, such as the endogenous ligand glutamate itself, or AMPA, a selective ligand which specifically binds to the AMPAR (but not the NMDAR). Glutamate is an endogenous excitatory neurotransmitter acting through both ionotropic (AMPA, NMDA and kinate receptor subtypes) and metabotropic glutamate receptors (mGluRs) in the CNS(Dingledine et al., 1999; Collingridge et al., 2004; Mayer and Armstrong, 2004). mGluRs are classified into eight subtypes (mGluR1-8), of which mGluR1 and mGluR5 are mainly located on postsynaptic sites(Conn and Pin, 1997). In order to test if glutamate mimics NMDA and APV in potentiating glycine currents via a previously undescribed binding site, we applied glutamate in the presence of CNQX (20 µM; to block AMPA and kinate glutamate receptors), MK-801 (10 µM; to block NMDARs), YM-298198 (10 µM; to block mGluR1 receptors) and MPEP (10 µM; to block mGluR5 receptors). As shown in Fig. 2A, under these conditions, glutamate (50 µM), like NMDA or APV, produced a reversible increase in the glycine-induced current (161.2 ± 26.7 %; n = 7; P < 0.001). Co-application of APV (50 M)  205  with glutamate did not prevent glutamate-induced potentiation, but instead resulted in a greater potentiation (200.3 ± 16.6 %, n = 5, P < 0.01, Fig. 2A) than observed with glutamate alone. Next, to determine whether glutamate exerts its effect via the same binding site as APV, we performed an occlusion experiment. We assumed that if two ligands share the same acting site, APV at the maximum concentration should be able to occlude the glutamate effects. We first determined the dose-response relationship of APV-induced potentiation on glycine currents. As illustrated in Fig. 2B, APV produced a dose-dependent potentiation of glycine-induced currents, with a respective EC50 at 49.4 ± 7.6 M and saturating concentration about 1 mM. At the saturating concentration (1 mM), APV resulted in a remarkable potentiation of the glycine current, and the potentiation was not further increased by addition of glutamate (50 µM; Fig. 2C). This suggests that glutamate and APV most likely act on the same binding site in potentiating glycine currents. Similar to glutamate, application of AMPA (50 µM) also resulted in the significant potentiation of glycine currents (141.3± 7.4 %, n = 7, P < 0.01, Fig. 2D) and this potentiation was occluded by the potentiation produced with a saturating concentration of APV (Fig. 2D). Together, these results strongly suggest that these glutamate ligands share a novel glutamate-binding site.  We next further explored the mechanisms underlying the potentiation of GlyR function using single-channel recordings under both outside-out and on-cell attached configurations. We used APV (but not glutamate itself) as the glutamate-like ligand in these experiments to avoid any potential complications due to the activation of any ionotropic  206  glutamate receptor-gated single channel activities. In excised outside-out patches held at -60mV, bath application of glycine (5 µM) evoked single channel activities which, as previously reported(Bormann et al., 1987),(Twyman and Macdonald, 1991), were composed of main and several sub-conductances, with the main conductance level being 46.8 ± 2.4 pS (Fig. 3A). Application of APV (50 µM) to the patch did not produce any single-channel activity on its own (data not shown), but instead substantially increased glycine-induced single-channel activities. APV enhancement manifested primarily as an increase in the channel open probability (Po) (Fig. 3A; the mean Po of glycine induced channel activities before and after APV being 0.035 ± 0.012 and 0.12 ± 0.03, respectively; n = 5, P < 0.01), without a significant alteration of the single-channel conductance (45.5 ± 2.4 pS after APV vs 46.2 ± 2.7 pS before APV; n = 5; P > 0.05). The glycine-induced single-channel activity in the absence or presence of APV could be completely blocked by addition of strychnine (1 M; Fig. 3A); confirming that the single-channel activities are the result of the opening of strychnine-sensitive GlyR-gated chloride channels. The observation that the potentiation can be detected in the excised patch strongly suggests that the putative glutamate-binding site responsible for the potentiation is either on the GlyR itself, or on an unidentified glutamate-binding receptor/protein closely associated with the GlyR. This is more clearly demonstrated by single-channel recordings under the on-cell attached configuration (Fig. 3B). In these recordings, glycine (5 µM) was included in recording pipettes filled with the extracellular solution at a pipette potential (Vp) of 0 mV. As illustrated in Fig. 3B, when APV (50 µM) perfused to the cell outside the recording pipette, it did not significantly affect glycine-induced  207  single-channel activities (Fig. 3B). The mean Po before and after APV was 0.02 ± 0.007 and 0.02 ± 0.008, respectively (n = 7; P > 0.05).  However, when APV was co-applied with  glycine to the inside of the recording pipette, the mean Po was increased to 0.08 ± 0.02 (n = 6, P < 0.01, Fig. 3B).  To further differentiate whether glutamate potentiation is mediated  through the ligand binding to the GlyR itself, or to a closely associated glutamate receptor/protein, we examined the potential regulation of GlyR function in HEK293 cells transiently overexpressing recombinant human GlyRs. Functional GlyRs are pentameric channel complexes formed with various subunit combinations from a total of four different  and one  subunit (Lynch, 2004; Betz and Laube, 2006). The  subunits can efficiently form functional homomeric GlyRs in recombinant expression systems, but the majority of native GlyRs in the CNS are believed to be heteromeric 1 and  receptors(Betz and Laube, 2006). Therefore, we transiently expressed recombinant human homomeric α1 (Fig. 4a-e) or heteromeric α1 and β GlyRs (Fig. 4f) in HEK293 cells. As shown in Fig 4a, under whole-cell recordings at a holding potential of -60 mV with a chloride-based intracellular solution, fast perfusions of glycine at concentrations of 10-100 M produced inward currents in a dose-dependent manner. These currents had a reversal potential close to the chloride equilibrium potential (0 mV; Fig. 4c) and were blocked by strychnine (1 M; Fig. 4a), confirming that the currents are gated through strychnine-sensitive GlyR-gated chloride channels. Fast perfusion of glutamate (100 M) alone produced no detectable currents, indicating the lack of expression of functional ionotropic glutamate receptors in these cells (Fig. 4a). But when co-applied with glycine, glutamate significantly potentiated the glycine-induced current. On average, currents  208  induced by glycine at 10 M were increased from -503 ± 156 pA in the absence of glutamate to -1095 ± 357 pA in the presence of glutamate (n = 6; p < 0.05). The potentiated currents are entirely mediated through GlyR-gated chloride channels, as currents in the presence of glutamate were also blocked by strychnine (1 M; Fig. 4a).  The glutamate-induced potentiation was more pronounced when the currents were induced at lower concentrations of glycine, and diminished at the saturating concentration of glycine (100M; Fig. 4a). The potentiation was also glutamate concentration-dependent. As shown in Fig. 4b, the potentiation occurred in a low micromolar range (about ~5 M) with a respective EC50 and saturating concentration around 20 M and 1 mM (Fig. 4b). To determine whether the glutamate-induced potentiation is due to a change in chloride driving force, or due to a change in the channel function, we examined the current-voltage (I-V) relationship by recording glycine-induced currents at various holding potentials prior to, and after, bath application of glutamate (100 M). As shown in Fig. 4c, the glutamate-induced potentiation was associated with a change of the slope of the I-V curve, without altering the reversal potential. This is consistent with an enhanced channel function, but not an alteration in chloride driving force.  To further examine whether glutamate induced potentiation of glycine currents can be mimicked by other glutamate like-ligands, we tested the effects of various subtype ionotropic glutamate receptor agonists including NMDA (100 M for NMDARs), quisqualate (100 M;  209  for AMPARs) and kainic acid (KA; 100 M for KARs) and antagonists including APV (100 M for NMDARs) and CNQX (100 M for AMPARs and KARs). None of these ligands generated detectable currents on their own, but all of them, with the exception of CNQX, mimicked the action of glutamate, resulting in a significant enhancement of glycine-induced currents. The potentiation is qualitatively similar for cells expressing either α1 homomeric (Fig. 4e) or α1 and β heteromeric (Fig. 4f) GlyRs, although it appeared more pronounced in cells expressing the homomeric receptors. In contrast to all other glutamate-like ligands tested, CNQX had little effect on glycine-induced currents at a concentration below 20 M (data not shown), but at 100 M, it reduced the currents by 26.6 ± 5.0 % (n=6; Fig. 4d and e) in cells expressing homomeric GlyRs and 26.2 ± 2.2 % (n=3) in cells expressing heteromeric GlyRs, respectively (Fig. 4f).  5.4. Discussion Our study revealed a previously unrecognized positive modulation of GlyR function by various glutamate-like ligands. The demonstration of potentiation using either excised outside-out patches or on-cell attached recordings when a glutamate ligand was co-applied with glycine into the recording pipette is consistent with an allosteric modulation by a direct binding of glutamate ligands to GlyRs. This was further supported by the observed similar modulation in HEK293 cells transiently transfected with recombinant GlyRs, but lacking any known functional glutamate receptors. The observation that the glutamate potentiation can be mimicked by most ionotropic glutamate receptor ligands tested thus far suggests that this putative glutamate-binding site on the GlyR has a novel pharmacological profile, and appears to  210  be less stringent than any glutamate-binding site on known ionotropic glutamate subtype receptors. However, it is interesting to note that CNQX, unlike all other glutamate-like ligands tested, has no positive modulation, and at high concentrations has a weaker inhibitory action on the GlyR-mediated currents. This suggests that the newly discovered glutamate-binding site has a pharmacological profile distinct enough from other known glutamate-binding sites, and such a distinctive profile may allow for the future development of agonists and/or antagonists that can specifically modulate the strength of glycine-receptor mediated synaptic inhibition.  This  glutamate allosteric regulation of GlyR function is also both mechanistically and functionally distinct from the previously NMDAR-mediated potentiation of GlyRs (Fucile et al., 2000; Xu et al., 2000; Zhu et al., 2002) mechanistically, this modulation, unlike the NMDAR-mediated one, appears not to require for activation of another receptor and its downstream of intracellular signalling(s); and as such, functionally this allosteric modulation may be much more rapid and efficient then the NMDAR-mediated modulation.  Glutamate and glycine are respectively the primary excitatory and inhibitory transmitters in the mammalian brainstem and spinal cord. However, the allosteric potentiation of GlyR by glutamate described in the present study, along with the previously discovered allosteric potentiation of NMDA subtype of glutamate receptors by glycine (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988), blurs the classic distinction between excitatory and inhibitory neurotransmitters. These positive cross-talks between glutamate and glycine transmitter systems may also suggest a novel and efficient mode of homeostatic regulation of  211  neuronal excitability via a reciprocal allosteric enhancement of each other’s receptor function. It is important to note that the EC50 of glutamate modulation of GlyRs observed in the present study is around 20 M, which is much higher than extracellular glutamate concentrations available under basal physiological conditions.  The higher EC50 than normal extracellular  glutamate concentration may impart the specific physiological and/or pathological significance of the glutamate homeostatic regulation of GlyRs.  For instance, at normal conditions,  efficient glial and/or para-synaptic neuronal glutamate transporters will keep presynaptically released glutamate to be concentrated at synaptic clefts of excitatory synapses, and limit the glutamate spilled-over to adjacent glycinergic synapses at concentrations below the threshold required to potentiate GlyR function. Therefore, under these basal physiological conditions, glutamate functions as a true excitatory transmitter, mediating synaptic excitation only. However, under certain conditions such as following high-frequency-stimulation of presynaptic inputs and/or during un-controlled seizure activity, repetitive firing of glutamatergic neurons can cause the amount of presynaptically released glutamate over pass the ability of glutamate transporter uptake, thereby transiently escaping from the uptake and spilling-over to the adjacent glycinergic synapses. When such a heterosynaptic glutamate spill-over(Vogt and Nicoll, 1999; Stafford et al., 2009) increases its concentration nearby glycinergic synapses at the level of a few M range, and only then, glutamate will then allosterically potentiate GlyR function, thereby strengthening glycine-mediated synaptic inhibition at these synapses. The enhanced inhibition may, in turn, rapidly and efficiently counteract glutamate-mediated excitation, thereby ensuring a tight control of neuronal  212  excitability under most physiological conditions. Vice versa, a similar heterosynaptic spill-over feedback mechanism can also take place via glycine potentiation of NMDARs when glycinergic neurons are firing extensively. Compared with other levels of homoestastic regulation such as neuronal network (Windhorst, 1996) or synaptic homoestasis (Turrigiano, 2007), this glutamate-glycine reciprocal receptor cross-talk mode is clearly much faster. Such a rapid mode of homoestatic regulation may be particularly important for some neuronal functions requiring the very tight timing of inputs, such as during synaptic plasticity or rhythmic generations. To this end, it is relevant to note that NMDAR-mediated excitation and glycine-mediated inhibition are thought to be the primary counteracting systems critically involved in the generation and maintenance of the rhythmicity of local motor activity in the spinal cord(Grillner, 2003). Such cross-talk may also be important under certain pathological conditions. For instance, under some pathological conditions such as neurotrauma, the excessive release of glutamate, in combination of compromised glutamate uptake, may transiently increase extracellular glutamate concentration to a level of hundreds of micromoles or even millimoles (Clements et al., 1992; Otis et al., 1996; Bergles et al., 1999). This increased glutamate concentration may significantly potentiate glycine-mediated chloride conductance, thereby protecting neurons from excessive glutamate-induced excitotoxic neuronal death.  213  Figure 5.1. APV and NMDA potentiate postsynaptic GlyR-mediated currents via a NMDAR-independent mechanism. Whole-cell patch-clamping of cultured spinal neurons were performed with a CsCl-based pipette solution at a holding potential of -60mV.  (A)  GlyR mediated mPISCs were isolated by addition of 0.5 µM TTX and 10 µM Bicuculline (a), 10 µM CNQX (b) and 100 µM APV (c). APV produced reversible potentiation of amplitudes of mIPSCs, which was blocked by 1uM strychnine (d). (f) and (g) were accumulative frequency and amplitude distribution of mIPSCs from the same cell. h, average mIPSCs before and after APV application, which were best fitted with the one-exponential function (dotted lines). The rising time and τD were 1.2 ms and 12.4 ms in control and 1.1 ms and 13.8 ms with APV. (B) APV (50 µM) reversibly potentiates postsynaptic glycine-induced currents. (C) NMDAR activation potentiates glycine-induced currents. (D) NMDA-induced potentiation does not require the opening of NMDAR-gated channel.  Blockade of NMDA channels was  produced by co-application of NMDA (50 µM) and MK-801(10 µM) (a), and subsequent application of NMDA did not alter the holding current (b).  On the right are representative  glycine-evoked current traces obtained before (Control) and after bath application of NMDA (50 µM) following the NMDAR blockade, indicating NMDA potentiation of glycine currents does not require the opening of NMDAR channels.  In C and D, the extracellular solution  contains 1.3 mM Ca2+ and 0 mM Mg2+. (E). APV (50 µM) and NMDA (50 µM) produced greater potentiation of glycine currents than APV alone. The extracellular solution contains 1 mM Mg2+, 0 mM Ca2+ and 5 mM EGTA (F). Bar graph summarizing the data from B-E showing effects of AVP and/or NMDA on glycine-induced currents under various conditions.  214  Number of cells in each group is indicated on the top of each bars. Error bars in this and following figures indicate SEM. ** < 0.01, *** < 0.001.  215  216  Figure 5.2. Potentiation of GlyR-mediated currents by glutamate and AMPA in cultured spinal neurons. Whole-cell GlyR-mediated currents evoked by glycine (100 M) were recorded with an intracellular solution supplemented with 20 mM BAPTA and in the presence of CNQX (20 M to block AMPARs and KARs), MK-801 (10 µM to block NMDARs), YM-298198 (10 µM to block mGluR1) and MPEP (10 µM to block mGluR5) in 5 mM EGTA and Ca2+-free and extracellular solution. (A) glutamate (50 M) reversibly potentiates glycine currents (Glu) and glutamate potentiation is additive to APV (50 µM; n=7) -induced potentiation (Glu+APV; n=7). (B) dose-response curve for APV-induced potentiation of glycine current (n = 6). Each data point is the mean ± SEM of normalized glycine currents at the indicated APV concentrations. The solid line is the best fit of the data to the Hill equation, which yields a mean EC50 of 49.4 ± 7.60 M and Hill coefficient of 1.96. (C) APV at the saturating concentration (1 mM) resulted in a pronounced potentiation of the glycine current and occluded further potentiation by glutamate (50 M) as quantified in the bar graph on the right (n = 5 for each group).  (D) AMPA (50 M) potentiates glycine-induced currents and the  potentiation is occluded by the potentiation induced with the saturating concentration of APV (1 mM). n = 6 for both groups. Error bars indicate SEM. ** p < 0.01, **p < 0.001.  217  218  Figure 5.3. APV increases the open probability of GlyR-mediated single-channel activities in cultured spinal neurons. (A) glycine-induced single-channel activity was increased by APV in outside-out patches at a holding potential of – 60 mV. Glycine (5 µM), APV (50 µM) and strychnine (1 µM) were applied to the patches through bath applications as indicated in the diagram on the left. Representative current traces were obtained in the absence of APV (Glycine), presence of APV (+APV) and presence of APV and strychnine (+APV/Strychnine).  APV significantly  increased the single-channel open probability as summarized in the bar graph on the right, but had little effect on the single-channel conductance  (44.7 pS and 44.2 pS for the main channel  conductances in the absence and presence of APV respectively; n=6 patches). (B) Effects of APV on glycine induced single-channel currents recorded under the on-attached mode at a pipette potential of 0 mV. As illustrated in the diagrams above the representative single-channel recording traces, glycine (5 M) was applied into the patches via the intra-pipette recording solution and APV (50 M) was applied either to the patched cells through bath applications or into the patches by including it, along with glycine in the intra-pipette recording solutions.  In this patch, the glycine channel Po was ~0.02 in the  absence of APV (traces on the left) and was only potentiated by APV applied through the recording pipette (traces on the right; Po: 0.07), but not when it was applied through the bath (traces in the middle; Po: 0.02). Bar graph on the right summarizing results from effects of APV on the mean Po of glycine-induced single channel activities from six individual patches. Error bars indicate SEM. ** < 0.01.  219  220  Figure 5.4. Glutamate-like ligands potentiate glycine currents in HEK293 cells expressing human recombinant GlyRs. HEK cells were transiently transfected with human α1 homomeric (A-E) or α1/β heteromeric (f) GlyRs. Whole-cell voltage-clamp recordings were performed with a Cl--based intracellular recording solution at a holding potential of -60 mV.  a. Glutamate potentiates  strychnine-sensitive GlyR-gated chloride currents in a glycine dose-dependent manner. Representative traces were currents induced by fast perfusion of various ligands in various combinations through a computer-driven multi-barrel fast perfusion system. Glycine induced fast inward currents from 10 to 100 M (Gly 10, Gly 30 and Gly 100) in a clear dose-dependent fashion. Fast perfusion of glutamate 100 µM (Glu) alone produced no detectable currents, but potentiated currents induced by glycine, with the potentiation being more pronounced at lower glycine concentrations. The glutamate potentiated glycine currents were blocked by addition of 1 µM strychnine. B. Glutamate potentiates currents induced by glycine in a dose-dependent manner. Dose-response curve was constructed for glycine (10 M)-induced currents with various glutamate concentrations (n = 8) and fitted with the Hill equation that yielded a Hill coefficient of 1.95 and EC50 of 19.9 µM. c. Glutamate potentiation of glycine currents is not associated with alteration of the reversal potential. Currents were induced by fast perfusion of glycine (15 M) without (top traces) or with glutamate (100 M; bottom traces) at various holding potentials from -60 to +60 mV, with a step interval of 20 mV. Current-Voltage (I-V) curves on the right were constructed by measuring the peak amplitude of the currents. d. Effects of various glutamate-like ligands on glycine-induced currents. Representative traces  221  were currents induced by glycine (10 M) with or without 100 M NMDA, APV and CNXQ. E and F. Normalized glycine current amplitude graphs summarizing effects of glutamate and its various analog ligands on currents induced by glycine in HEK293 cells overexpressing 1 homomeric (e) and  heteromeric (f) GlyRs.  The amplitudes of peak currents were  normalized to that of currents induced by glycine alone and the number of cells in each of the groups is indicated on top of each bar.  222  223  5.5. 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Vogt KE, Nicoll RA (1999) Glutamate and gamma-aminobutyric acid mediate a heterosynaptic depression at mossy fiber synapses in the hippocampus. Proc Natl Acad Sci U S A 96:1118-1122.  Windhorst U (1996) On the role of recurrent inhibitory feedback in motor control. Prog Neurobiol 49:517-587.  225  Wong EH, Kemp JA, Priestley T, Knight AR, Woodruff GN, Iversen LL (1986) The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist. Proc Natl Acad Sci U S A 83:7104-7108.  Xu TL, Dong XP, Wang DS (2000) N-methyl-D-aspartate enhancement of the glycine response in the rat sacral dorsal commissural neurons. Eur J Neurosci 12:1647-1653.  Zhu L, Krnjevic K, Jiang Z, McArdle JJ, Ye JH (2002) Ethanol suppresses fast potentiation of glycine currents by glutamate. J Pharmacol Exp Ther 302:1193-1200.  226  6. DISCUSSION AND FUTURE DIRECTIONS  The primary findings of this thesis are 1. NR2A and NR2B subunits play different roles in LTP and LTD induction in the lateral amygdala, respectively; 2. AMPAR insertion and internalization are respectively responsible for LTP and LTD expression in LA; 3. excitatory transmitter glutamate allosterically potentiates  the function of inhibitory GABAARs and  GlyRs.  6.1. NMDAR Subunits Have Differential Roles in the Induction of LA-LTP and LTD Calcium theory is the most popular hypothesis about induction of synaptic plasticity. The key point of this theory is calcium concentration determines the direction of synaptic plasticity (Malenka and Nicoll, 1993). For instance, transient and high calcium concentration is required for LTP induction, whereas long-lasting and low calcium concentration is responsible for LTD.  Different induction protocols for LTP and LTD in different brain  regions including hippocampus and lateral amygdala strongly support the calcium theory (Liu et al., 2004; Brebner et al., 2005; Rumpel et al., 2005; Yu et al., 2008). The obvious character of HFS (100 Hz, 1 s) induction protocol for LTP is the intensive stimulation which can induce massive glutamate release and accumulation in synaptic cleft. Consequently, it will in turn cause strong cell membrane depolarization as a result of excessive activation of AMPARs and thereby completely relieve NMDARs from Mg2+ blockade, leading to a large amount of  227  Ca2+ influx in a very short time scale. Pairing short term (1 - 2 mins) presynaptic stimulation at 2 Hz with artificial strong depolarization by holding postsynaptic membrane at 0 mV also can effectively induce LTP (Yu et al., 2008). On the contrary, classical LTD induction protocol of 1 Hz stimulation for 15 mins cannot maintain high concentration of glutamate in synaptic cleft. This weak activation of AMPARs causes a relatively smaller membrane depolarization and thereby partially relieves NMDARs from Mg2+ blockade, leading to a less amount of Ca2+ influx persisted for a longer period of time. Pairing a longer term (5 - 8 mins) and less frequent (1 Hz) presynaptic simulation with postsynaptic holding at -45 mV to mimic weak depolarization can induce LTD in different brain areas (Yu et al., 2008). Moreover, low dose of APV (0.5 µM) blocks 30-40% of the NMDAR synaptic component and also blocks LTP and leaves LTD intact (Liu et al., 2004), consistent with the calcium theory. However, some experimental results can not be interpreted by the calcium theory.  Liu et al found that 0.5 µM  NR2B-containing NMDAR antagonist Ro, like the low concentration of APV, blocks 30-40% of the NMDAR-mediated synaptic currents.  However, unlike the low dose of APV, it  abolishes LTD, leaves LTP intact in hippocampal slices (Liu et al., 2004). Furthermore, the selective blocker of NR2A-containing NMDAR, NVP, can abolish LTP induction and leave LTD intact (Liu et al., 2004), providing a new theory for controlling direction of synaptic plasticity, the NMDAR subunit theory. According to this theory, activation of NR2A-containing NMDARs is required for LTP induction, whereas activation of NR2B-containing NMDARs is required for LTD induction. The NR2 subunit theory is further supported by experiments using pharmacological and genetic approaches to define different  228  roles of NR2A- or NR2B-containing NMDARs in LTP and LTD in hippocampal and cortical slices (Liu et al., 2004; Massey et al., 2004; Woo et al., 2005; Yang et al., 2005). However, different brain areas may have different underlying mechanisms of induction of synaptic plasticity. For instance, in bed nucleus, activation of NR2A-containing NMDARs is not obligatory for LTP induction (Weitlauf et al., 2005).  In this thesis, we confirm that NMDARs  are required for LTP and LTD induction in lateral amygdala and find that NR2A-containing NMDARs are required for LTP induction whereas NR2B-containing NMDARs are required for LTD induction, proving that LTP and LTD induction in LA are following the rules of the NR2 subunit theory and providing potential tools for dissociate the roles of LTP and LTD in fear memory.  6.2. AMPAR Trafficking is Critical for the Expression of LTP and LTD in the LA Besides the LTP and LTD inductions, their expressions are also intensively studied in the hippocampus. AMPAR trafficking is considered as the underlying mechanism of expression of LTP (AMPARs exocytosis) and LTD (AMPARs endocytosis). Accumulating evidence strongly suggests that GluR1 and GluR2 subunits are involved in AMPAR insertion and internalization, respectively (Man et al., 2000; Shi et al., 2001). In cultured hippocampal slices, Shi et al. reported that LTP is impaired after infection of C-terminus of GluR1 whereas LTP is even enhanced after infection of C-terminus of GluR2, indicating GluR1 C-terminus but not GluR2 C-terminus is required for LTP expression in hippocampus (Shi et al., 2001). Man et al. showed that insulin can induce clathrin-dependent AMPAR internalization (Man et  229  al., 2000). This process requires GluR2 C-terminus and occludes LTD expression in hippocampal slices (Man et al., 2000). A following study further found that tyrosine phosphorylation at GluR2 C-terminus is required for AMPAR internalization and GluR2 C-terminal interfere peptide (GluR2-3Y, which is designed to interfere with GluR2 C-terminal tyrosine phosphorylation) blocks AMPAR internalization and LTD expression in hippocampal slices (Ahmadian et al., 2004), clearly demonstrating that GluR2 C-terminal phosphorylation is required for LTD expression. More recently, our laboratory found that GluR2-3Y peptide can block LTD expression in acute accumbens slices (Brebner et al., 2005). This result extends the role of GluR2 in LTD expression from hippocampus to accumbens, indicating that GluR2 mediated internalization may also play an important role in other brain area LTD expression.  In lateral amygdala, the studies related to AMPAR trafficking remain lacking. Rumpel reported recently that viral infection of GluR1 C-terminus blocks LTP expression in LA and also impairs acquisition of fear memory (Rumpel et al., 2005), indicating for the first time that GluR1 is required for both LTP induction and fear memory formation. However, whether AMPAR insertion is involved in this process and how AMPARs are inserted into cell membrane is still unclear. Our findings show that AMPARs insertion exists after LTP induction and this insertion is in a SNARE and GluR1 dependent manner (Yu et al., 2008), confirming the role of GluR1 in LTP expression in the LA and providing more details for the mechanism on AMPARs insertion in the LA.  230  Besides AMPAR insertion in amygdala, our findings also show that AMPAR internalization involves LTD expression here. AMPARs internalization occurs after LTD induction. And furthermore, we also found that AMPAR internalization is a GluR2 dependent process, which is confirmed by using the GluR2-3Y peptide.  Synaptic plasticity in the LA has been proposed as a prominent cellular mechanism responsible for certain forms of learning and memory encoded by neurons in this area. Consistent with this idea, evidence accumulated in recent years has provided much support for a critical role of NMDARs in fear-conditioning related learning and memory (Rodrigues et al., 2004). However, since NMDARs participate in mediating synaptic transmission and are also required for both LTP and LTD, these previous studies provided few clues about the exact roles of LTP or LTD in this learning paradigm. The availability of molecule specific reagents, such NVP, Ro and the GluR2-3Y peptide used in chapter 2 and 3, has allowed investigators to begin addressing the detailed roles of LTP or LTD in some learning and memory-related behaviors.  6.3. Cross-talk between Excitatory and Inhibitory Synaptic Transmission via Glutamate-Mediated Allasteric Modulation of GABAARs and GlyRs In addition to modulation of excitatory receptors (AMAPRs and NMDARs), modulation of inhibitory receptors (like GABAARs and GlyRs) is also important in synaptic plasticity and maintenance of basal level of neuronal excitability. Like excitatory receptors,  231  dynamic modulation of inhibitory receptor function can be achieved by controlling the number of the receptors on the membrane surface  and modulating their channel properties. The later  has been proven to be one of the most important targets for psychoactive drug development.  In CNS, excitatory signal will follow specific pathways to transmit from a specific input site to a specific receiving site. During this process, feedback signal from synaptic inhibition is necessary to control this specific transmission. There are three levels of regulations between excitatory and inhibitory transmission, including the regulation at the network level, the cellular level and the receptor level. Regulations at the network and cellular levels are common and well studied. For instance, at the network level, the same motor neuron (excitatory output) innervates muscles to produce muscle activity while innervating the inhibitory Renshaw cell to produce a negative-feedback (Hochman, 2007). At the cellular level, excitatory neurotransmitter glutamate can activate AMPARs to produce excitatory signals while activating mGluRs to inhibits GABAAR-mediated current via second messengers (like Ca2+) (Chen et al., 1990; Zhou and Hablitz, 1997). The receptor level regulation, on the other hand, is clearly understudied.  The only example is that glycine, the inhibitory  transmitter, potentiates NMDAR mediated currents via allosteric modulation (Johnson and Ascher, 1987). However, in the present studies, we show that there exists receptor level interaction between the major excitatory transmitter glutamate and inhibitory receptor GABAAR and GlyRs in CNS.  232  Glutamate can potentiate both GABAARs and GlyRs mediated currents via allosteric modulation. This phenomenon reflects a fine, fast, negative modulation between excitatory and inhibitory transmissions. For instance, the repetitive firing of glutamatergic neurons may produce heterosynaptic spill-over of glutamate on adjacent inhibitory synapses, thereby strengthening inhibition of these synapses. The outcome may increase singal-noise ratio rapidly and perhaps facilitate synaptic plasticity formation.  Our findings and previous  findings blur the boundary of excitatory and inhibitory transmitters and ascribe inhibitory or excitatory outcome to inhibitory or excitatory receptor but not transmitter, strongly increasing our understanding of mutual actions between excitation and inhibition.  6.4. Future Directions Our findings, however, raise more questions about modulation of excitatory and inhibitory transmissions that need to be answered in the future direction.  For example, what is the role of different NR2 subunits-containing NMDARs in synaptic plasticity in cortical pathway in the LA? NMDAR-dependent synaptic plasticity not only exists in thalamic pathway but also in cortical pathway in lateral amygdala. In cortical pathway, NMDAR-dependent synaptic plasticity happens at presynaptic site. It would be very interesting to investigate whether different NR2 subunit-containing NDMARs also play different roles in synaptic plasticity at presynaptic sites of LA and what the underlying  233  mechanism of this presynaptic plasticity is.  Secondly, does inhibitory transmission plasticity exist in amygdala?  Given that  inhibitory transmission is very important in amygdala related fear memory extinction, it is possible that LTP of inhibition (iLTP) can be induced during extinction and also GABA ARs trafficking may be involved in iLTP.  Thirdly, how does glutamate interact with GABAARs/GlyRs? We speculate several possible mechanisms that glutamate potentiates GABAARs/GlyRs-mediated currents, 1. Glutamate interacts with adjacent area of GABAARs/GlyRs ligand binding sites and affect the structure of binding pocket; 2. Given GABAARs/GlyRs have two ligand binding sites, full activation of GABAARs/GlyRs need two sites to be occupied by GABA/glycine. Low concentration of GABA/glycine may bind to one binding site and in turn change the other binding site structure. As a consequence, glutamate can mimic GABA/glycine to occupy that site and enhance low concentration of GABA/glycine induced currents.  3. Other unknown  protein may mediate glutamate potentiation on GABAARs/GlyRs mediated currents.  Fourthly, what is the physiological role of potentiation of GABAAR or GlyRs-mediated currents by glutamate? A recent paper indicates that fluctuation of synaptic excitation is immediately and proportionally counterbalanced by inhibition, indicating the allosteric modulation of glutamate on GABAAR or GlyR may be involved in gamma oscillation in brain  234  (Atallah and Scanziani M, 2009).  Lastly, can GABA allosterically modulate AMPARs or NMDARs? Previous findings that glycine enhances NMDAR-mediate currents and our findings that glutamate enhances GABAAR and GlyR mediated currents indicate that receptor level interaction between excitatory and inhibitory transmissions is a general phenomenon. So GABA, the major inhibitory transmitter, may also allosterically modulate AMPARs or NMDARs.  235  6.5. References Ahmadian G, Ju W, Liu L, Wyszynski M, Lee SH, Dunah AW, Taghibiglou C, Wang Y, Lu J, Wong TP, Sheng M, Wang YT (2004) Tyrosine phosphorylation of GluR2 is required for insulin-stimulated AMPA receptor endocytosis and LTD. Embo J 23:1040-1050.  Atallah BV, Scanziani M (2009) Instantaneous modulation of gamma oscillation frequency by balancing excitation with inhibition. Neuron 62:566-577.  Brebner K, Wong TP, Liu L, Liu Y, Campsall P, Gray S, Phelps L, Phillips AG, Wang YT (2005) Nucleus accumbens long-term depression and the expression of behavioral sensitization. Science 310:1340-1343.  Chen QX, Stelzer A, Kay AR, Wong RK (1990) GABAA receptor function is regulated by phosphorylation in acutely dissociated guinea-pig hippocampal neurones. J Physiol 420:207-221.  Hochman S (2007) Spinal cord. current biology 17:R950-955.  Johnson JW, Ascher P (1987) Glycine potentiates the NMDA response in cultured mouse brain neurons. Nature 325:529-531.  Liu L, Wong TP, Pozza MF, Lingenhoehl K, Wang Y, Sheng M, Auberson YP, Wang YT (2004) Role of NMDA receptor subtypes in governing the direction of hippocampal synaptic plasticity. Science 304:1021-1024.  Malenka RC, Nicoll RA (1993) NMDA-receptor-dependent synaptic plasticity: multiple forms and mechanisms. Trends Neurosci 16:521-527.  Man HY, Lin JW, Ju WH, Ahmadian G, Liu L, Becker LE, Sheng M, Wang YT (2000) Regulation of AMPA receptor-mediated synaptic transmission by clathrin-dependent receptor internalization. Neuron 25:649-662.  236  Massey PV, Johnson BE, Moult PR, Auberson YP, Brown MW, Molnar E, Collingridge GL, Bashir ZI (2004) Differential roles of NR2A and NR2B-containing NMDA receptors in cortical long-term potentiation and long-term depression. J Neurosci 24:7821-7828.  Rodrigues SM, Schafe GE, LeDoux JE (2004) Molecular mechanisms underlying emotional learning and memory in the lateral amygdala. Neuron 44:75-91.  Rumpel S, LeDoux J, Zador A, Malinow R (2005) Postsynaptic receptor trafficking underlying a form of associative learning. Science 308:83-88.  Shi S, Hayashi Y, Esteban JA, Malinow R (2001) Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons. Cell 105:331-343.  Weitlauf C, Honse Y, Auberson YP, Mishina M, Lovinger DM, Winder DG (2005) Activation of NR2A-containing NMDA receptors is not obligatory for NMDA receptor-dependent long-term potentiation. J Neurosci 25:8386-8390.  Woo NH, Teng HK, Siao CJ, Chiaruttini C, Pang PT, Milner TA, Hempstead BL, Lu B (2005) Activation of p75NTR by proBDNF facilitates hippocampal long-term depression. Nat Neurosci 8:1069-1077.  Yang CH, Huang CC, Hsu KS (2005) Behavioral stress enhances hippocampal CA1 long-term depression through the blockade of the glutamate uptake. J Neurosci 25:4288-4293.  Yu SY, Wu DC, Liu L, Ge Y, Wang YT (2008) Role of AMPA receptor trafficking in NMDA receptor-dependent synaptic plasticity in the rat lateral amygdala. J Neurochem 106:889-899.  Zhou FM, Hablitz JJ (1997) Metabotropic glutamate receptor enhancement of spontaneous IPSCs in neocortical interneurons. J Neurophysiol 78:2287-2295.  237  APPENDICES  Appendix A: Publications  I. Published papers 1. Liu J*, Wu DC*, Wang YT. (* Equal contribution) Allosteric potentiation of glycine receptor chloride channels by glutamate. Nat Neurosci, 2010, under revision. 2. Migues PV, Hardt O, Wu DC, Gamache K, Sacktor TC, Wang YT, Nader K. Nat Neurosci, 2010, 13(5):630-4. Epub 2010 Apr 11 3. Yu SY*, Wu DC*, Liu L, Ge Y, Wang YT. (* Equal contribution) Role of AMPA receptor trafficking in NMDA receptor-dependent synaptic plasticity in the rat lateral amygdala. J Neurochem. 2008, 106(2):889-99. 4. Liu Y, Wong TP, Aarts M, Rooyakkers A, Liu L, Lai TW, Wu DC, Lu J, Tymianski M, Craig AM, Wang YT. NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo. J Neurosci. 2007, 27(11):2846-57. 5. Peineau S, Taghibiglou C, Bradley C, Wong TP, Liu L, Lu J, Lo E, Wu D, Saule E, Bouschet T, Matthews P, Isaac JT, Bortolotto ZA, Wang YT, Collingridge GL. LTP inhibits LTD in the hippocampus via regulation of GSK3beta. Neuron. 2007, 53(5):703-17. 6. Ryu J, Liu L, Wong TP, Wu DC, Burette A, Weinberg R, Wang YT, Sheng M. A critical role for myosin IIb in dendritic spine morphology and synaptic function. Neuron. 2006, 49(2):175-82.  238  II. Papers in preparation 1. Wu DC, Liu J, Lu J, Wang YT. Allosteric potentiation of GABAAR mediated current by glutamate. In preparation. 2. Ge Y, Lu J, Liu L, Wong TP, Wu DC, Cho T, Lin S, Kast J, Wang YT. Specific modulation of homomeric GluR1 receptors by p97 (VCP). In preparation.  239  Appendix B: UBC Research Ethics Board Certificate of Approval  240  THE UNIVERSITY OF BRITISH COLUMBIA  ANIMAL CARE CERTIFICATE Application Number: A08-0807 Investigator or Course Director: Yu Tian Wang Department: Medicine, Department of Animals:  Rats Sprague Dawley & Wistar 400 Start Date:  May 21, 2009  Approval Date: June 15, 2009  Funding Sources: Funding Agency: Funding Title:  Canadian Stroke Network (CSN) - Networks of Centres of Excellence (NCE) Targeting cell death cascades in the neuro vascular-inflammatory unit  Funding Agency: Funding Title:  Various Sources Molecular mechanisms of AMPA receptor cycling  Funding Agency: Funding Title:  Howard Hughes Medical Institute Investigation into the molecular mechanisms underlying synaptic plasticity  Funding Agency:  Canadian Institutes of Health Research (CIHR) A new therapeutic that protects the brain against excitotoxic and ischemic neuronal injuries  Funding Title: Funding Agency: Funding Title:  Canadian Institutes of Health Research (CIHR) Investigation into molecular mechanisms and physiological roles of activity-dependent AMPA receptor insertion  Funding Agency: Funding Title:  Canadian Institutes of Health Research (CIHR) Role of hippocampal synaptic plasticity in cue-induced relapsing  Funding Agency: Funding Title:  Canadian Institutes of Health Research (CIHR) Regulation of postsynaptic expression and function of GABA-A receptors  Funding Agency: Funding Title:  Heart and Stroke Foundation of British Columbia and Yukon Investigation into the molecular mechanisms mediating excitotoxicity - developing novel  post-stroke therapies Funding Agency: Funding Title:  National Natural Science Foundation of China Role of hippocampal synaptic plasticity in cue-induced relapsing  Funding Agency: Funding Title:  Canadian Stroke Network (CSN) - Networks of Centres of Excellence (NCE) Development of a stroke model in non-human primates  Funding Agency: Funding Title:  UBC Faculty of Medicine New faculty start-up grant  Funding Agency:  CHDI Foundation TREAT-HD: Translational Research on Excitotoxicity to Accelerate Therapies for Huntington's Disease  Funding Title:  Unfunded title:  N/A  The Animal Care Committee has examined and approved the use of animals for the above experimental project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies.  A copy of this certificate must be displayed in your animal facility.  Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093  

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