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THE DYNAMIC SYNAPSE
MOLECULAR METHODS IN
IONOTROPIC RECEPTOR BI0LOGY
© 2006 by Taylor & Francis Group, LLC

FRONTIERS IN NEUROSCIENCE
Series Editors
Sidney A. Simon, Ph.D.
Miguel A.L. Nicolelis, M.D., Ph.D.
Published Titles
Apoptosis in Neurobiology
Yusuf A. Hannun, M.D., Professor of Biomedical Research and Chairman/Department
of Biochemistry and Molecular Biology, Medical University of South Carolina
Rose-Mary Boustany, M.D., tenured Associate Professor of Pediatrics and Neurobiology,
Duke University Medical Center
Methods for Neural Ensemble Recordings
Miguel A.L. Nicolelis, M.D., Ph.D., Professor of Neurobiology and Biomedical Engineering,
Duke University Medical Center
Methods of Behavioral Analysis in Neuroscience
Jerry J. Buccafusco, Ph.D., Alzheimer’s Research Center, Professor of Pharmacology and
Toxicology, Professor of Psychiatry and Health Behavior, Medical College of Georgia
Neural Prostheses for Restoration of Sensory and Motor Function
John K. Chapin, Ph.D., Professor of Physiology and Pharmacology, State University of
New York Health Science Center
Karen A. Moxon, Ph.D., Assistant Professor/School of Biomedical Engineering, Science,
and Health Systems, Drexel University
Computational Neuroscience: Realistic Modeling for Experimentalists
Eric DeSchutter, M.D., Ph.D., Professor/Department of Medicine, University of Antwerp
Methods in Pain Research
Lawrence Kruger, Ph.D., Professor of Neurobiology (Emeritus), UCLA School of Medicine
and Brain Research Institute
Motor Neurobiology of the Spinal Cord
Timothy C. Cope, Ph.D., Professor of Physiology, Emory University School of Medicine
Nicotinic Receptors in the Nervous System
Edward D. Levin, Ph.D., Associate Professor/Department of Psychiatry and Pharmacology
and Molecular Cancer Biology and Department of Psychiatry and Behavioral
Sciences, Duke University School of Medicine
Methods in Genomic Neuroscience
Helmin R. Chin, Ph.D., Genetics Research Branch, NIMH, NIH
Steven O. Moldin, Ph.D, Genetics Research Branch, NIMH, NIH
Methods in Chemosensory Research
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Miguel A.L. Nicolelis, M.D., Ph.D., Professor of Neurobiology and Biomedical Engineering,
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The Somatosensory System: Deciphering the Brain’s Own Body Image
Randall J. Nelson, Ph.D., Professor of Anatomy and Neurobiology,
University of Tennessee Health Sciences Center

The Superior Colliculus: New Approaches for Studying Sensorimotor Integration
William C. Hall, Ph.D., Department of Neuroscience, Duke University
Adonis Moschovakis, Ph.D., Institute of Applied and Computational Mathematics, Crete
New Concepts in Cerebral Ischemia
Rick C. S. Lin, Ph.D., Professor of Anatomy, University of Mississippi Medical Center
DNA Arrays: Technologies and Experimental Strategies
Elena Grigorenko, Ph.D., Technology Development Group, Millennium Pharmaceuticals
Methods for Alcohol-Related Neuroscience Research
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of Health
David M. Lovinger, Ph.D., Laboratory of Integrative Neuroscience, NIAAA
In Vivo Optical Imaging of Brain Function
Ron Frostig, Ph.D., Associate Professor/Department of Psychobiology,
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Primate Audition: Behavior and Neurobiology
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The Primate Visual System
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Christine Collins, Department of Psychology, Vanderbilt University
Neurosteroid Effects in the Central Nervous System
Sheryl S. Smith, Ph.D., Department of Physiology, SUNY Health Science Center
Modern Neurosurgery: Clinical Translation of Neuroscience Advances
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Methods in Insect Sensory Neuroscience
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of Arizona, Tucson, AZ
Motor Cortex in Voluntary Movements
Alexa Riehle, INCM-CNRS, Marseille, France
Eilon Vaadia, The Hebrew University, Jeruselum, Israel
Neural Plasticity in Adult Somatic Sensory-Motor Systems
Ford F. Ebner, Vanderbilit University, Nashville, TN
Advances in Vagal Afferent Neurobiology
Bradley J. Undem, Johns Hopkins Asthma Center, Baltimore, MD
Daniel Weinreich, University of Maryland, Baltimore, MD
The Dynamic Synapse: Molecular Methods in Ionotropic Receptor Biology
Josef T. Kittler, University College London
Stephen J. Moss, University of Pennsylvania

THE DYNAMIC SYNAPSE
MOLECULAR METHODS IN
IONOTROPIC RECEPTOR BI0LOGY
Edited by
Josef T. Kittler
University College London
Stephen J. Moss
University of Pennsylvania
Boca Raton London New York
CRC is an imprint of the Taylor & Francis Group,

an informa business
1891_Discl.fm Page 1 Tuesday, February 7, 2006 4:35 PM
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Library of Congress Cataloging-in-Publication Data
The dynamic synapse : molecular methods in ionotropic receptor biology / [edited by] Josef T. Kittler
and Stephen J. Moss.
p. ; cm. -- (Frontiers in neuroscience)
Includes bibliographical references and index.
ISBN 0-8493-1891-2
1. Synapses. 2. Neuroplasticity. 3. Neural transmission. 4. Neurotransmitters.
[DNLM: 1. Synapses--physiology. 2. Carrier Proteins--physiology. 3. Neuronal
Plasticity--physiology. 4. receptors, Amino Acid--physiology. 5. Synaptic Transmission--physiology.
WL 102.8 D997 2006] I. Kittler, Josef T. II. Moss, Stephen J., 1962- III. Title. IV. Series: Frontiers
in neuroscience (Boca Raton, Fla.)
QP364.D96 2006
612.8--dc22 2005025723
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1891_C000.fm Page vii Friday, February 10, 2006 10:43 AM
Preface
Nerve cells in the brain communicate with each other at synapses, specialized sites
of cell-cell contact formed between a pre-synaptic nerve terminal and a post-synaptic
neuron. The properties of these synaptic connections determines, in part, how infor-
mation in the brain is processed and changes in the strength of these connections
(synaptic plasticity) is belie ved to be the molecular basis of information storage in
the brain. At synapses, information is passed in the form of neurotransmitters that
are released from the pre-synaptic terminal and dif fuse across the synaptic cleft to
activate post-synaptic neurotransmitter g ated ion channels (also called ionotropic
receptors). Activation of ionotropic receptors elicits the electroph ysiological
responses essential for f ast synaptic transmission and changes in the acti vity and
distribution of these receptors play a critical role in re gulating the strength of
synapses.
In this v olume in the CRC Press Methods and Ne w Frontiers in Neur oscience
series, we focus on tools and technologies to study the acti vity and functional
regulation of these important receptors and introduce the application of cutting-edge
approaches to the study of synapse and receptor biology . The recognition that
synapses can be studied in man y w ays and using a lar ge number of tools and
approaches, from molecular biology and protein biochemistry to imaging, electro-
physiology and in vivo studies, stimulated the production of this book, which aims
to provide a resource covering many of the methods that have become most relevant
to those interested in studying the synapse.
The first t o chapters provide broad overviews of the excitatory and inhibitory
synapse with Chapter 1 describing the v arious methods that ha ve been important
for establishing the critical role of AMPA receptor traf ficking in the plasticity o
excitatory synapses, and Chapter 2 focusing on the plasticity of inhibitory syn-
apses. Chapter 3, Chapter 4 and Chapter 5 focus on approaches for studying the
biochemical properties of the receptors and other proteins of the post-synaptic
domain. Chapter 3 describes recent advances in the use of proteomics approaches
for studying the complement of proteins in the post-synaptic density , whereas
Chapters 4 and 5 focus on tools for studying post-translational modification o
synaptic proteins by phosphorylation and palmito ylation, respectively. In Chapter
6 through Chapter 9, various state-of-the-art methods for studying the membrane
trafficking and sur ace localization of receptors are described, including biochem-
ical (Chapter 6), optical (Chapter 6, Chapter 7 and Chapter 8) and electrophysio-
logical (Chapter 9) approaches. Chapter 10 through Chapter 14 cover established
and newly emerging approaches for interfering with protein acti vity inside cells
from neuronal transfection and transduction to the genetic manipulation of neurons
by homologous recombination of ES cells. Chapter 10 focuses on the use of RNAi
technologies for knocking do wn e xpression le vels of tar get proteins of interest,
1891_C000.fm Page viii Friday, February 10, 2006 10:43 AM
whereas Chapter 11 through Chapter 13 focus on v arious approaches for trans-

fecting neural tissue, from the introduction of cDN As into primary neuronal
cultures to transfecting neurons in brain slices and nerve cells in vivo. In particular,
Chapter 12 and Chapter 13 describe ele gant advances in the use of viral v ectors
to transfect neurons in vivo . In addition, Chapter 13 and Chapter 14 focus on
methods for achieving long-term alterations in the genetic complement of neurons
either using the emer ging po wer of lenti viral v ector systems for the ef fective
delivery and expression of cDNAs and shRNAis to neurons in vivo (Chapter 13),
or by homologous recombination and “knockin” approaches (Chapter 14). Finally,
the last chapter explores the applicability of the “post-genomic” resources that are
now increasingly available to biologist and neuroscientists for the study of receptor
and synapse biology . Man y of the approaches described here are rele vant well
beyond studies of neurotransmitter receptors and are thus applicable to studies of
other important molecular and cellular components of the nerv ous system. We
therefore hope that this book will be useful to both ne wcomers and e xperienced
synaptic physiologists, in addition to the wider neuroscience community .
We are particularly grateful to the participating authors for their hard w ork and
excellent chapter contrib utions. We are also v ery grateful to Lorena Arancibia-
Carcamo, whose help with the formatting of chapters pro ved invaluable. We also
thank Pavel Osten, Jeremy Henley, Helene Marie, Sabine Levi and Lorena Arancibia-
Carcamo for their contrib utions to the front co ver art. We also thank the staf f of
CRC Press, in particular Barbara Norwitz, Robert Sims and Jill Jur gensen, for their
help, patience and encouragement.
Josef T. Kittler, Ph.D.

London
Stephen J. Moss, Ph.D .

Philadelphia
1891_C000.fm Page ix Friday, February 10, 2006 10:43 AM
The Editors
Josef T. Kittler, Ph.D., is a MRC fellow and Principal Investigator in the Department
of Physiology, University College London. Dr. Kittler received a B.Sc. in Biochem-
istry from the Uni versity of Bath, U.K., and carried out predoctoral research in the
Department of Ph ysiology and Pharmacology , Wake F orest Uni versity, Winston-
Salem, North Carolina, U.S.A. Dr . Kittler carried out doctoral studies in Molecular
Neuroscience in the MRC Laboratory for Molecular Cell Biology and postdoctoral
studies in the MRC Laboratory for Molecular Cell Biology and in the Departments
of Physiology and Pharmacology, University College London, U.K. Dr . Kittler has
published over 30 papers and book chapters in the field of molecular and cellula
neuroscience with a major focus on neuronal cell biology and the re gulation of
inhibitory synapses by membrane traf ficking and recepto -associated proteins.
Stephen J. Moss, Ph.D., is Professor of Neuroscience in the Department of
Neuroscience at the Uni versity of Pennsylv ania. Dr . Moss recei ved a B.Sc. in
Biochemistry from the University of Bath, U.K., and carried out his doctoral studies
in Neurobiology at the Medical Research Council (MRC) Laboratory for Molecular
Biology, Cambridge, U.K. Dr. Moss carried out postdoctoral studies on ion channel
phosphorylation at the Ho ward Hughes Medical Institute, Department of Neuro-
science, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A.
Following his postdoctoral w ork, Dr. Moss w as appointed as a lecturer and group
leader in the Department of Pharmacology and MRC Laboratory for Molecular Cell
Biology, University College London, U.K. In 2000, Dr . Moss w as appointed Pro-
fessor of Molecular Pharmacology and Cell Biology in the Pharmacology Depart-
ment at University College London. In 2003 Dr. Moss moved to the Department of
Neuroscience at the Uni versity of Pennsylv ania. Dr. Moss has published o ver 100
publications on the re gulation of neurotransmitter receptor acti vity and traf ficking
In addition, he has presented man y invited lectures and has been in volved in the
organization of several symposia and international meetings in the areas of synapse
function and ion channel re gulation.
1891_C000.fm Page xi Friday, February 10, 2006 10:43 AM
Contributors
I. Lorena Arancibia-Cárcamo Masaki Fukata
Department of Pharmacology Laboratory of Genomics and
University College London Proteomics
London, U.K. National Institute for Longe vity
Sciences
Yehezkel Ben-Ari Obu, Aichi, Japan
Institut de Neurobiologie de la
Méditerranée (INMED) Yuko Fukata
Institut National de la Santé et de la Laboratory of Genomics and
Recherche Médicale (INSERM) Proteomics
Marseille, France National Institute for Longe vity
Sciences
Obu, Aichi, Japan
David S. Br edt
Department of Ph ysiology
University of California at San Jean-Luc Gaiarsa
Francisco Institut de Neurobiologie de la
San Francisco, CA, U.S.A. Méditerranée (INMED)
Institut National de la Santé et de la
Recherche Médicale (INSERM)
Tanjew Dittgen Marseille, France
Department of Molecular Neurobiology
Max Planck Institute for Medical Jonathan G. Hanley
Research MRC Centre for Synaptic Plasticity
Heidelberg, Germany Department of Anatomy
School of Medical Sciences
Benjamin P. Fairfax University of Bristol
John Radcliffe Hospital Bristol, U.K.
Headley Way, Headington
Oxford, U.K. Jeremy M. Henley
MRC Centre for Synaptic Plasticity
Brian D. Fernholz Department of Anatomy
Department of Biochemistry School of Medical Sciences
NYU School of Medicine University of Bristol
New York, NY, U.S.A. Bristol, U.K.
1891_C000.fm Page xii Friday, February 10, 2006 10:43 AM
John T. R. Isaac Robert C. Malenka

Developmental Synaptic Plasticity Unit, Nancy Pritzker Laboratory
NINDS Department of Psychiatry and
Porter Neuroscience Research Center Behavioral Sciences
Bethesda, MD, U.S.A. Stanford University School of Medicine
Palo Alto, CA, U.S.A.
Tija C. J acob
Department of Pharmacology Hélène Marie
University College London European Brain Research Institute
London, U.K. Fondazioni Rita Levi-Montalcini
Rome, Italy
Bryen A. Jordan
Department of Biochemistry Stephen J. Moss
NYU School of Medicine Department of Neuroscience
New York, NY, U.S.A. School of Medicine
University of Pennsylvania
Jasmina N. J ovanovic Philadelphia, PA, U.S.A.
Department of Pharmacology
School of Pharmac y Thomas A. Neubert
University of London Department of Biochemistry
London, U.K. NYU School of Medicine
New York, NY, U.S.A.
Josef T. Kittler
Department of Ph ysiology Peter L. Oli ver
University College London MRC Functional Genetics Unit
London, U.K. Department of Human Anatomy &
Genetics
University of Oxford
Hey-Kyoung Lee
Oxford, U.K.
Department of Biology
Neuroscience and Cognitive Sciences
Pavel Osten
(NACS) Program
Department of Molecular Neurobiology
University of Maryland
Max Planck Institute for Medical
College Park, MD, U.S.A. Research
Heidelberg, Germany
Sabine Lévi
INSERM U.497 Pavel V. Perestenko
Ecole Normale Supérieure MRC Anatomical Neuropharmacology
Paris, France Unit
Oxford, U.K.
Pawel Licznerski
Department of Molecular Neurobiology Sophie Restituito
Max Planck Institute for Medical Department of Biochemistry
Research NYU School of Medicine
Heidelberg, Germany New York, NY, U.S.A.
1891_C000.fm Page xiii Friday, February 10, 2006 10:43 AM
Trevor G. Smart Antoine Triller

Department of Pharmacology INSERM U.497
University College London Ecole Normale Supérieure
London, U.K. Paris, France
Philip Thomas Edward B. Ziff

Department of Pharmacology Department of Biochemistry
University College London NYU School of Medicine
London, U.K. New York, NY, U.S.A.
1891_C000.fm Page xv Friday, February 10, 2006 10:43 AM
Table of Contents
Chapter 1
Methods for Unco vering the Mechanisms of AMPA Receptor Traffickin .............1
Sophie Restituito and Edwar d B. Zif f
Chapter 2
Long-Term Plasticity at Inhibitory Synapses: A Phenomenon That
Has Been Ov erlooked..............................................................................................23
Jean-Luc Gaiarsa and Yehezkel Ben-Ari
Chapter 3
New Tricks for an Old Dog: Proteomics of the PSD .............................................37
Bryen A. Jordan, Brian D. F ernholz, Thomas A. Neubert, and
Edward B. Zif f
Chapter 4
Phosphorylation Site-Specific Antibodies as Research Tools in
Studies of Native GABAA Receptors ......................................................................57
Jasmina N. Jovanovic
Chapter 5
Protein Palmitoylation by DHHC Protein F amily ..................................................83
Yuko Fukata, David S. Br edt, and Masaki Fukata
Chapter 6
Studying the Localization, Surf ace Stability and Endoc ytosis of
Neurotransmitter Receptors by Antibody Labeling and
Biotinylation Approaches ........................................................................................91
I. Lorena Arancibia-Cárcamo, Benjamin P. Fairfax,
Stephen J. Moss, and J osef T. Kittler
Chapter 7
Visualization of AMPAR Trafficking and Sur ace Expression.............................119
Pavel V. Perestenko and Jeremy M. Henley
Chapter 8
Neurotransmitter Dynamics ...................................................................................143
Sabine Lévi and Antoine Triller
1891_C000.fm Page xvi Friday, February 10, 2006 10:43 AM
Chapter 9
Receptor Dynamics at the Cell Surf ace Studied Using Functional Tagging .......155
Philip Thomas and Trevor G. Smart
Chapter 10
RNAi and Applications in Neurobiology ..............................................................177
Tija C. Jacob
Chapter 11
Transfecting and Transducing Neurons with Synthetic Nucleic Acids and
Biologically Active Macromolecules ....................................................................205
Josef T. Kittler, Jonathan G. Hanley, and John T. R. Isaac
Chapter 12
Acute In Vivo Expression of Recombinant Proteins in Rat Brain
Using Sindbis Virus ...............................................................................................241
Hélène Marie and Robert C. Malenka
Chapter 13
Lentivirus-Based Genetic Manipulations in Neurons In Vivo ..............................249
Pavel Osten, Tanjew Dittgen, and Pawel Licznerski
Chapter 14
AMPA Receptor Phosphorylation in Synaptic Plasticity:
Insights from Knockin Mice ................................................................................ 261
Hey-Kyoung Lee
Chapter 15
Genomic and Post-Genomic Tools for Studying Synapse Biology .....................279
Josef T. Kittler and P eter L. Oliver
1891_C001.fm Page 1 Friday, February 10, 2006 10:49 AM
1 Methods for Uncovering

the Mechanisms of
AMPA Receptor
Trafficking
Sophie Restituito and Edward B. Ziff
CONTENTS
1.1 Introduction ......................................................................................................1

1.2 Approaches Used in the Study of AMPA Receptor Traffickin .....................3
1.2.1 Protein Interaction Assays ...................................................................3
1.2.2 Cell Biology .........................................................................................7
1.2.2.1 Microscopy ...........................................................................7
1.2.2.2 Cell Biology Assays ...........................................................11
1.2.3 Biochemistry ......................................................................................12
1.2.3.1 Biochemical Assays ............................................................12
1.2.3.2 Phosphorylation ..................................................................12
1.2.4 Electrophysiology...............................................................................13
1.2.4.1 Blocking Peptide.................................................................13
1.2.4.2 Rectification Ind x..............................................................13
1.2.4.3 Agonists and Toxins ...........................................................14
1.2.5 Pharmacology.....................................................................................15
1.2.6 Genetic Approach...............................................................................15
1.2.6.1 Murine Models.................................................................... 15
1.2.6.2 Knockout Mice ...................................................................15
1.2.6.3 C. Elegans ...........................................................................16
1.3 Synthesis, Summary and Speculation ............................................................16
References................................................................................................................17
1.1 INTRODUCTION
Information storage in the brain involves alterations in the strength of communication
between neurons. This requires activity-dependent, long-lasting changes in synaptic
transmission. Long-term potentiation (LTP) is a long lasting use-dependent increase
1
2 The Dynamic Synapse
in the ef ficien y of e xcitatory synaptic transmission that has been suggested to

underlie certain forms of learning and memory [1]. The induction of L TP requires
Ca2+ entry through the N-meth yl-D-aspartate receptor (NMD AR). Ho wever, the
region within the synapse whose regulation results in LTP is still controversial. Some
groups suggest a pre-synaptic modification that results in an increase in the amoun
of glutamate released, whereas others suggest a post-synaptic modification, such a
an increase in the number of receptors or a change in receptor properties [2].
Interestingly, the description of the silent synapse, synapses that contain NMD AR
only b ut could acquire α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
receptor (AMPAR) as a result of synaptic acti vity, support a post-synaptic mecha-
nism [3–5].
Glutamate receptors mediate most excitatory synaptic transmission in the brain.
The ionotropic glutamate receptors are clustered with associated do wnstream sig-
nalling molecules that are found in the post-synaptic density (PSD), and the synaptic
clustering of these receptors is critical for rapid and ef ficient synaptic transmission
This comple x of receptors with signalling molecules can also under go dynamic
changes. In particular , changing the number of glutamate receptors at the synaptic
membrane could constitute a critical mechanism for rapidly altering synaptic
strength.
Two major classes of ionotropic glutamate receptors exist, the NMDAR and the
AMPAR. A third class, the kainate receptors, will not be discussed here as it has
been reviewed elsewhere [6]. Both NMD AR and AMPAR are highly concentrated
at e xcitatory synapses link ed to the PSD b ut the y interact with dif ferent sets of
scaffolding proteins. In addition, whereas NMD AR are v ery stably localized at the
PSD, AMPAR cycle rapidly to and from the synaptic membrane. This difference in
trafficking beh vior of NMD AR versus AMPAR may reflect their use of di ferent
mechanisms for anchoring to the PSD.
The conversion of a silent synapse to a synapse with AMPAR, a change that
creates a functional synapse, has been proposed as one of the main mechanisms for
LTP induction. F or these reasons, studies of the traf ficking of AMPAR and their
cycling in and out of the synapse ha ve provided a large step in understanding L TP.
Synaptic organization, assembly and traf ficking h ve been studied e xtensively for
other receptors, such as the acetylcholine receptor at the neuromuscular junction
[7]. AMPAR trafficking has been one of the most xtensively studied properties of
the excitatory synapse because of its implication in synaptic plasticity .
The complexity of the or ganization of the brain mak es assaying receptor prop-
erties without disrupting the system dif ficult. A complete picture of synaptic re gu-
lation comes from combining a wide range of methodological approaches, such as
electrophysiology, cell biology, biochemistry and genetics. The development of new
methods or the adaptation of methods used in other systems has allo wed a better
understanding of the traf ficking of AMPAR. Mechanisms of AMPAR traf fickin
have been reviewed elsewhere [3,8,9], and this chapter will present an o verview of
the different approaches that have been used to study the trafficking of AMPAR and
its implication for synaptic plasticity , focusing on the contrib ution and the impor -
tance of each method.
Methods for Uncovering the Mechanisms of AMPA Receptor Trafficking 3
1.2 APPROACHES USED IN THE STUDY OF AMPA

RECEPTOR TRAFFICKING
AMPARs are hetero-oligomeric proteins composed of subunits GluR1–4 [10]. Each
receptor complex contains four sub units [11]. In an adult hippocampus, tw o heter-
omeric receptor comple xes predominate: GluR1/2 and GluR2/3 [12]. All subunits
have an extracellular domain, three transmembrane domains, a membrane re-entrant
hairpin that forms the pore and a c ytoplasmic C-terminal tail. The tail can either be
short or long. GluR1, GluR4 and one of the splice v ariants of GluR2 ha ve long,
highly homologous cytoplasmic tails containing around 80 amino acids. In contrast,
the predominant forms of GluR2 and GluR3 and one GluR4 splice v ariant ha ve
short, homologous c ytoplasmic tails of around 50 amino acids [8]. Through their
cytoplasmic tails, each sub unit interacts with specific ytoplasmic proteins. Man y
of these interacting proteins ha ve single or multiple PDZ domains, which mediate
protein-protein interactions with the e xtreme C-termini of the tar get proteins. Two
classes of PDZ domains have been defined based on their binding specificities. Cla
I PDZ domains ha ve a histidine at the αB1 position, b ut in class II, this residue is
hydrophobic [13]. GluR1 has a class I PDZ lig and whereas GluR2, GluR3 and
GluR4c have class II PDZ lig ands.
Channels containing exclusively GluR1, GluR3 and GluR4 are Ca 2+ permeable,
whereas inclusion of GluR2 makes a channel Ca2+ impermeable. This specific chang
in permeability imparted by GluR2 is the result of a post-transcriptional modificatio
introduced by RN A editing of the GluR2 mRN A residues at the Q/R site in the
channel pore. Glutamine Q607 is replaced by an arginine in 99% of post-natal GluR2
[14]. Interestingly , this property of GluR2 dominates the permeability of the
AMPAR. Indeed, its presence in a comple x confers Ca 2+ impermeability to the
AMPAR [15]. The existence of different AMPAR heterotetramers will then generate
channels with different electrophysiologic and trafficking properties
1.2.1 PROTEIN INTERACTION ASSAYS

The use of protein interaction assays, such as the yeast tw o-hybrid screen, the co-
immunoprecipitation assay and the pull do wn assay , has allo wed a great break-
through in the characterization of partners ofAMPAR involved in receptor anchoring,
trafficking and egulation ( Table 1.1). Indeed, these approaches h ave all owed the
characterization of protein comple xes involving multiple partners, highlighting the
complex regulation of AMPAR trafficking.
The yeast two-hybrid screen is a potent technique to identify protein interactors
in vivo [16,17]. This well-known technique consists of fusing the protein of interest
to a DNA-binding domain and e xpressing it in a yeast host cell carrying a reporter
gene under the control of the transcriptional acti vity of the DN A-binding domain.
The fusion protein cannot acti vate transcription on its o wn, b ut it can be used as
“bait” to screen a library of cDN A clones encoding proteins that are fused to an
activation domain. The cDNA clones within the library that encode a fusion protein
capable of interaction with the “bait” are identified by their ability to act vate
expression of the reporter gene.
4
TABLE 1.1
Summary of the Various Approaches Used to Characterize Proteins Interacting with AMPARs
Two-Hybrid Co IP/ Murine
Screen Biochemistry Mutagenesis Microscopy Physiology Models
ABP/GRIP Interaction with Interaction with Mutation of Subcellular localization and co- Regulation of GluR2
GluR2 GluR2 [20,21,23] interacting localization with AMPAR by trafficking [58
[20,21,23] Interaction regulated motif [28] confocal microscopy [20,23]
Interaction with by phosphorylation Subcellular localization and EM
liprin- and LAR [53,54] [21,22]
[31] Localization, EM of GRIP [32]
Colocalization with liprin [31]
Regulation of GluR2 surf ace
accumulation [28]
PICK1 Interaction with Interaction with Subcellular localization and co-
GluR2 [24] GluR2 [19,24] localization with GluR2 [24]
NSF Interaction with Interaction with Colocalization with GluR2 [27] Regulation of GluR2
GluR2 [25–27] GluR2, SNAP [27] trafficking [26,55–57
and PICK1 [19]
SAP97 Interaction with
GluR1 [18]
The Dynamic Synapse

RIL Interaction with Interaction with
GluR1 [30] GluR1 [30]
4.1N Interaction with Interaction with Colocalization with AMPAR [29]
GluR1 [29] GluR1 [29]
STG Interaction with Subcellular localization and co- Regulation of AMPAR Cloning [65]
AMPAR [65] localization with GluR4 [65] trafficking [65

This interaction is usually confirmed by biochemical methods, such as co

immunoprecipitation. This standard technique is based on the formation of a comple x
between an antibody specific to a protein of interest, the protein of interest and al
the proteins that interact with it. The complex is then isolated using beads to which
the antibody is link ed [18]. A pull-do wn assay can also be used to confirm a
interaction between two proteins. This method is an in vitro method very similar to
co-immunoprecipitation, e xcept that a tagged bait protein is used instead of an
antibody [19].
The major proteins interacting with the C-terminal domains of AMPAR subunits
were identified using a yeast t o-hybrid screen. Thus, Dong et al. identified a n vel
protein, GRIP (glutamate receptor -interacting protein), interacting with the C-ter -
minal tails of GluR2 and GluR3 [20] (Table 1.1). GRIP contains seven PDZ domains,
is enriched in the brain and co-localizes with AMPAR at e xcitatory synapses. The
interaction is between PDZ4 and 5 of GRIP and the last seven amino acids of GluR2
and GluR3, b ut not GluR1. Subsequently , Srivastava et al., using the same assay ,
identified another partner of GluR2, ABP (AMPA receptor-binding protein, Table
1.1) [21], which is closely related to GRIP . ABP has splice v ariants with either six
or seven PDZ domains and also interacts with the C-terminal tails of GluR2 and
GluR3 but not GluR4, GluR1 or NMDA-NR2A [21,22]. The interaction is between
the C-terminal motif, VKI of GluR2 and PDZ5 of ABP. ABP and GRIP e xhibit 64
to 93% amino acid homology in the six PDZ domains.They have a similar structural
organization, including tw o clusters of three PDZ domains, with each cluster fol-
lowed by linker regions. These studies suggest that GRIP andABP could be involved
in the ta rgeting of AMPAR to the synapse ( Figure 1.1). A palmit oylated form of
ABP, pABP, w as also identified ( able 1.1) [23]. This splice v ariant is present in
spines, whereas non-palmitoylated ABP and GRIP are found in the dendritic shaft.
The authors propose that pABP andABP provide alternative anchorages for AMPAR
at the synapse or intracellular membranes during receptor trafficking (Figure 1.1[3b
and Figure 1.1[8b]). Xia et al. identified a n vel protein that also interacts with the
C-terminal tail of GluR2, PICK1 (protein interacting with C kinase, Table 1.1) [24].
Through its PDZ domain, PICK1 interacts with the last seven amino acids of GluR2,
GluR3 and GluR4, b ut not with GluR1. PICK1 is thought to function in the traf-
ficking phase of AMPAR transport (Figure 1.1[3b] and Figure 1.1[5b]).
The yeast two-hybrid approach has also identified r gulatory proteins associated
with AMPARs. Several groups showed that NSF (N-ethylmaleimide sensitive fusion
protein) interacts with the C-terminal tail of GluR2 and interacts weakly with GluR3
but not with GluR1 or GluR4 (Table 1.1) [25–27]. NSF is a homomeric ATPase and
a component of the protein machinery responsible for v arious membrane fusion
events. It interacts with α and β SNAP and disassembles the SN ARE comple x,
driven by ATP h ydrolysis [19,28]. Osten et al. and Hanle y et al. suggest that the
NSF/SNAP comple x could serv e as a chaperone in dissociating PICK1-AMP AR
complexes involved in AMPAR cycling (Figure 1.1[3b] and Figure 1.1[6b]) [19,28].
Several proteins ha ve thus been sho wn to interact with the C-terminal domain
of GluR2. To dissect the function and the implication of each protein in this complex,
Osten et al. used mutagenesis of the C-terminal tail of GluR2 (T able 1.1) [28].
By removing the binding site for ABP/GRIP, Osten et al. demonstrated that the
4b
5b
4a
3a 3b
8b 6b
2a 7b
ER
golgi 9b
1 2b 10b
GluR1/2 GluR2/3 GluR1/2+GluR2/3 NMDAR
actin RIL 4.1 SAP97 PKC PICK1 NSF SNAP ABP/GRIP PSD95 Stg
activity dependent constitutive NMDAR synaptic activity
FIGURE 1.1 (Color figure fol ows page 176.) Schematic representation of AMPAR traffick
ing. Pathway (a) represents GluR1 traf ficking and path ay (b) represents GluR2 traf ficking
AMPAR complexes exit from the ER/Golgi associated with star gazin (1). GluR1-containing
AMPAR traf fic through the secretory path ay (2a) and are inserted at e xtrasynaptic sites
through an acti vity-dependent mechanism (3a). Star gazin binds PSD95, which localizes the
complex at the synapse where the receptor can bind other scaf folding proteins such as RIL
and 4.1N (4a). Stargazin can then be released. GluR2-containing AMPAR traffic through th
secretory pathway (2b) and are inserted at synaptic sites through a constituti ve mechanism
(3b). PICK1 f acilitates the transport of the receptor possibly both to and from the plasma
membrane but is remo ved from the AMPAR by the NSF/SN AP when the receptor reaches
the plasma membrane. NSF/SNAP/ PICK1 forms a transient complex with GluR2-containing
AMPAR (not shown on this schematic).AMPAR are stabilized at the membrane by scaffolding
proteins. Phosphorylation by PKC pre vents interaction between ABP/GRIP and GluR2 (4b)
and favors the PICK1/GluR2 interaction (5b). The AMPAR are then internalized follo wing a
coupling with PICK1, and AMPAR/PICK1 complex are destabilized by NSF/SNAP when the
receptor reaches its destination (6b). GluR2-containing AMPAR can then interact with an
intracellular pool of ABP/GRIP, possibly in the endosome (7b), and get rec ycled to the
membrane through interaction with PICK1 (8b) or tar geted to lysosomes (9b) and de graded
(10b).
plasma-membrane tar geting of the receptor w as not dependent on PDZ-mediated

binding but that the PDZ binding motif w as required for surf ace accumulation of
GluR2-containing receptor at the synapse. They proposed that the binding of a PDZ
protein, ABP or GRIP, but not PICK1, mediated accumulation of the AMPAR at the
synapse by regulating the receptor’s local endocytic synaptic turnover (Figure 1.1).
In a tw o-hybrid screen using the C-terminal domain of GluR1 as bait, Shen et

al. sho wed an interaction between the membrane proximal re gion of C-terminal
domains of GluR1 and protein 4.1N, a protein critical for the or ganization and
maintenance of the spectrin-actin cytoskeleton (Table 1.1) [29]. The authors suggest
that protein 4.1N might play a role in the anchoring of AMPAR to the actin c yto-
skeleton and stabilizing the sur face expression of the receptors ( Figure 1.1).
However, Schulz et al. found that the C-terminal tail of GluR1 is highly toxic
to yeast [30]. To circumvent this condition, the y used the C-terminal tail of GluR2
fused to the last 10 amino acids of GluR1 as a bait in a two-hybrid screen and found
an interaction between GluR1 and RIL (re version-induced LIM protein, Table 1.1)
[30]. RIL belongs to a f amily of proteins that has been proposed to function as
adaptors linking proteins to the actin c ytoskeleton. RIL can bind α-actinin via a
PDZ domain and GluR1 by a LIM domain (Figure 1.1). Co-immunoprecipitation
or pull-down assays from transfected cells expressing the proteins of interest or from
brain lysate extracts were used to further characterize all of these interactions (Table
1.1) [21–24,29,30].
Leonard et al. chose a co-immunoprecipitation approach using various extraction
conditions to identify partners of the GluR1 sub unit (Table 1.1) [18]. They charac-
terized an interaction between SAP97, a member of the SAP f amily (synapse-
associated proteins), and GluR1. The SAP97 f amily of proteins clusters NMD ARs
at the postsynaptic membrane, and SAP97 could play a similar role for the AMPAR
(Figure 1.1).
These assays have also allowed the identification of la ger complexes involving
multiple proteins. Using a tw o-hybrid screen, Wyszynski et al. identified a n vel
partner for GRIP , liprin- α (Table 1.1) [31]. Liprin had first been identified by i
interaction with a receptor tyrosine phosphatase, LAR. Liprin- α interacts through
its C-terminal domain, with the PDZ6 of GRIP . The authors were able to co-
immunoprecipitate a comple x containing GRIP, liprin- α, GluR2/3 and LAR. Their
data implicate liprin and LAR in the surf ace expression or the synaptic tar geting of
AMPAR. Similarly , Hanle y et al. were able to sho w, using pull-do wn and co-
immunoprecipitation approaches, that GluR2, NSF, PICK1 and SNAP form a com-
plex (Table 1.1) [19]. They showed that the NSF/GluR2 interaction stabilizes the
AMPAR at the surf ace by NSF’ s ability to disrupt the PICK1/GluR2 comple x,
preventing PICK1 dependent-endoc ytosis of the AMPAR (Figure 1.1[3b], Figure
1.1[8b], Figure 1.1[5b] and Figure 1.1[6b]).Together, these approaches have allowed
the identification of la ge protein comple xes involved in the synaptic localisation,
stabilization or re gulation of AMPAR at the synapse.
1.2.2 CELL BIOLOGY

1.2.2.1 Microscopy
While biochemical assays allo wed the identification of components of AMPAR asso-
ciated complexes, a further critical step w as to study the function of these proteins in
more physiological systems. The use of classical techniques such as light and electron
microscopy allowed the visualization of the proteins in tissues or culture.
Electron microscopy, a classical technique that uses a beam of highly ener getic
electrons to examine objects on a very fine scale, all ws the localization of a protein
at an ultra-structural le vel. Using immunoperoxidase electron microscop y, Dong et
al. showed that GRIP1 is present in the dendrite and dendritic spine, close to the
PSD, b ut is also found in the endoplasmic reticulum (ER) and Golgi ( Table 1.1)
[22]. These various localizations suggest a function of this protein at different points
in the trafficking path ay and could implicate this protein in theAMPAR trafficking
The results were confirmed using immunogold electron microsco y, which allo ws
a higher resolution (T able 1.1) [21,22]. Similarly , Wyszynski et al. sho wed a pre-
and post-synaptic localization of liprin- α and GRIP, raising potential new functions
for these tw o proteins (T able 1.1) [31,32]. Using post embedding immunogold
electron microscopy, Takumi et al. performed a very precise study of the relationship
between AMPAR and NM DAR density at specific synapses Table 1.2) [33]. They
were able to detect synapses without an y AMPAR staining, consistent with the
existence of silent, AMPAR-deficient synapses. They also sho wed that the number
of AMPAR in a synapse is proportional to the PSD area, whereas NMD AR number
increases v ery little with synapse size. They sho wed a v ery lar ge v ariability in
AMPAR number and heterogeneity in synapse size, with which it can be correlated
and suggest that the ratio between AMPAR and NMDAR depends critically on PSD
diameter. Thus, electron microscop y studies ha ve allowed the visualization of the
ultra-structure of the PSD. They also gi ve an idea of the precise localizations of
receptors at an ultra-structural le vel.
Electron microscop y can be used for more comple x studies. Kiro v et al. took
advantage of this technique to study changes in the number of spines during synaptic
activity using hippocampal slices in culture. Using serial electron microscop y anal-
ysis, the y sho wed that a blockade of acti vity increases spine number [34]. They
suggest that neurons can re gulate spine number depending on the le vel of activity.
Confocal microscopy, a well-known technique that uses spatial filtering to elim
inate out-of-focus light, has been widely used for a better characterization of protein
subcellular localization, for the co-localization of se veral proteins and for definin
possible functions of these proteins. F or example, by confocal microscop y, Xia et
al. showed a colocalization of PICK1, GluR2 and protein kinase C (PKC) in hippo-
campal neurons in culture (T able 1.1) [24].
The use of recombinant tagged receptors and the development of overexpression
techniques that may be emplo yed together with confocal microscop y studies ha ve
allowed a more complex investigation of the AMPAR trafficking in neuronal models
Specially, tagging with green fluorescent protein (GFP) has pr vided a major insight
into the dynamics of AMPAR trafficking in l ving cells. Shi et al. o verexpressed N-
terminally GFP-tagged GluR1 sub units in hippocampal neurons in culture or in
organotypic slices using a Sindbis virus system and e xamined the beha vior of the
subunit after tetanic stimulation (T able 1.2) [35]. The change in distrib ution w as
monitored by time-lapse, tw o-photon laser scanning microscop y. GluR1-GFP w as
mainly intracellular in the dendritic shaft. Interestingly , redistribution to the spine
was observed after tetanic stimulation.
However, GFP does not distinguish surf ace from intracellular AMPAR. To cir-
cumvent this problem, Passafaro et al. have used a thrombin surf ace cleavage assay
Methods for Uncovering the Mechanisms of AMPA Receptor Trafficking
TABLE 1.2
Summary of the Various Approaches Used to Study Trafficking of AMPARs
Physiology/ Physiology/
Microscopy Biochemistry Peptide Block/Drug Rectification Pharmacology Animal Models
GluR1 Activity-dependant Phosphorylation [52] Regulation of GluR1 Activity- Function of GluR1

insertion [35,36] trafficking by dependent [67]
Local synthesis [38] phosphorylation insertion
[52] [59,60]
GluR2 Constitutive insertion Trafficking of GluR2 Regulation of GluR2 Constitutive Incorporation Function of GluR2
at synapse [36] through secretory trafficking by NSF insertion at of GluR2 at [68–71]
Trafficking of GluR2 pathway [48,49] [26,55–57] synapse synapse [61]
in live neuron [37] Phosphorylation Regulation of GluR2 [60,61]
Local synthesis [38] [53,54] trafficking by
Movement of single ABP/GRIP [58]
receptor [40–42]
AMPARs EM relationship AMPAR movement Trafficking of Function of GluR2
AMPAR and in/out surface AMPAR [64] and GluR3 [72]
NMDAR [33] [45–47] Function of GluR [73]
9
to study AMPAR surface delivery (Table 1.2) [36]. AMPAR subunits tagged at the
N-terminal domain with a hemagglutinin (HA) tag followed by a thrombin cleavage
site were transfected into hippocampal neurons and the localization of the sub units
was assessed by confocal microscopy. They were able to show that GluR1 and GluR2
have distinctive roles in AMPAR surface accumulation and synaptic plasticity ( Fig-
ure 1.1[2a] and Figure 1.1[2b]). HA-GluR1 has a much slower time course of surface
reinsertion than HA-GluR2. Activity promotes GluR1 reinsertion b ut not GluR2
(Figure 1.1[3a] and Figure 1.1[3b]). Ho wever, GluR1 acts dominantly o ver GluR2,
and GluR1 properties will predominate when it is in a comple x with GluR2. Using
a synaptic marker, the authors were able to sho w that most HA-GluR1 accumulates
during the first f w minutes follo wing reinsertion in a non-synaptic area of neuron
surface, whereas ne wly inserted HA-GluR2 accumulates directly at the synapse.
They concluded that GluR2 e xocytosis is rapid and constituti ve and GluR1 is slo w
and inducible. They also concluded that GluR1 determines the rate and site of
exocytosis and GluR2 controls the recycling and endocytosis. For the first time, thi
approach has allowed the study of reinsertion of AMPAR; however, it presents some
limitations in temporal resolution.
Ashby et al. used a modified GFP to assess the m vement of AMPAR at the
cell surface (Table 1.2) [37]. The pH-sensitive GFP (ecliptic pHluorin) is nonfluo
rescent at pH less than 6.0 and its brightness increases up to pH 8.5. Placed on the
N-terminal domain of GluR2, the protein will be fluorescent at the cell sur ace and
not inside the cell, where the en vironment is mainly acidic. Using this mutant GFP ,
surface GluR2 was then assessed by live cell confocal microscopy. The authors were
able to sho w that synaptic and e xtrasynaptic AMPAR act dif ferently follo wing
NMDAR acti vation. They could sho w a rapid internalization of e xtrasynaptic
AMPAR that preceded the internalization of synaptic AMPAR.
Recently, Ju et al. took adv antage of properties of no vel dyes to study the local
synthesis of AMPAR (Table 1.2) [38]. Local synthesis has been broadly studied and
reviewed else where [39]. The biarsenical dyes bind to short peptide sequences
containing four c ysteine residues. They are nonfluorescent until th y bind to the
tetracysteine motif and then become green (FlAsH-EDT 2) or red (ReAsH-EDT 2).
The authors introduced the tetrac ysteine motif in the C-terminal tails of GluR1 and
GluR2, HA/thrombin N-terminally tagged (GluR1c y4 and GluR2c y4). Using this
technique, they could compare the dendritic traf ficking of pre- xisting and recently
synthesized AMPAR subunits. They were able to sho w that a chronic blockade of
activity, which enhances synaptic strength, causes an increase in the amount of both
pre-existing and recently synthesized GluR1c y4 but not GluR2cy4. However, high-
K+ depolarization that w ould mimic L TP increased both recently synthesized
GluR1cy4 and GluR2cy4. They concluded that AMPAR subunit mRNAs are targeted
to proximal and distal dendrites where the sub units can be locally translated, pro-
cessed and inserted at or near synaptic sites. This novel approach could be used to
study other aspects of AMPAR trafficking
Borgdorff et al. de veloped a v ery original approach based on confocal micro-
scopy to study the mo vement of single endogenous receptors at the cell surf ace
(Table 1.2) [40]. They used 0.5 µm late x beads coated with antibodies ag ainst the
N-terminal domain of GluR2 to study the lateral mobility of nati ve GluR2-containing
AMPAR [40]. Each bead was held in contact with the surface of a neurite with laser
tweezers for 5 to 10 sec to allo w binding to GluR2. The bead’ s trajectory w as
followed by video microscop y for 200 sec with a spatial resolution of 5 to 10 nm.
They found that the GluR2-containingAMPAR exists in two states, one characterized
by a rapid dif fusion movement and the other a confined, sl w moving state. They
showed that surrounding the synapse w as a region of low receptor diffusion. Basal
neuronal activity or Ca2+ regulate the movements of AMPAR. However, using caged
Ca2+, which allo ws a local increase of Ca 2+, they could sho w that local Ca 2+ influ
triggers lateral GluR2 mo vement. The same group adapted this technique to study
AMPAR movement within the synaptic membrane ( Table 1.2) [41,42]. They used
Cy3-coupled antibodies or quantum dot coupled antibodies to label the receptor and
to follow its mo vement inside the synapse. Quantum dots are fluorescent semicon
ductor nanocrystals that are encapsulated in phospholipids, allo wing their use as
probes in biological imaging [43,44]. The authors were able to sho w that AMPAR
are mobile between synaptic and extrasynaptic locations (Figure 1.1) [41]. Changes
in neuronal activity modified AMPAR but not NMDAR localization [42].
Thus, complementary approaches have been employed to understand the move-
ment of AMPAR at the synapse. All suggest a v ery high mobility of the receptor at
the surf ace, even if the receptor is part of a lar ge complex of proteins. They also
showed that this mo vement seems to be dependent on the sub unit composition of
the heterotetramer and that the insertion and exit of the receptor from the cell surface
takes place at specific sites on the plasma membrane
1.2.2.2 Cell Biology Assays
Other approaches, combining microscop y and biochemistry , ha ve been used to

understand the mechanisms that re gulate the mo vement of AMPAR in and out of
the synapse (Table 1.2) [45–47]. Ehlers used an antibody-feeding assay to study this
aspect of trafficking [45]. Hippocampal neurons in culture were stained, while lving,
with an antibody ag ainst GluR1 or GluR2 and internalized receptors were detected
after acidic treatment that remo ves all the receptors remaining at the cell surf ace.
This technique allowed the authors to determine the rate of endocytosis. Co-staining
with specific mar ers of the endoc ytosis and de gradation pathways allowed Ehlers
to identify the pathways [45]. Ehlers and Lin et al. used also a biotinylation approach
to complement the antibody-feeding approach [45,46]. Biotin is a small molecule
that can be conjug ated to a protein of interest. It can then interact with strepta vidin
and the comple x can be isolated and detected. This approach is more quantitati ve
and more sensitive than using immunofluorescent staining. Using these approaches
several distinct pathw ays for AMPAR endoc ytosis ha ve been described. AMPAR
can be endoc ytosed via NMD A-dependent or independent pathw ays [45,47] or an
insulin-dependent pathway [46,47]. AMPAR endocytosed through a NMDA-depen-
dent pathw ay go to the rec ycling endosome and are reinserted into the plasma
membrane (Figure 1.1[7b] and Figure 1.1[8b]). This pathway involved Ca 2+ flu es
and protein dephosphorylation [45,47], whereas AMPAR endoc ytosed through
AMPA-dependent internalization are de graded [45] (Figure 1.1, Figure 1.1[9b] and
Figure 1.1[10b]).
1.2.3 BIOCHEMISTRY
1.2.3.1 Biochemical Assays
Greger et al. chose a biochemical approach to study the traf ficking of AMPAR
through the secretory path way (Table 1.2) [48,49]. Taking advantage of subcellular
and sucrose gradient fractionations, the y identified t o forms of GluR2, one that is
mature and present in the synaptic fraction and a second that is immature and
enriched in the ER [49]. Using de glycosylation methods and pulse chase e xperi-
ments, they were able to sho w that the majority of GluR2 is immature, retained in
the ER and not associated with GluR1 ( Figure 1.1). By mutagenesis, they identifie
several regions in the C-terminal tail of GluR2 in volved in the ER retention. Most
significantl , they also sho wed that R607, at the Q/R editing site, also controls ER
retention [49]. Using h ydrodynamic methods and nati ve P AGE gels that mak e
possible the molecular weight characterization of comple xes, the y sho wed that
tetramerisation but not dimerization is controlled by the re-entrant pore loop con-
taining the Q/R editing site [48]. They proposed that the edited R-sub units remain
mainly dimeric and ER-retained unless comple xed with unedited Q sub units,
whereas the unedited Q-subunits can tetramerize and traffic to the cell surace (Figure
1.1). Thus, the Q/R site affects subunit stoichiometry and, in turn, channel function.
This editing site affects the functional properties ofAMPAR at two levels: one during
assembly, by restricting the number of GluR2 incorporated into tetramers, and the
other during ion conduction by controlling major AMPAR transmission properties,
in particular Ca2+ permeability [48]. Thus, biochemical and cell biologic approaches
have allowed a dissection at a molecular le vel of the traf ficking of the AMPAR.
1.2.3.2 Phosphorylation
Phosphorylation is one of the major mechanisms of re gulation of AMPAR function.

Its importance has also been highlighted in L TP and long-term depression (L TD)
by se veral groups [3,8,50,51]. A classic approach to study phosphorylation is to
label the cells with 32P orthophosphate and immunoprecipitate the protein of interest,
as done by Roche et al., to identify the phosphorylation sites of the GluR1 sub unit
[52]. Two-dimensional phosphopeptide mapping after trypsin digestion and treat-
ment with drugs to stimulate the various kinases are also commonly used to identify
possible phosphorylation sites of a protein [52]. Potential phosphorylation sites can
then be confirmed by mutagenesis [52]. Using these approaches, Roche et al. shwed
that GluR1 w as phosphorylated at S845 by protein kinase A (PKA) and S831 by
PKC [52]. They showed a potentiation of the peak amplitude of whole cell glutamate-
gated current after stimulation by purified PKA. This effect disappeared after muta-
tion of S845. Using a phosphorylation assay , Matsuda et al. sho wed that phospho-
rylation of S880 of the GluR2 sub unit by PKC reduced the af finity of GluR2 fo
GRIP, and thus could be important in controlling the stabilization of the receptor at
the synapse [53]. Hayashi et al. chose a different approach to study the phosphylation
of the GluR2 sub unit [54]. They generated a phospho-specific antibody a ainst a
sequence surrounding T876, which is very similar to the tar geting sequence for Src
family of tyrosine kinases, and sho wed that GluR2 w as tyrosine phosphorylated.
They also showed that the Lyn, Src and Fyn kinases could phosphorylate the GluR2
C-terminal tail. Tyrosine phosphorylation of GluR2 could regulate AMPAR traffick
ing to the cell surf ace. This study suggests that GluR2 is phosphorylated on T876
in vivo and in vitro by the Src f amily of tyrosine kinases. The authors suggest that
the ef fect on GluR2 traf ficking ould be through a destabilization of the
GluR2/GRIP interaction.
1.2.4 ELECTROPHYSIOLOGY
1.2.4.1 Blocking Peptide
Another approach to understand the mechanism of AMPAR traf ficking and it

physiological implication w as to block the interaction of a sub unit with its protein
partners in vivo in hippocampal slices to e xamine the ef fects on basal synaptic
transmission and synaptic plasticity . Nishimune et al. first characterized th
GluR2/NSF interaction by two-hybrid screen and by biochemical approaches (Table
1.1) [26]. They then characterized the p hysiological function by infusing a peptide
that blocks the GluR2/NSF interaction, or a specific antibody of NS , into a patch
pipette and recorded evoked excitatory post-synaptic current (EPSC). They described
a reduction of the EPSC amplitude and suggested that a rapid NSF-dependent
insertion exists of GluR2-containing AMPAR at the post-synaptic membrane (Figure
1.1[3b]). Using the same blocking peptide, Noel et al. further characterized the role
of this re gulation in AMPA mEPSC and sho wed a reduction in mEPSC frequenc y
(Table 1.1) [55]. They also sho wed a reduction of AMPAR surf ace le vels upon
expression of this peptide in cultured hippocampal neurons. Luthi et al. and Luscher
et al. analyzed the effects on plasticity using, respectively, the NSF/GluR2 blocking
peptide, or agents known to block various steps of exocytosis, and endocytosis (Table
1.1) [56,57]. Their experiments suggest that AMPAR cycle in and out of the synapse
and that certain forms of plasticity such as LTD could utilize this mechanism for its
expression (Figure 1.1[3b]). Similarly , Da w et al. block ed the interaction of
PICK1/ABP/GRIP1 and GluR2 with a peptide and sho wed an increase in basal
AMPAR-mediated transmission and a block of the generation of L TD (Table 1.1)
[58]. The authors proposed a model in which the proteins interacting with the C-
terminal domain of GluR2 are involved in the expression of LTD. Thus, this approach
complemented microscopy data obtained at the same time and presented previously.
1.2.4.2 Rectification Index
The current-voltage (IV) relationship of AMPAR is determined largely by the GluR2

subunit. AMPAR with GluR2 sho w a linear IV relation whereas AMPAR without
GluR2 conduct little outward current at +40 mV. Taking advantage of this property,
Hayashi et al. studied the importance of GluR1 in synaptic act ivity (Table 1.2) [59].
The recombinant AMPAR lacked GluR2. Thus, the incorporation of this recombinant
receptor into a synapse w ould increase rectification of synaptic responses. The
overexpression of GluR1 in or ganotypic hippocampal slices did not ha ve any effect
on the amplitude or rectification of synaptic transmission in the absence of act vity.
However, expression of GluR1 and CaMKII resulted in an increase of rectification
suggesting an insertion of homomeric GluR1-GFP into the synapse ( Figure 1.1 and
Figure 1.1[3a]). Interestingly , mutation of the PDZ binding site present on the C-
terminal domain of GluR1 could block the CaMKII ef fect and L TP, suggesting a
requirement for an interacting protein.Thus, the authors suggested that incorporation
of GluR1-containing AMPAR into the synapse is a major mechanism underlying
plasticity. Taking advantage of the same IV relationship properties, Shi et al. studied
the molecular determinant on AMPAR that re gulates their synaptic deli very (Table
1.2) [60]. Unlike GluR1, the expression of homomeric GluR2 was sufficient for it
incorporation at the synapse. To determine the requirements for GluR2 traf fickin
in synaptic transmission, Shi et al. mutated GluR2-R to Q at the pore apex, allowing
a distinct “electrophysiological signature.” These receptors showed a marked inward
rectification in voked AMPAR-mediated synaptic transmission. With this tool, the
authors were able to sho w that no activity was required for the continuous synaptic
replacement of GluR2, unlike GluR1 (Figure 1.1 and Figure 1.1[3b]). The incorpo-
ration of this subunit was accompanied by a remo val of a fraction of the pre viously
existing receptor. They also showed that GluR2 was not delivered to silent synapses,
whereas GluR1 was able to do so when coexpressed with an active form of CaMKII.
Thus, the y suggested tw o distinct synaptic deli very processes controlled by the
subunit composition of the receptor . The first one requires a su unit with a long C-
tail, such as GluR1, and participates in plasticity (Figure 1.1[3a]), and the second
requires subunits with short C-tails, such as GluR2, and replaces synaptic receptors
independent of synaptic acti vity (Figure 1.1[3b]). Ho wever, GluR1 properties are
dominant when expressed in a heteromer. Thus, GluR1 + GluR2 require activity for
their synaptic delivery and GluR2 + GluR3 comple xes are formed and deli vered to
synapses continuously.
1.2.4.3 Agonists and Toxins
Using specific bloc ers and taking adv antage of the dif ference in the inw ardly
rectifying IV relationship observ ed when GluR2 is present or not in AMPAR, Liu
et al. studied the composition of AMPAR at the parallel fibre stellate cell synaps
during acti vity (T able 1.2) [61]. The authors used spermine, which is kno wn to
confer voltage-dependent block of AMPAR lacking GluR2, Joro spider toxin (JST),
a sub unit specific bloc er of GluR2-lacking AMPAR, and pentobarbital, which
selectively inhibits GluR2-containing AMPAR at a specific concentration, to sh w
that stellate cells e xpress a population of GluR2-containing AMPAR but predomi-
nantly at e xtrasynaptic sites. Using dif ferent frequencies of stimulation, the y were
able to show that the level of GluR2-containing AMPAR at the parallel fibre stellat
cell synapse undergo dynamic changes controlled by local Ca 2+ influx. In the basa
state, synaptic AMPAR are mainly Ca 2+ permeable and so lack the edited GluR2.
During high frequenc y stimulation, the increased Ca 2+ entry through synaptic
AMPAR triggers the insertion of GluR2-containing AMPAR into the synapse and
the removal of GluR2-lacking AMPAR. This results in a reduction of EPSC ampli-
tude at +60 mV and a change in Ca 2+ permeability.
1.2.5 PHARMACOLOGY
The pharmacology of AMPAR has been e xtensively studied and agonists or antag-
onists of AMPAR or NMD AR have been broadly used to analyze the functions of
these receptors [62,63]. Here, we described an e xample of the use of AMPAR
pharmacology in the study of the traf ficking of this recepto . Using antagonists of
NMDAR or AMPAR (APV and CNQX, respecti vely), Liao et al. sho wed that the
level of AMPAR and NMD AR acti vity could re gulate the proportion of putati ve
silent synapses (Table 1.2) [64]. The authors suggested that most excitatory synapses
physically contain NMD AR but not AMPAR. Thus, the modulation of the endog-
enous activity of AMPAR and NMDAR regulates the number of silent synapses.
1.2.6 GENETIC APPROACH

1.2.6.1 Murine Models
Use of mutant or genetically modified mice represents another means for iden
tifying novel interacting components or to better understand receptor functions.
Thus, Chen et al. took adv antage of an ataxic and epileptic mutant mouse,
stargazer, to identify a ne w pathw ay re gulating AMPAR trafficking Table 1.1)
[65]. The stargazer mouse (stg/stg) exhibit seizures and cerebellar ataxia and lack
of functional AMPAR on granule cells [66]. The defective protein, stargazin (stg),
exhibits a low homology with the sub unit of Ca 2+ channels. Chen et al. sho wed
that stg/stg mice present no spontaneous acti vity, have lower GluR4 staining in
cerebellar culture and no GluR2/3 staining in granule cells. They were able to
co-immunoprecipitate stg with GluR4 and GluR2/3. Through its PDZ domain,
stg can also interact with PSD95 and SAP97. The authors suggested that stg has
two distinct roles. First, stg can re gulate the deli very of AMPAR to the cell
surface (Figure 1.1). This does not require interaction of the C-terminal stg PDZ-
binding domain with a PDZ protein. But stg can also mediate the synaptic
targeting of the receptor (Figure 1.1[2a], Figure 1.1[2b], Figure 1.1[3a] and Figure
1.1[3b]). This effect requires a PDZ interaction.
1.2.6.2 Knockout Mice
Several groups ha ve used knock out approaches to understand the importance of

GluR1 [67], GluR2 [68–71] and GluR3 sub units (Table 1.2) [72].
Mice lacking GluR1 exhibit AMPA-mediated neurotransmission but fail to gen-
erate LTP. They present a selecti ve loss of e xtrasynaptic AMPAR. However, spatial
learning is normal [67]. Using mice lacking the GluR2 sub unit, Jia et al. showed an
increase in Ca2+ permeability and an enhancement of LTP. They suggested that GluR2
was not required for L TP but seems to play a ne gative regulatory role [70]. Later ,
Sans et al. showed aberrant formation of GluR1/3 heteromers and GluR1 and GluR3
homomers in mice lacking the GluR2 sub unit, suggesting that GluR2 plays an
important role in controlling the assembly of AMPAR [71]. Brusa et al. and Feld-
meyer et al. used mice not able to edit GluR2 to sho w the importance of the edited
GluR2 subunit in the AMPAR function [68,69]. Mice with unedited GluR2 present
a higher Ca2+ permeability and develop seizures. The generation of a double-knock-

out mouse lacking GluR2 and GluR3 confirmed the importance of GluR1 in th
generation of LTP and the critical involvement of GluR2 and GluR3 in maintaining
basal synaptic transmission [72].
Thus, modification at the l vel of the whole animal can help an understanding
of the function of a receptor in a more inte grated system. Ho wever, mice are v ery
complex animals to handle; modifications ta e time. In addition, the e xistence of
several genes implicated in the same pathway can lead to compensatory effects when
the knockout is restricted to one of them. These compensatory effects can mask the
function of a protein.
1.2.6.3 C. Elegans
Simpler systems such as Caenorhabditis elegans have also been used to characterize
the function of AMPAR in the whole animal. Genetic modifications are easier an
quicker to assess in such systems. Thus, random mutations can allo w the iden-
tification of a n w pathw ay. In addition, less redundanc y is observ ed in simpler
systems. Finally , the identification of a gene conser ed from w orm to mammal
highlights the primordial function of such a gene. Thus, Hart et al. identified GLR
1, a receptor 40% identical to an AMPAR subunit, during a screen for a mutation
that disrupts ASH-mediated nose-touch sensitivity [73]. The authors suggested that
GLR-1 acts in synaptic tar gets of the ASH neurons.
1.3 SYNTHESIS, SUMMARY AND SPECULATION

The use of the v arious methods discussed in this chapter has allo wed a better
understanding of the traf ficking of AMPAR. AMPAR comple xes e xit from the
ER/Golgi associated with stargazin (Figure 1.1; Table 1.1) [65]. GluR1-containing
AMPAR traf fic through the secretory path ay (Figure 1.1[2a]) [48,49] and are
inserted at e xtrasynaptic sites through an acti vity-dependent mechanism (Figure
1.1[3a]; Table 1.2) [35,36]. Sta rgazin binds PSD95, which localizes the compl ex
at the synapse where the receptor can bind other scaf folding proteins such as RIL
and 4.1N (Figure 1.1[4a]; Table 1.1) [29,30]. GluR2-containing AMPAR traf fi
through the secretory pathw ay (Figure 1.1[2b]) and are inserted at synaptic sites
through a constitutive mechanism (Figure 1.1[3b]; Table 1.2) [59,60]. PICK1 [24]
facilitates the transport of the receptor , possibly both to and from the plasma
membrane, but it is removed from the AMPAR by the NSF/SNAP when the receptor
reaches the plasma membrane. NSF/SNAP/PICK1 forms a transient comple x with
GluR2-containing AMPAR (Table 1.2) [19,25–27,55–57]. AMPAR are stabilized
at the membrane by scaf folding proteins [20–22,58]. Phosphorylation by PKC
prevents interaction between ABP/GRIP and GluR2 (Figure 1.1[4b]; Table 1.2)
[53] and favors PICK1/GluR2 interaction (Figure 1.1[5b]; Table 1.2). The AMPAR
are then internalized follo wing a coupling with PICK1, and AMPAR/PICK1 com-
plexes are destabilized by NSF/SN AP when the receptor reaches its destination
(Figure 1.1[6b]; Table 1.1) [19,27]. GluR2-containing AMPAR can then interact
with an intracellular pool of ABP/GRIP, possibly in the endosome (Figure 1.1[7b];
Table 1.2) [23] and get re cycled to the membrane through interaction with PICK1
(Figure 1.1[8b]; Table 1.2) or get targeted to lysosomes (Figure 1.1[9b]; Table 1.2)
and degraded (Figure 1.1[10b]; Table 1.2) [45–47].
Research on the trafficking of AMPAR has led to pioneering studies in the fiel
of the dynamics of synaptic proteins because of the importance of AMPAR traffick
ing in synaptic plasticity . Ho wever, similar techniques and approaches ha ve been
used to understand the dynamics of other neuronal proteins, such as other channels
or receptors that determine and control properties and specificity of a neuron. The
next phase will involve development of new and more sophisticated techniques and
approaches that will allow linking properties of a protein to its functions in the whole
animal.
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1891_C002.fm Page 23 Monday, February 6, 2006 9:59 AM
2 Long-Term Plasticity at
Inhibitory Synapses: A
Phenomenon That Has
Been Overlooked
Jean-Luc Gaiarsa and Yehezkel Ben-Ari
CONTENTS
2.1 Introduction ....................................................................................................23

2.2 Long-Term Changes in the Strength of Inhibitory Synapses .......................24
2.2.1 Minimal Requirements and Characteristics .......................................24
2.2.2 Expression ..........................................................................................29
2.2.3 Functional Relevance .........................................................................31
2.3 Conclusion......................................................................................................32
References................................................................................................................33
2.1 INTRODUCTION
Synaptic plasticity describes the ability of individual synapses to alter their strength
of transmission in response to dif ferent stimuli or en vironmental cues. Persistent
activity-dependent changes are often referred to as long-term potentiation (LTP) and
long-term depression (LTD), and represent respecti vely an increase and a decrease
in the effica y of synaptic transmission. Initially studied as a model for learning and
memory, L TP and L TD are also thought to play a crucial role in the netw ork
hyperexcitability observ ed in pathological conditions and in the establishment of
appropriate synaptic connections.
The most extensive characterization of the cellular mechanisms in volved in the
induction and maintenance of long-term plasticity has been undertaken at glutamater-
gic synapses. However, considering the ubiquitous distribution of inhibitory synapses
and their role in shaping indi vidual and population acti vity, acti vity-dependent
changes in the strength of inhibitory synapses w ould have important consequences
on the development and the proper functioning of neuronal networks and, ultimately,
on cognitive processes. Thus, information on long-term plasticity at inhibitory syn-
apses is required to fully understand ho w acti vity-dependent changes in synaptic
23
effica y contribute to brain de velopment and function in concert with plasticity at

glutamatergic synapses.
Surprisingly, plasticity at inhibitory synapses has only recently been reported.
In this chapter, we summarize our current understandings of the cellular and molec-
ular processes underlying inhibitory synaptic plasticity and the possible functions
that this plasticity might serv e in the de veloping and adult nerv ous system.
2.2 LONG-TERM CHANGES IN THE STRENGTH OF

INHIBITORY SYNAPSES
2.2.1 MINIMAL REQUIREMENTS AND CHARACTERISTICS
In the past decade, long-term changes in the strength of inhibitory synapses ha ve
been reported in a large number of developing and adult structures, including Mauth-
ner cells of the goldfish [34], the hippocampus [9,22,24,40,41,49,54,56], the cort x
[21,30], the cerebellum [23], the deep cerebellar nucleus [43], the lateral superior
olive [36], lateral amygdal [2] and the brain stem [4,17] (Figure 2.1 and Figure 2.2).
In all these structures, attention had be focused to demonstrate that GAB Aergic and
glycinergic synapses themselv es under go long-term changes in synaptic ef fica y.
Long-term plasticity was observed on pharmacologically isolated inhibitory postsyn-
aptic potentials (IPSPs) or currents (IPSCs) or on unitary IPSCs e voked by direct
stimulation of the interneurons.
To date, most studies on plasticity at GAB Aergic synapses ha ve focused on
GABAA-receptor mediated postsynaptic potentials (GAB AA-PSPs). However, activa-
tion of either pre- or post-synaptic GABAB receptors during the conditioning protocol
appears to have a key role in the induction of long-term plasticity at both GABAAergic
and glycinergic synapses (Figure 2.1a and Figure 2.2c) [4,22,31,35,49,53], although
in one case activation of GABAB receptors has been reported to pre vent the induction
of GABAergic synaptic plasticity [25,26].
In most studies, plasticity at inhibitory synapses can be induced by high- or low-
frequency stimulations [15]. Although such conditioning protocol pro vides a good
tool to study the mechanisms by which plasticity are triggered and e xpressed, such
patterns of acti vity are rather unlik ely to occur in vivo . Thus, the rele vance of the
conditioning protocol to physiological or pathological conditions must be taken into
account to gain insight into the role that this plasticity might serve in the developing
and adult nerv ous system. Compelling e vidence shows that conditioning protocols
relevant to ph ysiological or pathological conditions can also induce plasticity at
GABAergic and glycinergic synapses. The best example was provided by Korn and
colleagues, who sho wed that auditory stimulations in the goldfish trigger TP of
glycinergic synapses in vivo [47] that shares similar properties with L TP induced
by tetanic stimulation [34,46]. More recently , Cherubini and colleagues ha ve
reported that spontaneously occurring netw ork oscillations, which constitute a hall-
mark of de veloping netw orks, can trigger L TPGABA-A in the de veloping rat hippo-
campus [24]. In the hippocampus and corte x, plasticity of inhibitory synapses can
also be triggered by post-synaptic firing of the ta get cells [5,38] or by coincidence
of back-propag ating action potentials with pre-synaptic stimulation of inhibitory
Long-Term Plasticity at Inhibitory Synapses 25
terminals [21,56]. Such conditioning protocols are related to the sort of acti vity that
pyramidal neurons might e xperience in vivo .
When stimulation of presynaptic inhibitory terminals is required, L TP and L TD
is only expressed by the conditioned fibers. In the adult rat hippocampus, TDGABA-A
is a highly localized phenomenon that spreads less than 20 µm a way from the condi-
tioned fibers along the apical dendrite [9]. In this particular xample, the local re gu-
lation of GABAergic synaptic strength is due to the restricted spread of the retrograde
signal required for LTDGABA-A induction to synapses impinging on a small portion of
the CA1 p yramidal cell dendrite. The interneurons e xhibited a great heterogeneity .
One of the most striking dif ferences is found on the restricted and specific lamina
distribution of their axonal terminals on dendritic and perisomatic re gions of their
target cells [14]. This heterogeneity provides the possibility that the different interneu-
ronal populations may selecti vely influence the e fica y of af ferent inputs and the
emergence and maintenance of netw ork oscillations. Thus, depending on the type of
interneurons, local changes in the ef fica y of inhibitory synapses will ha ve different
consequences on the input-output relationship of the tar get neurons.
Both homosynaptic and heterosynaptic plasticity at inhibitory synapses ha ve
been reported ( Figure 2.1 and Figure 2.2 ). In the neonatal rat hippocampus, the
induction of L TPGABA-A requires a membrane depolarization, pro vided by the acti-
vation of GABAA receptors during the conditioning protocol [18,41]. This depolar-
ization is strong enough to allo w activation of voltage-dependent calcium channels
(VDCCs), which will in turn trigger a cascade of e vents leading to homosynaptic
LTPGABA-A (Figure 2.1a). In the adult and neonatal rat hippocampus, tetanic stimu-
lation triggers heterosynaptic LTDGABA-A. In both cases, LTDGABA-A is triggered post-
synaptically via the acti vation of glutamater gic receptors during the conditioning
protocol. That is, glutamate released during repetiti ve stimulation activates group I
metabotropic glutamate receptors [9] on adult CA1 p yramidal cells (Figure 2.2a) or
N-methyl-D-aspartate (NMDA) receptors on neonatal CA3 p yramidal cells [6,42]
(Figure 2.1a) or adult CA1 pyramidal cells (Figure 2.2b) [40,55]. Activation of these
receptors will, in turn, trigger LTDGABA-A. Such heterosynaptic plasticity will locally
affect dendritic integration of synaptic input, for instance, by decreasing or increasing
the inhibitory shunt of neighboring glutamatergic afferents. Moreover, several studies
have reported that a conditioning protocol can trigger L TP at e xcitatory synapses
and, concomitantly, LTD at inhibitory synapses [40,55]. This opposite change in
excitatory and inhibitory synaptic strength can change the ability of the e xcitatory
synaptic potential to dischar ge an action potential [40,55], thereby changing the
excitability of neuronal netw orks.
As with glutamatergic synaptic plasticity, a rise in intracellular Ca 2+ concentra-
tion is important in shaping the strength of inhibitory synapses, although the source
and location of this calcium rise and the consequences on inhibitory synaptic strength
will vary depending on the structure considered and the conditioning protocol used
(Figure 2.1 and Figure 2.2). In the neonatal rat hippocampus, the induction of
LTPGABA-A requires a calcium influx through postsynaptic VDCCs (Figure 2.1a)
[5,18], while in the corte x (Figure 2.1c) [31] and cerebellum (Figure 2.2d) [20],
activation of postsynaptic calcium stores is required. An influx of calcium throug
VDCCs triggers L TD in the corte x [38] and deep cerebellar nuclei (Figure 2.1b)
+ −
Mg 2+
VDCCs GABAA NMDA
Pyr.
cell
Ca2+ Cl− Ca2+
Depolarization
RyR-Ca2+
stores
(a)
Mg2+
VDCCs GABAA NMDA
Pyr.
cell
Ca2+ Cl− Ca2+
LTDGABA−A LTDGABA−A
(b)
AP
VDCCs
α1 GABA B GABAA
PLC +
Pyr.
cell IP3
Cl−
Ca2+ stores
Ca2+
LTPGABA-A
(c)
FIGURE 2.1 Long-term changes in the strength of inhibitory synapses in the de veloping
brain. (a) In the neonatal rat hippocampus, high-frequency stimulation leads to LTD and LTP
of GABAergic synapses. Both forms of synaptic plasticity require a membrane depolarization
provided by activation of GABAA receptors during the stimulation. This depolarization leads
to a postsynaptic influx of calcium through oltage-dependent calcium channels (VDCCs)
and the induction of LTP. The depolarization also removes the Mg2+ block of NMDA channels,
leading to an influx of calcium through these channels. This calcium increase, amplified b
the acti vation of ryanodine (RyR)-sensiti ve calcium stores, triggers L TD. Both forms of
plasticity are e xpressed as a modification in the probability of GA A release through yet
unknown mechanisms. (b) In the de veloping neocortex, high-frequency stimulation triggers
heterosynaptic LTD through the activation of NMDA receptors. LTD of GABAergic synaptic
transmission can also be triggers by post-synaptic firing of the ta get cells. The mechanisms
underlying the expression of this LTD are presently unknown. (c) In the developing neocortex,
tetanic stimulation triggers LTP. The induction of LTP requires the activation of postsynaptic
GABAB receptors. Activation of these receptors f avors the release of calcium from post-
synaptic IP3-sensitive stores induced by α1 adrenoreceptors.
[44]. An influx of calcium through postsynaptic NM A channels also triggers

LTDGABA-A in the neonatal and adult rat hippocampus (Figure 2.1a and Figure 2.2b)
[6,40,55] as well as in the neocorte x (Figure 2.1b) [32]. Interestingly , the initial
depolarization required for the remo val of the Mg 2+ block of NMD A channels
depends on the de velopmental stage. This depolarization is pro vided by acti vation
of postsynaptic GABAA receptors in the neonates (Figure 2.1a) and by α-amino-3-
hydroxy-5-methyl-4-isoxazole propionic acid (AMP A) receptors (Figure 2.2b) in
the adult. Release of calcium by intracellular calcium stores could also trigger
LTPGABA-A in the neocortex (Figure 2.1c) [31] and hippocampus (Figure 2.2c) [22].
In these peculiar e xamples, GABA released during the conditioning protocol acti-
vates postsynaptic metabotropic GAB AB receptors, which then causes or f avors
intracellular release of calcium.
In most cases, the calcium rise that triggers plasticity of inhibitory synapses
occurs in the post-synaptic tar get cells, as post-synaptic loading with a calcium
chelator prevents its induction. Ho wever, the calcium rise could also occur in the
presynaptic terminal [7,16] or in neighboring astrocytes (Figure 2.2c) [22]. Thus, in
the adult hippocampus, repetiti ve firing of a single interneuron triggers a calcium
dependent LTPGABA-A on pyramidal cells. The calcium rise is triggered on neighbor -
ing astroc ytes follo wing the acti vation of GAB AB receptors by GAB A released
during the conditioning protocol [22]. This calcium rise then causes the release of
a retrograde messenger acting on GAB Aergic terminals [22].
Interestingly, the same conditioning protocol can lead to both L TP and L TD,
depending on the experimental conditions (Figure 2.1a). The source or the magnitude
of the postsynaptic Ca 2+ rise has been proposed to determine the polarity of the
plasticity. In the neonatal rat hippocampus, high-frequenc y stimulation can lead to
either LTPGABA-A or LTDGABA-A, depending on whether or not NMDA receptors were
activated during the conditioning protocol [41]. Gi ven that both forms of plasticity
require a postsynaptic rise in calcium [41], these observ ations suggest that the
polarity of synaptic changes might be determined by the source of calcium influx
i.e., an influx through VDCCs triggers L TPGABA-A [5], whereas an influx throug
Heterosynaptic LTDGABA-A Heterosynaptic LTDGABA-A
Schaffer col. Schaffer col.
Mg2+
NMDA AMPA
DAG
Depol.
PLC DAG-L CB1 Ca2+
mGluR ?
2-AG -
GABAA GABAA
Pyr. cell Pyr. cell Calcineurin − P

Cl− Cl−
(a) (b)
LTPGABA-A Rebound potentiation

+ Climbing
Astrocyte fiber
Ca2+
GABAB
GABAA GABAA VDCCs AMPA
Pyr. cell Purkinje P

cell
Cl− Cl− Ca2+ Depolarization
+
CaMKII IP3 Ca2+ stores
(c) (d)
FIGURE 2.2 Long-term changes in the strength of inhibitory synapses in the adult brain. (a)
In the adult rat hippocampus, repetiti ve activation of Schaffer collaterals triggers heterosyn-
aptic LTD that is induced via the acti vation of postsynaptic group I metabotropic glutamate
receptors (mGluR). Activation of mGluR leads to PLC activation, formation of diacyl-glyceral
(DAG) that is converted by DAG lipase (DAG-L) into 2-arachidonoyl glycerol (2-AG). 2-AG
acting on presynaptic CB1 receptors decreases GAB A release. (b) In the adult rat hippocam-
pus, high-frequenc y stimulation of Schaf fer collaterals triggers heterosynaptic L TD that is
induced via the activation of AMPA and NMDA receptors. The calcium influx through NM A
receptors likely leads to a dephosphorylation of post-synaptic GAB AA receptors. (c) In the
adult rat hippocampus, repetitive pre-synaptic firing of GA Aergic interneurons triggers LTP
that is induced by the acti vation of GAB AB receptors on neighboring astroc ytes. Activation
of GABAB receptors leads to a calcium rise that causes the release of retrograde messenger
acting on GABAergic terminals. (d) In the cerebellum, direct depolarization of Purkinje cells
or stimulation of climbing fibers triggers a long lasting potentiation of GA Aergic synapses,
termed rebound potentiation. The induction of this potentiation requires a post-synaptic rise
in calcium, amplified by the act vation of IP3-sensiti ve stores and leading to the acti vation
of the calcium/calmodulin-dependent kinase II (CaMKII).
NMDA channels triggers LTDGABA-A (Figure 2.1a) [6]. Alternatively, the magnitude
of the calcium rise could determine the polarity of synaptic changes, as documented
in the deep cerebellar nucleus. In this structure, the number of spikes and the related
amount of Ca 2+ that enters the post-synaptic neurons during the post-inhibitory
rebound depolarization seems to determine whether L TPGABA-A or L TDGABA-A is
induced with lar ge numbers of spik e leading to LTPGABA-A [1].
2.2.2 EXPRESSION
Long-term changes in the strength of synaptic ef fica y can be accounted for by at
least four none xclusive mechanisms: modifications in the number or properties o
receptors at functional synapses; changes in the re versal potential of post-synaptic
responses; modifications in the probability of transmitter release; and modificatio
in the number of functional synapses though either pre- or post-synaptic changes.
Probably because fewer studies have been performed to date, the locus of expression
of plasticity at inhibitory synapses appears less controversial than at their excitatory
counterparts.
If the amount of GAB A released during synaptic stimulation reaches the con-
centration that saturates postsynaptic GAB AA receptors, the total number or prop-
erties of receptors at functional synapses will be an important limiting f actor. In the
adult dentate gyrus, a direct relationship between synaptic GABAA receptor number
and quantal size at potentiated GAB Aergic synapses has been reported in an e xper-
imental model of temporal lobe epilepsy [45]. In this model, insertion of ne w GABAA
receptors underlies the increase in amplitude of unitary IPSCs. Other studies ha ve
reported that the pathw ay through which this Ca 2+ rise is translated into long-term
changes of synaptic ef fica y in volves changes in the properties of post-synaptic
GABAA receptors through activation of protein kinases or phosphatases [29]. Thus,
in the adult hippocampus, the e xpression of LTDGABA-A involves a do wn-regulation
of GABAA receptors by the calcium-sensitive phosphatase, calcineurin (Figure 2.2b)
[40,55]. In the cerebellum, the e xpression of L TPGABA-A required the acti vation of
post-synaptic calcium-calmodulin-dependent kinase II (CaMKII, Figure 2.2d)
[23,26]. In the lateral superior olive, both post-synaptic CaMKII and protein kinases
A and C participate in the L TD of inhibitory synapses [37].
Changes in the strength of inhibitory inputs can also result from modificatio
in the re versal potential of GAB Aergic synaptic responses, as documented in the
hippocampus [56]. In this structure, coincident pre- and post-synaptic spiking leads
to a persistent decrease in GAB Aergic synaptic strength associated with a depolar -
izing shift of the reversal potential of GABAA receptor-mediated synaptic potentials
(EGABA). Similarly, pairing e xogenously applied GAB A with postsynaptic depolar -
ization leads to a long-lasting transformation of h yperpolarizing GAB Aergic
responses into depolarizing responses [12]. GAB AA receptors are permeable to
chloride and the intracellular concentration is regulated by different chloride cotrans-
porters [50]. In their study, Woodin et al. have shown that coincident pre- and post-
synaptic spiking decreases the cation/chloride cotransporter KCC2 activity, resulting
in the shift of EGABA to more positive values [56]. Long-lasting change in EGABA have
been also reported in epileptic tissue [10,27], supporting the contrib ution of such
phenomenon in the emer gence and maintenance of pathological netw ork activity.
However, recent studies have reported that saturation of GABAA receptors does
not occur at all sites of GAB A release [19]. In this case, change in the probability
of GAB A release or in the number of functional release sites might underlie the
expression of synaptic plasticity . Compelling e vidence has accumulated in support
of this h ypothesis. Thus, long-term changes in the strength of inhibitory synapses
are often associated with changes in the f ailure probability of unitary inhibitory
postsynaptic currents (IPSCs) [22,24,46]; changes in the coef ficient of ariation of
evoked IPSCs [6,16,46]; changes in the paired pulse ratio of e voked IPSCs [9,24];
and changes in the frequenc y, b ut not amplitude, of miniature IPSCs [5,22] or
asynchronous quantal IPSCs e voked in the presence of strontium [6].
A modification in the number of functional release sites has been directl
demonstrated in the goldfish [8]. Thus, in dual recordings of presynaptic glycinergic
interneurons and postsynaptic Mauthner cells, 25% of the interneurons produced no
detectable postsynaptic response. Morphological examination revealed that the num-
ber of synaptic contacts made by these “silent” interneurons is similar to that of
“functional” interneurons. After a tetanic stimulation of the VIIIth nerve, that pro-
duces LTPgly , the “silent” interneurons become functional. Similarly, in the neonatal
rat hippocampus, postsynaptic application of a conditioning protocol leading to
LTPGABA-A leads to the appearance of functional GAB Aergic synapses in previously
“silent” CA3 p yramidal neurons [18]. It remains to be determined whether the
modifications in the number of functional release sites is due to all-or none modi
fications in the number of postsynaptic receptors or to presynaptic switching on i
transmitter release.
If plasticity is e xpressed at the postsynaptic le vel while induction requires a
postsynaptic rise in calcium, the information should be transmitted back from the
postsynaptic cell to the presynaptic inhibitory terminal. Such synaptic feedback
provided by retrograde messengers has been demonstrated in the rat hippocampus.
Chevaleyre and Castillo [9] recorded from adult hippocampal slices, and found that
high frequenc y stimulation induces an NMD A-independent L TDGABA-A. This
LT-GABA-A is triggered postsynaptically via acti vation of group I metabotropic
glutamatergic receptors (mGluRs), b ut is e xpressed presynaptically (Figure 2A).
Thus activation of postsynaptic group I mGluRs leads to the release of endocanna-
binoids, which then causes a persistent reduction of GABA release. The implication
of a trans-synaptic messenger in the induction of L TPGABA-A in the rat hippocampus
has also been demonstrated in an ele gant study by Kang and collaborators [22]
(Figure 2.2c ). In this stud y, repetit ive firing of hippocampal interneurons leads t
the acti vation of GAB AB receptors on astroc ytes, and a subsequent increase in
intracellular calcium concentration. This calcium rise causes the release of a mes-
senger from astroc ytes, probably glutamate, which in turn triggers an increase in
the probability of GABA release. Similar feedback regulation of inhibitory synaptic
transmission has been reported in the rat cerebellum, although this control occurs
in a short (seconds to minutes) time scale. Thus, short depolarization of Purkinje
cells leads to a transient (tens of seconds) decrease follo wed by a short-term (10 to
15 minutes) increase in the frequency of miniature inhibitory post-synaptic currents
(mIPSCs). Both phenomena, termed respecti vely depolarization-induced suppres-

sion of inhibition (DSI) [39] and depolarization-induced potentiation of inhibition
(DPI) [13], require a post-synaptic rise in calcium concentration and are mediated
presynaptically. In DSI, the post-synaptic calcium rise initiates endocannabinoid
synthesis that activates presynaptic CB1 receptors, resulting in inhibition of GAB A
release for tens of seconds [48]. In DPI, the post-synaptic rise in calcium induces
the release of glutamate from Purkinje cells [13]. Glutamate acti vates pre-synaptic
NMDA receptors, leading to calcium influx into the pre-synaptic terminal that, wit
the activation of ryanodine-sensitive calcium stores, induces a short-lasting increase
of GABA release.
In all studies, modified synaptic strength seems to be maintained by a mechanis
independent of neuronal activity. In contrast, in the developing rat visual cortex, the
maintenance of L TPGABA-A requires firing of pre-synaptic inhibitory terminals an
pre-synaptic calcium influx throug VDCCs (Figure 2.1c) [33]. Thus, if stimulation
of the test pathw ay w as stopped after L TPGABA-A induction, potentiated responses
returned to a baseline level. Because this plasticity could underlie experience-depen-
dent refinement of visual inputs early in life, this obser ation suggests that this
refinement might not persist unless strengthened synapses are act vated by visual
stimulation.
2.2.3 FUNCTIONAL RELEVANCE

Most of our interests on long term changes in synaptic ef fica y stem from the possible
functions that LTP and LTD might serve in the developing and adult nervous system.
In the adult nerv ous system, the role of plasticity at inhibitory synapses w as
previously thought to re gulate the occurrence of plasticity at e xcitatory synapses.
However, plasticity of inhibitory synapses, in parallel with plasticity at e xcitatory
synapses, can also affect the probability of the target neurons to fire action potentials
and will contribute to hyperexcitability of neuronal networks observed in patholog-
ical conditions. The balance between excitation and inhibition in neuronal networks
can critically influence the l vel and type of spontaneous synaptic acti vity within
the netw ork, and changes in synaptic strength could lead to profound netw ork
modifications. or instance, in the adult rat hippocampus, L TDGABA-A induced by
tetanic stimulation re vealed latent e xcitatory synaptic connections between hippo-
campal pyramidal cells. Such phenomenon lik ely contributes to the emer gence or
maintenance of epileptiform acti vity [54]. Plasticity at inhibitory synapses alone,
without changes in glutamater gic synaptic strength, could also ha ve beha vioral
consequences. This w as directly addressed in the goldfish with the plasticity a
glycinergic synapses onto Mauthner cells. Activation of the Mauthner cell system
by sound is kno wn to contrib ute to an escape reaction that orients the fish way
from the predator [59]. In an ele gant study performed in vivo , sound stimulation
sufficient to produce TP of glyciner gic synapses, b ut inef ficient to modify th
excitatory inputs onto Mauthner cells, w as reported to decrease the probability to
escape in the conditioned goldfish [57,58], whereas the basic properties of the escap
refl x were not modified
Plasticity at inhibitory synapses could also play a crucial role in the de veloping
brain. Thus, both L TP and L TD of inhibitory synapses ha ve been described in
different developing brain regions, including the lateral superior olive [36], the cortex
[30] and the hippocampus [41]. In all these structures, plasticity is induced during
a restricted period of de velopment that closely matches the period of functional
synaptic maturation. In the auditory system, the tonotopic organization of glycinergic
projections is achieved through synapses elimination [51], a process involving activ-
ity-dependent mechanisms [52]. The structural refinement of axonal arbors eme ges
gradually and is preceded by a functional elimination and strengthening of
GABA/glycine connections [28]. The period during which L TD is induced in this
structure coincides with the period of functional elimination of inhibitory synapses
[36].
To strengthen the link between activity-dependent maturation of inhibitory syn-
apses and long-term plasticity showing that the activity involved in the development
of inhibitory synapses is able to produce modifications in inhibitory synaptic strengt
is necessary. The presence of spontaneous pattered synaptic acti vity is a hallmark
of the de veloping network [3]. In the neonatal hippocampus, this netw ork activity,
termed giant depolarizing potentials (GDPs), consists of b ursts of action potentials
associated with post-synaptic influx of calcium through VDCCs. Thus, the minimal
requirements to induce LTPGABA-A in the neonatal rat hippocampus are present during
GDPs. This activity could therefore represent the ph ysiological pattern of acti vity
leading to the functional maturation of inhibitory synapses through L TP/LTD-like
mechanisms [18]. In agreement with this h ypothesis, pharmacological blockade of
the GDPs pre vents the functional maturation of GAB Aergic synapses [11], and
application of a conditioning protocol that mimics, at least in part, the post-synaptic
consequences of GDPs triggers L TPGABA-A during a narro w postnatal time windo w
[18]. Moreo ver, the same protocol also leads to the appearance of functional
GABAergic synapses on previously silent cells, thus mimicking the functional mat-
uration of GAB Aergic synapses occurring in vivo [5,18]. Finally, pairing e voked
GABAergic synaptic responses with GDPs leads to L TPGABA-A in the neonatal rat
hippocampus [24].
A key issue that might unco ver the link between acti vity-dependent functional
maturation and synaptic plasticity is to sho w that the same cellular mechanisms are
involved in both phenomena. In this context, neurotrophins and related Trk receptor-
coupled protein tyrosine kinases (PTKs) ha ve been implicated in synapse de velop-
ment and plasticity, and could likely represent the signal linking long-term plasticity
and acti vity-dependent maturation of inhibitory synapses. In agreement with this
hypothesis, recent results suggest that TrkB receptors participate in the induction of
LTDGly in the developing auditory brain stem [35] and L TPGABA-A in the developing
rat hippocampus (Gaiarsa and Gubellini, unpublished results).
2.3 CONCLUSION
The data reviewed here indicate that inhibitory synapses undergo calcium-dependent,
long-term changes in synaptic ef fica y. This plasticity can be triggered by condi-
tioning protocol rele vant to ph ysiological and pathological conditions, pointing to
a possible contribution in the de veloping and adult brain. Moreo ver, although most
studies have been performed in vitro, a study has reported that similar process occurs
in vivo , and demonstrates behavioral consequences following the induction of plas-
ticity at glyciner gic synapses. Thus, we must consider plasticity at inhibitory syn-
apses to fully understand the consequences of acti vity-dependent plasticity on the
development and function of the neuronal netw ork.
However, one should consider the heterogeneity of GAB Aergic interneurons
[14]. In the adult hippocampus, different interneurons impinge precisely on specifie
areas and differentially control the excitability of their target cells. Persistent strength
modifications of di ferent types of interneurons will likely produce different changes
on integrative functions and in future studies, inserting the heterogeneity of inter -
neuronal types in this general scheme will be important. For instance, heterosynaptic
plasticity that results from interactions with glutamatergic synapses will be expressed
by dendritic synapses where glutamatergic inputs impinge on target cells. Therefore,
to completely understand the o verall ef fect of long-term plasticity at inhibitory
synapses on the acti vity generated by a neuronal netw ork, the morphological iden-
tification of the interneurons underlying this plasticity will be required
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Another random document with
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Egerton, emancipated from the trammels that had bound his will
while his party was in office, desired, in the turn of events, to be
nominee of no other man—desired to stand at least freely and singly
on the ground of his own services, be guided by his own penetration;
no law for action, but his strong sense and his stout English heart.
Therefore he had declined all offers from those who could still
bestow seats in Parliament. Those he could purchase with hard gold
were yet open to him. And the £5000 he had borrowed from Levy
were yet untouched.
To this lone public man, public life, as we have seen, was the all in
all. But now more than ever it was vital to his very wants. Around
him yawned ruin. He knew that it was in Levy’s power at any
moment to foreclose on his mortgaged lands—to pour in the bonds
and the bills which lay within those rosewood receptacles that lined
the fatal lair of the sleek usurer—to seize on the very house in which
now moved all the pomp of a retinue that vied with the valetaille of
dukes—to advertise for public auction, under execution, “the costly
effects of the Right Hon. Audley Egerton.” But, consummate in his
knowledge of the world, Egerton felt assured that Levy would not
adopt these measures against him while he could still tower in the
van of political war—while he could still see before him the full
chance of restoration to power, perhaps to power still higher than
before—perhaps to power the highest of all beneath the throne. That
Levy, whose hate he divined, though he did not conjecture all its
causes, had hitherto delayed even a visit, even a menace, seemed to
him to show that Levy still thought him one “to be helped,” or, at
least, one too powerful to crush. To secure his position in Parliament
unshackled, unfallen, if but for another year,—new combinations of
party might arise, new reactions take place, in public opinion! And,
with his hand pressed to his heart, the stern firm man muttered,—“If
not, I ask but to die in my harness, and that men may not know that I
am a pauper, until all that I need from my country is a grave.”
Scarce had these words died upon his lips ere two quick knocks in
succession resounded at the street door. In another moment Harley
entered, and, at the same time, the servant in attendance approached
Audley, and announced Baron Levy.
“Beg the Baron to wait, unless he would prefer to name his own
hour to call again,” answered Egerton, with the slightest possible
change of colour. “You can say I am now with Lord L’Estrange.”
“I had hoped you had done for ever with that deluder of youth,”
said Harley, as soon as the groom of the chambers had withdrawn. “I
remember that you saw too much of him in the gay time, ere wild
oats are sown; but now surely you can never need a loan; and if so, is
not Harley L’Estrange by your side?”
Egerton.—“My dear Harley!—doubtless he but comes to talk to
me of some borough. He has much to do with those delicate
negotiations.”
Harley.—“And I have come on the same business. I claim the
priority. I not only hear in the world, but I see by the papers, that
Josiah Jenkins, Esq., known to fame as an orator who leaves out his
h’s, and young Lord Willoughby Whiggolin, who is just now made a
Lord of the Admiralty, because his health is too delicate for the army,
are certain to come in for the city which you and your present
colleague will as certainly vacate. That is true, is it not?”
Egerton.—“My old committee now vote for Jenkins and
Whiggolin. And I suppose there will not be even a contest. Go on.”
“So my father and I are agreed that you must condescend, for the
sake of old friendship, to be once more member for Lansmere!”
“Harley,” exclaimed Egerton, changing countenance far more than
he had done at the announcement of Levy’s portentous visit—“Harley
—No, no!”
“No! But why? Wherefore such emotion?” asked L’Estrange, in
surprise.
Audley was silent.
Harley.—“I suggested the idea to two or three of the late
Ministers; they all concur in advising you to accede. In the first place,
if declining to stand for the place which tempted you from Lansmere,
what more natural than that you should fall back on that earlier
representation? In the second place, Lansmere is neither a rotten
borough, to be bought, nor a close borough, under one man’s
nomination. It is a tolerably large constituency. My father, it is true,
has considerable interest in it, but only what is called the legitimate
influence of property. At all events, it is more secure than a contest
for a larger town, more dignified than a seat for a smaller. Hesitating
still? Even my mother entreats me to say how she desires you to
renew that connection.”
“Harley,” again exclaimed Egerton; and, fixing upon his friend’s
earnest face, eyes which, when softened by emotion, were strangely
beautiful in their expression—“Harley, if you could but read my heart
at this moment, you would—you would—” His voice faltered, and he
fairly bent his proud head upon Harley’s shoulder; grasping the hand
he had caught, nervously, clingingly—“Oh Harley, if I ever lose your
love, your friendship!—nothing else is left to me in the world.”
“Audley, my dear dear Audley, is it you who speak to me thus?
You, my school friend, my life’s confidant—you?”
“I am grown very weak and foolish,” said Egerton, trying to smile.
“I do not know myself. I, too, whom you have so often called ‘Stoic,’
and likened to the Iron Man in the poem which you used to read by
the riverside at Eton.”
“But even then, my Audley, I knew that a warm human heart (do
what you would to keep it down) beat strong under the iron ribs. And
I often marvel now, to think you have gone through life so free from
the wilder passions. Happier so!”
Egerton, who had turned his face from his friend’s gaze, remained
silent for a few moments, and he then sought to divert the
conversation, and roused himself to ask Harley how he had
succeeded in his views upon Beatrice, and his watch on the Count.
“With regard to Peschiera,” answered Harley, “I think we must
have overrated the danger we apprehended, and that his wagers were
but an idle boast. He has remained quiet enough, and seems devoted
to play. His sister has shut her doors both on myself and my young
associate during the last few days. I almost fear that, in spite of very
sage warnings of mine, she must have turned his poet’s head, and
that either he has met with some scornful rebuff to incautious
admiration, or that he himself has grown aware of peril, and declines
to face it; for he is very much embarrassed when I speak to him
respecting her. But if the Count is not formidable, why, his sister is
not needed; and I hope yet to get justice for my Italian friend
through the ordinary channels. I have secured an ally in a young
Austrian prince, who is now in London, and who has promised to
back, with all his influence, a memorial I shall transmit to Vienna.
Apropos, my dear Audley, now that you have a little breathing-time,
you must fix an hour for me to present to you my young poet, the son
of her sister. At moments the expression of his face is so like hers.”
“Ay, ay,” answered Egerton quickly, “I will see him as you wish, but
later. I have not yet that breathing-time you speak of; but you say he
has prospered; and, with your friendship, he is secure from fortune. I
rejoice to think so.”
“And your own protégé, this Randal Leslie, whom you forbid me to
dislike—hard task!—what has he decided?”
“To adhere to my fate. Harley, if it please Heaven that I do not live
to return to power, and provide adequately for that young man, do
not forget that he clung to me in my fall.”
“If he still cling to you faithfully, I will never forget it. I will forget
only all that now makes me doubt him. But you talk of not living,
Audley! Pooh!—your frame is that of a predestined octogenarian.”
“Nay,” answered Audley, “I was but uttering one of those vague
generalities which are common upon all mortal lips. And now
farewell—I must see this Baron.”
“Not yet, until you have promised to consent to my proposal, and
be once more member for Lansmere. Tut! don’t shake your head. I
cannot be denied. I claim your promise in right of our friendship,
and shall be seriously hurt if you even pause to reflect on it.”
“Well, well, I know not how to refuse you, Harley; but you have not
been to Lansmere yourself since—since that sad event. You must not
revive the old wound—you must not go; and—and I own it, Harley;
the remembrance of it pains even me. I would rather not go to
Lansmere.”
“Ah! my friend, this is an excess of sympathy, and I cannot listen
to it. I begin even to blame my own weakness, and to feel that we
have no right to make ourselves the soft slaves of the past.”
“You do appear to me of late to have changed,” cried Egerton
suddenly, and with a brightening aspect. “Do tell me that you are
happy in the contemplation of your new ties—that I shall live to see
you once more restored to your former self.”
“All I can answer, Audley,” said L’Estrange, with a thoughtful
brow, “is, that you are right in one thing—I am changed; and I am
struggling to gain strength for duty and for honour. Adieu! I shall tell
my father that you accede to our wishes.”
CHAPTER VI.
When Harley was gone, Egerton sunk back on his chair, as if in
extreme physical or mental exhaustion, all the lines of his
countenance relaxed and jaded.
“To go back to that place—there—there—where—Courage, courage
—what is another pang?”
He rose with an effort, and folding his arms tightly across his
breast, paced slowly to and fro the large, mournful, solitary room.
Gradually his countenance assumed its usual cold and austere
composure—the secret eye, the guarded lip, the haughty collected
front. The man of the world was himself once more.
“Now to gain time, and to baffle the usurer,” murmured Egerton,
with that low tone of easy scorn, which bespoke consciousness of
superior power and the familiar mastery over hostile natures. He
rang the bell: the servant entered.
“Is Baron Levy still waiting?”
“Yes, sir.”
“Admit him.”
Levy entered.
“I beg your pardon, Levy,” said the ex-minister, “for having so long
detained you. I am now at your commands.”
“My dear fellow,” returned the Baron, “no apologies between
friends so old as we are; and I fear that my business is not so
agreeable as to make you impatient to discuss it.”
Egerton, (with perfect composure.)—“I am to conclude, then, that
you wish to bring our accounts to a close. Whenever you will, Levy.”
The Baron, (disconcerted and surprised.)—“Peste! mon cher, you
take things coolly. But if our accounts are closed, I fear you will have
but little to live upon.”
Egerton.—“I can continue to live on the salary of a Cabinet
Minister.”
Baron.—“Possibly; but you are no longer a Cabinet Minister.”
Egerton.—“You have never found me deceived in a political
prediction. Within twelve months, (should life be spared to me) I
shall be in office again. If the same to you, I would rather wait till
then, formally and amicably to resign to you my lands and this
house. If you grant that reprieve, our connection can thus close,
without the éclat and noise, which may be invidious to you, as it
would be disagreeable to me. But if that delay be inconvenient, I will
appoint a lawyer to examine your accounts, and adjust my
liabilities.”
The Baron, (soliloquising.)—“I don’t like this. A lawyer! That may
be awkward.”
Egerton, (observing the Baron, with a curl of his lip.)—“Well,
Levy, how shall it be?”
The Baron.—“You know, my dear fellow, it is not my character to
be hard on any one, least of all upon an old friend. And if you really
think there is a chance of your return to office, which you apprehend
that an esclandre as to your affairs at present might damage, why, let
us see if we can conciliate matters. But, first, mon cher, in order to
become a Minister, you must at least have a seat in Parliament; and,
pardon me the question, how the deuce are you to find one?”
Egerton.—“It is found.”
The Baron.—“Ah, I forgot the £5000 you last borrowed.”
Egerton.—“No; I reserve that sum for another purpose.”
The Baron, (with a forced laugh.)—“Perhaps to defend yourself
against the actions you apprehend from me?”
Egerton.—“You are mistaken. But to soothe your suspicions, I will
tell you plainly, that finding any sum I might have insured on my life
would be liable to debts preincurred, and (as you will be my sole
creditor) might thus at my death pass back to you; and doubting
whether, indeed, any office would accept my insurance, I appropriate
that sum to the relief of my conscience. I intend to bestow it, while
yet in life, upon my late wife’s kinsman, Randal Leslie. And it is
solely the wish to do what I consider an act of justice, that has
prevailed with me to accept a favour from the hands of Harley
L’Estrange, and to become again the member for Lansmere.”
The Baron.—“Ha!—Lansmere! You will stand for Lansmere?”
Egerton, (wincing.)—“I propose to do so.”
The Baron.—“I believe you will be opposed, subjected to even a
sharp contest. Perhaps you may lose your election.”
Egerton.—“If so, I resign myself, and you can foreclose on my
estates.”
The Baron, (his brow colouring.)—“Look you, Egerton, I shall be
too happy to do you a favour.”
Egerton, (with stateliness.)—“Favour! No, Baron Levy, I ask from
you no favour. Dismiss all thought of rendering me one. It is but a
consideration of business on both sides. If you think it better that we
shall at once settle our accounts, my lawyer shall investigate them. If
you agree to the delay I request, my lawyer shall give you no trouble;
and all that I have, except hope and character, pass to your hands
without a struggle.”
The Baron.—“Inflexible and ungracious, favour or not—put it as
you will—I accede, provided, first, that you allow me to draw up a
fresh deed, which will accomplish your part of the compact;—and
secondly, that we saddle the proposed delay with the condition that
you do not lose your election.”
Egerton.—“Agreed. Have you anything further to say?”
The Baron.—“Nothing, except that, if you require more money, I
am still at your service.”
Egerton.—“I thank you. No; I owe no man aught except yourself. I
shall take the occasion of my retirement from office to reduce my
establishment. I have calculated already, and provided for the
expenditure I need, up to the date I have specified, and I shall have
no occasion to touch the £5000 that I still retain.”
“Your young friend, Mr Leslie, ought to be very grateful to you,”
said the Baron, rising. “I have met him in the world—a lad of much
promise and talent. You should try and get him also into
Parliament.”
Egerton, (thoughtfully.)—“You are a good judge of the practical
abilities and merits of men, as regards worldly success. Do you really
think Randal Leslie calculated for public life—for a Parliamentary
career?”
The Baron.—“Indeed I do.”
Egerton, (speaking more to himself than Levy.)—“Parliament
without fortune—’tis a sharp trial; still he is prudent, abstemious,
energetic, persevering; and at the onset, under my auspices and
advice, he might establish a position beyond his years.”
The Baron.—“It strikes me that we might possibly get him into the
next Parliament; or, as that is not likely to last long, at all events into
the Parliament to follow—not for one of the boroughs which will be
swept away, but for a permanent seat, and without expense.”
Egerton.—“Ay—and how?”
The Baron.—“Give me a few days to consider. An idea has
occurred to me. I will call again if I find it practicable. Good day to
you, Egerton, and success to your election for Lansmere.”
CHAPTER VII.
Peschiera had not been so inactive as he had appeared to Harley
and the reader. On the contrary, he had prepared the way for his
ultimate design, with all the craft and the unscrupulous resolution
which belonged to his nature. His object was to compel Riccabocca
into assenting to the Count’s marriage with Violante, or, failing that,
to ruin all chance of his kinsman’s restoration. Quietly and secretly
he had sought out, amongst the most needy and unprincipled of his
own countrymen, those whom he could suborn to depose to
Riccabocca’s participation in plots and conspiracies against the
Austrian dominions. These his former connection with the Carbonari
enabled him to track in their refuge in London; and his knowledge of
the characters he had to deal with fitted him well for the villanous
task he undertook.
He had, therefore, already collected witnesses sufficient for his
purposes, making up in number for their defects in quality.
Meanwhile, he had (as Harley had suspected he would) set spies
upon Randal’s movements; and the day before that young traitor
confided to him Violante’s retreat, he had, at least, got scent of her
father’s.
The discovery that Violante was under a roof so honoured, and
seemingly so safe as Lord Lansmere’s, did not discourage this bold
and desperate adventurer. We have seen him set forth to reconnoitre
the house at Knightsbridge. He had examined it well, and discovered
the quarter which he judged favourable to a coup-de-main, should
that become necessary.
Lord Lansmere’s house and grounds were surrounded by a wall,
the entrance being to the high-road, and by a porter’s lodge. At the
rear there lay fields crossed by a lane or by-road. To these fields a
small door in the wall, which was used by the gardeners in passing to
and from their work, gave communication. This door was usually
kept locked; but the lock was of the rude and simple description
common to such entrances, and easily opened by a skeleton key. So
far there was no obstacle which Peschiera’s experience in conspiracy
and gallantry did not disdain as trivial. But the Count was not
disposed to abrupt and violent means in the first instance. He had a
confidence in his personal gifts, in his address, in his previous
triumphs over the sex, which made him naturally desire to hazard
the effect of a personal interview; and on this he resolved with his
wonted audacity. Randal’s description of Violante’s personal
appearance, and such suggestions as to her character and the
motives most likely to influence her actions, as that young lynx-eyed
observer could bestow, were all that the Count required of present
aid from his accomplice.
Meanwhile we return to Violante herself. We see her now seated in
the gardens at Knightsbridge, side by side with Helen. The place was
retired, and out of sight from the windows of the house.
Violante.—“But why will you not tell me more of that early time?
You are less communicative even than Leonard.”
Helen, (looking down, and hesitatingly.)—“Indeed there is
nothing to tell you that you do not know; and it is so long since, and
things are so changed now.”
The tone of the last words was mournful, and the words ended
with a sigh.
Violante, (with enthusiasm.)—“How I envy you that past which
you treat so lightly! To have been something, even in childhood, to
the formation of a noble nature; to have borne on those slight
shoulders half the load of a man’s grand labour. And now to see
Genius moving calm in its clear career; and to say inly, ‘Of that
genius I am a part!’”
“Helen, (sadly and humbly.)—“A part! Oh, no! A part? I don’t
understand you.”
Violante.—“Take the child Beatrice from Dante’s life, and should
we have a Dante? What is a poet’s genius but the voice of its
emotions? All things in life and in Nature influence genius; but what
influences it the most, are its sorrows and affections.”
Helen looks softly into Violante’s eloquent face, and draws nearer
to her in tender silence.
Violante, (suddenly.)—“Yes, Helen, yes—I know by my own heart
how to read yours. Such memories are ineffaceable. Few guess what
strange self-weavers of our own destinies we women are in our
veriest childhood!” She sunk her voice into a whisper: “How could
Leonard fail to be dear to you—dear as you to him—dearer than all
others?”
Helen, (shrinking back, and greatly disturbed.)—“Hush, hush! you
must not speak to me thus; it is wicked—I cannot bear it. I would not
have it be so—it must not be—it cannot!”
She clasped her hands over her eyes for a moment, and then lifted
her face, and the face was very sad, but very calm.
Violante, (twining her arm round Helen’s waist.)—“How have I
wounded you?—how offended? Forgive me—but why is this wicked?
Why must it not be? Is it because he is below you in birth?”
Helen.—“No, no—I never thought of that. And what am I? Don’t
ask me—I cannot answer. You are wrong, quite wrong, as to me. I
can only look on Leonard as—as a brother. But—but, you can speak
to him more freely than I can. I would not have him waste his heart
on me, nor yet think me unkind and distant, as I seem. I know not
what I say. But—but—break to him—indirectly—gently—that duty in
both forbids us both to—to be more than friends—than——”
“Helen, Helen!” cried Violante, in her warm, generous passion,
“your heart betrays you in every word you say. You weep; lean on me,
whisper to me; why—why is this? Do you fear that your guardian
would not consent? He not consent! He who—”
Helen.—“Cease—cease—cease.”
Violante.—“What! You can fear Harley—Lord L’Estrange? Fie;
you do not know him.”
Helen, (rising suddenly.)—“Violante, hold; I am engaged to
another.”
Violante rose also, and stood still, as if turned to stone; pale as
death, till the blood came, at first slowly, then with suddenness from
her heart, and one deep glow suffused her whole countenance. She
caught Helen’s hand firmly, and said, in a hollow voice—
“Another! Engaged to another! One word, Helen—not to him—not
to—Harley—to——”
“I cannot say—I must not. I have promised,” cried poor Helen, and
as Violante let fall her hand, she hurried away.
Violante sate down, mechanically. She felt as if stunned by a
mortal blow. She closed her eyes, and breathed hard. A deadly
faintness seized her; and when it passed away, it seemed to her as if
she were no longer the same being, nor the world around her the
same world—as if she were but one sense of intense, hopeless misery,
and as if the universe were but one inanimate void. So strangely
immaterial are we really—we human beings, with flesh and blood—
that if you suddenly abstract from us but a single, impalpable, airy
thought, which our souls have cherished, you seem to curdle the air,
to extinguish the sun, to snap every link that connects us to matter,
and to benumb everything into death, except woe.
And this warm, young, southern nature, but a moment before was
so full of joy and life, and vigorous, lofty hope. It never till now had
known its own intensity and depth. The virgin had never lifted the
veil from her own soul of woman. What, till then, had Harley
L’Estrange been to Violante? An ideal—a dream of some imagined
excellence—a type of poetry in the midst of the common world. It
had not been Harley the Man—it had been Harley the Phantom. She
had never said to herself, “He is identified with my love, my hopes,
my home, my future.” How could she? Of such, he himself had never
spoken; an internal voice, indeed, had vaguely, yet irresistibly,
whispered to her that, despite his light words, his feelings towards
her were grave and deep. O false voice! how it had deceived her. Her
quick convictions seized the all that Helen had left unsaid. And now
suddenly she felt what it is to love, and what it is to despair. So she
sate, crushed and solitary, neither murmuring nor weeping, only now
and then passing her hand across her brow, as if to clear away some
cloud that would not be dispersed; or heaving a deep sigh, as if to
throw off some load that no time henceforth could remove. There are
certain moments in life in which we say to ourselves, “All is over; no
matter what else changes, that which I have made my all is gone
evermore—evermore.” And our own thought rings back in our ears,
“Evermore—evermore!”
CHAPTER VIII.
As Violante thus sate, a stranger, passing stealthily through the
trees, stood between herself and the evening sun. She saw him not.
He paused a moment, and then spoke low, in her native tongue,
addressing her by the name which she had borne in Italy. He spoke
as a relation, and excused his intrusion: “For,” said he, “I come to
suggest to the daughter the means by which she can restore to her
father his country and his honours.”
At the word “father” Violante roused herself, and all her love for
that father rushed back upon her with double force. It does so ever—
we love most our parents at the moment when some tie less holy is
abruptly broken; and when the conscience says, “There, at least, is a
love that never has deceived thee!”
She saw before her a man of mild aspect and princely form.
Peschiera (for it was he) had banished from his dress, as from his
countenance, all that betrayed the worldly levity of his character. He
was acting a part, and he dressed and looked it.
“My father!” she said quickly, and in Italian. “What of him? And
who are you, signior? I know you not.”
Peschiera smiled benignly, and replied in a tone in which great
respect was softened by a kind of parental tenderness.
“Suffer me to explain, and listen to me while I speak.” Then,
quietly seating himself on the bench beside her, he looked into her
eyes, and resumed.
“Doubtless, you have heard of the Count di Peschiera?”
Violante.—“I heard that name, as a child, when in Italy. And
when she with whom I then dwelt, (my father’s aunt,) fell ill and
died, I was told that my home in Italy was gone, that it had passed to
the Count di Peschiera—my father’s foe.”
Peschiera.—“And your father, since then, has taught you to hate
this fancied foe?”
Violante.—“Nay; my father did but forbid me ever to breathe his
name.”
Peschiera.—“Alas! what years of suffering and exile might have
been saved your father, had he but been more just to his early friend
and kinsman; nay, had he but less cruelly concealed the secret of his
retreat. Fair child, I am that Giulio Franzini, that Count di Peschiera.
I am the man you have been told to regard as your father’s foe. I am
the man on whom the Austrian emperor bestowed his lands. And
now judge if I am in truth the foe. I have come hither to seek your
father, in order to dispossess myself of my sovereign’s gift. I have
come but with one desire, to restore Alphonso to his native land, and
to surrender the heritage that was forced upon me.”
Violante.—“My father, my dear father! His grand heart will have
room once more. Oh! this is noble enmity, true revenge. I understand
it, signior, and so will my father, for such would have been his
revenge on you. You have seen him?”
Peschiera.—“No, not yet. I would not see him till I had seen
yourself; for you, in truth, are the arbiter of his destinies, as of mine.”
Violante.—“I—Count? I—arbiter of my father’s destinies? Is it
possible!”
Peschiera, (with a look of compassionate admiration, and in a
tone yet more emphatically parental.)—How lovely is that innocent
joy; but do not indulge it yet. Perhaps it is a sacrifice which is asked
from you—a sacrifice too hard to bear. Do not interrupt me. Listen
still, and you will see why I could not speak to your father until I had
obtained an interview with yourself. See why a word from you may
continue still to banish me from his presence. You know, doubtless,
that your father was one of the chiefs of a party that sought to free
Northern Italy from the Austrians. I myself was at the onset a warm
participator in that scheme. In a sudden moment I discovered that
some of its more active projectors had coupled with a patriotic
enterprise schemes of a dark nature—and that the conspiracy itself
was about to be betrayed to the government. I wished to consult with
your father; but he was at a distance. I learned that his life was
condemned. Not an hour was to be lost. I took a bold resolve, that
has exposed me to his suspicions, and to my country’s wrath. But my
main idea was to save him, my early friend, from death, and my
country from fruitless massacre. I withdrew from the intended
revolt. I sought at once the head of the Austrian government in Italy,
and made terms for the lives of Alphonso and of the other more
illustrious chiefs, which otherwise would have been forfeited. I
obtained permission to undertake myself the charge of securing my
kinsman in order to place him in safety, and to conduct him to a
foreign land, in an exile that would cease when the danger was
dispelled. But unhappily he deemed that I only sought to destroy
him. He fled from my friendly pursuit. The soldiers with me were
attacked by an intermeddling Englishman; your father escaped from
Italy—concealing his retreat; and the character of his flight
counteracted my efforts to obtain his pardon. The government
conferred on me half his revenues, holding the other at its pleasure. I
accepted the offer to save his whole heritage from confiscation. That
I did not convey to him, what I pined to do—viz., the information
that I held but in trust what was bestowed by the government, and
the full explanation of what seemed blamable in my conduct—was
necessarily owing to the secresy he maintained. I could not discover
his refuge; but I never ceased to plead for his recall. This year only I
have partially succeeded. He can be restored to his heritage and
rank, on one proviso—a guarantee for his loyalty. That guarantee the
government has named: it is the alliance of his only child with one
whom the government can trust. It was the interest of all Italian
nobility, that the representation of a house so great falling to a
female, should not pass away wholly from the direct line;—in a word,
that you should ally yourself with a kinsman. But one kinsman, and
he the next in blood, presented himself. Brief—Alphonso regains all
that he lost on the day in which his daughter gives her hand to Giulio
Franzini, Count di Peschiera. “Ah,” continued the Count, mournfully,
“you shrink—you recoil. He thus submitted to your choice is indeed
unworthy of you. You are scarce in the spring of life. He is in its
waning autumn. Youth loves youth. He does not aspire to your love.
All that he can say is, love is not the only joy of the heart—it is joy to
raise from ruin a beloved father—joy to restore, to a land poor in all
but memories, a chief in whom it reverences a line of heroes. These
are the joys I offer to you—you, a daughter, and an Italian maid. Still
silent! Oh speak to me!”
Certainly this Count Peschiera knew well how woman is to be
wooed and won; and never was woman more sensitive to those high
appeals which most move all true earnest womanhood, than was the
young Violante. Fortune favoured him in the moment chosen. Harley
was wrenched away from her hopes, and love a word erased from her
language. In the void of the world, her father’s image alone stood
clear and visible. And she who from infancy had so pined to serve
that father, who had first learned to dream of Harley as that father’s
friend! She could restore to him all for which the exile sighed; and by
a sacrifice of self! Self-sacrifice, ever in itself such a temptation to the
noble! Still, in the midst of the confusion and disturbance of her
mind, the idea of marriage with another seemed so terrible and
revolting, that she could not at once conceive it; and still that instinct
of openness and honour, which pervaded all her character, warned
even her inexperience that there was something wrong in this
clandestine appeal to herself.
Again the Count besought her to speak; and with an effort she said,
irresolutely—
“If it be as you say, it is not for me to answer you; it is for my
father.”
“Nay,” replied Peschiera. “Pardon, if I contradict you. Do you know
so little of your father as to suppose that he will suffer his interest to
dictate to his pride. He would refuse, perhaps, even to receive my
visit—to hear my explanations; but certainly he would refuse to buy
back his inheritance by the sacrifice of his daughter to one whom he
has deemed his foe, and whom the mere disparity of years would
incline the world to say he had made the barter of his personal
ambition. But if I could go to him sanctioned by you—if I could say
your daughter overlooks what the father might deem an obstacle—
she has consented to accept my hand of her own free choice—she
unites her happiness, and blends her prayers, with mine,—then,
indeed, I could not fail of success: and Italy would pardon my errors,
and bless your name. Ah! Signorina, do not think of me save as an
instrument towards the fulfilment of duties so high and sacred—
think but of your ancestors, your father, your native land, and reject
not the proud occasion to prove how you revere them all!”
Violante’s heart was touched at the right chord. Her head rose—
her colour came back to her pale cheek—she turned the glorious
beauty of her countenance towards the wily tempter. She was about
to answer, and to seal her fate, when at that instant Harley’s voice
was heard at a little distance, and Nero came bounding towards her,
and thrust himself, with rough familiarity, between herself and
Peschiera. The Count drew back, and Violante, whose eyes were still
fixed on his face, started at the change that passed there. One quick
gleam of rage sufficed in an instant to light up the sinister secrets of
his nature—it was the face of the baffled gladiator. He had time but
for few words.
“I must not be seen here,” he muttered; “but to-morrow—in these
gardens—about this hour. I implore you, for the sake of your father—
his hopes, fortunes, his very life, to guard the secret of this interview
—to meet me again. Adieu!”
He vanished amidst the trees, and was gone—noiselessly,
mysteriously, as he had come.
CHAPTER IX.
The last words of Peschiera were still ringing in Violante’s ears
when Harley appeared in sight, and the sound of his voice dispelled
the vague and dreamy stupor which had crept over her senses. At
that voice there returned the consciousness of a mighty loss, the
sting of an intolerable anguish. To meet Harley there, and thus,
seemed impossible. She turned abruptly away, and hurried towards
the house. Harley called to her by name, but she would not answer,
and only quickened her steps. He paused a moment in surprise, and
then hastened after her.
“Under what strange taboo am I placed?” said he gaily, as he laid
his hand on her shrinking arm. “I inquire for Helen—she is ill, and
cannot see me. I come to sun myself in your presence, and you fly me
as if gods and men had set their mark on my brow. Child!—child!—
what is this? You are weeping?”
“Do not stay me now—do not speak to me,” answered Violante
through her stifling sobs, as she broke from his hand and made
towards the house.
“Have you a grief, and under the shelter of my father’s roof? A grief
that you will not tell to me? Cruel!” cried Harley, with inexpressible
tenderness of reproach in his soft tones.
Violante could not trust herself to reply. Ashamed of her self-
betrayal—softened yet more by his pleading voice—she could have
prayed to the earth to swallow her. At length, checking back her tears
by a heroic effort, she said, almost calmly, “Noble friend, forgive me.
I have no grief, believe me, which—which I can tell to you. I was but
thinking of my poor father when you came up; alarming myself about
him, it may be, with vain superstitious fears; and so—even a slight
surprise—your abrupt appearance, has sufficed to make me thus
weak and foolish; but I wish to see my father!—to go home—home!”
“Your father is well, believe me, and pleased that you are here. No
danger threatens him; and you, here, are safe.”
“I safe—and from what?”
Harley mused irresolute. He inclined to confide to her the danger
which her father had concealed; but had he the right to do so against
her father’s will?
“Give me,” he said, “time to reflect, and to obtain permission to
intrust you with a secret which, in my judgment, you should know.
Meanwhile, this much I may say, that rather than you should incur
the danger that I believe he exaggerates, your father would have
given you a protector—even in Randal Leslie.”
Violante started.
“But,” resumed Harley, with a calm, in which a certain deep
mournfulness was apparent, unconsciously to himself—“but I trust
you are reserved for a fairer fate, and a nobler spouse. I have vowed
to live henceforth in the common workday world. But for you, bright
child, for you, I am a dreamer still!”
Violante turned her eyes for one instant towards the melancholy
speaker. The look thrilled to his heart. He bowed his face
involuntarily. When he looked up, she had left his side. He did not
this time attempt to follow her, but moved away and plunged amidst
the leafless trees.
An hour afterwards he re-entered the house, and again sought to
see Helen. She had now recovered sufficiently to give him the
interview he requested.
He approached her with a grave and serious gentleness.
“My dear Helen,” said he, “you have consented to be my wife, my
life’s mild companion; let it be soon—soon—for I need you. I need all
the strength of that holy tie. Helen, let me press you to fix the time.”
“I owe you too much,” answered Helen, looking down, “to have a
will but yours. But your mother,” she added, perhaps clinging to the
idea of some reprieve—“your mother has not yet—”
“My mother—true. I will speak first to her. You shall receive from
my family all honour due to your gentle virtues. Helen, by the way,
have you mentioned to Violante the bond between us?”
“No—that is, I fear I may have unguardedly betrayed it, against
Lady Lansmere’s commands too—but—but—”
“So, Lady Lansmere forbade you to name it to Violante. This
should not be. I will answer for her permission to revoke that
interdict. It is due to Violante and to you. Tell your young friend all.
Ah, Helen, if I am at times cold or wayward, bear with me—bear with
me; for you love me, do you not?”
CHAPTER X.
That same evening Randal heard from Levy (at whose house he
staid late) of that self-introduction to Violante which (thanks to his
skeleton key) Peschiera had contrived to effect; and the Count
seemed more than sanguine—he seemed assured as to the full and
speedy success of his matrimonial enterprise. “Therefore,” said Levy,
“I trust I may very soon congratulate you on the acquisition of your
family estates.”
“Strange!” answered Randal, “strange that my fortunes seem so
bound up with the fate of a foreigner like Beatrice di Negra and her
connection with Frank Hazeldean.” He looked up at the clock as he
spoke, and added—
“Frank, by this time, has told his father of his engagement.”
“And you feel sure that the Squire cannot be coaxed into consent?”
“No; but I feel sure that the Squire will be so choleric at the first
intelligence, that Frank will not have the self-control necessary for
coaxing; and, perhaps, before the Squire can relent upon this point,
he may, by some accident, learn his grievances on another, which
would exasperate him still more.”
“Ay, I understand—the post obit?”
Randal nodded.
“And what then?” asked Levy.
“The next of kin to the lands of Hazeldean may have his day.”
The Baron smiled.
“You have good prospects in that direction, Leslie: look now to
another. I spoke to you of the borough of Lansmere. Your patron,
Audley Egerton, intends to stand for it.”
Randal’s heart had of late been so set upon other and more
avaricious schemes, that a seat in Parliament had sunk into a
secondary object; nevertheless, his ambitious and all-grasping nature
felt a bitter pang, when he heard that Egerton thus interposed
between himself and any chance of advancement.
“So!” he muttered sullenly—“so. This man, who pretends to be my
benefactor, squanders away the wealth of my forefathers—throws me
penniless on the world; and, while still encouraging me to exertion
and public life, robs me himself of—”
“No!” interrupted Levy—“not robs you; we may prevent that. The
Lansmere interest is not so strong in the borough as Dick Avenel’s.”
“But I cannot stand against Egerton.”
“Assuredly not—you may stand with him.”
“How?”
“Dick Avenel will never suffer Egerton to come in; and though he
cannot, perhaps, carry two of his own politics, he can split his votes
upon you.”
Randal’s eyes flashed. He saw at a glance, that if Avenel did not
overrate the relative strength of parties, his seat could be secured.
“But,” he said, “Egerton has not spoken to me on such a subject;
nor can you expect that he would propose to me to stand with him, if
he foresaw the chance of being ousted by the very candidate he
himself introduced.”
“Neither he nor his party will anticipate that possibility. If he asks
you, agree to stand—leave the rest to me.”
“You must hate Egerton bitterly,” said Randal; “for I am not vain
enough to think that you thus scheme but from pure love to me.”
“The motives of men are intricate and complicated,” answered
Levy, with unusual seriousness. “It suffices to the wise to profit by
the actions, and leave the motives in shade.”
There was silence for some minutes. Then the two drew closer
towards each other, and began to discuss details in their joint
designs.
Randal walked home slowly. It was a cold moonlit night. Young
idlers of his own years and rank passed him by, on their way from
the haunts of social pleasure. They were yet in the first fair holiday of
life. Life’s holiday had gone from him for ever. Graver men, in the
various callings of masculine labour—professions, trade, the state—
passed him also. Their steps might be sober, and their faces
careworn; but no step had the furtive stealth of his—no face the same
contracted, sinister, suspicious gloom. Only once, in a lonely
thoroughfare, and on the opposite side of the way, fell a foot-fall, and
glanced an eye, that seemed to betray a soul in sympathy with
Randal Leslie’s.
And Randal, who had heeded none of the other passengers by the
way, as if instinctively, took note of this one. His nerves crisped at
the noiseless slide of that form, as it stalked on from lamp to lamp,
keeping pace with his own. He felt a sort of awe, as if he had beheld
the wraith of himself; and ever, as he glanced suspiciously at the
stranger, the stranger glanced at him. He was inexpressibly relieved
when the figure turned down another street and vanished.
That man was a felon, as yet undetected. Between him and his kind
there stood but a thought—a veil airspun, but impassable, as the veil
of the Image at Sais.
And thus moved and thus looked Randal Leslie, a thing of dark
and secret mischief—within the pale of the law, but equally removed
from man by the vague consciousness that at his heart lay that which
the eyes of man would abhor and loathe. Solitary amidst the vast city,
and on through the machinery of Civilisation, went the still spirit of
Intellectual Evil.
CHAPTER XI.
Early the next morning Randal received two notes—one from
Frank, written in great agitation, begging Randal to see and
propitiate his father, whom he feared he had grievously offended;
and then running off, rather incoherently, into protestations that his
honour as well as his affections were engaged irrevocably to Beatrice,
and that her, at least, he could never abandon.
And the second note was from the Squire himself—short, and far
less cordial than usual—requesting Mr Leslie to call on him.
Randal dressed in haste, and went at once to Limmer’s hotel.
He found the Parson with Mr Hazeldean, and endeavouring in
vain to soothe him. The Squire had not slept all night, and his
appearance was almost haggard.
“Oho! Mr young Leslie,” said he, throwing himself back in his chair
as Randal entered—“I thought you were a friend—I thought you were
Frank’s adviser. Explain, sir; explain.”
“Gently, my dear Mr Hazeldean,” said the Parson. “You do but
surprise and alarm Mr Leslie. Tell him more distinctly what he has to
explain.”
Squire.—“Did you or did you not tell me or Mrs Hazeldean, that
Frank was in love with Violante Rickeybockey?”
Randal, (as in amaze.)—“I! Never, sir! I feared, on the contrary,
that he was somewhat enamoured of a very different person. I hinted
at that possibility. I could not do more, for I did not know how far
Frank’s affections were seriously engaged. And indeed, sir, Mrs
Hazeldean, though not encouraging the idea that your son could
marry a foreigner and a Roman Catholic, did not appear to consider
such objections insuperable, if Frank’s happiness were really at
stake.”
Here the poor Squire gave way to a burst of passion, that involved,
in one tempest, Frank, Randal, Harry herself, and the whole race of
foreigners, Roman Catholics, and women. While the Squire himself
was still incapable of hearing reason, the Parson, taking aside
Randal, convinced himself that the whole affair, so far as Randal was
concerned, had its origin in a very natural mistake; and that while
that young gentleman had been hinting at Beatrice, Mrs Hazeldean
had been thinking of Violante. With considerable difficulty he
succeeded in conveying this explanation to the Squire, and somewhat
appeasing his wrath against Randal. And the Dissimulator, seizing
his occasion, then expressed so much grief and astonishment at
learning that matters had gone as far as the Parson informed him—
that Frank had actually proposed to Beatrice, been accepted, and
engaged himself, before even communicating with his father; he
declared so earnestly, that he could never conjecture such evil—that
he had had Frank’s positive promise to take no step without the
sanction of his parents; he professed such sympathy with the
Squire’s wounded feelings, and such regret at Frank’s involvement,
that Mr Hazeldean at last yielded up his honest heart to his consoler
—and griping Randal’s hand, said, “Well, well, I wronged you—beg
your pardon. What now is to be done?”
“Why, you cannot consent to this marriage—impossible,” replied
Randal; “and we must hope therefore to influence Frank by his sense
of duty.”
“That’s it,” said the Squire; “for I’ll not give way. Pretty pass things
have come to, indeed! A widow too, I hear. Artful jade—thought, no
doubt, to catch a Hazeldean of Hazeldean. My estates go to an
outlandish Papistical set of mongrel brats! No, no, never!”
“But,” said the Parson, mildly, “perhaps we may be unjustly
prejudiced against this lady. We should have consented to Violante—
why not to her? She is of good family?”
“Certainly,” said Randal.
“And good character?”
Randal shook his head, and sighed. The Squire caught him roughly
by the arm—“Answer the Parson!” cried he, vehemently.
“Indeed, sir, I cannot speak ill of the character of a woman, who
may, too, be Frank’s wife; and the world is ill-natured, and not to be
believed. But you can judge for yourself, my dear Mr Hazeldean. Ask
your brother whether Madame di Negra is one whom he would
advise his nephew to marry.”
“My brother!” exclaimed the Squire furiously. “Consult my distant
brother on the affairs of my own son!”
“He is a man of the world,” put in Randal.
“And of feeling and honour,” said the Parson; “and, perhaps,
through him, we may be enabled to enlighten Frank, and save him
from what appears to be the snare of an artful woman.”
“Meanwhile,” said Randal, “I will seek Frank, and do my best with
him. Let me go now—I will return in an hour or so.”
“I will accompany you,” said the Parson.
“Nay, pardon me, but I think we two young men can talk more
openly without a third person, even so wise and kind as you.”
“Let Randal go,” growled the Squire. And Randal went.
He spent some time with Frank, and the reader will easily divine
how that time was employed. As he left Frank’s lodgings, he found
himself suddenly seized by the Squire himself.
“I was too impatient to stay at home and listen to the Parson’s
prosing,” said Mr Hazeldean, nervously. “I have shaken Dale off. Tell
me what has passed. Oh! don’t fear—I’m a man, and can bear the
worst.”
Randal drew the Squire’s arm within his, and led him into the
adjacent park.
“My dear sir,” said he, sorrowfully, “this is very confidential what I
am about to say. I must repeat it to you, because without such
confidence, I see not how to advise you on the proper course to take.
But if I betray Frank, it is for his good, and to his own father;—only
do not tell him. He would never forgive me—it would for ever destroy
my influence over him.”
“Go on, go on,” gasped the Squire; “speak out. I’ll never tell the
ungrateful boy that I learned his secrets from another.”
“Then,” said Randal, “the secret of his entanglement with Madame
di Negra is simply this—he found her in debt—nay, on the point of
being arrested—”
“Debt!—arrested! Jezabel!”
“And in paying the debt himself, and saving her from arrest, he
conferred on her the obligation which no woman of honour could
accept save from her affianced husband. Poor Frank!—if sadly taken
in, still we must pity and forgive him!”
Suddenly, to Randal’s great surprise, the Squire’s whole face
brightened up.
“I see, I see!” he exclaimed, slapping his thigh. “I have it—I have it.
’Tis an affair of money! I can buy her off. If she took money from
him, the mercenary, painted baggage! why, then, she’ll take it from
me. I don’t care what it costs—half my fortune—all! I’d be content
never to see Hazeldean Hall again, if I could save my son, my own
son, from disgrace and misery; for miserable he will be, when he
knows he has broken my heart and his mother’s. And for a creature
like that! My boy, a thousand hearty thanks to you. Where does the
wretch live? I’ll go to her at once.” And as he spoke, the Squire
actually pulled out his pocket-book and began turning over and
counting the bank-notes in it.
Randal at first tried to combat this bold resolution on the part of
the Squire; but Mr Hazeldean had seized on it with all the obstinacy
of his straightforward English mind. He cut Randal’s persuasive
eloquence off in the midst.
“Don’t waste your breath. I’ve settled it; and if you don’t tell me
where she lives, ’tis easily found out, I suppose.”
Randal mused a moment. “After all,” thought he, “why not? He
will be sure so to speak as to enlist her pride against himself, and to
irritate Frank to the utmost. Let him go.”
Accordingly, he gave the information required; and, insisting with
great earnestness on the Squire’s promise not to mention to Madame
di Negra his knowledge of Frank’s pecuniary aid, (for that would
betray Randal as the informant); and satisfying himself as he best
might with the Squire’s prompt assurance, “that he knew how to
settle matters, without saying why or wherefore, as long as he opened
his purse wide enough,” he accompanied Mr Hazeldean back into the
streets, and there left him—fixing an hour in the evening for an
interview at Limmer’s, and hinting that it would be best to have that
interview without the presence of the Parson. “Excellent good man,”
said Randal, “but not with sufficient knowledge of the world for
affairs of this kind, which you understand so well.”
“I should think so,” quoth the Squire, who had quite recovered his
good-humour. “And the Parson is as soft as buttermilk. We must be
firm here—firm, sir.” And the Squire struck the end of his stick on
the pavement, nodded to Randal, and went on to Mayfair as sturdily
and as confidently as if to purchase a prize cow at a cattle show.
CHAPTER XII.
“Bring the light nearer,” said John Burley—“nearer still.”
Leonard obeyed, and placed the candle on a little table by the sick
man’s bedside.
Burley’s mind was partially wandering; but there was method in
his madness. Horace Walpole said that “his stomach would survive
all the rest of him.” That which in Burley survived the last was his
quaint wild genius. He looked wistfully at the still flame of the
candle: “It lives ever in the air!” said he.
“What lives ever?”
Burley’s voice swelled—“Light!” He turned from Leonard, and
again contemplated the little flame. “In the fixed star, in the Will-o’-
the-wisp, in the great sun that illumes half a world, or the farthing
rushlight by which the ragged student strains his eyes—still the same
flower of the elements. Light in the universe, thought in the soul—ay
—ay—Go on with the simile. My head swims. Extinguish the light!
You cannot; fool, it vanishes from your eye, but it is still in the space.
Worlds must perish, suns shrivel up, matter and spirit both fall into
nothingness, before the combinations whose union makes that little
flame, which the breath of a babe can restore to darkness, shall lose
the power to unite into light once more. Lose the power!—no, the
necessity:—it is the one Must in creation. Ay, ay, very dark riddles
grow clear now—now when I could not cast up an addition sum in
the baker’s bill! What wise man denied that two and two made four?
Do they not make four? I can’t answer him. But I could answer a
question that some wise men have contrived to make much knottier.”
He smiled softly, and turned his face for some minutes to the wall.
This was the second night on which Leonard had watched by his
bedside, and Burley’s state had grown rapidly worse. He could not
last many days, perhaps many hours. But he had evinced an emotion
beyond mere delight at seeing Leonard again. He had since then
been calmer, more himself. “I feared I might have ruined you by my
bad example,” he said, with a touch of humour that became pathos as
he added, “That idea preyed on me.”

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whereas Chapter 11 through Chapter 13 focus on v arious approaches for trans-

Josef T. Kittler, Ph.D.

Stephen J. Moss, Ph.D .

John T. R. Isaac Robert C. Malenka

Trevor G. Smart Antoine Triller

Philip Thomas Edward B. Ziff

1 Methods for Uncovering

1.1 Introduction ......................................................................................................1

2 The Dynamic Synapse

in the ef ficien y of e xcitatory synaptic transmission that has been suggested to

Methods for Uncovering the Mechanisms of AMPA Receptor Trafficking 3

1.2 APPROACHES USED IN THE STUDY OF AMPA

1.2.1 PROTEIN INTERACTION ASSAYS

The Dynamic Synapse

© 2006 by Taylor & Francis Group, LLC

Methods for Uncovering the Mechanisms of AMPA Receptor Trafficking 5

This interaction is usually confirmed by biochemical methods, such as co

6 The Dynamic Synapse

GluR1/2 GluR2/3 GluR1/2+GluR2/3 NMDAR

activity dependent constitutive NMDAR synaptic activity

plasma-membrane tar geting of the receptor w as not dependent on PDZ-mediated

Methods for Uncovering the Mechanisms of AMPA Receptor Trafficking 7

In a tw o-hybrid screen using the C-terminal domain of GluR1 as bait, Shen et

1.2.2 CELL BIOLOGY

8 The Dynamic Synapse

GluR1 Activity-dependant Phosphorylation [52] Regulation of GluR1 Activity- Function of GluR1

10 The Dynamic Synapse

Methods for Uncovering the Mechanisms of AMPA Receptor Trafficking 11

1.2.2.2 Cell Biology Assays

Other approaches, combining microscop y and biochemistry , ha ve been used to

12 The Dynamic Synapse

Phosphorylation is one of the major mechanisms of re gulation of AMPAR function.

Methods for Uncovering the Mechanisms of AMPA Receptor Trafficking 13

Another approach to understand the mechanism of AMPAR traf ficking and it

1.2.4.2 Rectification Index

The current-voltage (IV) relationship of AMPAR is determined largely by the GluR2

14 The Dynamic Synapse

1.2.4.3 Agonists and Toxins

Methods for Uncovering the Mechanisms of AMPA Receptor Trafficking 15