The Glutamate Receptors

THE GLUTAMATE RECEPTORS

T H E R E C E PT O R S KIM A. NEVE, SERIES EDITOR The Glutamate Receptors, EDITED BY Robert W. Gereau, IV, and Geoffrey T. Swanson, 2008 The Chemokine Receptors,

Jeffrey K. Harrison, 2007

EDITED BY

The GABA Receptors, Third Edition, Hanns Möhler, 2007

EDITED BY

S. J. Enna and

The Serotonin Receptors: From Molecular Pharmacology to Human Therapeutics, EDITED BY Bryan L. Roth, 2006 The Adrenergic Receptors: In the 21st Century, M. Perez, 2005 The Melanocortin Receptors,

EDITED BY

The GABA Receptors, Second Edition, Norman G. Bowery, 1997

EDITED BY

Dianne

Roger D. Cone, 2000 EDITED BY

S. J. Enna and

The Ionotropic Glutamate Receptors, EDITED BY Daniel T. Monaghan and Robert Wenthold, 1997 The Dopamine Receptors, L. Neve, 1997

Kim A. Neve and Rachael

EDITED BY

The Metabotropic Glutamate Receptors, and Jitendra Patel, 1994 The Tachykinin Receptors,

EDITED BY

The Beta-Adrenergic Receptors,

EDITED BY

The Serotonin Receptors,

EDITED BY

EDITED BY

The Alpha-2 Adrenergic Receptors, The Opiate Receptors,

EDITED BY

P. Jeffrey Conn

Stephen H. Buck, 1994

Adenosine and Adenosine Receptors, 1990 The Muscarinic Receptors,

EDITED BY

John P. Perkins, 1991

EDITED BY

Michael Williams,

Joan Heller Brown, 1989

Elaine Sanders-Bush, 1988 EDITED BY

Lee Limbird, 1988

Gavril W. Pasternak, 1988

The Glutamate Receptors Edited by

Robert W. Gereau, IV, PhD Washington University Pain Center, Department of Anesthesiology and Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO and

Geoffrey T. Swanson, PhD Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL

Editors Robert W. Gereau, IV Washington University Pain Center Department of Anesthesiology and Biological Chemistry Department of Anatomy and Neurobiology Washington University School of Medicine St. Louis, MO

Geoffrey T. Swanson Department of Molecular Pharmacology Northwestern University Feinberg School of Medicine Chicago, IL

Series Editor Kim A. Neve Senior Research Career Scientist Research Service, VAMC Department of Behavioral Neuroscience Oregon Health and Science University Portland, OR

ISBN: 978-1-58829-792-1

e-ISBN: 978-1-59745-055-3

Library of Congress Control Number: 2007941658 ©2008 Humana Press, a part of Springer Science+Business Media, LLC All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, 999 Riverview Drive, Suite 208, Totowa, NJ 07512 USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Cover illustration: Fig. 2B, C from Chapter 6, “Structural Correlates of Ionotropic Glutamate Receptor Function,” by Anders S. Kristensen et al. Printed on acid-free paper 987654321 springer.com

Preface The abundant amino acid glutamate is the principal excitatory neurotransmitter in the mammalian central nervous system. Glutamate exerts its actions on cells via activation of two main classes of receptors. One class, known as the ionotropic glutamate receptors, includes a diverse group of ion channels that, in most cases, are directly gated by glutamate binding. The second class of glutamate receptors, known as metabotropic glutamate receptors, is made up of seven transmembrane-domain proteins that couple to intracellular signaling pathways via heterotrimeric guanosine triphosphate (GTP)-binding proteins. In rodents, at least 22 distinct gene products comprise these two classes of glutamate receptors. In addition to having both ion channels and G proteincoupled receptors, this broad superfamily of receptors encompasses several subunit proteins that do not, in fact, exhibit an affinity for glutamate. These gene products are quite obviously structurally related to other family members and subserve roles in excitatory neurotransmission, and for that reason warrant discussion in a review of the field. Glutamate receptors are critically important molecules for normal brain function. They transduce the vast majority of excitatory neurotransmission and regulate the strength of both excitatory and inhibitory transmission in the nervous system. Glutamatergic systems are dysfunctional in most neuropathologies, and aberrant receptor function appears to have causative roles in many neurologic diseases. Therefore, it is desirable for all neuroscientists to have a good working knowledge of the general structural and functional properties of these receptors. The Glutamate Receptors comprises a series of chapters by experts in the study of glutamate receptor function. This book serves as an update to two excellent previous books, The Ionotropic Glutamate Receptors and The Metabotropic Glutamate Receptors, and is intended to serve as a comprehensive primer on the field of glutamate receptors. In the decade since publication of these earlier volumes, an extraordinary amount of research has produced an abundance of insights into nearly every aspect of glutamate receptor function. This book is intended to cover the significant developments in this fertile period and to give a snapshot of how prominent scientists in the field look to the future of glutamate receptor research. The amount of material covered is vast, and thus in order to facilitate location of similar aspects of the various receptor subfamilies, we have organized the v

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book into a series of chapters that follow a similar format. The two main groups of receptors are discussed in separate chapters covering the structure of the receptors, their roles in synaptic plasticity, and the potential therapeutic utility of glutamate receptor ligands. Each subgroup of receptors is discussed in individual chapters covering major areas of emphasis including structure, function, pharmacology, protein–protein interactions, and roles in synaptic transmission and neuromodulation. The editors hope that this collection will serve as a valuable resource for scientists and students. Robert W. Gereau, IV, PhD Geoffrey T. Swanson, PhD

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. AMPA Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Michael C. Ashby, Michael I. Daw, and John T. R. Isaac

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2. NMDA Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ronald S. Petralia and Robert J. Wenthold

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3. Kainate Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anis Contractor and Geoffrey T. Swanson

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4. Delta Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 Michisuke Yuzaki 5. Ionotropic Glutamate Receptors in Synaptic Plasticity . . . . . . . . 179 Kenneth A. Pelkey and Chris J. McBain 6. Structural Correlates of Ionotropic Glutamate Receptor Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 Anders S. Kristensen, Kasper B. Hansen, Lonnie P. Wollmuth, Jan Egebjerg, and Stephen F. Traynelis 7. Positive Modulators of AMPA-Type Glutamate Receptors: Progress and Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Gary Lynch and Christine M. Gall 8. Clinically Tolerated Strategies for NMDA Receptor Antagonism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 Huei-Sheng Vincent Chen, Dongxian Zhang, and Stuart A. Lipton 9. The Structures of Metabotropic Glutamate Receptors . . . . . . . . . 363 David R. Hampson, Erin M. Rose, and Jordan E. Antflick 10. Group I Metabotropic Glutamate Receptors (mGlu1 and mGlu5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387 Julie Anne Saugstad and Susan Lynn Ingram vii

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11. Group II Metabotropic Glutamate Receptors (mGlu2 and mGlu3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465 Michael P. Johnson and Darryle D. Schoepp 12. Group III Metabotropic Glutamate Receptors (mGlu4, mGlu6, mGlu7, and mGlu8) . . . . . . . . . . . . . . . . . . . 489 Volker Neugebauer 13. Metabotropic Glutamate Receptor-Dependent Synaptic Plasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509 Stephen M. Fitzjohn and Zafar I. Bashir 14. Metabotropic Glutamate Receptor Ligands as Novel Therapeutic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529 Ashley E. Brady and P. Jeffrey Conn Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565

Contributors Jordan E. Antflick • Department of Pharmaceutical Sciences and Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada Michael C. Ashby • Developmental Synaptic Plasticity Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD Zafar I. Bashir • MRC Centre for Synaptic Plasticity, Department of Anatomy, University of Bristol, Bristol, UK Ashley E. Brady • Department of Pharmacology and VICB Program in Drug Discovery, Vanderbilt University Medical Center, Nashville, TN Huei-Sheng Vincent Chen • Center for Neurosciences, Aging and Stem Cell Research, Burnham Institute for Medical Research and University of California, San Diego, La Jolla, CA P. Jeffrey Conn • Department of Pharmacology and VICB Program in Drug Discovery, Vanderbilt University Medical Center, Nashville, TN Anis Contractor • Department of Physiology, Northwestern University Feinberg School of Medicine, Chicago, IL Michael I. Daw • MRC Centre for Synaptic Plasticity, Department of Anatomy, University of Bristol, Bristol, UK Jan Egebjerg • Department of Molecular Biology, H. Lundbeck A/S, Valby, Denmark Stephen M. Fitzjohn • MRC Centre for Synaptic Plasticity, Department of Anatomy, University of Bristol, Bristol, UK Christine M. Gall • Department of Anatomy and Neurobiology, University of California, Irvine, CA Robert W. Gereau, IV • Washington University Pain Center, Department of Anesthesiology and Department of Anatomy and Neurobiology, Washington University School of Medicine, St Louis, MO David R. Hampson • Department of Pharmaceutical Sciences and Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada Kasper B. Hansen • Department of Molecular Biology, H. Lundbeck A/S, Valby, Denmark ix

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Susan Lynn Ingram • Department of Psychology, Washington State University Vancouver, Vancouver, WA John T. R. Isaac • Developmental Synaptic Plasticity Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD Michael P. Johnson • Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN Anders S. Kristensen • Department of Pharmacology, Emory University School of Medicine, Rollins Research Center, Atlanta, GA Stuart A. Lipton • Center for Neurosciences, Aging and Stem Cell Research, Burnham Institute for Medical Research and University of California, San Diego, La Jolla, CA Gary Lynch • Department of Psychiatry and Human Behavior, University of California, Irvine, CA Chris J. McBain • Laboratory of Cellular and Synaptic Neurophysiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD Volker Neugebauer • Department of Neuroscience & Cell Biology, The University of Texas Medical Branch, Galveston, TX Kenneth A. Pelkey • Laboratory of Cellular and Synaptic Neurophysiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD Ronald S. Petralia • Laboratory of Neurochemistry, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD Erin M. Rose • Department of Pharmaceutical Sciences and Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada Julie Anne Saugstad • Robert S. Dow Neurobiology Laboratories, Legacy Clinical Research and Technology Center, Portland, OR Darryle D. Schoepp • Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN Geoffrey T. Swanson • Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL Stephen F. Traynelis • Department of Pharmacology, Emory University School of Medicine, Rollins Research Center, Atlanta, GA Robert J. Wenthold • Laboratory of Neurochemistry, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD

Contributors

Lonnie P. Wollmuth • Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, New York, NY Michisuke Yuzaki • Department of Physiology, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan Dongxian Zhang • Center for Neurosciences, Aging and Stem Cell Research, Burnham Institute for Medical Research and University of California, San Diego, La Jolla, CA

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1 AMPA Receptors Michael C. Ashby, Michael I. Daw, and John T. R. Isaac

Summary -Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMP ARs) are glutamate-gated ion channels. They are the neurotransmitter receptors that mediate the great majority of fast excitatory synaptic transmission in the mammalian brain and are found throughout the animal kingdom in organisms as diverse as rodents, honeybees, nematode worms, and humans. They are absolutely critical for brain function; for example, infusion of a selective AMPAR antagonist into the rat hippocampus in vivo completely silences excitatory transmission in that region (1). AMPARs are also required for adaptive changes in the brain, mediating the expression of forms of long-term and short-term synaptic plasticity that are believed to underlie learning and memory, development, and certain neurologic diseases (2–5). Thus, AMPARs play a central role in brain function, and consequently there is great interest in the development of novel therapies directed at modulating AMPAR function for treatment of neurologic disorders, such as Alzheimer disease and stroke. Key Words: Glutamate; Ion channel; Excitatory synaptic transmission; Synaptic plasticity; Receptor phosphorylation; Receptor trafficking; Hippocampus.

1. Structure 1.1. Genes There is remarkable homology among all of the ionotropic glutamate receptor genes that have been identified, suggesting that they may have arisen From: The Receptors: The Glutamate Receptors Edited by: R. W. Gereau and G. T. Swanson © Humana Press, Totowa, NJ

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from a common ancestral gene. There are 16 distinct mammalian genes, 4 genes from non-mammalian vertebrates, at least 6 genes from invertebrates, and several genes from plants. A prokaryotic protein called GluR0, which is a glutamate-activated K+ channel, has substantial homology with the ion channel of glutamate receptors from higher organisms and thus may represent the common ancestor (6). In mammals, there are four different -amino-3-hydroxy-5-methyl-4isoxazolepropionic acid receptor (AMPAR) subunits: GluR1, GluR2, GluR3, and GluR4 (these subunits are also known as GluR-A, GluR-B, GluR-C. and GluR-D or GLUA1 , GLUA2 , GLUA3 , and GLUA4 ) (7). The four different mammalian AMPAR subunits are encoded by separate but related genes that form a single gene family. Although the classification of glutamate receptors was initially based on their pharmacologic properties, the AMPA, kainate, and N-methyl-d-aspartate (NMDA) receptor subunits are encoded by similarly distinct families of genes. Although the mammalian AMPAR subunits share ∼70% homology, they vary much more from the other ionotropic glutamate receptor subunits (∼20%–40% homology). The genes encoding GluR1–4 are named GRIA1–4. They contain multiple intron–exon repeats (17 in mouse GRIA2) and share similar overall structure (7). The overall size of the genes is likely to be >200 kilobases, whereas the translated protein subunits contain only ∼850–900 amino acids (8). 1.2. Topology and Stoichiometry There was initially substantial uncertainty about the topology of AMPAR subunits and the other ionotropic glutamate receptors. However, domain mapping of glycosylation and phosphorylation sites and antibody targeting revealed the topology of AMPAR subunits in the membrane (9,10). This was confirmed by high-resolution structural analyses (11). All the AMPAR subunit proteins have an extracellular amino (NH3 ) terminal and four membraneassociated hydrophobic domains (M1–4). Three of these domains are transmembrane (M1, 3, and 4), and the other forms a reentrant loop that enters and exits the membrane on the cytoplasmic side without traversing the membrane (M2). This arrangement of M2 means that the C-terminal tail of the protein is intracellular. Transmembrane AMPAR regulatory proteins (TARPs) also are coassembled stoichiometrically with native AMPARs (12) (Fig. 1). The AMPAR proteins, similar to the other mammalian ionotropic glutamate receptors, have likely evolved through fusion of three gene segments that were once individual bacterial proteins. The amino-terminal domain (NTD) is homologous to the bacterial leucine-isoleucine-valine–binding protein (LIVBP) and forms a large fraction of the total size of the protein (∼400 amino acids). Residues within the NTD are important for receptor assembly, may have roles in modulating channel kinetics (13,14), and potentially play a role in

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Fig. 1. Topology of the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR). A. Schematic of an AMPAR subunit in the plasma membrane in association with a transmembrane AMPA receptor regulatory protein (TARP). Glycosylation and palmitoylation sites are indicated. The N-terminal domain (NTD), extracellular ligand-binding domains (S1 and S2), transmembrane domains (M1–4), the flip/flop alternative splicing site, and the RNA editing sites (Q/R and R/G) are also shown. B. Three-dimensional representation of the AMPAR complex depicting the arrangement of one subunit within the complex and showing that the M2 region lines the channel.

transsynaptic interactions and the regulation of spine morphology. In this latter respect, overexpression of the GluR2 NTD in isolation can induce changes in neuronal morphology (15). The ligand-binding domain, which resembles the bacterial lysine-arginine-orthinine binding protein (LAOBP), comprises two separate segments, named S1 and S2. These extracellular polypeptides are interrupted by the ion channel pore, which is structurally similar to bacterial K+ channels (in particular, the GluR0 protein) (11). The reentrant M2 transmembrane loop forms the lining of the channel pore, and amino acids in this region determine the selectivity of the ion channel (16) (Fig. 1). The Cterminal tail is the most variable region between the AMPAR subunits and is the site of subunit-specific protein interactions and phosphorylation sites that modulate AMPAR function (17–19) (as discussed in more detail later in this chapter). Recently, the structure of the native AMPAR complex has been visualized directly using single-particle electron microscopy (Fig. 2). This reveals an asymmetric organization of the extracellular N-terminal domains of heteromeric receptor complexes, the tight association with TARPs and shows that a conformational change of the extracellular region of the receptor is associated with ligand binding and desensitization (20,21).

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Fig. 2. The structure of the native -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) analyzed using single-particle electron microscopy. A. AMPAR purified from brain in type I (nondesensitized) conformation. Top left: Two panels showing the averaged image. Top right: Schematic of the arrangement of the domains of the native AMPAR–transmembrane AMPA receptor protein (TARP) complex in this type I configuration. Bottom: Three-dimensional reconstruction of the AMPAR in the type I conformation. LBD, ligand-binding domain (equivalent to S1 and S2); NTD, N-terminal domain; TMD, transmembrane domain (equivalent to M1– M4). B. AMPAR in the two type II (desensitized) conformations (panels as for part A). C. Superimposition of related known crystal structures onto the type I AMPAR image. Crystals used are extracellular domain of mGluR1 (NTD2 ), ligand-binding domain of

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Despite initial controversy, the consensus is that mature functional AMPARs are tetramers (22,23). Each receptor is formed in the endoplasmic reticulum as a dimer of dimers (11). That is, the initial stage of formation is the dimerization of two subunits that is dependent on the interactions in the NTD (24). This is followed by a second dimerization step mediated by associations at the ligandbinding and membrane domains in a process that is also dependent on Q/R editing in M2 (25) (discussed in detail later). The formation and stabilization of the tetramer is further promoted by NTD interactions. 1.3. Diversity 1.3.1. RNA Splice Variants and Editing The functional diversity of AMPARs is increased by alternative splicing and editing of subunit RNA. These posttranscriptional modifications generate multiple isoforms of each subunit, producing varied structural and functional properties (Fig. 3). The pre-mRNA transcripts of all of the AMPAR subunits can be alternatively spliced to produce either “flip” or “flop” isoforms (26). This alternative splicing of adjacent exons results in variation within a 38amino acid sequence in the extracellular region of the protein, close to the final transmembrane domain (M4). The two isoforms have different expression patterns, channel kinetics, and pharmacologic profiles. Generally, flip variants are expressed early in development, whereas flop isoforms are initially expressed in low abundance and are upregulated in adult animals (26,121). There are also cell type– and subunit-specific differences in the ratios of flip and flop isoforms, and levels of expression can be modulated by activity and following injury and during disease. However, little is known about regulation of the flip/flop splicing in neurons. Since the flip and flop isoforms can influence receptor formation and stoichiometry (27), splicing may be important in determining the AMPAR subunit composition. The major functional difference is that desensitization of flip AMPARs in response to glutamate is markedly reduced and slower compared to that of flop-containing receptors, leading to larger steady-state currents (28). This may be caused by amino acid differences in regions that influence the ligand-binding domain (29). Splicing of GluR1, 2, and 4 mRNA at a 5’ donor recognition site just after the M4 sequence is responsible for producing variations in the C-terminal tail of these subunits (7,8) (Fig. 3). GluR2 and GluR4 are expressed as both short- and long-tailed proteins; this is dependent on differential splicing between exons 16  Fig. 2. (Continued) GluR2 (LBD2 ), and transmembrane segment of KcsA (TMD4 ). Adapted from Nakagawa T, et al. Structure and different conformational states of native AMPA receptor complexes. Nature 2005;433(7025):545–549.

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Fig. 3. Sequence alignments of the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits showing the membrane-spanning regions and RNA editing sites and highlighting the alternatively spliced regions, phosphorylation sites, and protein–protein interactions in the C-terminus.

and 17 (short-tailed forms are also referred to as GluR2c and GluR4c). In adult brain, >90% of GluR2 subunits are of the short form (8), whereas the GluR4 subunit is usually, but not exclusively, expressed as the long-tailed form (30). GluR1 and GluR3 are not alternatively spliced in their C-terminal domains and have long and short tails, respectively. The cytoplasmic C-terminal tails of AMPAR subunits contain a number of residues that are biochemically modified and amino acid sequences that participate in protein–protein interactions. Both of these mechanisms can regulate receptor localization and function (17,19). Therefore, C-terminal splicing plays an important role in the generation of AMPAR subunits that exhibit distinct regulatory mechanisms. A good example of this is the differential regulation of GluR2 short, the predominant splice variant of this subunit in adult brain, and GluR2 long, which is highly expressed early in development in forebrain and throughout life in olfactory bulb (208). AMPAR subunits also undergo RNA editing (Figs. 1 and 3). The most functionally significant editing is that described for the GluR2 subunit. Most mature GluR2 protein contains an arginine residue (R) within the reentrant M2 membrane loop region at position 607 that is genomically encoded to be glutamine (Q) (28). This change is effected by hydrolytic editing of a single adenosine base in the pre-mRNA to an inosine by the adenosine deaminase enzyme, ADAR2 (31). The inosine-containing codon is read as an R at residue 607 rather than the genomically encoded Q by the translation machinery. Although this residue is conserved throughout the AMPAR subunit genes,

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Q/R editing is restricted to GluR2 because it is dependent on a 10-nucleotide sequence (the editing complementary site [ECS]) specifically found in the intron that precedes the exon encoding the Q/R site of GluR2. In the healthy adult brain, the vast majority of GluR2 subunits are Q/R edited (32). However, during early development and in certain neurons and glial cells, Q/R editing of GluR2 is not so complete (33–35). Q/R editing has several effects on the function of GluR2-containing receptors that will be discussed in the section on ion channel function. The importance of these effects on channel function is shown by the fact that transgenic mutation of the ECS site results in loss of editing of GluR2, and these mice are susceptible to seizures and die by 3 weeks of age (36). A reduction in Q/R editing efficiency has also been linked to several diseases. Spinal cord motor neurons taken from patients with amyotrophic lateral sclerosis (ALS) exhibit a marked reduction in editing of GluR2 (37,38), and a reduction in ADAR2 expression and Q/R editing correlates strongly with increased neuronal susceptibility to cerebral ischemia (39,40). These findings suggest that aberrant regulation of ADAR2 levels or activity may be an important contributor to neuronal dysfunction and excitotoxicity in these disorders. The potential mechanisms underlying regulation of editing and the relative importance of deficient editing compared to reduced GluR2 expression in forming Ca2+ -permeable AMPARs remain to be determined. In GluR2, 3, and 4 pre-mRNAs another adenosine, which is located directly before the flip/flop alternative splice region, can also undergo nuclear editing (41). The editing causes a change from arginine (R) to glycine (G) and can be mediated by ADAR2 acting at the junction of exon and intron 13 (42). The R/G editing produces channels that desensitize faster and recover more rapidly from desensitization (41,43). Although not as complete as Q/R editing, R/G-edited subunits form the majority of AMPARs in adult mouse brain (41). Changes in the fraction of R/G-edited subunits have been found in hippocampal tissue from epileptic patients (44) and following ischemia in rats (45). 1.3.2. Heteromeric Subunit Diversity The great majority of AMPARs in the central nervous system are thought to exist as heteromers (46,47). AMPAR subunits only assemble with other AMPAR subunits, and this exclusivity of assembly is determined by the specificity of interactions within the NTD (13,14). The formation of specific AMPAR heteromeric combinations is likely under the control of several factors. In cells in which GluR2 is expressed, the great majority of the AMPARs contain this subunit, and the preferred organization of receptor complexes containing GluR2 is a symmetric heteromer (48). This is likely linked to Q/R editing in the pore loop of GluR2, which regulates receptor assembly and transit of GluR2-containing dimers out of the endoplasmic reticulum (ER) (25,47). It was

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suggested that a large pool of available, unassembled GluR2 resides in the ER, and this excess of GluR2 ensures that the great majority of AMPARs include GluR2. The result is that there is a predominance of GluR1/2- and GluR2/3containing receptors in GluR2-expressing cells such as principal neurons (46). The importance of GluR2 is further highlighted by GluR2-knockout mice, in which a profound disruption in the subunit composition of AMPARs is observed (49). Therefore, in cells in which Q/R editing of GluR2 is almost complete, the incorporation of GluR2 into functional AMPARs seems simply to depend on the level of GluR2 expression. In this regard, the expression of GluR2 is highly regulated at the transcriptional level (50–52), and there is evidence that a loss of this regulation contributes to excitotoxicity mediated by pathologic expression of calcium-permeable, GluR2-lacking AMPARs during cerebral ischemia (53–55). However, certain cell types exhibit calcium-permeable, GluR2-lacking AMPARs under physiologic conditions, and these cells typically exhibit low levels of GluR2 expression (56,57). Moreover, there is evidence that cells expressing high levels of GluR2 (e.g., cortical pyramidal neurons) express a minor population of GluR2-lacking, calcium-permeable AMPARs. These receptors can be incorporated at synapses under certain conditions (58,59) and are involved in the expression of long-term synaptic plasticity (60,61). 1.4. Posttranslational Modifications 1.4.1. Phosphorylation Several serine (S), threonine (T), and tyrosine (Y) amino acid residues in the C-terminal tail of AMPARs are targets for phosphorylation (Fig. 3). Details of these sites are listed in the following paragraph, and the implications for AMPAR function are discussed later in the chapter. GluR1 is phosphorylated in vitro and in vivo at the S831 position by PKC and CaMKII and at S845 by protein kinase A (PKA) (62). The predominant, short-tailed GluR2 splice variant is phosphorylated at S880 by protein kinase C (PKC) (63). A third site on the C-terminal tail of GluR2, S863, can also be phosphorylated in vitro by PKC, although its direct effect on receptor function is unclear (63). Phosphorylation of the predominant GluR4 splice variant (longtailed) occurs at S842 and can be mediated by PKA, PKC, and CaMKII in vitro (64). Several other consensus sequences for phosphorylation exist within the cytoplasmic domains of the various AMPAR subunits that can be phosphorylated in vitro. Some of these sites are conserved, such as the potential PKC target sequence around the T830 residue in GluR3 and long-tailed variants of GluR4 and GluR2 (64). However, there is inherent danger in extrapolating in vitro information to phosphorylation of AMPARs in the brain. This is exemplified by the early identification of several AMPAR phosphorylation

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sites that subsequently were identified on regions of the protein located on the extracellular side of the plasma membrane (65). Therefore, further work is needed to elucidate the sites of phosphorylation on the AMPAR subunits in the brain and to understand the effects in vivo of such phosphorylation on channel function and trafficking on the characteristics of synaptic receptors. This need is highlighted by the fact that almost nothing is known about phosphorylation of GluR3. 1.4.2. Palmitoylation Palmitoylation is the reversible addition of the 16-carbon fatty acid palmitate to cysteine amino acids. AMPAR subunits can be palmitoylated at two intracellular cysteine residues, one close to M2 on the intracellular loop and the other in the C-terminal tail proximal to M4 (66) (Fig. 1). The Golgi-associated palmitoyl transferase GODZ palmitoylates the first of these sites (66). Palmitoylation promotes association of proteins with specialized membrane domains and thus may be involved in controlling AMPAR association with particular membrane compartments. 1.4.3. Glycosylation All of the AMPAR subunits have between four and six consensus sites for N-linked glycosylation, at which carbohydrate chains can be added onto extracellular residues of the protein (67) (Fig. 1). The sites reside in conserved positions of the NTD and the first ligand-binding domain, S1, although GluR2 lacks the sites on the extreme NTD. There is a progressive glycosylation of AMPARs as they pass through the secretory pathway such that mature AMPARs at the plasma membrane exhibit substantial glycosylation, as shown by a decrease in molecular weight of ∼4 kDa after in vitro removal of oligosaccharides from native proteins (68,69). Although the oligosaccharides have been identified (70) and they are known to be sulfated (in GluR2 at least), it is not known which sites on the AMPAR subunits are glycosylated in the mature protein. Moreover, the role of glycosylation is unclear for AMPARs: Glycosylation is not absolutely required for receptor expression, trafficking, ligand binding, or channel function, but does have an effect on ligand binding and is likely to influence other characteristics (68,71,72). In this regard, incomplete glycosylation of GluR3 can result in cleavage of the protein by granzyme B that may be involved in generating the autoimmune response underlying Rasmussen syndrome (73).

2. Function 2.1. In Vitro AMPARs have a relatively small single-channel conductance and fast kinetics, and they rapidly inactivate and desensitize in the presence of agonists such as glutamate or AMPA (74). The affinity for the natural agonist,

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l-glutamate, is relatively low (compared to NMDA receptors), with a halfmaximal effective concentration (EC50 ) of ∼0.5 mM. These biophysical properties result in a fast transient macroscopic current on agonist application; this is observed both for recombinant homomeric AMPARs expressed in heterologous cells such as HEK293 and for native AMPARs in patches excised from neurons (Fig. 4). The kinetics and desensitization of the channel depend on subunit composition, splice variant, and RNA editing. For example, the desensitization properties of AMPARs depend on splicing at the flip/flop site: Flip-variant receptor subunits exhibit slower desensitization than the flop variants and have a nondesensitizing low-conductance state (26). The AMPAR channel opens to a number of subconductance states between 7 and 50 pS, with those 1000 m−2 , whereas the density of extrasynaptic receptors is likely tens to hundreds of times lower (Fig. 5). The density of AMPARs at synapses can vary greatly among cell types and developmental stages. The best-characterized example of this is for the hippocampus, where synapses onto CA1 pyramidal cells show highly variable numbers of AMPARs and the average number of receptors increases during development (140,146,150,151,154). In an extreme example of this variation, it is clear that there is a fraction of synapses lacking AMPARs (but containing NMDARs), which are termed “silent” synapses. Silent synapses are particularly evident early in development and can be unsilenced acutely during LTP (154). In addition, there are differences in AMPAR distribution along dendrites. In particular, this has been described for CA1 pyramidal cells, in which a distance-dependent scaling of synaptic strength and AMPAR number at synapses along the apical dendrites is observed (155). This is mediated by increasing numbers of GluR1-containing AMPARs at progressively more distal synapses (156) and is believed to normalize synaptic strength by compensating for increased dendritic filtering of synapses more distal to the cell body.  Fig. 5. (Contiuned) and lower-density labeling of extrasynaptic membrane (small closed arrows). Spines lacking AMPARs are also evident (open arrows, s− ). Scale bar = 200 nm. D. Functional mapping of surface AMPAR distribution in cultured hippocampal neurons using two-photon glutamate uncaging. Top left: Fluorescence image of a neuron with the region of interest highlighted (box). Top right: Highermagnification image of the region of interest. Bottom left: AMPAR-mediated current (2pEPSC) evoked by two-photon glutamate uncaging on a spine; false color scale of the current amplitude is indicated. Bottom right: False color image map of 2pEPSC amplitude superimposed on the region of interest in experiments in which glutamate is uncaged at numerous locations on the dendrite. This demonstrates that “hot spots” of response to glutamate exist on dendrites. E. Top: Three-dimensional reconstruction of the fluorescence image of a region of dendrite from a cultured hippocampal neuron. Bottom: Superimposition of a false color image map of 2pEPSC amplitude showing that hot spots correlate with large spines. A: Reproduced from Richmond SA, et al. Localization of the glutamate receptor subunit GluR1 on the surface of living and within cultured hippocampal neurons. Neuroscience 1996;75(1):69–82 (see original publication for color images). B: Modified from Tanaka J, et al. Number and density of AMPA receptors in single synapses in immature cerebellum. J Neurosci 2005;25(4):799–807. C: Reproduced from Nusser Z, et al. Cell type and pathway dependence of synaptic AMPA receptor number and variability in the hippocampus. Neuron 1998;21(3):545–559. D, E: Reproduced from Matsuzaki M, et al. Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons. Nat Neurosci 2001;4(11):1086–1092. (see original publication for color images).

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The AMPARs present intracellularly are found at different stages of the canonical secretory pathway. As described previously, the assembly of AMPARs in the ER is influenced by subunit-specific interactions and editing of the Q/R site in GluR2 (14,25,47). The regulated ER exit of GluR2 results in a large GluR2 pool in the ER relative to the other subunits. ER export of glutamate receptors is influenced by the unfolded protein response (UPR) (157,158), and a subpopulation of AMPARs is known to associate with the ER chaperones BiP and calnexin, which may influence receptor folding or maturation (159,160). It was recently shown that the UPR is induced in the absence of the AMPAR-binding protein stargazin, suggesting that stargazin acts to promote transit of mature receptors through the ER (157). Stargazin may also influence subsequent transit of AMPARs through the Golgi apparatus via an interaction with the Golgi-enriched protein nPIST (161). Post-Golgi vesicular trafficking of AMPARs to the cell surface has been the subject of intense investigation (18,19,162). AMPARs are continually delivered to the surface of neurons (163,164) on relatively rapid time scales. Furthermore, synaptic AMPAR responses are rapidly decreased on infusion of antibodies that block the function of N-ethylmaleimide–sensitive fusion protein (NSF) (165) or toxins that cleave proteins mediating vesicular fusion (166). Such toxins also block the induction of forms of LTP that are dependent on AMPAR delivery to synapses (167). The vesicles on which these toxins act have not been identified, but recent evidence suggests that AMPAR exocytosis promoted during LTP (168) delivers receptors that have come through recycling endosomes (169,170). This suggests that there is a pool of AMPARs that recycle rapidly between the plasma membrane and internal vesicles, and that exocytosis and endocytosis have major roles in transport of AMPARs to and from synapses. A recent study using a photoactivatable irreversible antagonist (ANQX) failed to detect such rapid recycling of native AMPARs (171), but instead supported a role for an alternative mode of AMPAR trafficking, lateral diffusion in the plasma membrane. Rapid lateral movement of AMPARs in the membrane of neurons was directly visualized for the first time recently (172), demonstrating that a significant proportion of surface AMPARs move around the plasma membrane at relatively rapid rates and can exchange between extrasynaptic and synaptic sites (173). The rate of AMPAR diffusion in the membrane can be influenced by activity, proximity to postsynaptic sites, changes in intracellular calcium concentration, and dendritic spine morphology (172,174,175). These findings, along with the studies indicating that AMPARs move laterally away from synapses prior to removal from the plasma membrane (164,176,177), suggest an important role for diffusion within the plasma membrane as a regulated trafficking mechanism for AMPARs. Overall, this leaves a complex picture of AMPAR trafficking in which the dynamic interplay of vesicular trafficking, lateral diffusion, and protein–protein interactions

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determines the distribution of AMPARs on the surface of neurons. In future work it will be important to elucidate the relative roles of these mechanisms and how they interact with one another.

4. Interaction Partners A number of proteins have been identified that bind directly to AMPARs. In the following we summarize interactors or classes of interacting proteins for which some function is well established. In addition to these are other, less-well-studied interactions, such as GluR2–Lyn kinase (178) and the GluR1 interaction with Gi (179), the significance of which is unclear. 4.1. N-Ethylamide-Sensitive Fusion Protein (NSF) and Adaptor Protein 2 (AP2) NSF is a protein that is known to be involved in membrane fusion (180); therefore it was of great interest when NSF was found to interact directly with the C-terminal of GluR2 (165,181,182) (Fig. 3). This interaction is at a membrane proximal site, and - and -SNAPs can also coassemble with the NSF-GluR2 complex (182). In whole-cell recordings from CA1 pyramidal cells, acute disruption of the NSF–GluR2 interaction using specific peptides, or blockade of NSF ATPase activity with a function-blocking antibody present in the whole-cell pipette causes a rapid depression in EPSC amplitude (165, 181). This reduction in synaptic AMPAR function is due to a loss of surface receptors (166,183,184). The effects of disrupting this interaction appear to be activity dependent (166,185). Taken together, these data suggest that the NSF–GluR2 interaction is important for maintaining AMPARs at synapses during synaptic transmission. The role of the NSF–GluR2 in the maintenance of basal transmission appears to be related to the mechanisms underlying synaptic plasticity because the decrease in EPSC amplitude caused by disrupting the NSF–GluR2 interaction is reversibly occluded by NMDAR-dependent LTD (166,184). These findings can be explained if the reduction in surface AMPARs caused by blocking the GluR2–NSF interaction results in the complete removal of a population of synaptic AMPARs available for internalization during LTD. There is also evidence that AP2, a protein critical for clathrin-dependent endocytosis that acts as an adaptor for cargo to be internalized (186), associates with GluR2 in the same region as NSF (187). Although AP2 coimmunoprecipitates with GluR2-containing AMPARs, a direct interaction has not been demonstrated; therefore it is unclear whether AMPARs are directly recruited by this interaction for clathrin-dependent endocytosis. However, there is good evidence that clathrin-mediated endocytosis is required for the internalization of AMPARs during NMDAR-dependent LTD (106), and the GluR2–AP2 association is required for LTD (187). Therefore, a simple

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hypothesis is that AP2 recruits AMPARs for clathrin-mediated endocytosis in response to NMDAR-mediated calcium influx. Recent work provides a mechanism by which NMDAR-mediated calcium influx triggers this AP2dependent recruitment of AMPARs. Hippocalcin, a calcium-sensing protein, has been found to bind AP2, and both this interaction and the calcium-sensing function of hippocalcin are required for LTD (188). 4.2. PDZ Interactions In 1995, an abundant PSD protein, PSD-95, was shown directly to interact with NMDARs. Due to the similarity of the interaction domain on PSD-95 to that on two other proteins (Discs-large and ZO-1), this type of interaction motif was named a PDZ (PSD-95/SAP-90, Discs-large, ZO-1 homologous) domain (189). A number of PDZ domain–containing proteins have been identified that interact with the intracellular C-terminus of AMPAR subunits (190,191). Glutamate receptor interacting protein (GRIP) was the first protein reported to interact directly with an AMPAR subunit and is a PDZ domain–containing protein (192). GRIP interacts with the last 10 amino acid residues of GluR2 and 3, which contain a PDZ-binding motif (Fig. 3). Subsequently two other proteins were shown to interact with the same 10 amino acid domain: AMPAR-binding protein (ABP or GRIP2) (193) and protein interacting with C-kinase-1 (PICK1) (194,195). GRIP and ABP are very similar, contain multiple PDZ domains, and may function primarily as scaffolds. Acute disruption of the GRIP/ABP– GluR2/3 interaction can have rapid effects on synaptic transmission (196,197). PICK1 contains a single PDZ domain, but can dimerize via a separate coiledcoil domain; PICK1 also interacts with PKC and is thought to chaperone PKC to AMPARs and mobilize them during synaptic plasticity (109,198–202). Probably the best-established role for PICK1–GluR2/3 interactions is in the expression of cerebellar Purkinje cell LTD, in which PICK1 interaction with GluR2 is required for the PKC-dependent depression of transmission involving the phosphorylation of serine 880 on GluR2 (203–205). Less well studied is synapse-associated protein 97 (SAP97), which binds to a PDZ motif at the extreme C-terminus of GluR1 (206) and a potential PDZ interaction with GluR4 (207). Although the roles of these interactions are not well understood, there is evidence that the long-tailed subunits (GluR1, GluR4-long, and GluR2-long) are rapidly incorporated at synapses during LTP in a mechanism requiring AMPAR–PDZ interactions (18,207–209). However, the importance of the PDZ interaction with the long-tailed AMPAR subunits is unclear because a transgenic mouse lacking the GluR1 PDZ domain exhibits normal LTP (210). Another interactor, mLIN-10, has been identified that is a PDZ domain–containing protein interacting with GluR1 and GluR2 (211). The role of this interaction is unclear, but, potentially, it may influence AMPAR surface expression by regulating sorting in the Golgi apparatus.

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4.3. Cytoskeletal Proteins Postsynaptic application of drugs that depolymerize actin cause a decrease in AMPA-mediated EPSC amplitude and block LTP (212,213). In addition, latrunculin A, which destabilizes actin, decreases the surface expression of AMPARs in cultured cortical neurons (214) and occludes AMPA-induced AMPAR internalization (215). These findings suggest a strong association of AMPARs with the cytoskeleton, and a number of direction interactions between AMPAR subunits and cytoskeletal proteins have been identified (Fig. 3). There is a specific interaction between GluR1 and the cytoskeletal protein 4.1 N, which binds actin (214). Disruption of this interaction causes a reduction in surface AMPARs in heterologous cells. GluR4 interacts with actinin, and this is regulated by GluR4 S842 phosphorylation (216). The GluR1 C-terminus also interacts with the protein RIL via an LIM domain interaction, and RIL also binds -actinin via a PDZ interaction (217). This interaction can regulate GluR1 expression in spines and synaptic strength. Thus, although the precise roles of these direct interactions are not known, the cytoskeleton clearly plays an important role in the surface expression of AMPARs and is required for synaptic plasticity. 4.4. Neuronal Activity-Regulated Pentraxin (NARP) Neuronal activity-regulated pentraxin (NARP) is a secreted immediate-early gene product whose expression is regulated by activity in the brain. It binds to the extracellular domain of all AMPAR subunits and is believed to cause their clustering during synaptogenesis (218,219). The precise role of NARP is unclear, and the mechanism is selective for certain neuronal subtypes. For example in hippocampus, NARP only promotes glutamate synaptogenesis onto GABAergic interneurons. 4.5. Stargazin and Transmembrane AMPA Receptor Regulatory Proteins Stargazin was originally identified as the mutated protein in the stargazer mouse, which exhibits a phenotype of absence seizures and cerebellar ataxia. Stargazin was found to be a four-transmembrane domain protein that interacts with all AMPAR subunits but not with other glutamate receptors (220). Cerebellar granule cells from stargazer mice exhibit a profound phenotype: They completely and specifically lack surface AMPARs, and this phenotype can be rescued by expression of recombinant stargazin. Stargazin is part of a larger family of proteins originally thought to be  calcium channel subunits. Stargazin is -2, and the other members of the family that regulate AMPARs are -3, -4, and -8 (221). These proteins have now been termed transmembrane AMPA receptor regulatory proteins (TARPs) and have been shown

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to associate stoichiometrically with AMPARs (12). TARPs appear to play a critical role in trafficking AMPARs to the plasma membrane of neurons in all parts of the mammalian brain (84). Furthermore, in addition to being required for plasma membrane expression, TARPs are required for synaptic incorporation of AMPARs via a PDZ domain interaction with PSD-95 (220,222). This PSD-95–dependent mechanism is also involved in regulating the number of synaptic AMPAR during hippocampal LTP and LTD (223–226). Although it is clear that TARPs have a very important role in regulating AMPARs in the brain, the mechanisms by which they act in neurons other than cerebellar granule cells is not fully understood. For example, knockout of -8, the predominant TARP expressed in hippocampus, only has a mild effect on AMPAR expression at synapses (224), in contrast to the complete lack of AMPARs on the surface of cerebellar granule cells in the stargazer mouse that lacks -2 (stargazin). However, -8 is required for the stability of AMPARs, because the knockout exhibits a strong reduction in AMPAR protein levels but normal mRNA levels for the subunits. An additional, very profound finding is that TARPs regulate AMPAR pharmacology, gating, and single-channel conductance (as detailed earlier in this chapter) (84). This role of interacting proteins regulating AMPARs may be a more general principle. Recently, a novel CUB domain–containing protein has been identified in Caenorhabditis elegans that is an auxiliary subunit of the worm AMPAR, GLR-1, and regulates gating (227,228). Moreover, stargazin-like proteins have now also been identified in nematodes, Drosophila, and honeybees that are required for invertebrate AMPAR function (229). Thus, regulation of AMPAR function by auxiliary transmembrane proteins is an extremely important new theme and is likely to continue to have profound implications for our understanding of physiologic and pathologic brain function.

5. Pharmacology Quisqualate, AMPA, and kainate all act as agonists at AMPARs. Their rank order of potency is quisqualate > AMPA > glutamate > kainate, with AMPA being the most selective (230). These are still the most commonly used agonists, although others have become available. One high-affinity series is based on willardiine, with (S)-5-fluorowillardiine being even more potent than AMPA (231). A series of quinoxalinedione derivatives comprised the first widely used selective, competitive AMPAR antagonists, with 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), 6,7-dinitro-quinoxaline-2,3dione (DNQX), and 2,3-dihydroxy-6-nitro-7-sulfamoy-benzo(F)quinoxaline (NBQX) becoming the standard tools for blocking AMPARs (232). CNQX and DNQX also act as antagonists of the glycine-binding site on NMDA receptors (233). Therefore, NBQX is the drug of choice and currently the most commonly used AMPAR antagonist.

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Although effective against AMPARs, the quinoxalinediones are also antagonists at kainate receptors. In some cases NBQX does exhibit functional selectivity for AMPARs over kainate receptors: for example, a low micromolar dose of NBQX appears selectively to block AMPARs in hippocampus without significantly antagonizing kainate receptors on CA1 interneurons (234,235). However, more-selective compounds have been developed based on the 2,3-benzodiazepines that are noncompetitive antagonists at AMPARs and show considerable selectivity over kainate receptors. The most useful compound in this series is 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl7,8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine (GYKI53655) (236), which has allowed the physiologic roles of kainate receptors to be investigated (237–240). Unfortunately, GYKI53655 is not commercially available, which precludes its widespread use as a potent and selective AMPA receptor antagonist. An additional class of noncompetitive antagonists, the 1,2dihydrophthalazines, for example, SYM2206, have also proved useful in selectively antagonizing AMPA but not kainate receptors (241,242). Open channel blockers are also useful selective noncompetitive AMPAR antagonists, such as the wasp toxin philanthotoxin and Joro spider toxin (243). These toxins act at the polyamine-binding site in AMPAR channels lacking edited GluR2; although they exhibit some selectivity for AMPARs, these toxins also block other polyamine-modulated receptors such as 7 nicotinic, kainate, and NMDA receptors (244). These toxins have been used to probe the GluR2 content of synaptic receptors (61,108,245). In addition to the noncompetitive antagonists, positive allosteric modulators have also been identified for AMPARs, which are characterized by an ability to increase the function of the receptor in the presence of agonist without activating it in the absence of agonist. The first of these compounds to be described was aniracetam (246,247), a pyrrolidone, which was shown to act by decreasing the deactivation rate of AMPA receptors (248). A second group of allosteric modulators, the benzothiazides, includes cyclothiazide (249), and these show a subtly different mode of action than the pyrrolidines in slowing the transition from activated to desensitized states (248). In addition to a different mode of action, these classes show a different preference to AMPAR splice variants, with aniracetam having a greater effect on the flip than on the flop isoform, whereas cyclothiazide prefers flop receptors (250). This preference has been isolated to a single residue in the spliced region, which is an arginine residue in the flop and a serine residue in the flip isoform (251). These compounds can also be used to distinguish between AMPA and kainate receptor receptors because kainate receptors are not modified by these reagents (252). A more recently developed group of modulators comprises the biarylpropylsulfonamides, including LY503430 (253); these act by speeding the recovery from desensitization (254). AMPAR modulators have recently

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gained attention as possible therapeutic drugs: CX 516, a relative of aniracetam, is undergoing clinical trials to counteract cognitive problems in schizophrenia and attention-deficit hyperactivity disorder (ADHD), and LY503430 has shown potential to slow the progress of Parkinson disease (253,255) and improve cognitive function in Alzheimer disease (256). Both of these benefits are proposed to be via an increase in brain-derived neurotrophic factor (BDNF) production, an effect that has lead to speculation that AMPA modulators may also show antidepressant action (257).

6. Modulation Phosphorylation has a profound influence on the function of AMPARs via direct and indirect mechanisms. The probability of channel opening and/or surface expression is increased by PKA phosphorylation of GluR1 at S845 (258), whereas S831 phosphorylation leads to an increase in single-channel conductance (82). The mechanism and physiologic relevance for this latter effect are somewhat unclear because S831 phosphorylation increases singlechannel conductance in homomeric GluR1 receptors, but not in GluR1/GluR2 heteromers (259). These phosphorylation sites on GluR1 have received considerable interest because there is good evidence that they are sites of direct modification of the AMPAR during expression of LTP and LTD (4,17,18,260). Whereas phosphorylation of GluR2 does not have any direct affect on channel function, it can influence GluR2-containing AMPAR trafficking. S880 forms part of the PDZ domain recognition site at the extreme C-terminus of GluR2, and S880 phosphorylation regulates interactions with the PDZ domain–containing proteins PICK1, GRIP, and ABP that bind GluR2 in this region. Phosphorylation of S880 prevents the interaction of GluR2 with GRIP1 and ABP but has no effect on PICK1 binding (198,199,261). In addition, ABP binding can itself prevent phosphorylation of S880 on the GluR2 Cterminus (262). Phosphorylation of the Y876 residue by Src tyrosine kinase also regulates the GRIP1/ABP interaction but has no effect on PICK1 (263). Both S880 and Y876 can be phosphorylated in vivo, and the regulation of the protein–protein interactions by these mechanisms is believed to influence the trafficking of GluR2- containing AMPARs to and from synapses during synaptic plasticity (204). PKA phosphorylation of the S842 site on GluR4 has been proposed to be a critical step for the synaptic incorporation of GluR4-containing AMPARs during LTP (207). In addition, in chick retinal amacrine cells, which express high levels of GluR4, activation of PKA or PKC leads to S842 phosphorylation and an increase in AMPAR function (264,265). This may be mediated by PKC, which binds directly to the proximal C-terminal tail of GluR4 and preferentially phosphorylates S842 (266).

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7. Genetic Studies The ability to produce directed knockout mice in which a single gene is removed or rendered nonfunctional has allowed the correlated study of the effects of that gene product on both whole-animal behavior and synaptic and receptor function. In the case of AMPA receptors, knockouts for all the subunits have been made, but detailed synaptic and behavioral studies only have been performed on the GluR1 (GluRA) and GluR2 (GluRB) knockouts. 7.1. GluR1 Knockout Mice The results of studies on GluR1 knockout mice were initially surprising. The animals have normal life expectancy and development and no detectible deficits in neuronal structure or brain anatomy (267). However, there is a striking redistribution of GluR2 subunits in hippocampal CA1 pyramidal neurons in the GluR1 knockout, so that they are largely restricted to the cell body. AMPA receptor-mediated EPSCs recorded in CA1 pyramidal neurons appear normal and contain GluR2; therefore, the small amount of GluR2 present in the dendrites in the absence of GluR1 is preferentially targeted to synapses. LTP is absent in adult mice lacking GluR1, indicating a critical role for this subunit in the expression of LTP. The lack of effect of the knockout on development and basal transmission suggests that developmental synaptic plasticity is unaffected in these animals; indeed, a subsequent study showed that LTP is normal in young GluR1 knockout mice (268). Although LTP is absent in the adult GluR1 knockout, there is no deficiency in spatial learning (267). Because this form of synaptic plasticity is widely believed to be the synaptic basis of spatial learning, this was unexpected. Subsequent studies, however, found that although spatial reference memory is unaffected by GluR1 knockout, spatial working memory is profoundly deficient (269–271). It is noteworthy that spatial working memory is also preferentially affected by bilateral hippocampal lesion (271), and both spatial working memory (272) and hippocampal LTP (273) can be rescued by transgenic expression of GluR1. Non–hippocampus-related behaviors are also affected by GluR1 deletion. Learning an association between a cue and reward (Pavlovian approach conditioning) in GluR1 knockout mice is not different than in wild-type mice; however, the ability to use this cue as a reward to learn a new behavior (conditional reinforcement) is lost (274). 7.2. GluR2 and GluR3 Knockouts Knockout of GluR2 produces a severe phenotype; homozygotes are born alive but are sickly, are smaller than wild types, exhibit a high mortality rate, and exhibit a decrease in exploration behavior and motor coordination (275).

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On a cellular level the Ca2+ permeability of AMPA receptors is increased ninefold, and the amount of LTP in CA1 pyramidal cells is doubled compared to wild types and can be induced by Ca2+ influx through the GluR2-lacking AMPA receptors (275). LTD can also be induced in the GluR2 knockout, although this is of smaller amplitude than in the wild type. A GluR3 and a GluR2/3 double knockout have also been investigated. Knockout of GluR3 has little detectable effect on synaptic transmission or synaptic plasticity in hippocampus and, surprisingly, LTP and LTD can still be readily induced in the GluR2/3 double knockout (276). Recently a forebrain-specific GluR2 knockout has also been studied. This exhibits deficits in synaptic transmission and some developmental abnormalities, but LTP can be induced, which is dependent on NMDA receptors (277). GluR2 knockout also affects AMPA-receptor subunit composition in hippocampal neurons, producing an increased number of GluR1 and GluR3 homomers and also GluR1/3 heteromers that are not thought to exist in wild type, suggesting a role for GluR2 in receptor assembly (49). In cerebellum, GluR2 knockout results in a decrease in synaptic AMPA receptors on Purkinje cells but a specific increase in expression of the 2 glutamate receptor subunit at climbing fiber synapses on the same cells (278). GluR2 knockout produces numerous behavioral abnormalities. Hippocampal place cells are affected, forming unstable place fields (279), and this may underlie the deficiency in hippocampus-dependent spatial memory that is observed in the forebrain-specific GluR2 knockout (277). Numerous other behavioral changes are observed in the GluR2 knockout including deficiencies in object exploration, rearing, grooming, eye closure, motor performance, spatial and nonspatial learning (280), and emotional response conditioning (281). In addition, the GluR2 knockout also exhibits altered reproductive behavior involving changes in hypothalamic and septal function (282). Other studies on AMPA-receptor–subunit knockout mice indicate a role for GluR3 in the plastic changes underlying alcohol seeking and relapse (283) and both GluR1 and GluR2 in dorsal horn synaptic plasticity underlying nociception (284). 7.3. GluR2 Q/R Editing Mutants Mutant forms of GluR2 lacking the Q/R editing site have also been expressed in mouse strains. This results in highly seizure prone animals that die within 3 weeks (36,285). These animals also show deficits in dendritic structure and an even greater increase in AMPA receptor Ca2+ permeability than that seen in the GluR2 knockout mice. Similar to the GluR2 knockout, the increase in Ca2+ permeability in the GluR2 Q/R editing mutant results in the ability to induce NMDA receptor–independent LTP (285). These studies point to a critical role of GluR2 editing in AMPA receptor function in vivo. This is supported by studies on another mutant mouse strain, in which the enzyme

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responsible for RNA editing that produces the Q/R switch in GluR2, ADAR2, was knocked out. This strain of mice exhibit a very similar phenotype to the GluR2 Q/R editing mutant and can be rescued by transgenic expression of edited GluR2 (286).

8. Future Directions This chapter has summarized a large amount of knowledge accumulated from many thousands of studies focused on AMPARs. However, much remains to be understood, and there are also likely to be unexpected challenges ahead. A good example of how present understanding can be rapidly redefined is the recent elucidation of the role of TARPs in regulating AMPAR function. Within a few years of their discovery, the importance of their role has become clear. However, a number of broadly unanswered questions remain concerning these accessory proteins. Perhaps the most obvious of these concerns the differential expression pattern of the different TARP members in the mouse brain (221). These striking variations perhaps suggest that differing characteristics of the various TARPs could have region-specific roles in controlling AMPAR function. If this is the case, what are the differing characteristics, and how do they relate to what is known about AMPAR function in different areas of the brain? If it is not, then why is there differential expression, and, which is important, how is the expression controlled? Naturally, given their influence on AMPAR channel properties and synaptic localization (84), the TARPs offer a potentially important pharmacologic and therapeutic target. A full understanding of the physical nature and the dynamics of the TARP– AMPAR interaction would facilitate development of this potential. In addition, are there further, as-yet-unidentified accessory proteins that influence AMPAR function in the mammal? Recent work in C. elegans demonstrates that this is the case for the worm AMPAR GLR-1 (227,228). There is a developing acknowledgment that activation of and/or calcium influx through GluR2-lacking or unedited AMPARs plays important physiologic and pathologic roles in the brain and spinal cord. Work on two key issues is required for further development of this field: first, elucidation of the mechanisms regulating the amount of the GluR2 subunit at synaptic receptors in differing cell types, and, second, studies of the mechanisms that regulate the Q/R editing process that defines AMPAR calcium permeability. The first of these also links to a wider need to understand the control of AMPAR subunit transcription. There are clearly developmental and cell-specific variations in subunit expression that define the characteristics of the AMPARs and hence the characteristics of synaptic transmission and plasticity. However, little is known

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about the mechanisms underlying differential transcription and the signaling pathways controlling such processes. The trafficking of AMPARs to and from synapses is central to many aspects of glutamatergic synaptic transmission including development, plasticity, and pathology (19,162). Many mechanisms have been proposed to play a role in trafficking under varying experimental and physiologic paradigms. In addition, huge efforts have been made toward identifying proteins that influence AMPAR trafficking. A difficulty facing this field is to make sense of the myriad data that exist. Although it is likely that there is no single overarching biochemical pathway that controls AMPAR number at synapses, it seems important to assess any potential mechanism within the framework of the many mechanisms that are known to exist. For example, the highly numerous, varied, and interconnected protein–protein interactions within the postsynaptic density make it very difficult to interpret the true physiology of a protein when focusing on only a single mechanism. It is, of course, absolutely necessary to identify potential mechanisms, but perhaps the goal of this field should move from the data obtained from reductionist approaches to an integrated assessment of AMPAR trafficking. The use of genetically manipulated mice is beginning to yield exciting and novel data on various aspects of AMPAR function. However, a well-known difficulty with this approach is the possibility of compensation or redundancy. Much of the work within the AMPAR field has shown how plastic the brain can be. This plasticity, when employed to adjust to genetically induced changes, can lead to difficulty in interpretation of experimental results from knockout animals. Targeting genetic modifications to specific cell types (or even single cells) and controlling the onset of the modifications will likely prove to be an important technical advance in designing experiments to assess AMPAR function in vivo. The pace of research on AMPARs remains very high, and this topic is likely to be intensely studied for many years to come. Although much is known about AMPARs, the continuing high rate of novel discoveries suggests that significant further progress can be made in our understanding of AMPAR structure, function, and regulation. The expectation is that this will lead to continuing progress in understanding their roles in brain function both under physiologic conditions and during disease.

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2 NMDA Receptors Ronald S. Petralia and Robert J. Wenthold

Summary One of the major classes of ionotropic glutamate receptors is made up of the N-methyl-D-aspartate (NMDA) receptors, which require two agonists, glycine and glutamate, for activation and can pass calcium ions that may mediate synaptic and neuronal plasticity. They are formed from complexes made by various combinations of the subunits NR1 (with eight isoforms), NR2A-D, and NR3A-B and are found in most neurons of the brain and in various other cells. During development, generally NMDA receptors with NR2B, NR2D, and NR3A are abundant and decrease during maturation, whereas those with NR2A and NR2C increase. The function of NMDA receptors has been explored with a wide range of in vitro and in vivo studies, employing both recombinant gene constructs and native receptors. NMDA receptor subunits contain various motifs that control retention in the endoplasmic reticulum and trafficking through Golgi and other organelles to reach the cell membrane. Association of NMDA receptors with PDZ domain–containing proteins such as PSD-95 and SAP102 may be particularly important to trafficking and/or stabilization and function on the cell membrane. NMDA receptors on the cell membrane are sequestered mainly to the postsynaptic membrane of synapses, but some populations remain in extrasynaptic domains, especially those receptors that contain NR2B or NR2D. Key Words: Endocytosis; Endosome; Exocytosis; Glutamate receptors; MAGUK; NR1; NR2; PDZ; PSD-95; SAP102; Trafficking.

From: The Receptors: The Glutamate Receptors Edited by: R. W. Gereau and G. T. Swanson © Humana Press, Totowa, NJ

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1. Introduction Localization of NMDA receptors to synapses or extrasynaptic domains involves interactions with various other proteins, including, as noted, PDZ proteins. NMDA receptors (NMDARs) are a class of ionotropic glutamate receptors that were identified initially by the sensitivity to the agonist NMDA (1). NMDARs are the only ligand-gated ion channels with a probability of opening that depends strongly, under physiologic conditions, on a change in voltage across the membrane that relieves a magnesium ion blockage of the ion channel. NMDARs also are unique in requiring two different agonists for activation—glycine and glutamate (2). When this double-agonist activation occurs under the proper membrane voltage, the ion channel opens and passes calcium into the cell (7%–18% of inward current (3,4)); the calcium then acts as a second messenger to modulate synaptic strength and alter neuronal functions in a variety of ways including mediation of neuronal plasticity. NMDAR activation leads to an excitatory postsynaptic potential, and this excitatory response is slow (in both its rise and its decay) compared to that of -amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs), which comprise the other major group of ionotropic glutamate receptors. Typically, glutamate neurotransmission begins with a fast response generated by AMPARs (mainly sodium influx); the resultant membrane depolarization changes the membrane potential to allow NMDAR channels to open and pass calcium into the neuron to elicit long-term changes.

2. Structure 2.1. Genes Functional NMDARs are found in neurons and glia throughout the brain and spinal cord (1,5–7), as well as in several other organs (8). Seven different NMDAR subunits are known: NR1, NR2A, NR2B, NR2C, NR2D, NR3A, and NR3B (5). These subunits differ in the length of their C-termini, with the longest ones found in NR2A and NR2B and the shortest in NR1 and NR3B. Functional NMDARs always contain NR1 that is combined with one or two subunits of NR2 or NR3. The mature protein size of NR1 can vary from 867 to 941 amino acids and has a molecular weight of 97,449 to 106,042 Da; the rat gene contains 22 exons (25 kb) and 21 introns (1,9). There also is some evidence for truncated N-terminal isoforms (exon 3 inclusion leads to a truncated receptor (1,9)), including recent evidence of an mRNA isoform with an insertion of an intron with an inframe stop codon that is found only in the embryonic brain; its expression is modulated by brain-derived neurotrophic factor (BDNF) and a metabotropic GluR agonist (10). The mature protein size of NR2 subunits varies from 1218 to 1456 amino acids with a molecular weight of 133,491 to

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163,385 Da; the mouse gene for NR2C contains 15 exons spanning about 20 kb (9). For NR3A, the mature protein has 1082 or 1089 amino acids and a molecular weight of 121,580 Da (9). NR3B has 1002 amino acids and is about 109 kDa (11). Splice variants have been reported for most NMDAR subunits (9,12,13) but are well known only for NR1. NR1 has three regions of alternative splicing including an N-terminal N1 cassette (exon 5) and two C-terminal regions: C1 (exon 21) and C2 (exon 22) cassettes. N1 and C1 can be present or absent, and C2 can alternate with C2’ (which contains a PDZ-binding domain; see Section 3.2.1), thus generating eight different isoforms: NR1-1a (C1C2), NR1-1b (N1C1C2), NR1-2a (C2), NR1-2b (N1C2), NR1-3a (C1C2’), NR1-3b (N1C1C2’), NR1-4a (C2’), and NR1-4b (N1C2’). These variants show a great deal of overlap in the brain throughout development and in the adult, although some differences in distribution do occur (14). The most extensive distributions are seen for NR1-a (i.e., based on in situ hybridization with an oligonucleotide probe that recognizes the a form of NR1-1-4) and NR1-2; overall, NR1-3 is the least abundant. Some differences in distribution are seen between NR1-a and NR1-b. In the hippocampus, NR1-a is high in all areas, whereas NR1-b is high mainly in the CA3 region. In the adult cerebellum, NR1-a is widely distributed at all postnatal ages. In contrast, NR1-b increases in the cerebellum with age and is the predominant form in adult stellate-basket and granule cells (see also ref. 15). 2.2. Topology and Stoichiometry The elucidation of tertiary and quaternary structure of NMDARs (and glutamate receptors in general) has been controversial over the years (1,16,17). NMDARs contain three kinds of subunits: NR1, NR2, and/or NR3. Each subunit starts with an extracellular amino (N-)-terminal region, followed by an S1 binding region, transmembrane region 1 (TM1), a P loop that goes partially through the membrane (TM2 with short intracellular portions on each side), TM3, extracellular S2 binding region, TM4, and an intracellular carboxy (C-)-terminal region. Each subunit forms a tertiary structure that consists of an amino-terminal domain (ATD), an S1S2 ligand-binding core, a transmembrane domain, and a carboxy-terminal domain. NMDARs are made up of heteromeric complexes of glycine-binding NR1 subunits combined with glutamate-binding NR2 subunits and glycine-binding NR3 subunits (17,18). These complexes may assemble as dimers of dimers held together via interactions of the ATD and ligand-binding domains; single NR1 and NR2 subunits can come together as dimers in an S1S2 heterodimer configuration, that is, with the NR1 and NR2 S1S2 ligand-binding core domains facing back to back (17). Two NR1-NR2 dimers then come together to form the tetrameric NMDAR complex, forming the ion channel between the two

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dimers. However, there also is evidence for the formation of NR1 dimers (e.g., ref. 19; see also ref. 20), so that the normal native NMDAR assembly sequence is still not clear. In any case, agonist-induced closure of each ligand-binding domain (glycine for NR1 and glutamate for NR2) causes a separation of the ion channel–proximal portions of the subunits and, thus, an opening of the ion channel. The unique ability of NMDARs to bind the agonist NMDA becomes clear when the binding site is examined in detail (17,21). NR2A has an aspartate acting as a crucial negatively charged residue that participates in the binding to the positively charged amino group of the agonist. In contrast, the equivalent residue in the AMPAR subunit, GluR2, is a glutamate; this larger residue would cause a steric clash with the N-methyl group of NMDA. Another residue in the NR2 subunit, a tyrosine amino acid (730 in NR2A), can form a van der Waals contact with glutamate; this interaction may contribute to the high-affinity binding of glutamate to NMDA receptors (i.e., compared to AMPA receptors). Finally, a tyrosine residue in NR1 (amino acid 535) may account for the slow deactivation of NMDARs. When the agonists glycine and glutamate bind to the ligand-binding domains of NR1 and NR2A, respectively, the agonist-induced closure of each ligand-binding domain shifts the position of this tyrosine. Consequently, the aromatic side chain of this tyrosine may bind into a primarily hydrophobic pocket on the NR2A; this pocket is exposed during the shift in position, and the pocket’s interaction with the NR1 tyrosine stabilizes the activated, glutamate-bound confirmation. 2.3. Diversity NMDAR functional characteristics vary according to subunit composition. For example, for complexes that include the most common NR1 variant, NR1-1a, NR1/NR2A has the shortest deactivation time constant (50 ms); NR1/NR2B and NR1/NR2C have intermediate deactivation time constants of around 300 ms; and NR1/NR2D has the longest deactivation time constant (1.7 sec (5)). In addition, NR1/NR2A and NR1/NR2B channels show relatively high single channel conductance while NR1/NR2C and NR1/NR2D channels show relatively low conductance and low magnesium sensitivity, although all four show high calcium permeability (5,22). Furthermore, the rate of magnesium unblock of the channel of NR1/NR2A and NR1/NR2B receptors has a slow component that is absent from NR1/NR2C and NR1/NR2D receptors; this slow component is slower for NR1/NR2B than for NR1/NR2A (23). NR1/NR2A/NR3A channels resemble NR1/NR2C and NR1/NR2D channels in their low conductance and low magnesium sensitivity but differ from all diheteromeric NR1/NR2 receptors due to much lower calcium permeability (22,24). Variation in NR1 splice variants also confers differences in NMDAR responses (5). Thus, the deactivation rate of NR1-1b/NR2B (containing exon

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5; i.e., the N1 cassette, indicated by the “b”) is much faster than that of NR11a/NR2B (lacking exon 5; indicated by the “a”) (5,25). NR1 subunits lacking exon 5 are inhibited by protons and zinc and are potentiated by polyamines. These actions do not occur when exon 5 is included in the NR1 molecule; this addition may form a surface loop that acts as a tethered modulator to shield the proton sensor and zinc-binding site. However, for NR1 subunits that lack exon 5, this sensitivity is much more evident for NR1/NR2A and NR1/NR2B than it is for NR1/NR2C and NR1/NR2D (5,26). Sensitivity to zinc also differs between NR1/NR2A and NR1/NR2B receptors, so that NR1/NR2A receptors are highly sensitive and subject to tonic (nanomolar) inhibition by zinc. In contrast, NR1/NR2B receptors are susceptible only to phasic (micromolar) inhibition by zinc, such as might occur due to co-release of zinc and glutamate at a synapse (27). It is interesting that triheteromeric NR1/NR2A/NR2B may be inhibited by both tonic and phasic levels of zinc (28). 2.4. Posttranslational Modification Following translation, NMDA receptors can be modified in a number of ways including phosphorylation, glycosylation, nitrosylation, ubiquitinization, and calpain cleavage. Phosphorylation of serine or tyrosine residues in the intracellular C-terminal portion of NMDAR subunits is a major mechanism for modulation of the function of NMDA receptors and is discussed in other sections, especially Section 6. The extracellular N-terminal portion of many proteins including NMDAR subunits is glycosylated after translation and during their passage through the ER and Golgi. NR1 has 12 consensus Nlinked glycosylation sites, and this extensive glycosylation may be necessary for proper oligomerization with NR2 subunits (29). Glycosylation of NR1 is atypical because it remains in a high-mannose form established in the endoplasmic reticulum (ER), and thus is still endoglycosidase H-sensitive after exiting the ER; NR2A also is highly sensitive to the enzyme but is not completely sensitive (reported for cerebellar granule cell cultures and cerebellum (30)). In contrast, most proteins undergo further maturation in the Golgi, and thus reach the plasma membrane in a form that is insensitive to this enzyme (e.g., in the latter study, labeling of membrane homogenates with antibodies to AMPA receptor subunits GluR2/3 and GluR4 revealed only a slight sensitivity to the enzyme). S-nitrosylation of cysteines of NR2A via nitric oxide (NO) can downregulate ion channel activity (31). NO can be produced in some neurons after NMDAR activation, and this may be facilitated by coupling of both the NMDAR and neuronal nitric oxide synthase (nNOS) to the synapse-localized membrane-associated guanylate kinase (MAGUK) postsynaptic density (PSD)-95 (see Section 4.1). The calcium-dependent protease calpain can cleave NR2 subunits in their C-terminal and may modulate NMDAR function and turnover. Susceptibility to calpain cleavage varies with

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neuronal maturity, and this process may be blocked by the association of NMDARs with the MAGUK PSD-95 at the synapse (32,33). Both calpain cleavage and ubiquitination are mechanisms for degrading and removing NMDARs from the surface and are discussed in more detail in Section 3.2.5 (internalization).

3. Function 3.1. In Vitro Studies of NMDAR function, as for that of other receptors, have been done mainly using biochemistry and whole-cell in vitro systems (i.e., outside of the living organisms, or “ex vivo,” and literally in a “glass” receptacle). Typically, the latter will include functional studies in nonneuronal heterologous cell lines such as HEK or COS, in oocytes, and in neurons. Recombinant gene constructs can be transfected into heterologous cells, which lack native NMDARs in most cases, to isolate NMDAR properties that could be masked if studies are done in neurons, which contain both native NMDARs and many other proteins that are connected intimately to NMDAR function. Then, NMDARs can be studied in neurons to elucidate NMDAR function as it is modified and modulated by native neuronal mechanisms. Oocytes from the frog Xenopus laevis can be injected with NMDAR construct RNAs and work well for electrophysiologic studies. However, their use for NMDAR function has been somewhat problematic because injection of NR1 alone can produce functional ion channels, even though in all other systems, NMDARs seem to require both NR1 and NR2s to form functional surface channels. This exception for oocytes originally was attributed to the formation of heteromeric complexes between the injected NR1 and an endogenous kainate-binding protein, XenU1. However, recent studies indicate that neither mammalian NR1 nor Xenopusspecific NR1 subunits bind to XenU1, suggesting that the introduced NR1 binds to a native NR2 subunit (one subunit, Xenopus-specific NR2B, was identified from brain cDNA (34,35)). The nature of these NMDARs in the oocytes is further complicated by their pharmacology; in addition to glutamate/glycineinduced currents, there also is a tiny current induced by glycine alone (35). Studies of recombinant NMDARs in heterologous cell cultures have been especially helpful in elucidating basic properties of native NMDARs such as ion permeation, blocking, and gating properties including deactivation and desensitization, and are discussed in other sections of this review (Sections 1.2, 1.3, and others). Desensitization of NMDARs (i.e., a reduction of macroscopic NMDA currents in the continuous presence of glutamate (36)) is particularly complicated and is found in at least three forms (37) based on in vitro studies of both native receptors in cultured neurons and recombinant receptors in heterologous cells. However, the results are not all consistent

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among studies, and sometimes it can be problematic to use recombinant methods in heterologous cells to help interpret the mechanisms of desensitization of native receptors (38). The first type, glycine-independent desensitization, is studied in the presence of saturating glycine concentrations and is present in NR1-NR2A and NR1-NR2B receptors but not those with NR2C and NR2D; it is mediated by two N-terminal regions of NR2 that are close to the ligand-binding site (37). Participation of at least one site, the pre-M1 region, was clarified recently by expressing cysteine mutants of NR2A with NR1 in HEK293 cells and using the “substituted-cysteine accessibility method” to determine accessibility of the substituted cysteines (39). This form of desensitization also is regulated by protein kinase C (PKC) phosphorylation of the NMDAR subunits (examined in HEK293 cells (40)). The second type of desensitization is glycine dependent, and thus it is observed in low glycine concentrations (37,38). Basically, the binding of the agonist to the NR2-glutamate site (such as seen for NMDA in studies with cultured neurons) causes the affinity at the NR1-glycine site to decrease, so that current decreases when the glycine concentration is low. Thus, in HEK293 cells, desensitization of recombinant NMDARs with NR1a/NR2A shows a greater sensitivity to glycine concentration than in those with NR1a/NR2B because cells expressing NR2A are known to have a much lower affinity for glycine than those expressing NR2B (38). The third type of desensitization is Ca2+ -dependent inactivation; it may depend on calcium entry into the cell from the NMDAR or from other calcium-permeable ion channels and has been demonstrated definitively for NMDARs with NR2A or NR2D but not NR2C (37). For NR2A, studies in HEK293 cells showed that inactivation involves both the M2-3 loop (i.e., the intracellular loop immediately distal to the pore-forming P-loop M2) and part of the C-terminal distal to M4 (41). Lack of inactivation of NR2Ccontaining receptors may involve amino acid differences in the M2-3 loop; for example, residue 619 is an isoleucine in NR2C but is a valine in the other NR2 subunits, and switching this amino acid in NR2A reduces inactivation without affecting calcium permeability. Ca2+ -dependent inactivation of NMDARs also directly involves the NR1 C-terminus, and this could involve modulation by the NR2 M2-3 loop (41) and/or Ca2+ -dependent binding of calmodulin (37). Presence of Ca2+ -dependent inactivation for NR1/NR2B has been controversial (37), and more recent work using HEK293 cells shows an involvement of CaMKII in this phenomenon; it either promotes Ca2+ dependent inactivation of NMDARs containing NR2B or induces a new type of mechanism for desensitization of NR2B-containing NMDARs (42). It is interesting that whereas CaMKII enhances the extent of desensitization of NR2Bcontaining NMDARs, it decreases the extent of desensitization of NR1/NR2A receptors.

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In vitro studies on neurons can involve either transfection/infection of recombinant gene constructs or examination of native NMDARs that are affected by pharmacologic agents or other manipulation of neuron cell function. The neurons used in these studies are either from dissociated cell cultures or in slices; slices are either acute slices or long-term cultured slices. Each method (biochemistry vs. whole cell, heterologous cells vs. neurons, recombinant genes vs. native NMDARs, dissociated culture vs. slice culture) has advantages and disadvantages, and any results obtained from these are open to criticisms that the paradigm used is not equivalent to a study of the normal in vivo system. Most of the experimental neuronal studies covered in this review involve various combinations of these in vitro methods and are discussed in other sections. 3.2. In Vivo As noted in Section 2.1, the goal of in vitro studies is to help elucidate in vivo function of NMDARs, that is, in the living animal. True in vivo studies of function can be problematic and thus are relatively uncommon. Thus, in most cases, the best way to come closest to understanding true in vivo function is to examine native receptors in neurons in vitro, using dissociated or slice cultures. Examination of the function of native NMDARs has revealed changes in NMDAR function during development as well as among different cell types, and has considered mainly gating, magnesium sensitivity, and single-channel conductance (37); variations in these properties in different kinds of NMDARs (described elsewhere in this chapter) can provide clues to the kinds and combinations of NMDARs found in vivo. For example, during development of cerebellar mossy fiber-granule cell synapses, studied in slices from P7-P40 rats, there appears to be a switch from NR2B- to NR2A-containing NMDARs in the second postnatal week, based in part on observations of a speeding up of NMDAR excitatory postsynaptic currents (EPSCs) as well as changes in sensitivity to various pharmacologic agents (43). This is followed by a slowing of decay kinetics by P40, indicating that there is an increase in NR1/NR2C receptors during maturation. It is curious that, prior to this slowing, by the end of the third week, there is a reduction in magnesium sensitivity, suggesting that the major receptors at this time contain both NR2A (for rapid decay kinetics) and NR2C (for reduced magnesium sensitivity). More recent work using dissociated granule cell cultures from wild-type (WT) and NR2A knockout (KO) mice have helped to clarify the early switch from NR2B- to NR2A-containing native NMDARs (44). Another work used autaptic synapses of cerebellar granule neurons (i.e., solitary neurons cultured in microislands) from WT, NR2A KO, and NR2C KO mice (45). Although this synaptic arrangement is abnormal for granule cells, developmental changes in NMDARs seen in this

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experimental system may be more similar in some ways to in vivo development compared to typical dissociated culture systems. The special role of the NR2C subunit in certain glutamatergic synapses also has been shown in tangential neocortical slices from P20-24 mice (46). These synapses interconnect spine stellate (SpS) neurons of the barrel cortex and have a very large NMDAR-mediated component with low magnesium sensitivity, probably due to NR1/NR2C NMDARs. However, there also is evidence that these neurons contain some separate synapses with NR2A/B-containing NMDARs. In fact, several examples have been reported of different NMDAR types in separate populations of synapses on a single neuron (47). For example, recordings from layer 5 pyramidal neurons in the agranular frontal cortex using in vitro coronal slices reveal that these neurons have mostly NR2B-containing (but not NR2Acontaining) NMDARs at intracortical inputs, and mainly NR2A-containing (but not NR2B-containing) ones at callosal inputs (48). NR2C/D-containing NMDARs are found in both kinds of synaptic inputs but may play a more prominent role at the intracortical ones. Thus, in vitro studies on neurons reveal a great variety of functional relationships among different kinds of NMDARs. The reasons for the presence of various kinds of NMDARs with different physiological properties are summarized by Monyer et al. (37); for example, NMDARs with low sensitivity to magnesium ions could help to generate excitatory postsynaptic potentials (EPSPs) at rest, and differences in deactivation time course and magnesium sensitivity may help to control time courses and thresholds for synaptic plasticity. In contrast to the in vitro studies described previously, in vivo studies (i.e., in living animals) of NMDAR function should approximate normal NMDAR function more closely and may give us a more accurate look at NMDAR function under true physiologic conditions. In some in vivo studies, manipulations are performed in the living animals, and then function is determined in vitro. For example, anesthetized rats (postnatal day 21–28) were infected with constructs of constitutively active forms of CaMKIV or CREB injected into the CA1 region of the hippocampus (49). Subsequent in vitro studies on CA1 pyramidal neurons in acute hippocampal slices showed that these compounds enhanced NMDAR-mediated synaptic responses and long-term potentiation (LTP). Furthermore, electrophysiologic and morphologic studies indicated that there was a generation of “silent synapses” (i.e., with NMDARs but few or no AMPARs). These may be new synapses that are amenable to further experience-dependent plasticity, important for maintenance and consolidation of memories. In other studies, neurophysiology is carried out directly in the brain of the live, anesthetized rat; only the subsequent morphologic analyses are done postmortem. Thus, Fan et al. (50) used this methodology to demonstrate that stimulation of inputs to some CA1 pyramidal neurons (70%) can induce late depolarizing postsynaptic potentials that are dependent on NMDARs and

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GABAA receptors. Other in vivo functional studies of NMDARs employ direct visualization of neurons in the living animal. Sullivan et al. (51) performed two-photon calcium imaging in the cerebellar cortex of rats (P21-28) and found prolonged calcium transients in interneurons that probably result from synaptic activation of NMDARs via glutamate spillover due to enhanced stimulation of the presynaptic parallel fibers (confirming previous in vitro studies). Confocal microscope analyses on the brains of living animals also include studies of NMDAR function in neurite outgrowth and synapse formation during development. With this methodology, retinal axon development in the brain was studied in frog tadpoles (52); NMDARs are involved directly in axon branch tip stabilization and elimination. A similar function for NMDARs was seen in the postnatal rat, using an in vivo lesioning and tracer injection protocol followed by in vitro analysis (53); NMDARs mediate suppression of synapse formation on sprouting axons. Results from both studies indicate that NMDARs eliminate weak connections at these early stages of circuitry formation.

4. Expression, Trafficking, and Targeting 4.1. Expression and Distribution NR2A and NR2B are the most common NR2 subunits in the adult forebrain. NR2B is the most common also in the early postnatal forebrain and is replaced to some extent by NR2A during development. NR2C is abundant in adult cerebellar granule cells. NR2D is most common in early postnatal development in the diencephalon and brainstem. Receptor subunit abundance also may vary with neuron type within a region; cortical parvalbuminpositive GABAergic interneurons at 21 days in vitro have a five-times-higher NR2A/NR2B ratio than pyramidal neurons (54). Within neurons, NMDAR complexes are found at the synapse and in extrasynaptic locations, with the latter made up mainly of NR1/NR2B and NR1/NR2D types; in fact, NR1/NR2D may never enter the synapse (55). In addition to diheteromeric NMDARs made up of two NR1 subunits and two NR2 subunits of one kind, triheteromeric NMDARs occur (5). Evidence for NMDARs containing two kinds of NR2 subunits has been shown for transfected cells and for a number of native receptors, including NR1/NR2A/NR2B receptors in hippocampal neuron synapses, NR1/NR2A/NR2C in cerebellar granule cell synapses, and NR1/NR2B/NR2D in substantia nigra-dopaminergic neurons (5,56). Much of the information that we have on the functional importance of these subunits comes from KO and other transgenic studies. These are described in Section 7. NMDARs that include NR3 subunits are not well known. NR3A is most common in the early postnatal brain and is less common in the adult, where it has been localized to the postsynaptic membrane of presumptive excitatory

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synapses (57,58). In contrast, NR3B remains high in adult brains and is most common in motor neurons of the brainstem and spinal cord (11,59,60). When NR3 subunits combine with NR2 subunits to form triheteromeric receptors (NR1/NR2/NR3; i.e., containing two NR1 subunits), activity of the channel is diminished (see prior discussion). Pérez-Otaño et al. (24) showed that NR3A reduces the single channel conductance and calcium permeability of NMDARs. There also is evidence that diheteromeric NMDARs containing only NR1 and NR3 subunits can occur, but this is controversial (11,59). NR3 subunits are reported to form functional excitatory glycine receptors following assembly with NR1 (5,11). In fact, NR3A has a high-affinity glycine-binding domain with a dissociation constant 650 times less than that for NR1 (18), and agonist binding to the glycine site of NR3 alone can activate a significant component of NR1/NR3 currents (61). As noted earlier, different kinds of NMDARs show various distributions in the nervous system and during development. During early postnatal development, NR2B-, NR2D-, and NR3A-containing NMDARs are abundant and decrease overall during maturation, whereas NR2A- and NR2C-containing NMDARs become abundant overall with maturation. Exceptions do occur; for example, some motoneurons express abundant NR2A early in development (62). The shift during development from a prevalence of NR2Bcontaining NMDARs to NR2A-containing NMDARs has been studied most extensively, and this occurs especially throughout the forebrain. For example, electron microscope immunogold analyses of the postnatal CA1 region of the hippocampus demonstrate that synaptic labeling for NR2B is highest at postnatal day 2 (P2) and shows a gradual decrease to about half as much by P35 (Fig. 1 (63)). In contrast, NR2A-immunogold labeling at the synapse is very low at P2 and increases by about 12 times at P35. These findings correlate also with western blot analyses (64). Although NMDARs are already prevalent at synapses at P2, AMPARs are not common at this early age; like NR2A, AMPARs increase in numbers at synapses during maturation (65). Many of the synapses in this case probably are “silent synapses” (66). Activation of the NMDARs at these synapses can lead to plasticity resulting in the upregulation of AMPARs, but the NMDARs cannot fire because the NMDAR channels are blocked by magnesium. But what can elicit a depolarization that can relieve the magnesium block of the NR1/NR2B receptors at these synapses of the early hippocampus and other parts of the forebrain? This is not clear. It could be due to activation of excitatory -aminobutyric acid (GABA) receptors that can occur in the early postnatal brain (67), although the latter action also has been implicated in an early form of long-term depression (LTD) (68). Alternatively, it could be caused by the presence of NMDARs with low sensitivity to magnesium, such as with NR2C-, NR2D-, or NR3Acontaining receptors (see Sections 1.3 and 2.2 (22)).

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Fig. 1. Immunogold labeling for NR2A and NR2B in synapses during postnatal development of hippocampus CA1 stratum radiatum. Micrographs illustrate the decrease in NR2B and increase in NR2A at synapses during development. For NR2B, there was a significant decrease from postnatal day 2 (P2) to P35 and from P10 to P35; 30%, 33%, and 23% of synapses were labeled for P2, P10, and P35, respectively. For NR2A, there was a significant increase from P2 to P10 and from P10 to P35; 3%, 11%, and 29% of synapses were labeled for P2, P10, and P35, respectively. The Y axis indicates gold per synapse or gold per synapse + 100 nm (measured from the postsynaptic membrane to 100 nm deep). Scale bars are 100 nm, arrows in micrographs indicate gold labeling associated with the postsynaptic density, and histograms show values plus standard errors. From Petralia RS, Sans N, Wang YX, et al. Ontogeny of postsynaptic density proteins at glutamatergic synapses. Mol Cell Neurosci 2005;29:436–452; legend slightly modified.

4.2. Trafficking and Targeting 4.2.1. Processing of NMDAR in the Endoplasmic Reticulum Most membrane proteins that are made in the ER undergo some kind of quality control that inhibits the export of misfolded or otherwise imperfect protein molecules. Multimeric proteins such as ion channels are assembled in

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the ER, and mechanisms must be employed to prevent the export of monomers and incompletely assembled complexes from the ER. Thus, for NMDARs, subunits of NR1, NR2, and NR3 are retained in the ER until they can assemble with each other to form the tetramers that make up the NMDAR ion channel complex. NR1 uses an RXR motif in the intracellular C-terminal region as an ER retention/retrieval factor; NR3B may use a similar motif for ER retention, and RXR motifs also are found in other NMDAR subunits, but their function in ER retention is not known (59). In fact, it is not clear whether NR2B has a functional ER retention factor (see later discussion (69)). For NR1 subunits, four of the eight variants contain a C1 cassette, and this cassette includes the RXR motif (RRR (6,59,70–72)). When subunits join together to form a complex, the ER retention must be negated somehow, either by steric masking of the ER retention site or by an export signal that somehow overrides the ER retention function. Thus, when expressed in heterologous cells, the NR11 variant, which contains the C1C2 C-terminus, is retained in the ER (70). In contrast, expression of any of the other three variants, NR1-2, NR1-3, or NR1-4, results in trafficking of these transfected proteins to the cell surface. Because both NR1-2 and NR1-4 lack the C1 cassette (and thus they lack the RXR motif), their trafficking to the cell surface is expected. In contrast, NR13 (C1C2’) carries the ER retention motif yet still goes to the surface. This suggests that the C2’ carries a signal that can mask or override the ER retention mechanism. Indeed, the last six amino acids of C’, which includes the PDZbinding domain STVV, are sufficient to suppress ER retention. In addition, soluble fusion proteins that contain the PDZ-binding domain of C2’ block the surface expression of NR1-4 (presumably due to saturation of the PDZ proteins by the mutant molecule), suggesting that some kind of PDZ-containing protein normally binds to the C2’-containing NR1 subunits (NR1-3 and NR1-4) very early in the secretory pathway, perhaps at the ER exit sites (73). Nevertheless, other factors may be involved in the release of NR1 from their ER retention. PKC phosphorylation of serines in the C1 cassette, near the RXR ER retention motif, can relieve this ER retention and elicit robust surface expression of NR1 (71). This relief from ER retention also may require phosphorylation of an adjacent serine by PKA and some kind of coordination between the actions of PKA and PKC (74). In addition, the relief from ER retention that is elicited by the C2’ cassette may not be due to the binding of a PDZ protein to the STVV C-terminus. Rather, the valine residues in this terminus may bind COPII proteins, which are found at ER exit sites, and it may be that this is a common mechanism for ER exit of any integral membrane proteins with type I PDZ-binding motifs (T(S)XV (75)). The functional significance of the absence of ER retention of some of the NR1 subunits remains unknown. Any mechanisms for exit of NR1 subunits from the ER in normal neurons must consider the association of the NR1 with NR2 and/or NR3 subunits

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because only these combinations are known to exit the ER and traffic to the plasma membrane of normal neurons. In mice having an NR1 deletion (in this case, restricted to the hippocampus CA1 region), there is an aggregation of NR2 subunits in intracisternal granules of the ER (76), supporting other studies that show that NR2 subunits are retained in the ER in the absence of NR1 (77). In expression studies in heterologous cells, NR3A can bind to either NR1 or NR2A in the ER, but only heteromeric complexes that contain NR1 can reach the cell surface; homomeric NR3A complexes and NR2A/NR3A complexes are retained in the ER (the authors note that they used the NR1-1a variant, which is retained in the ER when transfected singly in heterologous cells) (24). NR3B seems to function similarly (59). Thus, even though all of the subunits, when alone, may manifest effective ER retention mechanisms, these mechanisms somehow are suppressed or overridden when they combine in the proper heteromeric complexes. Presumably, this is related to sustained conformational changes that occur with quaternary folding of the complex; retention signals may be masked and/or exit signals may be enhanced. For NR1/NR2 complexes, a potential exit signal is the HLFY motif, which is found in the proximal C-terminal region, immediately following the last transmembrane domain of the NR2 subunit. This motif is required for exit of the assembled NMDAR from the ER (69). When this motif is mutated, NR1 and NR2 subunits still assemble into functional complexes in the ER, but they cannot exit. 4.2.2. Golgi/Trans-Golgi Network to Synapse After an NMDAR-containing complex exits the ER, it is modified in the Golgi apparatus and then sorted in the trans-Golgi network (TGN), where it is packaged into various kinds of vesicular or tubulovesicular carriers. From there it either is transported directly to the plasma membrane or fuses with endosomes; transport is via kinesin motor proteins on polarized microtubules. Nascent NMDARs that reach endosomes may mix with NMDARs that have recycled from the surface; in any case, the endosomes are a site, like the TGN, where proteins can be sorted further. Eventually, NMDARs reach the surface of the neurons, with the final stage probably involving myosin motors traveling on actin filaments. Nascent NMDARs may first enter the extrasynaptic membrane and then proceed to the synapse, but it also is possible that they enter the postsynaptic membrane directly. For NMDARs and neuronal proteins in general, many of these steps are not clear. Studies in young cortical neuron cultures have elucidated some of the intermediate steps in this trafficking (78,79). Both NMDARs and AMPARs travel in mobile transport packets in neurons before and during synaptogenesis. Most mobile NMDAR clusters do not contain AMPARs, and they move rapidly relative to those that contain AMPARs but lack any NMDARs. This suggests that there are at least two kinds of carrier vesicles or tubulovesicular organelles and that one of these may be specialized

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for rapid trafficking of NMDARs. As NMDARs travel along the dendrites of these young neurons, prior to synapse formation, they go through cycles of exocytosis to the dendrite surface and endocytosis from it. Exocytosis is dependent on the SNARE protein SNAP-23, and transport in the dendrites is in large tubulovesicular organelles that move along microtubules. These organelles contain early endosomal antigen 1 (EEA1), indicating that they are early endosomes. They also contain the MAGUK SAP102. The distinctive sequential cycling of nascent NMDARs to and from the surface while traveling seems to be specific for a presynapse stage; this sequential cycling has not been demonstrated for the trafficking of nascent NMDARs following synapse formation or in adults. Preliminary immunogold studies in the developing and adult hippocampus support some of these findings (80,81). These latter studies suggest that there may be some selective cargo sorting between NMDARs and AMPARs at the TGN and perhaps at endosomes also. In fact, some recycling endosomes may contain AMPARs, along with their associated PDZ proteins, GRIP and PICK, but no NMDARs (82). However, there is little evidence that NR2A-containing NMDARs are sorted differently from NR2B-containing NMDARs at the TGN. Comparisons of binding of NR2A and NR2B C-termini with adaptor medium subunits (these adaptors are made from one medium, one small, and two large subunits) that may be involved in this transport show only minor differences between the two NR2 subunits (83). These adaptor medium subunits include μ1, μ3, and μ4, found in AP-1, AP-3, and AP-4 adaptor proteins, respectively; μ1 and μ3 are involved in clathrin-mediated cargo selection and transport from the TGN and endosomes; μ4 is involved in similar functions at the TGN, but clathrin is not involved (84). NR2A and NR2B C-termini bind strongly and equally to μ1 and μ4, whereas they bind relatively weakly to μ3; the latter binding is more prevalent for NR2B than for NR2A. As noted in Section 4.4, SAP102 is the common MAGUK associated with the major NMDAR of the early postnatal forebrain—NR1/NR2B complexes. Indeed, trafficking of NMDARs and their delivery to the cell surface and then to the synapse employs a large complex of proteins (85,86). Thus, the NMDAR, carried on the membrane of a transport vesicle or tubulovesicular carrier, is bound to SAP102 (or other MAGUKs), which in turn binds the exocyst component Sec8 and mPins (mammalian homologue of Drosophila melanogaster partner of inscuteable), and the latter binds to the G-protein subunit Gi. The binding to Gi may mediate G-protein signaling by inhibiting binding of Gi to the other G-protein subunit, G, thus enhancing G signaling. This complex of proteins may form in the first stages of the secretory pathway, in the ER, Golgi, or TGN. The exocyst or Sec6/8 complex consists of eight proteins. The exocyst has been studied in both yeast and in mammalian cells and is believed to direct intracellular membrane vesicles to their sites

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Fig. 2. A. The exocyst complex and the delivery of N-methyl-d-aspartate receptors (NMDARs) to the cell surface. Under normal conditions, Sec8 and the NMDAR associate with a PDZ protein. If the Sec8 interaction is blocked by the introduction of a dominant-negative form of Sec8 (left), delivery of NMDAR is blocked. However, if the NMDAR interaction with the PDZ protein is blocked by deletion of the PDZ-binding domain on the NMDAR (right), the NMDAR can be delivered to the cell surface by an exocyst-independent process, although synaptic delivery is compromised. B. Proposed model. Summary of the role of SAP102, Sec8/exocyst, and mPins/Gi complexes in the delivery of NMDARs to the cell surface. In the absence of the complete complex, the proteins may not reach the plasma membrane and instead become sequestered into cytoplasmic inclusion bodies. ER, endoplasmic reticulum.

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of fusion with the plasma membrane. Both Sec8 and the NR2 subunits of NMDARs bind to the same region of SAP102, mainly PDZ domains 1 and 2 (Fig. 2A). If the Sec8 interaction with SAP102 is blocked by the introduction of a dominant-negative form of Sec8 that lacks a PDZ-binding domain, delivery of NMDARs to the cell surface is blocked. Thus, surface delivery of NMDARs requires both SAP102 and Sec8. However, if the PDZ-binding domain of the NR2B subunit of the NMDAR is deleted, the NMDAR can be delivered to the cell surface by an exocyst (and MAGUK)-independent process, although these mutated NR2B-containing NMDARs cannot enter the synapse. This phenomenon supports the idea that extrasynaptic NMDARs are sorted independent of synaptic NMDARs, which may require a MAGUK association for entry into the synapse (this is not true for the NR2A subunit, as discussed in Sections 3.2.5 and 4.1). mPins is the mammalian form of Pins, which is a protein in Drosophila that regulates cell polarity and asymmetric cell division. In neurons, mPins interacts with the SH3/GK domains of SAP102, and this interaction influences the trafficking of NMDARs. In hippocampal neurons in culture, expression of dominant-negative constructs of mPins decreases native SAP102 in dendrites, and both these constructs and short-interfering RNA (siRNA)-mediated knockdown of mPins reduce the density of surface puncta and the intensity of staining per labeled surface punctum for transfected NR2B constructs. This suggests that the mPins/SAP102 complex promotes the efficient targeting of NMDARs to the cell surface (Fig. 2B). Overall, these studies suggest that SAP102 and mPins bind together in a closed or inactive state; subsequently, binding of NMDARs to this complex may open it up. In addition, interaction of Gi with mPins in this complex may help to stabilize the complex, facilitating its proper folding and targeting to the cell surface. During trafficking, the complex described so far, NR1/NR2B/SAP102/Sec 8/mPins, probably must associate with kinesin motors to travel along microtubules in dendrites (87). Indeed, NR2B appears to bind to a complex of other proteins, mLin-7/mLin-2/mLin-10, that link the NMDAR to a kinesin, KIF17; this complex can move the NMDAR (bound in the membrane of a carrier vesicle or tubulovesicular organelle) along the dendrite (88,89). NR2B expression and synaptic localization are impaired if KIF17 is knocked down  Fig. 2. (Continued) A: From Sans N, Prybylowski K, Petralia RS, et al. NMDA receptor trafficking through an interaction between PDZ proteins and the exocyst complex. Nat Cell Biol 2003;5:520–530. B: From Sans N, Wang PY, Du Q, et al. mPins modulates PSD-95 and SAP102 trafficking and influences NMDA receptor surface expression. Nat Cell Biol 2005;7:1179–1190. These diagrams were made by Dr. Nathalie Sans and were published originally in color.

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or blocked. It is interesting that this decrease in synaptic NR2B is followed by a corresponding increase in NR2A at synapses. This suggests that the replacement of NR2B- with NR2A-containing receptors is under some kind of reciprocal control, such that NR2A-containing receptors may replace lost NR2B-containing ones at the synapse, perhaps to maintain a certain density of NMDARs at the synapse. These experiments also indicate that the mechanism of transport for NR2A is different from that for NR2B. Another kinesin, KIF1B, associates with several MAGUKs including PSD-95 and SAP97 and the related protein S-SCAM, but its role in NMDAR transport, if any, is not known (90). This latter kinesin association may be a better candidate for motor movement of the NR1/NR2B/SAP102/Sec8/mPins complex because the former complex, NR1/NR2B/mLin-7/mLin-2/mLin-10/KIF17, already includes a nonMAGUK PDZ-protein link to NR2B. Once NMDARs reach the end of the microtubule tracks, they probably switch to myosin motors on actin filaments for the final transport to the cell surface (e.g., ref. 91) and for any transport within a postsynaptic spine (92). NMDARs are regulated by myosin light chain kinase (93) and have a direct interaction with myosin regulatory light chain (94). Finally, in considering the trafficking of nascent NMDARs to synapses, it is generally assumed that passage of the receptors from ER export sites, through the Golgi/TGN, and into carriers occurs in the cell body; then the NMDARs with their numerous associated proteins are transported along the dendrites and to the synapses. In addition, there is some evidence that NMDARs could be released from ER export sites within dendrites (95,96) or even synthesized locally near synapses from mRNAs that have been transported from the cell body (97). Perforant path transection induces the trafficking of NR1 mRNA into the dendrites of dentate gyrus granule cells, probably in response to increased terminal proliferation and sprouting (98). There is even more evidence that some of the cytoplasmic proteins associated with NMDAR function, such as CaMKII or Arc, can be synthesized locally to exert more precise control at individual synapses (97). 4.2.3. Exocytosis and Lateral Movement The site for exocytosis of NMDARs and other glutamate receptors at synapses is not clear (99). For AMPARs, labeled pitlike structures, without evident clathrin coats, are seen often on the sides of spines in the adult (80,100,101) and resemble the noncoated pits seen in structural studies in these locations (102). It is not clear whether these represent exocytotic sites or noncoated endocytotic sites, but preliminary data indicate that the SNARE SNAP-23 is concentrated in this area, suggesting that these are sites of exocytosis or sites of lipid raft/caveoli (see Section 3.2.5) involved in some kind of receptor regulation (see discussions of glutamate receptors and lipid rafts in refs. 103 and 104, as well as discussion of GLUT4 associations with lipid

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rafts, SNAP23, TC10, and the exocyst in ref. 105). Although this spine side-site of possible exocytosis may be specific for AMPARs (see also ref. 106), preliminary studies indicate that immunogold-labeled vesicles and pits are also in the perisynaptic region at the side of the PSD and possibly within the PSD itself (80,81); these are labeled with antibodies to either AMPARs or NMDARs. Although there is some evidence that newly exocytosed NMDARs may form extrasynaptic clusters first (107), other studies suggest that NMDARs could be incorporated more directly into synapses, presumably via actin/myosinmediated transport (89); see Section 3.2.2 for discussions on actin and myosin. NMDARs do not need to enter synapses directly because they can be very mobile in the surface membrane (108). Thus, whereas changes in neuronal activity modify AMPAR mobility but not NMDAR mobility, activation of PKC modifies mobility for both (109,110). 4.2.4. Extrasynaptic NMDARs The typical mature synapse has mainly NR2A-containing NMDARs, with NR2B-containing NMDARs remaining relatively prominent in the extrasynaptic membrane (111–113). This model also is consistent with evidence and suggestions that NR2B-containing NMDARs tend to be more readily removed from synapses than NR2A-containing NMDARs, with the latter tending to form a more stable association with PSD-95 at the synapse (113,114). This separation of NR2A in the synapse and NR2B in the extrasynaptic membrane in adults is not absolute; some NR2B still is found in the synapses in adults (63), as either NR1/NR2B or NR1/NR2A/NR2B (see also Section 4.4). Conversely, NR2A-containing NMDARs can be extrasynaptic (115,116). The composition of synaptic NMDARs varies throughout the brain. Thus, cerebellar granule cells lose all of their NR2B eventually in later maturation and have both NR2A and NR2C in the adult; in these cells, even the extrasynaptic NR2Bcontaining NMDARs must eventually be replaced by other kinds (44,115). Nevertheless, in adults, this predominant separation of NMDARs into synaptic NR2A-containing and extrasynaptic NR2B-containing receptors must have some function. Synaptic receptors would be activated by precise release of glutamate at the synapse, whereas extrasynaptic receptors would be activated only after extensive release of glutamate followed by spillover into the extrasynaptic spaces. Thus, extrasynaptic NMDARs may be adapted to elicit plastic changes to compensate for synapse overactivity. NR2A was proposed to be associated mainly with LTP and NR2B mainly with LTD (117,118), although some studies show that both NR2A and NR2B can induce LTP (119,120); results for NR2A are not clear due to problems in specificity of the antagonist used in these studies (see Section 5). In rat olfactory bulb granule cells, activation of extrasynaptic NMDARs generates inhibitory currents via BK-type calcium-activated potassium channels (121). In addition, synaptic and extrasynaptic NMDARs mediate opposite long-term changes in neuron gene

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expression: Synaptic ones promote CREB activation and induction of BDNF gene expression, whereas extrasynaptic ones shut off CREB and inhibit BDNF gene expression (122). 4.2.5. Internalization Cycling of NMDARs to and from the synapse includes both constitutive and regulated pathways; regulation involves a number of factors and depends largely on synapse activity (reviewed in refs. 22 and 123; see also the chapter in this book on plasticity). Regulated internalization of NMDARs can depend on direct effects of agonist binding during activity via mechanisms discussed later (124,125). Activity-based internalization can be controlled selectively, such as for differential regulation of synaptic and extrasynaptic NMDARs (126), or through an indirect route, via activation of metabotropic glutamate receptors (127). Internalization depends largely on the availability of specific internalization motifs on the NMDAR C-termini (reviewed in refs. 6, 22, and 123). NR2B contains a tyrosine-based endocytotic motif, YEKL, in its C-terminus, and this motif can bind to the medium subunit (2) of the AP-2 adaptor protein (83,128,129); this adaptor is involved in clathrin-mediated endocytosis from the cell surface. NR2A has a similar tyrosine-based motif, YKKM, but it is not involved in this endocytosis function. Instead, endocytosis of NR2A seems to involve a dileucine motif in the C-terminus. Both NR2A and NR2B can bind to the scaffolding MAGUK PSD-95 at the end of their C-termini (ESDV), and it is this interaction with PSD-95 that may regulate internalization, that is, the binding of PSD-95 to the C-terminus of either NR2A or NR2B may inhibit clathrin-mediated endocytosis of the NMDARs. However, the importance of this binding to NR2A is not clear because its binding to a PDZ protein is not required for synaptic localization, as discussed again later (114,116,130). Endocytosed NR2A-containing and NR2B-containing NMDARs both initially enter early endosomes, but then they diverge; NR2A tends to traffic to late endosomes for degradation, whereas NR2B prefers to enter recycling endosomes for recycling of the receptor to the cell surface (83,129). It is interesting that activation of the NMDAR/CaMKII pathway regulates casein kinase II (CK2) phosphorylation of the serine of the MAGUK-PDZ binding motif at the end of the C-terminus (131); this phosphorylation disrupts the interaction of NR2B with PSD-95 and SAP102 and consequently decreases surface expression of NR2B in neurons. Thus, this kinase could destabilize the connection of the NMDAR with the scaffold and allow internalization of the NMDAR from the surface; such a control also might operate internally to disrupt the connection of NMDARs with the MAGUK-centered protein complex described in Section 3.2.2 (86), leading to a natural disruption in forward trafficking of NMDARs to the surface (131). Whereas phosphory-

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lation of the serine in the PDZ-binding domain (ESDV) of NR2B (but not that of NR2A) may induce internalization of the NMDAR, phosphorylation of the tyrosine in the AP-2 binding site (YEKL) of NR2B, mediated by Fyn kinase that binds to the MAGUK, can prevent AP-2 binding and thus promote retention of NR2B-containing NMDARs at the synapse (130). Thus, the major function of MAGUK binding may be to keep Fyn kinase in close proximity to the AP-2 binding site of NR2B rather than to be just a mechanical scaffold for anchoring of the receptor at the synapse. The role of Fyn is complex. In the striatum, activation of dopamine D1 receptors induces a Fyn-dependent redistribution of NMDARs (132), and RACK1 binds to Fyn and prevents it from phosphorylating NR2B. In contrast to NR2B, synaptic localization of NR2A does not require interactions with PDZ proteins or AP-2 binding to its YKKM motif (as noted previously (130)), although PSD-95 promotes Fyn-mediated tyrosine phosphorylation of NR2A (but the specific tyrosine residues involved were not identified (133)), and, as noted previously, coexpression with PSD-95 inhibits NR2A-mediated endocytosis (83); its regulation must be very different from that of NR2B-containing receptors, as noted earlier (116,130). Another tyrosine residue-containing, four-residue endocytotic motif, in addition to the one near the distal C-terminus of NR2B, is involved in internalization of NMDARs. This one is found in the proximal C-terminus, near the last transmembrane domain in NR1 and in all NR2 subunits (134). In this motif, the three residues following the tyrosine vary and NR1 has an additional endocytotic motif (VWRK) near the first one (YKRH). Unlike the distal motif of NR2B that may target endocytosed NR2B-containing NMDARs to recycling endosomes and back to the surface, the proximal motifs appear to be involved in targeting NMDARs to degradation via late endosomes (134); also see the prior comment for NR2A. Although the mechanism is not clear, it may involve dephosphorylation of this proximal tyrosine residue (possibly phosphorylated by Src kinase), followed by binding of the AP-2 adaptor and clathrin-mediated endocytosis, as shown for NR1/NR2A NMDARs (124). It is interesting that agonist binding, independent of ion flow, seems to prime this NMDAR internalization (124,125); in fact, the glycine binding alone may be sufficient to prime the receptor for internalization by enhancing the association of AP-2 with the NMDAR, although both glycine and glutamate binding are required for endocytosis (125). In addition, some other proteins associated with clathrin-dependent endocytosis associate with NMDARs. CPG2 (candidate plasticity gene 2 protein) is localized specifically to the postsynaptic endocytotic zone of excitatory synapses (described later); RNAi knockdown of this protein can increase the number of postsynaptic clathrin-coated vesicles, including some that contain NMDARs and increase the number of surface NR1 and AMPAR GluR2 molecules (135). GIPC is associated with endocytosis and contains a PDZ domain that binds

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to the C-terminal ESDV domain of NR2B; preliminary studies indicate that GIPC helps to regulate the surface stabilization, endocytosis, and recycling of NMDARs (136,137). The adaptor PACSIN1/syndapin1binds to the C-terminal of the NR3A, as well as to dynamin and the actin-organizing protein N-WASP, and mediates the selective endocytosis of NR3A-containing NMDARs during postnatal development; this mechanism is activity dependent and may help to regulate synaptic maturation (138). The site of clathrin-mediated endocytosis of NMDARs (and glutamate receptors in general) commonly is found along the extrasynaptic membrane on the side of the spine (100,139,140). This location for endocytosis is typical for mature synaptic spines; the position is more variable in immature synapses and early postnatal ages, and often it is located in the perisynaptic membrane at the border of the young PSD, which typically is formed directly on a dendrite shaft (100). In immunolabeling analyses, it is difficult to find labeling for NMDARs within identified clathrin-coated pits, but a few examples have been found (Fig. 3 (100,135)) including occasional ones near the synapse seen during CPG2 knockdown in vitro (135) (see prior discussion) and in normal neurons (R. S. Petralia et al., unpublished data). Clathrin-mediated endocytosis may not be the only way that NMDARs and other glutamate receptors are internalized. A number of proteins, including some receptors, are internalized via clathrin-independent endocytosis, and some proteins can be endocytosed by either clathrin-dependent or clathrinindependent mechanisms (141,142). For example, epidermal growth factor (EGF) receptors are endocytosed by clathrin-coated pits if the EGFR is exposed to low levels of EGF. In contrast, higher levels of ligand cause EGFR to be ubiquitinated, and these receptors then are endocytosed via a lipid raftassociated, clathrin-independent pathway (both low and high levels of EGF are physiologically relevant (143)). Regulation of a number of PSD proteins, including PSD-95, Shank, guanylate kinase-associated protein (GKAP), and AKAP79/109, can involve ubiquitination. Although the mechanism is not fully understood, this ubiquitination could be involved in activity-dependent changes in NMDARs at synapses, particularly the switch from NR2B-containing to NR2A-containing NMDARs (113,144). Ubiquitination is important for direct regulation of NMDARs, although again the mechanism is not clear. F-box protein 2 (Fbx2) binds to high-mannose glycans of the N-terminal extracellular domain of NR1 following retrotranslocation of the N-terminal to the cytoplasm; Fbx2 induces ubiquitination of NR1 via linkage of ubiquitin-transferring enzymes, resulting in degradation by the proteasome (145). Such a mechanism probably requires some additional, unidentified proteins that can direct the Nterminal extracellular domain of NR1 (and the other, attached subunits of the NMDAR) through the membrane and into the cytoplasm (146); for now, then, this mechanism remains highly speculative. Overexpression of an Fbx2 mutant

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Fig. 3. Double immunogold labeling of clathrin-coated pits/vesicles (CCP/Vs; arrowheads) associated with bare densities (panels A, B; arrows) and extrasynaptic membrane regions (panels C–F) in the P2 hippocampus CA1 stratum radiatum with NR1 (panels A–C, F) or NR2A/B (panels D, E) antibody (5 nm gold) and clathrin (panels A–E) or adaptin  (panel F) (i.e., the  subunit of the AP-2 adaptor protein complex) antibody (10 nm gold). A, B: These two “bare” densities on dendrites actually show fairly close associations with adjacent processes. In both micrographs, a definitive CCP/V is seen in the vicinity of the density, and a second probable CCP/V is evident closer to the density. C, F: In panel C, NR1 and clathrin antibodies label an early, flat CCP/V adjacent to a CCP/V that is pinching off, and NR1 and adaptin  label a better developed CCP/V in panel F (both are dendrites). D, E: NR2A/B and clathrin antibodies label a newly formed CCV in panel E and another CCV in panel D in a process at a point where the latter is contacted by another process. Scale bars, 100 nm. Scale in panel E is valid for micrographs in panels A–E. From Petralia RS, Wang YX, Wenthold RJ. Internalization at glutamatergic synapses during development. Eur J Neurosci 2003;18:3207–3217; legend modified slightly.

accompanied by augmented activity (using a GABAA-receptor antagonist) increases the density of extrasynaptic NMDARs, suggesting that normally a ubiquitin-based degradation mechanism somehow regulates activity-dependent recycling of NMDARs between the cell surface and internal compartments. In addition, prolonged activation of NMDARs leads to downregulation in NMDAR currents that is calpain dependent, involves the degradation of

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NR2A and NR2B, and is independent of dynamin (147). Dynamin is a component of clathrin-dependent and some forms of clathrin-independent endocytosis. These data suggest that during overactivity, calpain can cleave NR2 subunits of NMDARs that are on the cell surface, leading to their destruction and loss from the surface via some form of clathrin-independent mechanism (see also Section 1.4); this may protect neurons from excitotoxicity. It is likely that lipid rafts and associated proteins such as flotillin and perhaps caveolin interact under some conditions with NMDARs and other glutamate receptors (103,104,148,149), and this may include forms of clathrin-independent endocytosis (148). The presence of caveolin in neurons is controversial, but immunogold labeling indicates that caveolin associated with postsynaptic structures in the hippocampus increases during development while the number of clathrin-coated pits decreases; this suggests that caveolin/lipid raft regulation of glutamate receptor trafficking may become more important with maturation (100).

5. Interaction Partners 5.1. Proteins That Interact with NMDARs NMDARs are associated with many proteins (Fig. 4; Tables 1 and 2; see also the chart in ref. 150 and discussion in other sections). These proteins make up a large portion of the PSD (6), and some of them interact directly with NMDARs. One group of proteins that interacts directly with NMDARs and that is among the most abundant components of the PSD contains the MAGUK proteins, including mainly a subgroup of four MAGUKs: PSD-95, PSD-93, SAP97, and SAP102 (151). These four MAGUKs have three PDZ (PSD-95/Dlg/ZO-1) domains, followed by an SH3 (Src-homology-3) domain and a GK (guanylate kinase) domain. All of these MAGUKs can bind directly to NMDARs through their PDZ domains, and they act as large scaffolding molecules that can associate with many other proteins in and around the PSD (6,150,152). In addition, at least two other kinds of large, PDZ domain– containing scaffolding molecules that associate similarly with NMDARs have been reported: S-SCAM (another kind of MAGUK (153)) and CIPP (154). CIPP has a limited distribution; it is abundant only in the thalamus, colliculi, cerebellum, and brainstem, and this distribution resembles that of NR2C + NR2D. The significance of these MAGUKs for NMDAR function has been clarified by a number of studies (6). Knockout of the MAGUK PSD-95 does not seem to affect NMDAR localization at synapses, suggesting either that the primary function of PSD-95 in the PSD is something other than being a physical scaffold for the anchoring of NMDARs (155) or the lack of PSD-95 is compensated by upregulation of SAP102 or another MAGUK. Because these

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Fig. 4. A hypothetical synapse at P2. Note that mature-appearing synapses at P2 contain most major component proteins of the postsynaptic density (PSD). The dashed outlines of -amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) and calcium/calmodulin-dependent protein kinase II (CaMKII) indicate their low density at this age. Transmembrane AMPAR regulatory proteins (TARPs), which may bind AMPARs to membrane-associated guanylate kinases (MAGUKs), are not discussed here. Not evident in this diagram are profound changes in MAGUKs and N-methyl-d-aspartate receptor (NMDAR) types that occur during further postnatal development; also not illustrated are the earlier stages in synaptogenesis when the only major proteins at the contact may be intercellular adhesion proteins (see text for details). BNDF, brain-derived neurotrophic factor; GKAP, guanylate kinase-associated protein; mGluR, metabotropic glutamate receptor; NCAM, neural cell adhesion molecule; SPAR, a Rap-specific, GTPase-activating protein; SynGAP, synaptic Ras-GTPase activating protein; TrkB, tyrosine receptor kinase B. (see also Table 1 and 2) Reprinted from Petralia RS, Sans N, Wang YX, et al. Ontogeny of postsynaptic density proteins at glutamatergic synapses. Mol Cell Neurosci 2005;29:436–452; slightly modified, with permission from Elsevier.

mice show enhanced LTP at different frequencies than those found in normal mice and they have severely impaired spatial learning, it is more likely that the major role of PSD-95 is to bring certain molecules in close proximity to favor particular mechanisms of plasticity. This enhanced LTP in the absence of PSD95 is supported by PSD-95–overexpression studies in which LTP is occluded and LTD is enhanced (156). These effects probably involve changes in AMPARs; an effect of overexpression of PSD-95 on NMDARs is less certain. Thus, PSD-95 overexpression in hippocampal slices does not change NMDAR EPSCs (157), whereas in cerebellar granule cell cultures, PSD-95 overexpression produces faster NMDAR EPSCs and appears to favor the changeover from NR2B to NR2A seen during maturation (158). The difference may be due to a higher expression of PSD-95 in the hippocampus compared to the cerebellum at the ages studied (158). Mice lacking the MAGUK PSD-93 show impairments in some systems but not in others. Thus, in cerebellar Purkinje cells, in which the only definitive PSD-95–group MAGUK is PSD-93, its

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Table 1 Ratio of Immunogold Labeling at Synapse/(Synapse + 100 nm) at Postnatal days 2, 10, and 35 for the N-Methyl-D-Aspartate Receptor-Homer Chain of Proteins Illustrated in Fig. 4 P2 NR2B (0.8), SAP102 (0.7), NR2A (0.6), GKAP (0.5), Shank/Homer (0.4)

P10

P35

NR2B(0.8), NR2A/SAP102/GKAP(0.7), Shank/Homer (0.5)

NR2B/NR2A/SAP102 (0.8), GKAP (0.7), Homer (0.5), Shank (0.4)

High ratios indicate that there was little gold subjacent to the postsynaptic density (PSD) (NR2A, NR2B, and SAP102), whereas low ratios indicate that there was substantial gold subjacent to the PSD (Shank and Homer). Guanylate kinase-associated protein (GKAP) shows a mid-value ratio, consistent with its middle position in the chain. P, Postnatal day.

absence does not affect the development or function of parallel fiber synapses, Purkinje cells, or cerebellum-dependent behaviors, suggesting that some other PDZ protein may substitute for the missing PSD-93 [159; see also 160]. In contrast, mice lacking this MAGUK show reduced surface expression of NR2A and NR2B and impaired NMDAR-mediated postsynaptic function in spinal dorsal horn and forebrain and show blunted NMDAR-dependent persistent pain (161). Whereas NMDARs can bind to MAGUKs, it is not clear whether this direct interaction is typical for synapses, especially for mature synapses. MAGUKs are very likely to have an important function at glutamatergic synapses because they are very abundant at these synapses at all ages (although the type varies with age, as discussed in Section 4.4). A direct interaction can occur between SAP102 and NR2B-containing NMDARs, especially during trafficking, as discussed in Section 3.2.2 (85). At the synapse, binding of NR2B to SAP102 or PSD-95 may be regulated by phosphorylation (131) (see Section 6), and binding is required for synaptic localization of NR2B-containing NMDARs (85,130) (see Sections 3.2.2 and 3.2.5). However, most of the controversy centers on the importance of a direct interaction, in late postnatal/adult synapses (especially in forebrain), between the most common MAGUK, PSD-95, and most common NR2 subunit, NR2A. Some studies suggest that NR2A-containing synapses do not require a PSD-95 interaction for localization of NR2A at the synapse, as discussed in Section 3.2.5 (130). Even if there is no direct binding of NR2A and PSD-95 in the mature synapse, it is likely that PSD-95 forms a major part of a scaffold of interlinked proteins. This scaffold appears to

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Table 2 Proteins That Bind Directly to N-Methyl-D-Aspartate Receptor Subunits NR1 PSD-95, SAP102, PSD-93, SAP97 (to NR1-3, NR1-4) (70) Calmodulin (262) Neurofilament-L (264) Yotiao (266) EphB receptors (185) Salm1 (188) Sec23/24 of COPII (75)

NR2

NR1 and NR2

PSD-95, SAP102, PSD-93, SAP97 (A, B) [257–260]

-Actinin (B) (261)

S-SCAM (=MAGI-2) (A, C) (165) CIPP (A–D) (154) mLin-7 (= Velis = Mals) (B) (267) Phospholipase C- (A, B) (268) Rack1 (B, not A) (245)  subunit of adaptor protein complexes (AP1-4) (A, B) (83) 1-chimerin (A) (174) RasGRF-1 (B, not A) (270) Cyclin-dependent kinase-5 (cdk5)a (A, not B–D) [271,272]

Tubulin (B) (263) Spectrin (A,B) (265) Myosin regulatory light chain (A, B) (94) Dopamine D1 (A) (269) CaMKII (B, not A) (234) NADH dehydrogenase subunit 2 (ND2)a (244)

In addition to the text, see reviews in refs. 6 and 150. The NR2 subunits that are known to bind are indicated in parentheses and in bold. a cdk5 may bind directly to NR2A, and ND2 appears to bind Src to N-methyl-daspartate receptors, but the exact nature of these interactions has not been determined.

hold kinases, GTPase activators and inhibitors, and cytoskeleton-associated proteins in close proximity to the surface receptors and cytoplasmic proteins, as discussed also in other sections. For example, PSD-95 can bind neuronal nitric oxide synthase (nNOS) via its PDZ2 domain and can bring nNOS into close proximity to the calcium pore of an NMDAR that is bound to its PDZ1 domain; in this arrangement, glutamate activation of the NMDAR would lead to specific activation of nNOS, causing the production of NO, which can modulate NMDAR signaling but also may underlie neuronal excitotoxicity (162). In addition, it is likely that a PSD-95 molecule in close proximity to an NR2A-containing NMDAR (that perhaps is not attached to the PSD-95) can regulate phosphorylation of NR2A via Fyn, Src, and other kinases. This phosphorylation can affect the function and/or trafficking of the receptor, as discussed in the sections on phosphorylation and internalization. Selective

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localization of NR2A-containing NMDARs at synapses, in the absence of NR2A binding to MAGUK PDZ domains (and assuming that NR2B is absent from this NMDAR complex), may rely on other proteins that can bind to the NMDAR (Table 2). Good examples include adhesion factors that bind to NR1 (see Section 4.3 on adhesion factors) and CaMKII (163,164) (see Section 6). CaMKII, like the MAGUKs, is one of the most abundant proteins of the postsynaptic density, and both CaMKII and MAGUKs interact with several other components of the density. 5.2. NMDAR/MAGUK-Associated Proteins of the PSD The basic scaffold of these PDZ-containing proteins can link to other proteins in chains that bind NMDARs to other glutamate receptors and other ion channels. Thus, GKAP binds to the GK-like domain of the MAGUKs and S-SCAM (165), and then GKAP binds to Shank, which binds to Homer dimers that bind to perisynaptic metabotropic glutamate receptors and TRPC cation channels (Fig. 4, Table 1 (63,92,166)). Shank holds a central position in these chains. It can bind through Homer dimers to inositol 1,4,5-triphosphate (IP3) receptors in smooth endoplasmic reticulum cisternae that extend into the spine. Shank also may be linked directly with metabotropic and delta glutamate receptors and indirectly to AMPARs via the PDZ protein GRIP (167,168). Chains also can be formed to link NMDARs and AMPARs via PSD-95, which links to AMPARs via stargazin (and other members of the stargazin family called transmembrane AMPAR regulatory proteins [TARPs] (169,170)). However, no evidence of this is seen in coimmunoprecipitation studies between AMPARs and NMDARs (64). Morphologic links between AMPARs and NMDARs also could occur via calcium/calmodulindependent protein kinase II (CaMKII) and an assembly of AMPAR-associated proteins including SAP97/GluR1, 4.1N protein, actinin, and actin (163). Such complexes that include a core of NMDARs and their associated MAGUKs are linked to the actin cytoskeleton of the synaptic spine (92); actin filaments control the overall structure of the postsynaptic spine and may form pathways for transport of proteins to and from the postsynaptic membrane. At least three such types of connections exist in addition to that associated with GluR1: NMDARs-actinin-actin, GKAP-Shank-cortactin-actin, and PSD-95SPAR-actin (171). SPAR is a Rap-specific, GTPase-activating protein; the latter proteins are implicated in regulation of MAP kinase cascades, cell adhesion, and activation of integrins. Like GKAP, SPAR binds to the GK domain of PSD-95 (172). It regulates spine morphology both via a direct interaction with F-actin and also probably via Rap signaling. Nevertheless, whereas actin–protein associations play important roles in synaptic structure and function, anchoring of NMDAR/PSD-95 complexes at synapses appears to be independent of actin associations (173). Finally, molecules connected to NMDAR-containing complexes (here defined as NMDARs bound to associated

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proteins) may regulate actin in spines indirectly. 1-Chimerin binds to the NR2A subunit and contains a GTPase-activating (GAP) domain that can inhibit Rac1 (Rho GTPase family member) function; Rac1 can promote actin polymerization, increase dendrite arbor complexity, and stimulate spine formation (174). Thus, 1-chimerin modulates dendrite spine morphology by inactivating local Rac1 following the binding of 1-chimerin to NMDARs. Citron is a PSD-95–associated protein; it is a target of Rho, which can regulate actin cytoskeleton organization (175,176). Citron is limited in distribution to certain specialized neurons, and it may mediate forms of NMDAR-dependent synaptic plasticity in these. Several proteins that are associated with the MAP kinase pathway can bind to PDZ domains of scaffolding proteins in the NMDAR-containing complexes. SynGAP (synaptic Ras-GTPase activating protein) binds to PSD-95 (177,178), nRap GEP (neural GDP/GTP exchange protein for Rap1 small G-protein) binds to S-SCAM (179), and MAGUIN binds to both PSD-95 and S-SCAM (180). SynGAP can catalyze rapid hydrolysis of Ras-GTP to Ras-GDP and thus may maintain a low steady-state level of active Ras near the synapse. Subsequently, calcium entry via NMDARs can activate CaMKII, which phosphorylates SynGAP, thus inactivating it. Free from SynGAP’s regulation, Ras-GTP can accumulate and increase activation of the MAP kinase cascade associated with LTP. Alternatively, SynGAP associates more with Rap than Ras, and inactivation of Rap by SynGAP results in a decrease in p38 MAP kinase activity and subsequent inhibition of AMPAR removal from synapses (181). In the latter model, SynGAP regulation depends on the following chain of interacting proteins: SynGAP/MUPP1 (a PDZ protein)/CaMKII/NMDAR. SynGAP is selectively associated with NR2B-containing NMDARs (182); it often is found at high levels at excitatory synapses in the hippocampus at P2, when most NMDARs contain NR2B (63) (see Section 4.4). It is the association of SynGAP with NR2B-containing NMDARs that couples NR2B to inhibition of the Ras-ERK pathway, which mediates surface delivery of GluR1 AMPARs; thus, NR2B/SynGAP associations may underlie the removal of AMPARs from synapses, causing a weakening of synaptic transmission (182). It is interesting that NR2A-containing NMDARs have the opposite effect, activating the RasERK pathway and promoting surface delivery of GluR1 (182). Related to this, Ivanov et al. (183) found that synaptic NMDARs activate ERK, whereas extrasynaptic NMDARs inactivate it (see discussion of extrasynaptic NMDARs in Section 3.2.4). 5.3. Adhesion Proteins Associated with NMDARs A number of adhesion proteins found both in presynaptic and postsynaptic sides of glutamatergic synapses are linked to NMDAR-containing complexes and may regulate them. Relatively few of these adhesion proteins bind directly to NMDARs. The Eph family of receptor tyrosine kinases may act like adhesion

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factors at the synapse (184). Ephrin-B ligands on the presynaptic terminal may bind to EphB2 receptors on the postsynaptic membrane and promote the binding of the extracellular domains of the EphB2 and NR1 (185). The activated EphB2 would now be in position to modulate NMDAR calcium fluxes either by its kinase action or by the action of kinases such as Src and Fyn that bind to Eph receptors. These calcium fluxes could control the Rhofamily GEFs kalirin 7 and Tiam that activate Rac, which is a regulator of actin polymerization and subsequent dendrite arborization (186,187). In addition, a family of five synaptic adhesion-like molecules (SALMs) interacts with NMDARs (188–191), and at least SALM1 can enhance surface expression of transfected NR2A. The first three SALMs have a C-terminal PDZ-binding domain ESTV, and at least SALM1 can bind to the MAGUKs, PSD-95, SAP102, and SAP97 (as described for NrCAM and SAP102 later). Preliminary studies suggest that SALMs adhere to NMDARs (via the extracellular domains as described for EphB2 earlier) in the synapse and may even interconnect with other adhesion proteins across the synaptic cleft. Several other adhesion proteins connect to NMDAR-containing complexes. Neuroligin binds to a PDZ domain of PSD-95 in the PSD and to neurexin in the synaptic cleft; neurexin is bound to PDZ domain containing proteins in the presynaptic terminal (63,192,193). Thus, the postsynaptic NMDARcontaining complexes can have a direct link with protein complexes in the presynaptic terminal. Neuroligin localization may be involved in specifying whether synapses will become excitatory or inhibitory (192,193). Neuroligin also binds to the PDZ domains of the MAGUK S-SCAM (153), whereas the PDZ domains of CIPP can bind to both neuroligins and neurexins (154). Both cadherins and catenins are associated with the NMDAR-containing complex and also form links between the presynaptic terminal and postsynaptic structures (194,195). Stability of the synaptic contact may be regulated directly by association because dimerization of cadherins is associated with NMDAR activation (195). NMDARs and associated PSD-95 also are found in cadherin-based attachment plaques in cerebellar glomeruli (196). Thus, glutamate spillover from adjacent synapses in the glomerulus may control the overall stability of the glomerulus. -Catenin binds directly to the MAGUK S-SCAM and may control synaptic targeting of S-SCAM (153). Adhesion proteins of the L1/NrCAM and NCAM families also are linked to NMDARcontaining synapses (63) and help to mediate synaptic plasticity (197) and modulate neuronal positioning and dendritic orientation (198). In hippocampus, NCAM180 is found in the central region of the postsynaptic membrane, where it associates with NR2A-containing NMDARs; this distribution changes following LTP (199). NrCAM is found on both the presynaptic and postsynaptic sides of glutamatergic synapses (63) and can link directly to the MAGUK SAP102 via its PDZ-binding domain (200); thus, it could be part of an

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NMDAR-containing complex of proteins at a synapse, and, like neuroligin, it could help to link NMDARs to presynaptic components of the synapse. Tyrosine receptor kinase B (TrkB) is found at glutamatergic synapses (63) and may control expression and function of NMDARs (201,202). It is interesting that TrkB might act as an adhesion factor across the synaptic cleft via a homophilic binding of two TrkB molecules that are linked by a dimer of their ligand, BDNF (as discussed in ref. 63). 5.4. Developmental Changes in NMDARs and Their Associated Proteins in the PSD Plasticity of glutamate receptors typically involves an interplay between NMDARs and AMPARs, with major changes in strength of the synapse, as in LTP and LTD, involving mostly a control of AMPAR numbers, although rapid changes in surface NMDARs due to LTP has been described in adult animals (203). Plasticity is described in more detail in another chapter in this book; here, we concentrate on changes in the NMDARs and their associated proteins during development. As noted in Sections 2.2 and 3.1, the best-studied change in NMDARs is the switch from primarily NR2B-containing NMDARs to a prevalence of NR2A-containing NMDARs, as found during maturation of excitatory synapses in the forebrain and other regions; this has been visualized in ultrastructural studies in the thalamus and cerebral cortex (204,205) and hippocampus (63). A number of studies demonstrate that this switchover is tied to learning experiences (113), such as visual experience during postnatal development (206) and rule learning for odor discrimination in adults (207). This change in NMDARs also is accompanied by an increase in PSD-95 at synapses (64,113,208). Another well-studied switchover is seen in cerebellar granular cells, in which NR2B is replaced by NR2C. After their migration and innervation by mossy fibers, granules cells downregulate NR2B and begin expressing NR2C (209); NR2A also is present at this time, as noted in Section 3 (44,115). NR2C expression is regulated by neuregulin that is secreted by the mossy fiber terminal. This secreted neuregulin interacts with its ErbB2 and ErbB4 receptors on granule cells to induce the NR2C expression. Because ErbB4 receptors can bind to MAGUKs, this induction of NR2C expression may involve an indirect structural link between NMDARs and neuregulin receptors (210,211). In the development of glutamatergic synapses, NMDAR-mediated plasticity plays an important role, and in most cases, synapse maturation involves a major change in NMDAR type, as described earlier for the switch from NR2Bcontaining to NR2A-containing NMDARs. Yet, NMDARs probably are not present in the earliest nascent excitatory synapses; other components come together first and are responsible for the initial steps in synaptogenesis (63, 212,213). The earliest components of a nascent synapse probably are some of

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the adhesion factors mentioned in Section 4.3; for example, NCAMs associate with both the presynaptic and postsynaptic sides in these nascent contacts and could play a major role in synaptogenesis and synapse maturation (214, 215). This is followed by delivery of presynaptic components in packets and subsequently by the postsynaptic MAGUKs and NMDARs (212,216). It is interesting that, even though the neuroligin/neurexin attachment across the synaptic cleft involves the postsynaptic anchoring of neuroligin to PSD-95, this association may not be necessary for the initial role of neuroligin in synapse formation. Targeting of neuroligin to synapses is independent of this binding; this supports suggestions that neuroligin is recruited early in synaptogenesis (193). Binding of presynaptic ephrins and postsynaptic EphB receptors also is believed to be involved in the early stages of synaptogenesis (184). Once the earliest nascent synapse is formed, other proteins enter the forming PSD relatively quickly, so that a distinctive PSD with multiple proteins linked directly or indirectly to NMDARs is formed in 80%), with the exception of transcripts in white matter (66%) and brainstem (55%) (64,65). GluR5 RNA exhibits a marked degree of regioselectivity in editing, ranging from 41% (in white matter) to 91% (thalamus) (66). It is important to note that these are proportional degrees of editing for populations of neurons and that more complex distributions might exist at the level of individual neurons (67); for example, Mackler and Eberwine found that GluR5 transcripts in juvenile CA1 pyramidal neurons were unedited (68). Because of the relevance of calcium signaling to several neurologic diseases, a number of studies have examined and found marked changes in Q/R-site editing of kainate receptor RNAs in pathologic states, including transient ischemia (69), epilepsy (70–72), and Down syndrome (73); the functional importance of these alterations to the disease processes, however, is not clear. 1.3.3. Genetic Polymorphisms Single-nucleotide polymorphisms that alter the predicted primary amino acid sequence exist in all human kainate receptor subunit genes, but, apart from a single instance in the GluR7 (GRIK3) gene, functional studies of these isoforms have not been undertaken. A thymine/guanine variation in human GluR7 cDNAs that produced either a serine or an alanine at residue 310 was proposed initially to result from a novel RNA-editing event (74). However, this variation was shown subsequently to occur naturally as a polymorphism that

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did not detectably alter homomeric GluR7a receptor function (75). In addition, the distinct alleles in heterozygous individuals were expressed unequally, suggesting that the gene is subject to imprinting. This polymorphism was used subsequently as a marker to demonstrate that the GluR7 gene was in linkage disequilibrium in patients with major depressive disorder (76) and schizophrenia in Italian patients (77) but not bipolar disease (type I) (76), obsessive-compulsive behavior (78), schizophrenia in the Japanese population (79), or alcoholism in Polish families (80). Polymorphisms in other kainate receptor subunits genes have been used similarly to explore their potential association with a variety of diseases. 1.4. Posttranslational Modification 1.4.1. N-Glycosylation Kainate receptor subunits undergo a number of posttranslational modifications after their synthesis and assembly into tetrameric receptors. The most thoroughly characterized process is that of N-glycosylation. All five subunits have a number of consensus sites for this form of modification. The relevance of glycosylation to receptor function, however, is unclear. For example, tunicamycin inhibition of core oligosaccharide addition did not eliminate GluR6 receptor plasma membrane expression in Xenopus oocytes (81) but did greatly reduce current amplitudes in mammalian expression systems (82). NGlycosylation is required for modulation of kainate receptors by certain plant lectins like concanavalin A, which greatly potentiates steady-state currents (see additional description in Section 5). In the GluR6 subunit, this potentiating activity is surprisingly position independent; that is, no particular subset of glycosylation sites is critical for lectin activity, and glycosylation of ectopic sites introduced by mutagenesis provides an effective substrate for concanavalin A binding (82). 1.4.2. Palmitoylation The GluR6 receptor subunit is fatty acylated on two cysteines in the cytoplasmic C-terminal domain (83). Palmitoylation of GluR6 does not alter gross functional properties of the kainate receptors but might reduce phosphorylation of nearby residues by protein kinase C (PKC). Mutation of one of the target cysteines (C871) reduced plasma membrane expression of the receptor by ∼30%, although it was not clear whether this effect was directly related to loss of the palmitoyl group (46). In AMPA receptor subunits such as GluR1, as well as receptor-associated proteins like PSD-95, palmitoylation is important for appropriate subcellular trafficking in neurons and targeting to synapses (84,85). The relevance of GluR6 palmitoylation to analogous processes for kainate receptors is unknown.

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1.4.3. Phosphorylation Kainate receptor subunits also contain consensus sites for modification by intracellular kinases, including cAMP-dependent protein kinase (PKA), Ca2+ and calmodulin-dependent kinase (CaMKII), and PKC. In some cases, these enzymes have been shown to modulate kainate receptor function (discussed further in Section 6). Although it is thought likely that kinase systems modify receptor function through phosphorylation of predicted sites on the cytoplasmic tail of the receptor subunits, this has not yet been formally demonstrated with phosphopeptide mapping.

2. Function 2.1. Biophysical Function Kainate receptors are glutamate-gated cation channels with many similarities in function to AMPA receptors. GluR5, GluR6, and GluR7 subunits expressed in heterologous cell lines produce homomeric receptors that gate current on application of glutamate. KA1 and KA2 subunits, in contrast, are efficiently retained in the endoplasmic reticulum when expressed alone (86) but can combine with GluR5–7 subunits to form functional heteromeric receptors with distinct physiologic properties (4). In addition, GluR5, -6, and -7 subunits can combine in heteromeric assemblies (58,87). Incorporation of multiple types of subunits has a marked effect on receptor biophysical and pharmacologic properties. Because of the nearly ubiquitous expression of the KA2 subunit in the nervous system (4) and the requirement of other subunits for functionality, it is thought that most neuronal kainate receptors are heteromers comprised of at least two, and possibly more, distinct types of kainate receptor subunits. Application of glutamate to recombinant kainate receptors elicits a rapidly activating and deactivating (or desensitizing) inward-directed current in normal artificial cerebrospinal fluid (ACSF). At hyperpolarized potentials these currents are carried largely by sodium ions, although receptors containing solely Q/R-site unedited subunits also are weakly permeable to calcium. Current– voltage relationships for kainate receptors are linear if an edited GluR5 or GluR6 subunit contributes to the channel but are strongly inwardly rectifying with only unedited subunits present in the receptor. The inward rectification of unedited kainate receptors results from occlusion of channel permeation at depolarized potentials by intracellular polyamines such as spermine or spermidine (56,57). Activation of kainate receptors by saturating concentrations of glutamate occurs with a submillisecond time course. Indeed, activation rates are almost as fast as the practical limits of current fast-switching drug application systems, even when measured in outside-out patches. For example, homomeric GluR6 receptors reportedly have a 10%–90% rise time (from baseline to the peak

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current) of 200–400 μs (88–90). An even faster activation rate of ∼120 μs (20%–80% rise time) was determined by Li and colleagues using laserpulse photolysis of caged glutamate (91), which was liberated at a rate not achievable with conventional rapid solution exchange systems. Rapid removal of agonist results in the deactivation of kainate receptors, which has a time constant of ∼2.5 ms for homomeric GluR6 receptors (88,91). Homomeric GluR6 receptors are the only type of kainate receptor whose activation and deactivation properties have been examined in detail. Kainate receptors desensitize profoundly in the continued presence of glutamate. When glutamate is rapidly applied at saturating concentration, peak current amplitudes are typically >95% larger than steady-state current amplitudes. The component subunits in kainate receptors strongly influence desensitization rates, ranging from a very fast time constant for GluR5/KA2 receptors (t ∼ 1.5 ms) (92) to the much slower homomeric GluR7 receptors (∼8 ms) (45). Slow applications and/or low concentrations of glutamate also desensitize kainate receptors without evoking significant peak currents. For GluR6 receptors and kainate receptors in cultured neurons this occurs with half-maximal inhibition constant (IC50 ) values of 0.3–0.4 and 2.8 μM, respectively (87,93), suggesting that synaptic kainate receptors could be partially desensitized by ambient levels of glutamate. Different receptor stoichiometries also differ in their rate of recovery from desensitization, an important biophysical parameter that controls how faithfully receptors follow high frequencies of transmission (at synapses) or agonist application (in cultured cells). In general, the time course of recovery of kainate receptors is significantly slower than that of AMPA receptors. For homomeric GluR5 receptors, recovery is biphasic, with rapid (50 ms) and slow (5 s) time constants in roughly equal proportion (43). GluR6 receptors recover from desensitization with a predominant time constant of 2–3 s (88,90,93). In contrast, recovery from desensitization for AMPA receptors occurs within several hundred milliseconds (or even faster) (94). Both entry into and recovery from desensitization are agonist-dependent processes; for example, recovery from desensitization of GluR6 kainate receptors induced by binding of the high-affinity agonists 2S,4R-4-methylglutamate (SYM 2081) and dysiherbaine occurs over a time period of minutes or hours (95,96). 2.2. Neuronal Function 2.2.1. Postsynaptic Kainate Receptors The advent of selective pharmacologic tools that discriminate between AMPA and kainate receptors allowed the first demonstration that kainate receptors contribute to fast excitatory synaptic transmission in the central nervous system. It had been assumed that these ionotropic glutamate-activated

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channels would be localized to postsynaptic sites and activated by synaptic release of glutamate similar to AMPA and NMDA receptors. However, the description of postsynaptic kainate receptors produced some surprises, not least of which is that although kainate receptor subunits are widely expressed in almost every cell type throughout the brain, localization to postsynaptic densities is far from ubiquitous. Indeed, kainate receptor distribution is highly compartmentalized in most neurons. For example, mossy fiber synapses on the proximal part of the dendrite of CA3 pyramidal neurons contain postsynaptic kainate receptors, but associational-commissural synapses on more distal dendritic regions of CA3 pyramidal neurons contain only AMPA and NMDA receptors (97,98). In general, kainate receptors play a wider variety of roles in the central nervous systems than do AMPA or NMDA receptors. Not only do they mediate point-to-point chemical transmission, they also have modulatory actions at synapses. For example, presynaptic kainate receptors influence the strength of both excitatory and inhibitory transmission and fine tune synaptic plasticity at a subset of central synapses, whereas postsynaptic receptors influence neuronal excitability through effects on voltage-gated ion channels. The first recordings of kainate-mediated excitatory postsynaptic currents (EPSCKA s) were reported simultaneously by the groups of Roger Nicoll and Graham Collingridge, and these seminal findings ushered in a new era of research interest in the neuronal function of kainate receptors. They were detected in the hippocampus at the mossy fiber synapse (97,98), which is formed between granule cell axons and the proximal dendrites of CA3 pyramidal neurons (99). The kainate component of the mossy fiber EPSC proved elusive and was detected initially by increasing the release probability of the synapse with short high-frequency trains of stimulation in the presence of a noncompetitive AMPA receptor antagonist. The mossy fiber EPSCKA had unexpectedly slow decay kinetics, with a time constant of approximately 100 ms, in contrast to the relatively fast ∼10-ms decay of the AMPA-mediated component at this synapse (Fig. 2A). The slow EPSCKA kinetics also contrasted with the properties of currents elicited by glutamate from recombinant kainate receptors, which, as mentioned in the previous section, decay with a time course of ∼2.5 ms (88,91). EPSCKA s were detected only at mossy fiber synapses in CA3 neurons (Fig. 2B); the observed absence from the abundant associational/commissural synapses in the same neurons was the first of many examples of the polarized distribution of neuronal kainate receptors. The slow kinetics and relatively small amplitude of evoked EPSCKA suggested that the receptors might be localized to peri- or extrasynaptic sites and thus sample a lower and broader glutamate transient than postsynaptic AMPA receptors. The evidence against this hypothesis, however, is substantial, consisting of immunolocalization at postsynaptic densities (100), insensitivity of EPSCKA

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A

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Fig. 2. A. Mossy fiber kainate-mediated excitatory postsynaptic current (EPSCKA ) activates and decays at a slower rate than -amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) EPSC. The GYKI-resistance component of the mossy fiber EPSC was scaled to the peak of the AMPA receptor component (97). B. GYKI 53655 largely blocked the response to single mossy fiber and associational/commissural stimulation, but repetitive stimulation (four stimuli at 200 Hz) evoked a slow, 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX)–sensitive EPSCKA only from mossy fiber synapses (97). C. Miniature EPSCs recorded from CA3 pyramidal neurons have relatively slow decay kinetics (black circles), mixed fast and slow components to the decay (gray circle), or only fast decay kinetics (open circles). Application of the moderately selective noncompetitive antagonist GYKI 52466 eliminates the fast and mixed miniature EPSCs, demonstrating that those with slow kinetics arise from synaptic kainate receptors. This was confirmed by inhibition with a high concentration of the nonselective antagonist CNQX (101). D. The mossy fiber EPSCKA is absent in GluR6−/− mice, whereas the AMPA receptor component is intact (103). E. Gene targeting of the KA2 kainate receptor subunit alters the decay rate of mossy fiber EPSCKA (104). Figures adapted from cited reports with permission.

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amplitude or kinetics to blockade of glutamate uptake (97,98), and, perhaps most convincingly, the demonstration that postsynaptic mossy fiber kainate receptors are activated by quantal release of glutamate (101). Cossart and colleagues described miniature events with slow kinetics mediated exclusively by kainate receptors (in the presence of an AMPA receptor antagonist) as well as mixed AMPA/kainate mEPSCs in CA3 pyramidal neurons, supporting the interpretation that kainate receptors are colocalized with AMPA receptors at some (but not all) mossy fiber synapses (Fig. 2C) (101). They also found that the time course of decay of quantal EPSCKA s, although faster than that of EPSCs evoked by bulk stimulation, was substantially slower than that of EPSCAMPA , supporting the interpretation that the current kinetics were determined by intrinsic characteristics of the synaptic receptors. In addition to their slow kinetics, EPSCKA s differ from EPSCAMPA s in that the former are markedly less sensitive to changes in mossy fiber release probability, have a lower coefficient of variance, and display less sensitivity to competitive inhibition by a low-affinity antagonist; these differences are consistent with the hypothesis that kainate receptors are preferentially (but not exclusively) localized to synapses with higher release probability (102). The subunit composition of mossy fiber kainate receptors has been the subject of some debate, but multiple lines of evidence support a growing consensus that postsynaptic kainate receptors are predominantly heteromeric combinations of the GluR6 and KA2 subunits (103,104). An early pharmacological study suggested that the postsynaptic kainate receptors were comprised of the GluR5 subunit (105), which was surprising considering that mRNA for GluR5 is expressed only weakly in hippocampal principal neurons after the first week of postnatal development (106,107). Subsequent studies in knockout mice instead demonstrated a critical role for the GluR6 subunit because the mossy fiber EPSCKA is absent in these animals (Fig. 2D) (103); this hypothesis is consistent with the robust expression of GluR6 mRNA in the CA3 region (106). Additional pharmacological experiments failed to reproduce the original demonstration of a role for GluR5-containing receptors in mediating the EPSCKA (108). mRNAs for the KA1 and KA2 subunits are also expressed at high levels in CA3 pyramidal neurons. The EPSCKA in KA2 knockout mice has significantly faster decay kinetics, suggesting that the inclusion of this subunit contributes to the biophysical properties of synaptic kainate receptors (Fig. 2E) (104). The EPSCKA in KA1-null mice also shows altered kinetics, indicative of a contribution by this subunit to postsynaptic receptors as well (A. Contractor unpublished observations). The subunit composition of mossy fiber kainate receptors as determined thus far from knockout studies remains to be confirmed with a complementary pharmacological analysis because antagonists that selectively inhibit GluR6 and other subunits have not been developed.

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Subsequent to these studies at mossy fiber synapses, kainate receptor– mediated postsynaptic currents were found at a number of other hippocampal excitatory synapses. Excitatory synapses onto a population of interneurons in the stratum oriens and radiatum in the CA1 region contain EPSCKA s with faster decay kinetics than those at mossy fiber synapses (101,109,110); nevertheless, interneuron EPSCKA s are much slower by comparison to the EPSCAMPA at the same synapses (101). The presence of relatively slow EPSCKA s in interneurons strongly suggested that this peculiar biophysical feature arose from intrinsic channel properties rather than the distinctive synaptic architecture of the mossy fiber–CA3 pyramidal cell synapse, and this idea has been further supported as more examples of synaptic kainate-mediated currents have been described. During early postnatal development, thalamocortical synapses onto layer IV neurons segregate AMPA and kainate receptors at synapses and thus exhibit exclusively EPSCKA or EPSCAMPA but not mixed synaptic currents (111,112). Thalamocortical EPSCKA s exhibit slow kinetics comparable to those at hippocampal mossy fiber synapses, and in a series of elegant studies John Isaac and colleagues demonstrated that, like mossy fiber kainate receptors, the kinetics are an intrinsic channel property rather than a result of extrasynaptic localization or electrotonic filtering of distal synaptic signals (111,112). Using minimal stimulation to activate single fibers or relatively few synapses, Isaac and colleagues observed that AMPA and kainate receptors were segregated into distinct AMPA-only or kainate-only synapses. It is interesting that thalamocortical kainate receptors are rapidly removed from postsynaptic sites in response to a stimulation paradigm that simultaneously potentiates AMPA receptor EPSCs, suggesting that kainate-only synapses are converted to AMPA-containing synapses after LTP (111). Kainate receptor currents also declined significantly over the first postnatal week, which correlated with the developmental refinement of synapses in this critical period. Activitydependent trafficking of kainate receptors has also been reported at synapses onto layer II/III neurons in the perirhinal cortex. Here long-term depression of the kainate-mediated synaptic responses is dependent on group I mGluR activation, PKC, and the glutamate receptor interacting protein PICK1, mechanisms that are distinct from the NMDA receptor–mediated LTD of AMPA receptors at the same synapses (113). PICK1 has also been implicated in maintaining kainate receptors at mossy fiber synapses and therefore might function as a common mediator of anchoring or trafficking of these receptors (114). A strict partition in the distribution of non-NMDA glutamate receptors also was observed at synapses between cone cells and “Off” bipolar cells in the retina (115). The cone cells tonically release glutamate in the dark, which causes a profound desensitization of postsynaptic kainate receptors. A switch to light conditions hyperpolarizes the cone and temporarily reduces or stops glutamate release, allowing the postsynaptic receptors to partially recover and

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thus produce a larger depolarization on resumption of transmitter release when darkness is restored (115). Kainate receptor–mediated postsynaptic responses have also been described in other cortical regions (116,117), the basolateral amygdala (118), thalamic neurons (119), layer 2 spinal cord neurons (120), and cerebellar Golgi and Purkinje cells (121,122). In each of these cases, the EPSCKA was relatively small in amplitude and had slower kinetics than the associated EPSCAMPA . The small contribution that kainate receptors make to the overall EPSC at most synapses raises the question of whether they participate significantly in ionotropic synaptic signaling. The answer to this question lies in their unusually slow decay kinetics, which prolongs depolarization beyond the brief temporal window mediated by AMPA receptor activation. This possibility was tested formally by modeling and comparing the integrative properties of AMPA and kainate receptor postsynaptic potentials in hippocampal CA1 interneurons (123). Frerking and Ohliger-Frerking found that activation of synaptic kainate receptors produced substantial tonic depolarization at physiologic presynaptic firing rates, which then resulted in enhanced action potential firing in the postsynaptic neuron. In contrast, the rapidly deactivating AMPA receptor component contributed phasic signaling without a tonic depolarization. In addition, the voltage response to synaptic kainate receptor activation (EPSPKA ) made a larger proportional contribution to the overall synaptic EPSP because the slow time course of the synaptic potential was attenuated to a lesser degree than the rapid EPSPAMPA by the passive properties of the neuronal membrane. Temporal summation of depolarization by kainate receptors also facilitates the reactivation of synaptic NMDA receptors, which require depolarization to relieve Mg2+ block. At corticothalamic synapses, this interaction between kainate and NMDA receptor–mediated depolarization results in a late onset and persistent pattern of action potential firing (119). Thus, one function of postsynaptic kainate receptors is likely to integrate changes in the frequency of afferent signals to alter neuronal excitability and firing patterns. 2.2.2. Postsynaptic Modulation of Intrinsic Conductances Postsynaptic and extrasynaptic kainate receptors also modulate neuronal excitability through actions on voltage-gated channels (124–126). Surprisingly, this appears to occur through a G protein–mediated process that has been referred to as “non-canonical” (126) because it contrasts with the traditional view of kainate receptors as ionotropic receptors. Originally proposed by Juan Lerma to account for the mechanism of action of presynaptic kainate receptors in CA1 interneurons (127) (as will be described in the following section), the hypothesis that kainate receptors have a “metabotropic” or G protein– mediated function was met initially with a healthy degree of skepticism. This skepticism has given way, however, in the face of increasing evidence from

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multiple laboratories that kainate receptors can activate G protein–mediated signaling pathways to effect changes in neuronal voltage-gated channels. In hippocampal CA1 pyramidal neurons, activation of kainate receptors increases CA1 pyramidal neuron excitability by pertussis toxin–sensitive G protein– and PKC-mediated inhibition of a slow afterhyperpolarizing current (IsAHP ) (124,125), which follows a burst of action potentials and is generated by a voltage-independent, Ca2+ -dependent potassium conductance (128). The postburst afterhyperpolarization activates for several seconds and limits further firing of the neuron, thereby contributing to the characteristic spike accommodation observed in pyramidal neurons. In contrast to mossy fiber–CA3 synapses, EPSCKA s are not observed at Schaffer collateral synapses with CA1 pyramidal neurons. Nevertheless, glutamate released synaptically during brief high-frequency stimulation of Schaffer collaterals in the slice preparation elicits a long-lasting inhibition of IsAHP (125), presumably through spillover-mediated activation of extrasynaptic receptors. Kainate receptor activation also inhibits IsAHP in CA3 pyramidal neurons (129,130). As with the postsynaptic EPSCKA , kainate receptor-mediated inhibition of IsAHP in CA3 pyramidal neurons is abrogated in GluR6−/− animals but is robust in GluR5−/− mice (130). The mechanism of kainate receptor–mediated inhibition of IsAHP is dependent on G protein and PKC signaling and can be mediated by synaptically activated receptors at mossy fiber inputs (130). However, mossy fiber kainate receptor inhibition of IsAHP following synaptic stimulation is transient and limited to a 5-sec window following activation, unlike the long-lasting effects induced by application of exogenous agonist (131). The idea of ionotropic receptors linked to G protein signaling pathways, although still unusual, is slowly becoming accepted; however, in a more recent report, an even more remarkable finding demonstrated that these divergent roles are played by distinct subunits within a single heteromeric kainate receptor complex. Christophe Mulle and colleagues examined kainate receptor– mediated inhibition of the IsAHP in gene-targeted mice and found that the modulatory activity was absent in KA2−/− knockout mice (131). The mossy fiber EPSCKA in these mice is intact, albeit with somewhat faster kinetics (104). This led the authors to conclude that metabotropic inhibition of IsAHP was not dependent on the ionotropic kainate receptor current (131). In contrast, elimination of EPSCKA s in GluR6−/− mice concurrently removed inhibition of I sAHP , presumably because KA2 subunits are retained intracellularly in the absence of GluR6 subunits. These observations led to the hypothesis that the ionotropic and metabotropic functions of postsynaptic mossy fiber kainate receptors are gated by distinct subunits within the same heteromeric complex (131); that is, activation of GluR6 subunits results in ionotropic gating, whereas the high-affinity KA2 subunits in the same receptor complex can link to G protein signaling pathways (131). Because the requirements for activation of

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the metabotropic function of kainate receptors are not known, it is possible that different patterns of presynaptic stimulation might preferentially engage one or the other mode of signaling. Similarly, the distinct pharmacologic profiles of the GluR6 and KA2 subunits suggest that selective activation (or antagonism) of one type of subunit could be used to test this intriguing hypothesis. In addition to metabotropic-mediated effects on this important potassium conductance underlying the IsAHP , kainate receptors modulate calcium currents through G protein signaling pathways in sensory neurons (126). Kainate receptor activation in dorsal root ganglion neurons has multiple effects mediated by second messengers, including a pertussis toxin–sensitive elevation in intracellular calcium concentration through release of calcium from internal stores (126). In addition, under conditions in which ionotropic activity of kainate receptors was not detectable, kainate receptor agonists caused a G protein–mediated inhibition of high-voltage activated calcium channels, an effect dependent on the GluR5 subunit (126). The mechanisms linking kainate receptors to metabotropic signaling pathways are just beginning to be defined. Future work will fully evaluate these unorthodox signaling cascades and how they link kainate receptors to physiologically relevant signaling mechanisms. Nevertheless, it is clear that the roles that kainate receptors play in the brain cannot be described completely without consideration of their metabotropic function. 2.2.3. Presynaptic Kainate Receptors 2.2.3.1. Presynaptic Receptors at Excitatory Synapses Ionotropic glutamate receptors have been traditionally described and thought of as postsynaptic mediators of synaptic signaling, but in fact non-NMDA and NMDA receptors have been localized to presynaptic terminals at a number of synapses, where their activation modulates the strength of transmitter release (132). The role of presynaptic kainate receptors in modulation of both excitatory and inhibitory neurotransmission has been most thoroughly explored at hippocampal synapses. Several early studies using isolated synaptosome preparations found that high concentrations of a kainate receptor agonist induced release of glutamate, suggesting that kainate receptors were localized in presynaptic terminals (133–135). These biochemical experiments were followed closely by a seminal study by Jeremy Henley and colleagues in which kainate both inhibited potassium-stimulated release of glutamate from hippocampal CA1 synaptosomes and caused pronounced and long-lasting depression of EPSCNMDA , mediated by a decrease in transmitter release, in CA1 pyramidal neurons in hippocampal slice preparations (Fig. 3A) (136). This result was counterintuitive, given that kainate receptor activation depolarizes membranes, and therefore the mechanism of inhibition was a topic of some uncertainty. Kamiya and Ozawa subsequently made the important observation

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Fig. 3. A. Top: N-methyl-d-aspartate (NMDA) excitatory postsynaptic currents (EPSCs) recorded in CA1 pyramidal neurons are selectively depressed by kainateselective agonist but not -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor activation. Bottom: Biphasic concentration-dependent effects of kainate; very low concentrations of kainate can facilitate release (136). B. Kainate receptor–mediated depression of CA1 EPSPs is dependent on G protein signaling. Activation of kainate receptors by domoate depresses the field EPSP (fEPSP) in control recordings but fails to have an effect in recordings from rats in which pertussis toxin (PTX) has been injected directly into the hippocampus 2–3 days prior to the experiments (138). C. Antidromic action potentials recorded in granule cells are enhanced by activation of kainate receptors. Whole-cell recording were made from granule cells in the dentate gyrus and antidromic action potentials evoked by stimulating the mossy fiber axons at an intensity that straddled threshold. Application of 0.5 μM kainate increased the success rate of antidromic events, suggesting that mossy fiber axon excitability is enhanced by kainate receptor activation (144). D. Frequency facilitation of mossy fiber synaptic transmission is impaired in GluR6-receptor knockout mice, suggesting that presynaptic GluR6-containing receptors are activated by homosynaptic release of glutamate and contribute to subsequent facilitation of transmitter release at mossy fiber synapses (146). WT, wild type. Figures adapted from cited reports with permission.

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that presynaptic calcium decreased in the presence of a kainate receptor agonist, indicating that the end result of presynaptic kainate receptor activation was to reduce terminal calcium influx and thereby affect transmitter release (137). Activation of kainate receptors did not have any effect on Schaffer collateral fiber volleys (105,137,138), suggesting that depolarizing block of axonal action potential propagation did not underlie the inhibition of EPSCs by kainate receptors. It was also noted in these early studies that kainate receptors are localized to somatodendritic domains of both principal cells and interneurons in the hippocampus, and therefore exogenous application of kainate receptor agonists induced depolarization that potentially triggered release of a variety of neuromodulators, which might be indirectly responsible for the depression of synaptic responses. This possibility was eliminated for a number of the likeliest candidates by including a cocktail of antagonists (136–138). Nonetheless, indirect effects are difficult to rule out completely because of the variety and ubiquity of systems that modulate transmitter release. In sum, however, these observations raised the possibility that kainate receptor inhibition of synaptic release was not due to the ionotropic action of these receptors but rather was via metabotropic pathways. This hypothesis had been explored previously with respect to kainate receptor actions on GABAergic synapses in CA1 (see additional discussion later) (127) and foreshadowed the discovery of G protein–mediated modulation of intrinsic conductances as discussed in the preceding section. At excitatory presynaptic terminals in CA1, inhibitors of Gi /Go G proteins blocked the effect of kainate receptor agonists but did not require PKC activation (Fig. 3B) (138), in contrast to either modulation of intrinsic conductances or inhibitory transmission. In summary, the evidence is substantial in support of kainate receptor-mediated metabotropic signaling through a G protein that ultimately modulates calcium influx by inhibiting Ca2+ channels in Schaffer collateral terminals. Presynaptic kainate receptors have been intensively characterized at the excitatory mossy fiber–CA3 pyramidal cell synapses in the hippocampus. Early autoradiographic data revealed an abundance of high-affinity binding sites for kainate in the mossy fiber termination zones (139) that were eliminated by selective ablation of granule cells (140). As with CA1 excitatory synaptic terminals, kainate receptors modulated transmitter release from isolated mossy fiber synaptosomes (135,141). Several groups demonstrated that relatively low concentrations of kainate, which predominantly activate kainate receptors, depressed excitatory transmission to CA3 pyramidal neurons (142–144). In addition, activation of kainate receptors had a biphasic effect on the mossy fiber presynaptic fiber volley and postsynaptic EPSC. Low nanomolar concentrations of agonist facilitated release (104,145), whereas higher concentrations depressed the EPSC through presynaptic actions (142,144). The biphasic concentration dependence of this response was mimicked by incrementally

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elevating extracellular potassium, suggesting that kainate receptor–mediated depolarization of the mossy fibers axons accounts for these effects on the fiber volley and transmitter release (145). The effect of low concentrations of kainate on antidromic action potentials recorded in granule cells of the dentate gyrus is particularly compelling because it directly demonstrates that mossy fiber excitability is enhanced by kainate receptor activation (144) (Fig. 3C). Presynaptic kainate receptors can be engaged by homosynaptic or heterosynaptic release of glutamate. Glutamate released during repetitive stimulation of mossy fiber axons acts on presynaptic kainate receptors to facilitate neurotransmitter release (Fig. 3D) (108,144,146). In addition, high-frequency stimulation of proximal associational/commissural inputs liberates sufficient glutamate to activate presynaptic mossy fiber kainate receptors and facilitate transmission, providing a frequency-dependent mechanism for heterosynaptic modulation (104,144). The presynaptic kainate receptors that mediate this facilitation have been proposed to be Ca2+ permeable because philanthotoxin, a polyamine that blocks unedited glutamate receptors, reduces frequency facilitation and agonist-induced, kainate receptor-mediated facilitation of mossy fiber EPSCs in conditions in which extracellular divalent ions are near physiologic concentrations (147). Moreover, these findings suggest that Ca2+ flux through these receptors is critical to synaptic facilitation (147). It should be noted, however, that this conclusion is predicated on the selectivity of philanthotoxin for kainate receptors, but it is known that this toxin also antagonizes Ca2+ channels and other receptors and channels (148–151). The hypothesis is attractive, however, because Ca2+ -permeable presynaptic kainate receptors would be ideally situated to modulate release directly. This mechanism would necessitate the existence of cellular processes for regulated and differential targeting of unedited receptors. Eighty-five percent of GluR6-subunit mRNAs are edited in granule cells in postnatal rats (59), and therefore if the presynaptic receptors are unedited as proposed (147), a small pool of unedited receptor subunits must be preferentially coassembled and targeted to presynaptic sites at mossy fiber terminals. Alternatively, GluR6 subunits expressed by dentate gyrus granule cells could be exclusively unedited, in contrast to the hippocampus as a whole. More experiments are required to differentiate between these possibilities. As with earlier studies that explored the subunit identity of postsynaptic mossy fiber kainate receptors, incongruity between pharmacologic and geneknockout approaches has generated debate regarding the subunit composition of presynaptic mossy fiber kainate receptors. mRNA localization suggests that the GluR6 receptor subunit is the most likely candidate for the principal subunit of presynaptic kainate receptors, and this is supported by studies in knockout mice (Fig. 3D) (142). However, pharmacologic reagents apparently selective for GluR5-containing receptors also occluded presynaptic facilitation (108), a result at odds with data from GluR5−/− mice, which exhibit normal frequency

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facilitation (146). To add fuel to the fire, a subsequent study was unable to replicate key effects on mossy fiber short-term plasticity with the GluR5 antagonist (152). The KA2 subunit clearly contributes to presynaptic kainate receptors because gene-targeted mice lacking this subunit have reduced sensitivity to heterosynaptic facilitation by glutamate released following stimulation of associational/commissural inputs (104). Finally, knockout mice lacking the GluR7 subunit exhibited marked reduction in short-term plasticity at mossy fiber synapses, demonstrating that this subunit, which has been difficult to characterize pharmacologically in neurons, contributes to the presynaptic kainate receptors (C. Mulle, personal communication). It remains to be clarified whether distinct receptor populations exist and perform distinct functions in mossy fiber axons and terminals, given that all five kainate receptor subunits have been implicated in the formation of presynaptic kainate receptors. 2.2.3.2. Presynaptic Receptors at Inhibitory Synapses

Kainate receptors also modulate inhibitory neurotransmission. This was first suggested more than two decades ago, when it was demonstrated that inhibitory postsynaptic potentials (IPSPs) in the CA1 region were depressed by exogenous kainate application (153). The mechanisms underlying this depression were not investigated until more than a decade later, and (as seems to be endemic to the field) these more recent studies generated controversy. Inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal neurons were shown to be depressed by low concentrations of kainate (154), and as mentioned previously, this presynaptic modulation of transmitter release was proposed to occur through a metabotropic activity following the observation that it was sensitive to occlusion by pertussis toxin and inhibition of PKC (127). This provocative study from Lerma’s laboratory also reported a kainate-mediated change in the frequency of action potential–independent (miniature [m]) IPSCs, further supporting a direct presynaptic action of kainate receptors at inhibitory terminals (127). Complicating this analysis, however, was the presence of kainate receptors on somatodendritic domains of CA1 interneurons. This receptor population depolarized interneurons on application of kainate receptor agonists, thereby increasing the spike rate and driving a dramatic increase in spontaneous GABAergic events onto their pyramidal neuron targets (109,110). The large increase in interneuron spiking caused a use-dependent depression of the evoked IPSC. These studies also failed to replicate the previously reported effects of kainate receptor activation on mIPSC frequency or amplitude or on paired pulse ratios of evoked IPSCs. Taken together, these latter results suggested that IPSC depression occurred through indirect mechanisms rather than through activation of presynaptic kainate receptors (109,110) and clearly contrasted with the earlier observations (154). Additional experiments using the relatively GluR5-selective agonist

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(RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propanoic acid (ATPA) supported the existence of at least two populations of kainate receptors in interneurons (155). These data also crystallized two opposing views of how kainate receptors in interneurons might function in the hippocampal network during induction of seizure by kainate receptor agonists (156,157). First, kainate receptor activation that decreases inhibition by presynaptic inhibition of GABA release is predicted to contribute to uncontrolled excitation of the network, as is the case during epileptiform activity. Alternatively, interneuron kainate receptors that increase inhibitory activity in the network through actions predominantly on somatodendritic receptors would have a dampening effect on network excitation. Because kainate receptor agonists are potent convulsants, the latter model suggests that the interneuron kainate receptors work in opposition to a distinct seizure-promoting population of kainate receptors (localized to pyramidal neurons in the CA3 of the hippocampus) (156). In support of this hypothesis, application of the GluR5-selective agonist ATPA depolarized interneurons, similar to previous studies with kainate itself, causing a large facilitation in interneuron spiking and concomitant depression of pyramidal neuron excitability (109). Ben-Ari and colleagues presented a more arresting example of this counterintuitive role of interneuron kainate receptors using a whole-hippocampal preparation in which the connection between hemispheres was maintained (and the bath perfusion of the hemispheres was controlled independently) (158). Drug-induced epileptiform activity in one hemisphere, which normally propagates to the contralateral hemisphere, was prevented by application of ATPA to the naive hippocampal hemisphere (158). These studies demonstrated that targeting specific populations of kainate receptors was a potentially viable therapeutic approach to controlling epileptiform activity. In addition to interneuron–pyramidal cell synapses, kainate receptors influence the strength of interneuron–interneuron signaling in the CA1 region. Presynaptic kainate receptors on GABAergic interneuron–interneuron terminals, which presumably are activated by spillover of glutamate from neighboring glutamatergic synapses, enhance release through a nonmetabotropic pathway (159). Quantal release of GABA at interneuron-tointerneuron synapses increased following kainate receptor activation, strongly suggesting that the receptors are indeed localized close to the presynaptic terminals (159,160). This kainate receptor–mediated increase in mIPSC frequency was not observed in all studies (161); Semyanov and Kullmann found instead that kainate receptor activation caused axonal depolarization and elicited ectopic action potentials, an effect that is difficult to reconcile with kainate receptor–mediated enhancement of evoked IPSCs in interneurons (159). Kainate receptor agonists also increased the probability of evoking unitary IPSCs in recordings from synaptically coupled stratum radiatum

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interneurons and pyramidal neurons (162). Conversely, inhibition of kainate receptors increased the number of failures of transmission, suggesting that tonic activation of kainate receptors increases axonal excitability (162). Tonic activation of axonal kainate receptors in the CA3 region of the hippocampus was also inferred by a reduction in spontaneous IPSCs in pyramidal neurons from GluR5-knockout mice (129). In summary, kainate receptor activation on interneuron terminals or axons will strengthen GABAergic transmission in local inhibitory circuits, although the mechanisms through which this occurs are a matter of debate. The subunit stoichiometry of the distinct populations of CA1 interneuron kainate receptors (e.g., somatodendritic vs. presynaptic) has been examined using both knockout animals and selective pharmacologic agents. The increase in spontaneous IPSCs observed in pyramidal neurons on application of low concentrations of a GluR5-selective agonist, ATPA, strongly suggests that GluR5 subunits contribute to interneuron somatodendritic receptors (109). This agonist-dependent increase in sIPSCs was reduced or absent in GluR5−/− and GluR6−/− mice (160). Conversely, Christensen and colleagues concluded that somatodendritic receptors were primarily composed of GluR6 and KA2 subunits, which are activated by higher concentrations of ATPA, because the agonist-evoked depolarization of interneurons was unaffected by a novel noncompetitive GluR5-selective antagonist (163). The putative presynaptic kainate receptors at interneuron–CA1 pyramidal neuron synapses, whose existence and precise functional localization have been a matter of much debate, are proposed to be heteromeric combinations of GluR5 and GluR6 (160) or GluR6 and KA2 (163) based on analyses using selective ligands and knockout animals. In contrast, those receptors at interneuron–interneuron synapses contain GluR6 but not GluR5 subunits (159,160). 2.2.3.3. Presynaptic Receptors at Nonhippocampal Synapses

A number of studies performed in additional brain regions have also addressed the cellular role of presynaptic kainate receptors. Excitatory synaptic transmission is modulated by presynaptic kainate receptors at cerebellar parallel fiber (164), cortico-accumbens synapses (165), and developing thalamocortical synapses onto layer IV spiny stellate neurons in the barrel cortex (166). In the cerebellum, the axons of granule cells form the parallel fibers and make excitatory synaptic connections onto Purkinje cells and interneurons in the molecular layer. Kainate receptor activation at these parallel fiber synapses facilitates or depresses evoked glutamate release in a concentration-dependent manner (164) similar to the biphasic activity observed at mossy fiber–CA3 pyramidal cell synapses (142–144). It is interesting that kainate receptors at Purkinje and stellate cell synapses exhibit a distinct concentration dependence for modulation of transmission; for example, 50 nM domoate enhances

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Purkinje cell EPSCs but depresses stellate cell EPSCs (164). This divergence in sensitivity of the biphasic effects of kainate receptor activation implies the existence of target-specific characteristics for presynaptic kainate receptors, perhaps through expression of receptors comprised of different heteromultimeric combinations of subunits (164). In contrast to this biphasic action in the cerebellum, activation of presynaptic kainate receptors with low concentrations of agonist exclusively depresses glutamate release at cortical inputs to the nucleus accumbus (165). Both GluR5 and GluR6 subunits contribute to this presynaptic activity. Similarly, synaptic release of glutamate activates presynaptic kainate receptors at thalamocortical synapses and contributes significantly to the short-term depression observed during brief high-frequency trains at early developmental stages (166). Glutamate and GABA release at synapses in the spinal cord are modulated by presynaptic kainate receptors. Dorsal root ganglia (DRG) contain a subpopulation of small- and medium-diameter nociceptive neurons that express kainate receptors as their predominant type of ionotropic glutamate receptor (167). It is not clear whether these neuronal receptors have a polarized distribution in situ because they are localized on cell bodies (167), dorsal root axons (168), sensory terminals in the skin (169,170) and at presynaptic sites in the spinal cord (171). Application of the GluR5-selective agonist ATPA suppressed glutamate release at the dorsal root–layer 2 neuron synapse (171), consistent with the strong expression of GluR5 mRNA in these cells (172) and profound reduction in DRG kainate receptor currents in GluR5−/− mice (160,173). In contrast, GluR6 rather than GluR5 subunits contributed significantly to kainate receptors in local interneurons in the spinal cord (173). The putative role for kainate receptors in modulation of pain processing inferred from these functional roles in nociceptive pathways has driven the interest in GluR5-selective compounds as potential anti-nociceptive agents (174,175). Presynaptic kainate receptors also influence inhibitory transmission between BLA interneurons and BLA pyramidal neurons in the basolateral amygdala, where the GluR5 subunit is expressed at high levels. Application of low concentrations of ATPA decreased the number of failures of evoked IPSCs, although it seems likely that this was due to depolarization of BLA interneurons via somatodendritic and axonal receptors rather than via presynaptic receptors (176). Higher concentrations of ATPA, however, inhibited evoked IPSCs in BLA neurons through activation of presynaptic receptors (176). Similarly, ATPA and glutamate had bidirectional concentration-dependent effects on miniature IPSCs, supporting the presence of presynaptic receptors on GABAergic terminals (176). This bi-directional activity is a unique feature of these kainate receptors and sets them apart from interneuron-to-pyramidal cell synapses in the hippocampus and neocortical synapses, where kainate receptors solely depress mIPSC frequency (117,154), or other inhibitory synapses at

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which kainate receptors facilitate GABA release (including the hypothalamus (177) and substantia nigra pars compacta (178)). 2.2.4. Kainate Receptors and Synaptic Plasticity The role of kainate receptors in long-term synaptic plasticity has been the subject of considerable research interest, and, as with many other aspects of synaptic kainate receptor function, has been best characterized at the mossy fiber synapse in the hippocampus. Graham Collingridge and colleagues first reported that a then-novel selective kainate receptor antagonist, LY382884, completely blocked the induction of NMDA receptor–independent LTP at mossy fiber synapses but had no effect on NMDA receptor–dependent LTP at the collateral synapses on the more distal dendrites of the CA3 neurons (Fig. 4A) (179). This finding was quite surprising and controversial because it was known that mossy fiber LTP could be induced in the presence of the nonselective AMPA/kainate receptor antagonist kynurenate or 6-cyano7-nitro-quinoxaline-2,3-dione (CNQX) (180–182). Contrary to these previous studies, however, Collingridge and colleagues found that, under their experimental conditions, relative low concentrations of CNQX occluded LTP (179). A second perplexing aspect to these results was that LY382884 was reportedly a GluR5-selective antagonist (183), and GluR5 mRNA is largely absent from the dentate gyrus and CA3 principal neuronal populations in the hippocampus after the first week of development (106,107). Furthermore, a subsequent report failed to observe any affect of LY382884 on mossy fiber LTP (152). Despite these controversies, additional support for a role of kainate receptors in mossy fiber LTP emerged with the characterization of kainate receptor knockout mice (146). GluR6−/− mice exhibited a profound deficit in mossy fiber LTP induced by high-frequency stimulation, but GluR5−/− mice had normal LTP (Fig. 4B) (146). Subsequent studies demonstrated that GluR6-containing receptors were not absolutely required for LTP, but, in a paradigm that elicited subsaturating potentiation, instead reduced the threshold for the induction of plasticity (152,184). That is, mossy fiber LTP was induced normally in the knockout mice if induction strength was increased or if terminals were depolarized with elevated extracellular K+ (184). In this way, kainate receptor activation could act as detectors of activity in neighboring synapses, thereby imparting an associativity to the induction of LTP at the mossy fiber synapse (Fig. 4C) (184). A few additional examples of kainate receptor involvement in LTP have been described in the CNS. LTP of excitatory transmission is absent both at thalamic inputs to lateral amygdala neurons and in auditory cortex neurons in GluR6-knockout mice, consistent with correlative behavioral studies of fear memory (185). In the basolateral amygdala, GluR5-containing kainate receptors mediate a long-term facilitation that is not input specific, unlike

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classically defined LTP (186). The synaptic and cellular mechanisms of kainate receptor involvement in these forms of plasticity are not known. 2.2.5. Kainate Receptors in Network Oscillations In the hippocampus, kainate receptors also play a role in supporting oscillatory activity in the neuronal network. Rhythmic network activity, particularly in the gamma frequency range, is thought to be required for normal brain function and cognitive processes. Kainate receptor activation has been a frequently used model to induce this oscillatory pattern of firing in vitro; very low concentrations of kainate receptor agonists induce oscillations in hippocampal slices that are long lasting and specifically require kainate receptor

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subunits for their induction and propagation (130). Overactivation of the receptors with higher concentrations of kainate in this model leads to epileptiform burst activity of the slice. GluR6-knockout mice lack kainate-induced oscillatory activity, whereas GluR5-null mice are more sensitive to induction of epileptiform activity (129), demonstrating that GluR6-containing receptors (localized predominantly on principal neurons) control excitatory dynamics of oscillations and subsequent seizures, whereas GluR5-containing receptors in interneurons set the inhibitory tone required to support the oscillations and increase the seizure threshold of the CA3 network (129). 2.2.6. Roles of Kainate Receptors in Synapse Development Kainate receptors have been suspected to play roles in developmental processes because of the marked changes in mRNA expression and editing status development of the GluR5 and GluR6 receptor subunits (59,70). For example, kainate receptors are present at thalamocortical synapses in the barrel cortex only during a restricted developmental period that coincides with the critical period for plasticity (111). These receptors contain unedited subunits, as demonstrated by their characteristic rectifying current–voltage relationships (111). After closure of the critical period, kainate receptors are no longer localized to synaptic sites and AMPA receptors primarily underlie fast synaptic transmission in these neurons. This switch in synaptic constituents, from a mixed population including slow kainate receptors to solely fast AMPA receptors, alters the kinetics of the EPSP and enhances EPSP-spike coupling by decreasing latency and jitter of synaptically evoked action potentials (187). In the neonatal hippocampus, presynaptic kainate receptors modulate network activity and establish low basal release probabilities at Schaffer collateral synapses. This occurs through tonic activation of presynaptic GluR5containing receptors by endogenous glutamate, which in turn reduces the basal glutamate release probability (188). Having a low probability of release is thought to permit a strong facilitation during high frequencies of transmission, and thus kainate receptors bias the hippocampal network to respond efficiently to characteristic bursts of activity that contribute to the maturation of the developing hippocampal network (189). Structural development of mossy fiber synapses is also influenced by kainate receptor activity (190). The motility of filopodial extrusions from mossy fiber axons decreases during development, which is thought to reflect a maturation process leading to the formation of stable synaptic contacts. This process appears to be dependent on kainate receptor activation, which plays multiple roles by first contributing to the motility of the filopodia as they “explore their environment” and then later in development reducing motility of the filopodia to help stabilize the synaptic contacts (190). Studies in knockout mice have also hinted at a role of kainate receptors in functional maturation of the mossy

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fiber synapse (191). AMPA receptor–mediated currents are reduced during early postnatal times in GluR5/GluR6 double-knockout mice, suggesting that functional synaptic kainate receptors are required for early maturation of these synapses (191). 2.2.7. Behavioral Studies Using Gene-Targeted Mice Characterization of mice with targeted mutations in kainate receptor subunit genes has yielded a wealth of information on their various roles in neurotransmission, but behavioral tests on these animals to determine their function in vivo has been less well explored. The initial description of the GluR6-null mice included the observation that these mutants were less susceptible to kainateinduced seizures and the associated excitotoxicity present in the hippocampus (103). This was an important finding because it was the first direct evidence of the seizurogenic potential of kainate receptors. In addition, mutant mice containing a deletion of the intronic GluR6 editing complementary site, which is required for Q/R-site RNA editing, were more sensitive to kainate-induced seizures, further supporting a critical role for GluR6 receptor subunits in this chemical form of seizure induction (192). In both knockout and editing GluR6 mutant mice a battery of standard behavioral tasks did not reveal any genotypespecific deficits (103,192). Similar general analyses are lacking in other kainate receptor mutant mice. The complex picture that has built up from recordings in slice preparations suggests that spatially restricted conditional knockout mice would provide greater insight into the involvement of kainate receptors in seizure generation and propagation. In addition to seizurogenic processes, kainate receptors have been proposed to play a role in nociception because receptor subunits are expressed widely in sensory neurons and afferent fibers in the spinal cord. Consistent with this hypothesis, knockout mice lacking the GluR5 subunit exhibit a profound deficit in pain-evoked behaviors following paw injection of formalin and capsaicin (185). Furthermore, relatively selective antagonists of GluR5containing receptors reduce nociceptive responses in several models of pain (see Section 5) (174,175,193,194). Finally, Zhuo and colleagues correlated a disruption in contextual and auditory fear memory in GluR6-knockout mice with a role for GluR6-containing receptors in synaptic transmission and plasticity in the amygdala (185). 2.3. Kainate Receptors and Disease Linkage studies of kainate receptor genes are beginning to identify potential disruptions or alterations in human subunit genes that might contribute to diseases with a hereditary component. These studies have supported an association between GRIK2 (GluR6) and schizophrenia (195) and autism (196).

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Polymorphisms have also been discovered in the GRIK2 gene linking it to Huntington disease (197) and in the GRIK3 gene linking it to schizophrenia (77), although this association could not be replicated in different populations (79,198). Alterations in RNA expression of kainate receptor subunits have been observed in a number of brain regions of schizophrenics (199–202), and changes in protein levels have been noted as well (203). Although these studies hint at the potential involvement of kainate receptors in some psychiatric disorders, there have been few definitive studies that positively determine how kainate receptor signaling is altered in these diseases and how this contributes to the progression of the disease. Recent work has specifically implicated kainate receptors expressed on oligodendrocytes in initiating activation of the complement system (204). Brief activation of kainate receptors, which in itself did not cause significant toxicity, increased the sensitivity of cultured oligodendrocytes to attack by complement (204), leading ultimately to formation of the membrane attack complex and osmotic lysis of the cells. Oligodendrocytes are known to be particularly vulnerable to complement attack in multiple sclerosis and other neurodegenerative diseases, suggesting that specific targeting of kainate receptors on oligodendrocytes represents a novel strategy for tackling these diseases. Kainate receptors also were linked to excitotoxicity and ischemia through an interaction with postsynaptic density (PSD)-95 and mixed lineage kinase 3 (MLK3), which mediates apoptosis through c-Jun NH2 terminal kinase (JNK) (205). Ischemic insult in rats caused an increase in the association of GluR6, PSD-95, and MLK3. The authors proposed a model in which association of these molecules leads to activation of MLK3, which then dissociates from the complex to activate JNK. Future studies in knockout mice will help to validate this model and clarify the specificity of the involvement of kainate receptors in excitotoxicity. As mentioned earlier, kainate receptors have long been associated with epileptogenic activity. It remains unknown, however, whether aberrant kainate receptor signaling contributes in any way to the disease process in humans. Peritoneal kainate injection in rodents is a well-established animal model for studying human temporal lobe epilepsy. It is now clear that activation of kainate receptors themselves is critical to induction of seizures with this compound (156). In particular, the high level of expression of kainate receptors in the CA3 region of the hippocampus makes this region one focal point for seizure generation and subsequent seizure-induced neuronal damage. Knockout mice in which the GluR6 receptor subunit (the principal subunit expressed in CA3 pyramidal neurons) is ablated have an increased tolerance to systemic kainate (103). Conversely, a mutant mouse strain in which the GluR6 receptor subunit cannot undergo post-transcriptional RNA editing modification has an increased susceptibility to kainate-induced seizures (192). Although there is little doubt

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about the consequences of systemic kainate administration, it is less clear how different populations of receptors—kainate receptors that modulate both excitatory and inhibitory transmission and AMPA receptors underlying fast transmission—produce the spectrum of behaviors characteristic of kainateinduced seizures.

3. Expression, Trafficking, and Targeting 3.1. mRNA Expression and Protein Distribution Kainate receptor mRNAs are expressed throughout the central and peripheral nervous systems (106,206,207). An exhaustive review of the distribution is beyond the scope of this chapter, but several generalizations can be made regarding their localization. GluR5, GluR6, and KA2 appear to be the “principal” receptor subunits expressed in the CNS, with GluR5 and GluR6 only rarely overlapping in neuronal expression. For example, in the hippocampus, GluR6 mRNA is primarily but not exclusively localized to the glutamatergic granule and pyramidal neurons, whereas GluR5 mRNA is predominantly found in interneurons (except early in development) (87,106). In the cerebellum, GluR5 mRNA is strongly expressed in Purkinje cells and GluR6 mRNA in granule neurons (106). There are certainly exceptions to this apparent mutual exclusivity, for example, in a subset of hippocampal interneurons (87). GluR5 is strongly developmentally regulated in many neuronal populations as compared to other kainate receptor subunit mRNAs, reaching peak expression levels in the hippocampus and cortical structures late in gestation and in the first week after birth (in rats) (106). On the other hand, in cerebellar Purkinje neurons, GluR5 mRNA is strongly expressed throughout development and into adulthood. Of the five family members, the KA2 subunit is expressed most ubiquitously in the CNS. Relatively few populations of neurons do not express KA2 mRNA; cerebellar Purkinje cells and interneurons are prominent exceptions (106). KA1 mRNA exhibits the most restricted distribution of the five subunits. Indeed, KA1 mRNA has been detected almost exclusively in CA3 pyramidal neurons, dentate gyrus granule cells, and subicular neurons (106,206,208). 3.2. Kainate Receptor Trafficking and Targeting Kainate receptor subunit proteins are assembled and targeted to a variety of functional domains in central and peripheral. Postsynaptic kainate receptors are relatively rare compared to postsynaptic AMPA and NMDA receptors, and even within single neuronal populations they are selectively targeted to a subset of synapses (e.g., mossy fiber synapses in CA3 neurons (97,98) or cerebellar climbing fiber synapses on Purkinje neurons (122)). Indeed, presynaptic kainate receptors, like those found at mossy fiber and Schaffer

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collateral synapses, appear to be more abundant in the CNS. Kainate receptors at nonsynaptic sites regulate axonal or neuronal excitability (124,143). The precise site of targeting does not obviously depend on subunit composition of the receptors. For example, GluR6 and KA2 subunit-containing receptors are targeted to postsynaptic mossy fiber synapses by CA3 pyramidal neurons and, on the other side of the synapse, axons and presynaptic boutons by granule cells (100,103,104,142). Despite this intriguing variety and tight regulation of their functional localization, however, the cellular mechanisms that selectively target kainate receptors to different functional sites in neurons are entirely unknown. Quite a bit more is understood about molecular and cellular mechanisms that control the biosynthesis and subcellular trafficking of kainate receptors. Homomeric kainate receptors are expressed on plasma membranes of transfected heterologous cells to differing degrees: extremely dense (e.g., GluR6a) or vanishingly sparse (e.g., KA2). As with other intrinsic membrane channels and receptors, many determinants exist within the receptor subunits themselves that control their subcellular fate and plasma membrane expression. These determinants often take the form of short, discrete sequences of amino acids in cytoplasmic domains of the receptor proteins. Trafficking signals found in the cytoplasmic domain of kainate receptor subunits have been generally categorized into two groups: arginine-rich endoplasmic reticulum (ER) retention/retrieval motifs and polybasic forwarding trafficking determinants (37). Those subunits that are strongly retained in the endoplasmic reticulum, such as GluR5-2b and KA2, have trafficking determinants composed of arginine-rich domains (41,42,86). For KA2, it has been demonstrated that ER sequestration is mediated by interactions between the polyarginine site and coat complex proteins COPI, a vesicular retrograde retrieval system (209). In contrast, the GluR6a and GluR7a receptor subunits contain a forward trafficking domain, the amino acids CQRRLKH, that efficiently exports assembled receptors to the plasma membrane (42,46). The cellular protein(s) that bind to this site have not been identified. To progress forward in the secretory pathway from the ER, new receptors also must pass a different type of quality control checkpoint that appears to be designed to assay the functional status of the receptor complex. Thus, elimination of glutamate-binding sites in AMPA and kainate receptor subunits causes sequestration of assembled receptors in the ER (33,210–212), as does mutation of sites that alter desensitization properties (213). Because of the presence of appropriately polarized transport across ER membranes (214), it is likely that glutamate concentrations are high enough to bind and induce conformational changes in newly assembled glutamate receptors. The precise mechanism of retention of these binding-incompetent receptors is not known, but one possibility consistent with the data is that ligand-associated

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conformational changes expose domains on the receptor subunits to cellular chaperones responsible for ER exit.

4. Interaction Partners Kainate receptor subunits interact with a number of proteins, some of which affect their function and subcellular distribution. Much of the research in this area has focused on the influence of PDZ-domain proteins, such as PSD95, because the majority of subunit splice isoforms contain interacting motifs at their carboxy-terminal tail. Other types of interacting proteins have been isolated through both hypothesis-driven assays and proteomic-based screens. With few exceptions, however, the functional significance of these interactions in neurons and at synapses is not well understood, and consequently we lack a well-developed understanding of the neuronal receptor—protein interactions analogous to those recently elucidated for AMPA and NMDA receptors. 4.1. PDZ Proteins A number of kainate receptor subunits interact with PDZ-domain proteins through canonical motifs at their carboxy termini as well as at less well-defined sites in the cytoplasmic domain. Some of these interactions influence receptor function in vitro, and occlusion of PDZ protein-subunit interactions can alter the stability of synaptic receptors. In the first study of its kind for kainate receptors, John Marshall and colleagues demonstrated that GluR6a subunits interacted in the rat brain (and in vitro) with a variety of PSD-95 family members, including PSD-95 itself, SAP97, and SAP 102 (215). The KA2 subunit, on the other hand, solely associated with PSD-95. For GluR6a, the terminal “ETMA” sequence acted as a binding motif for the PDZ1 domain in PSD-95, whereas proline-rich sequences in KA2 interacted with the SH3 and guanylate kinase domains in PSD-95. Interactions with the PDZ proteins had marked effects on clustering of kainate receptors in transfected heterologous cells. Coexpression of GluR6 and KA2 with PSD-95 also significantly reduced the degree of desensitization of glutamate-evoked currents in this study, suggesting that the PSD-95-induced clustering of the receptors strongly influenced their biophysical properties. A subsequent study from the same group (in part), however, observed a much more subtle functional modification of receptor currents by PSD-95 (216), The roles that PSD-95 family members might play in shaping synaptic kainate receptor function are unknown. The class II PDZ domain proteins GRIP, PICK1, and syntenin interact to differing degrees with a variety of kainate receptor subunits, including GluR5-2b, GluR5-2c, and GluR6a (114). PICK1 and GRIP association with GluR5-2b is mediated by the carboxy-terminal “ETVA” sequence. Syntenin, in

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contrast, appears to require multiple distributed domains that includes elements of the terminal PDZ-binding motif and consequently does not interact with the GluR5-2c subunit, which contains the same 20 residues at the carboxy terminal as the GluR5-2b isoform. Interaction with GRIP and PICK1 proteins appear to stabilize kainate receptors at mossy fiber synapses because KA-EPSC amplitudes decrease in the presence of peptides or recombinant proteins that interfere with the PDZ-subunit association (114). 4.2. BTB-kelch Proteins A pair of recent reports described the interaction of two related proteins, actinfilin and KRIP6 (for “kainate receptor interacting protein for GluR6”), with the GluR6a kainate subunit (217,218). These molecules belong to a family of multifunctional proteins that contain interaction domains known as BTB/POZ (“bric a brac, tramtrack, broad complex/poxvirus and zinc finger”) and kelch motifs. Actinfilin is associated with actin, as the name suggests, and couples the kainate receptor subunit to the E3 ubiquitin ligase Cul3, thereby targeting receptors for degradation (218). Acute reductions in actinfilin using RNAi markedly increased immunofluorescent localization of GluR6 subunits at synapses in cultured hippocampal neurons, supporting a role for this pathway in dynamic regulation of kainate receptors in the plasma membrane (218). Both actinfilin and KRIP6 bind to GluR6a subunits at a distributed domain in the cytoplasmic C-terminal tail. The role of KRIP6 in GluR6a trafficking or function was less clear; overexpression of the interacting protein reduced kainate receptor peak current density while increasing steady-state current amplitudes in transfected mammalian cells and in cultured hippocampal neurons (217). Unlike actinfilin, KRIP6 is not thought to associate with actin and thus might play a fundamentally distinct role in relation to GluR6containing kainate receptor function (217). 4.3. Proteomic Analysis of GluR6-Interacting Proteins Additional GluR6 subunit-interacting proteins were identified with biochemical assays following immunoprecipitation from transgenic mice overexpressing a myc-tagged GluR6a subunit (219). These studies found an association with the cadherin/catenin complex of adhesion molecules. Myc-GluR6 also co-precipitated with the cytoplasmic proteins CASK, Velis, and Mint, which are known to form macromolecular complexes with the cadherin/catenin molecules (219). Activation of cadherins in a transfected heterologous cell line caused a redistribution of myc-GluR6, suggesting that this interaction in neurons might play a role in synaptic localization of kainate receptors.

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In a follow-up study, Christophe Mulle and colleagues carried out the first proteomic screen of proteins associated with the two GluR6 subunit isoforms (44). A variety of techniques, including immunoprecipitation with antibodies selective for the GluR6a (that also recognize GluR7a) and GluR6b isoforms, pull-down assays, mass spectroscopy, and differential screening of the mycGluR6a transgenic mouse, were used to identify a set of proteins that interacted with the receptor subunits. Several proteins associated with trafficking processes, such as spectrin and dynamin-1, immunoprecipitated with GluR6a, as did the important signaling molecule calmodulin. The latter also interacted with GluR6b protein; other Ca2+ -sensitive molecules isolated included protein phosphatase 2B (calcineurin) and visinin-like proteins (VILIPs) (44). It is interesting that the authors did not detect PDZ proteins known to interact with neuronal kainate receptors (e.g., PSD-95), possibly because a number of proteins isolated on gels were not subsequently identifiable by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry in the proteomic analysis. 4.4. Proteins Involved in Trafficking of Kainate Receptor Subunits The intracellular proteins and pathways that regulate kainate receptor trafficking through interactions with critical carboxy-terminal determinants remain largely unidentified. The exception to this generalization is the mechanism of retention/retrieval of KA2 subunits; the coatomer protein complex 1 (COPI), an important system for retrieval of ER-resident proteins, was shown to interact with the arginine-rich domain in KA2 to prevent forward trafficking of homomeric assemblies of this subunit (209). Coassembly of KA2 with GluR5 or GluR6 subunits greatly reduces (or eliminates) interaction between KA2 and COPI, thereby releasing the heteromeric receptor to proceed through the secretory pathway.

5. Kainate Receptor Pharmacology Kainate receptors are the target of a diverse group of natural products and synthetic analogs that have played important roles in the development of our current appreciation for the diversity of glutamatergic neurotransmission. Kainic acid, derived from the red algae Digenia simplex (220), was used to demonstrate unequivocally the presence of a population of non-NMDA receptors distinct from AMPA receptors (which at that time were known as quisqualate receptors) in the dorsal root (167,221,222). Interest in kainic acid was stimulated by early observations that it was a potent convulsant and produced pathologic alterations in rat brain similar in many ways to those observed in humans with mesial temporal lobe epilepsy (TLE) (221,223). The “kainate model” of TLE developed subsequently led to many important

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insights regarding the function of neuronal kainate receptors, particularly those at hippocampal mossy fiber synapses (156), and is still used as one of several chemical models for testing the efficacy of potential new anticonvulsant agents. It is worth noting, however, that kainate is an effective agonist for AMPA receptors as well as kainate receptors, and both receptor systems are likely engaged when the compound is used in high doses in vivo or in vitro. The pathology observed in the kainate model of TLE therefore does not solely result from kainate receptor activation, and seizures are elicited by the drug even in the absence of functional kainate receptors in principle hippocampal neurons (e.g., in GluR6−/− mice) (103). All kainate receptor agonists that activate receptors containing the GluR6 subunit are potent convulsants, in large part because activation of these receptors in hippocampal CA3 pyramidal neurons effectively initiates synchronized firing of the recurrent CA3 network. The cloning of kainate receptor subunits and subsequent analysis of their ligand-binding properties revealed that they have diverse pharmacologic profiles (224), which in turn has spurred synthetic attempts to generate subunitselective compounds. The success of some of these efforts, in particular the generation of GluR5-selective compounds, is discussed in the following sections. Creation of these new tools represents one of the significant advances in kainate receptor pharmacology in the last decade. The recent resolution of ligand-binding domain structures for kainate receptors also provided key insights into the molecular interactions between ligands and receptors that conferred subunit specificity (20–23), as has molecular modeling based on the resolved structures (225). Despite these advances, however, problematic holes still exist in the pharmacologic toolbox for kainate receptors. In particular, there is no means selectively to activate or inhibit GluR6-containing kainate receptors without simultaneously affecting other types of kainate receptor subunits. In addition, many of the compounds that effectively inhibit kainate receptors also occlude AMPA receptor activation with similar (or greater) potency, including quinoxalinedione compounds such as CNQX and 2,3-dihydroxy-6nitro-7-sulfamoy-benzo(F)quinoxaline (NBQX). 5.1. Nonselective Kainate Receptor Agonists The endogenous excitatory neurotransmitter l-glutamate is a low-affinity and nonselective agonist that elicits rapidly activating and desensitizing currents from recombinant kainate receptors. The EC50 values for homomeric GluR5 and GluR6 receptors are similar to those observed for AMPA receptors (0.5–0.6 mM) when l-glutamate is rapidly applied to evoke nonequilibrium currents (4,89), which most accurately mimics synaptic activation of the receptors. In contrast, GluR7 receptors are relatively insensitive to l-glutamate (EC50 ∼ 6 mM) (45); this “reluctance” to gate current arises not from an inability to bind to the transmitter, but rather because residues unique to the

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GluR7 subunit impair gating in response to ligand binding (226). The potency of l-glutamate for heteromeric receptors containing KA1 or KA2 subunits has not been reported. Native kainate receptors in cultured hippocampal neurons exhibit a similar sensitivity to glutamate (0.3 mM) (227). In addition to l-glutamate, AMPA and kainate receptors are jointly activated by a number of agonists with differing degrees of potency and efficacy. For example, the eponymous ligands AMPA and kainate are only moderately selectively for their receptor families. AMPA is a low-affinity agonist for GluR5and KA2-containing kainate receptors but does not bind GluR6 or GluR7 subunits, a difference that is attributable to a single amino acid difference in the S2 domain (27). Kainate is a potent agonist for most kainate receptors, again excepting GluR7-containing receptors. Kainate-evoked currents from GluR6or KA2-containing receptors are rapidly desensitizing, similar to those elicited by l-glutamate (4,49). Homomeric GluR5 receptors, on the other hand, exhibit a distinct and slowly desensitizing current in response to kainate that bears a striking resemblance to that observed in a subset of neurons from dorsal root ganglia (6,43). Several agonists that activate kainate receptors to a much greater degree than AMPA receptors are among the most potent convulsants identified. Kainate itself has been used extensively to generate seizures in an animal model of temporal lobe epilepsy, but two other natural marine toxins are substantially more potent in eliciting acute convulsions in laboratory animals. Domoic acid is a high-affinity kainate receptor agonist that is produced by pennate diatoms of the genus Pseudonitzschia, which can be concentrated in shellfish. Domoic acid levels in shellfish have been monitored routinely following an incident of human poisoning in 1987 (228,229); the consequent pathology was named amnesiac shellfish poisoning, and long-term sequelae included a number of neurologic disturbances. Periodic die-offs of marine birds and mammals still occur despite the monitoring efforts (230,231). More recently, a novel di-amino, di-acid analog of glutamate, dysiherbaine, was isolated from the marine sponge Dysidea herbacea on the basis of its seizurogenic activity (232). Dysiherbaine is the most potent convulsant excitatory amino acid yet described and has a particularly high affinity for certain kainate receptor subunits; on homomeric GluR5 receptors, for example, the binding affinity (Ki value for displacement of kainate) was determined to be approximately 0.5 nM (233). An interesting and potentially useful aspect of the pharmacologic profile of dysiherbaine is that its affinity for the kainate receptor subunits GluR5 and GluR6 (and presumably GluR7) is four orders of magnitude higher than that for the accessory receptor subunits KA1 and KA2. This marked difference in affinity is inverted compared to that for the majority of other kainate receptor agonists, such as kainate and domoate, which have a greater binding affinity for KA1 and KA2. In addition to the natural compounds

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domoic acid and dysiherbaine, the synthetic agonist 2S,4R-4-methylglutamate (SYM 2081) exhibits very high affinity for kainate receptors (95,148). The receptors desensitize in the continued presence of very low concentrations of 4-methylglutamate, allowing it to be used effectively in a number of studies as a functional antagonist [e.g., 234]. 5.2. Nonselective Kainate Receptor Antagonists With few exceptions (as discussed later), all competitive antagonists that inhibit activation of kainate receptors also inhibit AMPA receptors with varying degrees of selectivity. For example, the well-characterized quinoxalinedione antagonist CNQX inhibits AMPA receptors with low-micromolar potency and kainate receptors with a wide range of IC50 values (4–76 μM) (3,235), depending on the subunit composition of the receptors. NBQX, in contrast, is ∼100-fold more selective for AMPA receptors and therefore can be used to isolate synaptic kainate receptor currents (107). A structurally related pyrrolylquinoxalinedione, LU 97175, was ∼3-fold selective for kainate sites compared to AMPA sites in radioligand-binding experiments from rat brain (236), but the potency of this compound for inhibition of receptor activation has not been reported. 5.3. Selective Agonists Characterization of a kainate receptor agonist as “selective” is complicated by the fact that most neuronal receptors appear to be assembled from more than a single type of subunit. Thus, compounds that are highly selective for defined recombinant receptors in heterologous cell lines often are significantly less selective when applied to neuronal kainate receptors, whose subunit stoichiometries are largely a matter of guesswork. Even with this caveat, however, it is clear that a number of agonists have been identified that are selective for receptors containing the GluR5 subunit. Most of these compounds are derived from synthetic modification of the nonselective agonists AMPA or willardiine, a heterocyclic amino acid that occurs naturally in Acacia and Mimosa seeds (237). The AMPA analog ATPA activates homomeric and heteromeric kainate receptors containing GluR5 subunits with low micromolar potency and at least 100-fold selectivity (235,238). ATPA sensitivity is used in testing for a role of GluR5-containing receptors in neuronal function (109,171,186,238), although interpretation of this pharmacologic activity has not been without controversy (144). Structural modification of the willardiine molecule has produced a family of agonists with a spectrum of selectivities for AMPA and kainate receptors. Application of these compounds to structural studies was integral to the construction of a physical model for full and partial agonism of ionotropic glutamate receptors (239). (S)-5-Iodowillardiine

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is highly selective for GluR5 subunit–containing kainate receptors and, similar to ATPA, elicits steady-state currents from these receptors with low micromolar potency (92,235,240). It should be emphasized that ATPA and (S)-5-iodowillardiine will activate all receptor assemblies containing GluR5 subunits—for example, GluR5, GluR5/KA2, and GluR5/GluR6 receptors— with roughly equivalent potencies (58,92,235), and thus the utility of the compounds is limited to assaying for the presence or absence of GluR5 subunits in a population of kainate receptors. 5.4. Selective Antagonists Most selective and competitive antagonists for GluR5-containing kainate receptors (KARs) have been generated from decahydroisoquinoline or willardiine (pyrimidine) templates. Three decahydroisoquinoline antagonists— LY293558, LY377770, and LY382884—show varying degrees of selectivity for GluR5-containing KARs compared to AMPA receptors; all three compounds exhibit little or no affinity for GluR6 or GluR7 subunits (183,194,235,241). LY293558 is roughly equipotent for inhibition of AMPA and GluR5 KARs (241,242), whereas LY382884 potently inhibits GluR5containing KARs but shows little affinity for AMPA receptors, making this compound a highly selective GluR5 antagonist (179,194,242,243). A new willardiine analog, UBP 302, selectively inhibits GluR5 receptors with little activity on AMPA or other kainate receptor subunits (244). UBP 302 inhibited glutamate-evoked calcium signals from GluR5-expressing cells with an IC50 of 3.5 μM, and 10 μM of the compound inhibited whole-cell currents by 83% (244). UBP 302 is the first potent and selective kainate receptor antagonist made available commercially. More recently, MSVIII-19, an analog of the convulsant dysiherbaine, was shown to be a potent GluR5 antagonist with an IC50 of 23 nM for inhibition of glutamate-evoked whole-cell currents (245). LY382884, UBP 302, and MSVIII-19 all exhibit >200-fold selectivity for inhibition of GluR5-containing kainate receptors compared to AMPA receptors. A nominally GluR6-selective kainate receptor antagonist, NS-102 (246), is commercially available but has been used in relatively few studies because of its limited solubility in aqueous solutions. Furthermore, the subunit selectivity of NS-102 for kainate receptors is not well characterized. A significant advance in kainate receptor pharmacology was made recently with the introduction of noncompetitive GluR5 receptor antagonists with varying degrees of selectivity and potency (247,248). The most potent analog of these 2-arylureidobenzoic acids, NS3763, inhibited GluR5 receptor—evoked calcium signals with an IC50 in the low micromolar range (249). Furthermore, NS3763 is selective for homomeric GluR5 receptors; coassembly with either GluR6 or KA2 subunits greatly reduced the potency of the compound for

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inhibition of receptor currents (163). The site of action of these noncompetitive antagonists is unknown, although two splice isoforms of GluR5 differing in both an extracellular and a cytoplasmic domain exhibited markedly different sensitivities to NS3763 (249), suggesting that one or both of these domains were involved in allosteric inhibition. NS3763 is available commercially and will likely prove a valuable tool for exploring kainate receptor stoichiometry and the role of homomeric GluR5 receptors in physiologic processes. 5.5. Allosteric Potentiation by Lectins A variety of plant lectins potentiate both peak and steady-state kainate receptor currents through allosteric mechanisms that are not well understood mechanistically. Lectins are proteins that bind to oligosaccharide groups covalently attached to proteins during posttranslational processing of receptor subunits; binding of multivalent lectins causes aggregation of glycoproteins. Kainate receptor modulators are primarily of the high mannose–binding family typified by concanavalin A (conA), a lectin from Canavalia ensiformis (the jack bean). Application of conA effectively potentiates steady-state glutamate-evoked currents from most kainate receptors by up to several orders of magnitude (172). The binding site for conA on kainate receptors is known to consist of Nlinked mannose groups, but the positioning of glycosylation sites in the receptor protein does not appear to be of particular importance (82). Furthermore, interaction of conA with kainate receptor subunits is state dependent; agonistinduced desensitization of receptors before application of conA occludes potentiation by the lectin (250,251). Unlike the action of benzothiazides and other allosteric modulators of AMPA receptors, conA-induced potentiation of kainate receptors is irreversible. This potentiating action has proved useful for characterizing pharmacologic properties of agonists in expression systems such as Xenopus oocytes, where fast application of agonists is problematic and extremely small steady-state currents (like those evoked by glutamate on kainate receptors) are difficult to measure accurately. In contrast, concanavalin A only weakly potentiates whole-cell currents elicited from cultured hippocampal neurons (227) and does not alter synaptic kainate receptor currents in acute brain slice preparations, which might be caused by additional processing of oligosaccharides on neuronal kainate receptor subunits or limited accessibility to the synapse. 5.6. Other Allosteric Modulators Monovalent ions play an important role in the biophysical operation of kainate receptors beyond simple permeation of the channel during receptor activation. In contrast to AMPA receptors, sodium and chloride appear to be integral to

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proper functioning of GluR6 kainate receptors (251,252); their absence “traps” the receptor in a high-affinity closed state (253). Protons influence kainate receptor function in a subunit-dependent manner. Physiologically relevant proton concentrations (pH ∼7) partially inhibit a number of recombinant kainate receptor combinations, including GluR6 and GluR6/KA2, but potentiate GluR6/KA1 receptors (254). Polyamines such as spermine also modulate kainate receptors by competing for the same site as protons and thereby relieving inhibition, although potentiation is only observed for Q/R-site edited (arginine-containing) receptor subunits because extracellular polyamines, like intracellular spermine, occlude channel permeation in receptors with unedited (calcium-permeable) subunits (254). The acute proton sensitivity of these subunits suggests that the function of synaptic kainate receptors will be dramatically altered during ischemic states, but a direct test of this hypothesis has not been reported. The Q/R editing site also plays a central role in determining the sensitivity of kainate receptors to inhibition by cis-unsaturated fatty acids such as arachidonic acid and docosahexanoic acid (255,256). Docosahexanoic acid potently inhibited homomeric edited GluR6(R) receptors and, at lower potencies, neuronal kainate receptors but did not occlude currents evoked from homomeric or heteromeric receptors containing unedited subunits (256). Despite the importance of the Q/R-site residue, which likely resides within the voltage field of the plasma membrane, fatty acid inhibition of GluR6(R) receptors was not voltage dependent, suggesting that the interaction with the editing-site residue might be mediated by an indirect mechanism.

6. Functional Modulation As with all other glutamate receptors, there has been considerable interest in post-translational modifications that might alter receptor function and contribute to plasticity at excitatory synapses. The biophysical properties of kainate receptors are modulated by a number of second-messenger systems, which can additionally alter trafficking of recombinant and neuronal receptors, but the significance to synaptic kainate receptor function remains largely undescribed. The GluR6a receptor subunit is phosphorylated by PKA (257,258), and this modification enhances macroscopic kainate receptor currents by increasing the open probability of the receptor channel (89). PKA has also been implicated the intracellular trafficking of kainate receptors following internalization induced by NMDA treatment of cultured hippocampal neurons, although it is not known whether this occurs through a direct phosphorylation of the receptors (259). Kainate receptors can also be modulated functionally by PKC. In cultured cortical neurons, receptor currents are enhanced upon activation of PKC by

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phorbol esters, and PKC is thought to mediate the heterologous modulation of kainate receptors by group 1 mGluRs (260). Phosphorylation by this kinase also may stabilize kainate receptors at hippocampal mossy fiber synapses, with the PDZ protein PICK1 acting as an intermediary to target the kinase to the receptor (114). In contrast, PKC-dependent processes mediate multiple forms of agonist-dependent internalization of extrasynaptic kainate receptors in cultured hippocampal neurons (259). Although it is clear that, in vitro, PKC can directly phosphorylate carboxy-terminal fragments of kainate receptor subunits (114), it is less evident which of these diverse functional activities result from direct phosphorylation of kainate receptors versus indirect modulation through ancillary proteins or signaling complexes. An early study demonstrated that whole-cell GluR6a receptor currents were enhanced by CaMKII, although this was attributed to modification of a serine now known to be on the extracellular domain of the receptor (261). In hippocampal neurons, kainate receptor function is transiently depressed by NMDA receptor activation in hippocampal neurons, an effect that depends on an increase in intracellular calcium. The recovery of kainate receptor currents after NMDA receptor treatment depended on CaMKII, whereas the transient depression was mediated by the phosphatase calcineurin (262), which was also shown to reduce the open probability of GluR6 receptor channels (89).

7. Genetic Studies Targeted mutagenesis of the mouse genome is a powerful tool for studying the function of genes. However, in many cases the analysis is complicated by functional or molecular compensation or by embryonic or early postnatal lethality. Although disruption of the individual kainate receptor genes has not produced any of the latter problems (in fact, disruption of multiple kainate receptor subunits is not lethal), the possibility of compensation has been debated in the literature and has been invoked in several cases to resolve apparent discrepancies between observations from pharmacologic and genetic studies. Regardless of this potentiality, kainate receptor–knockout mice have been central to furthering our understanding of the role of these receptors at synapses. 7.1. GluR5 Knockouts The GluR5-knockout mouse (along with all the other kainate receptor subunit knockouts) was generated in the laboratory of Stephen Heinemann and was first used to dissect out the subunit composition of kainate receptors at hippocampal synapses (142,146,160). No overt behavioral or developmental phenotype was noted in these initial studies. Several other laboratories have also

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used these mice to examine the subunit composition of both pre- and postsynaptic receptors in brain regions outside of the hippocampus (116,122,173). As noted previously, GluR5 is expressed in the sensory dorsal root ganglion neurons and sensory afferent fibers, and, furthermore, selective GluR5 antagonists produce antinociceptive responses in some pain models (194). In a series of behavioral tests designed to monitor responses to noxious stimuli, the GluR5knockout mice were deficient in their response to models of prolonged noxious stimulus and inflammatory pain but performed normally in their response to an acute thermal insult (185). 7.2. GluR6 Knockouts The initial description of GluR6-knockout mice helped to confirm the central role of kainate receptors in seizurogenic activity. Knockout mice had a reduced threshold for seizures induced by intraperitoneal injections of kainic acid and a marked reduction in the expression of immediate-early genes in the hippocampus subsequent to kainic acid (103). These mice have also been used extensively to study the subunit composition of different populations of receptors (103,129,130,142) and the role of kainate receptors in synaptic plasticity (146,152,184,185). GluR6-knockout mice have a reduction in both contextual and auditory fear memory along with a correlated reduction in LTP in the amygdala, a structure thought to be central to these forms of learning (185). 7.3. GluR7 Knockouts Knockouts of the GluR7 receptor were generated a number of years ago; however, at the time of writing there have been no published analyses of these mice. 7.4. KA1 Knockouts This strain also awaits characterization. 7.5. KA2 Knockouts No behavioral experiments have been reported for these mice, although they exhibit normal fertility and morbidity rates. However, these mice have been used to test the contribution of KA2 subunits to heteromeric receptors and their role in synaptic function in the hippocampus (104,131). 7.6. Kainate Receptor Editing Mutants As outlined earlier, the kainate receptor subunits GluR5 and GluR6 undergo RNA editing that is both developmentally and regionally regulated. The most

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important site for RNA editing of both subunits is in the pore of the ion channel. Editing at this site reduces both the Ca2+ permeability and the single-channel conductance; therefore when generating editing-mutant mice, the focus has been on Q/R editing in the pore. Ablation of the editing complimentary site (ECS) for GluR6 Q/R editing resulted in an almost complete loss of editing at the GluR6 Q/R site without affecting editing of the other editing sites on GluR6 or GluR5 (192). A battery of standard behavioral tests including the rotarod, elevated plus maze, and open-field activity found no differences between wild-type and mutant mice; editing-mutant mice, however, were more susceptible to seizures following systemically administered kainic acid, further underlining the significance of kainate receptors in seizurogenic activity. A second mutant mouse engineered to express only fully edited GluR5 receptors has also been reported (263). In this case a point mutation was introduced into the GluR5 gene at the Q/R site, resulting in expression of receptors that contain the arginine residue at this site. Again a battery of standard behavioral tests found no deficits in these mice, and in addition, seizure threshold and intensity induced by systemic kainic acid were not perturbed. This study also tested responses to noxious thermal and chemical stimuli and found no alterations in these knockin mice (263).

8. Future Directions Our understanding of the many and varied roles that kainate receptors play in the central and peripheral nervous systems has undergone a sea change in the last 10 years. They now can be detected and characterized reliably at synapses, thanks in large part to the development of pharmacologic agents that selectively target AMPA receptors. As is typical, some neurons and synaptic connections have received most of the attention in the initial rush to understand kainate receptor function. New insights continue to emerge from even wellworked-over areas of the brain, like the recent elucidation of a role for kainate receptors in the development of synaptic connections in the hippocampus (189). Despite the rapid progress, many challenges remain to understanding and manipulating kainate receptor function. Several of these topics were mentioned in the preceding sections and are revisited briefly here. A number of aspects of neuronal kainate receptor function are poorly understood. For example, the striking polarization of kainate receptor localization— to some postsynaptic densities but not others in the same population of neurons—presumably results from interaction with cellular proteins, but neither putative determinants in the receptor subunits nor their cellular partners have been elucidated. The difficulty in resolving these mechanisms lies, in part, in the absence of significant polarized targeting in cultured neurons. Resolution of the subunit-dependent signals and systems that control targeting will be

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particularly valuable in constructing a model for how different receptor assemblies are used for distinct functional roles in neurons (if, in fact, such a model can be constructed). For those interested in channel biophysics as well as the neuronal function of kainate receptors, the unusually slow activation and deactivation kinetics of many postsynaptic EPSCKA s, first described almost 10 years ago at the hippocampal mossy fiber synapse (97,98) and present in quantal synaptic currents (101), remains an enigma. What intrinsic or cellular mechanisms produce this striking alteration from the properties of recombinant kainate receptors (or the AMPA receptors located at the same set of synapses)? More generally, is the slow kinetics important to the appropriate function of the kainate receptors at these particular synapses? As with the preceding question about targeting, these questions can only be addressed by delivering the receptors to their “natural” environment, or at least the approximation used in brain slice experiments, because the unique functional properties are only observed in that context. The potential involvement of aberrant kainate receptor signaling in disease states is a mostly unexplored area, despite the long historical association of receptor overactivation with induction of seizure states. Do kainate receptors contribute to the pathologies observed in epilepsy or chronic pain states? Do they represent useful therapeutic targets? Certainly the extensive research with GluR5-selective antagonists suggests that targeting this population of receptors, at least, has potential clinical benefit. The gap in our ability to manipulate kainate receptors pharmacologically remains, however, and it will be difficult to test the importance of other receptor populations (e.g., those containing the GluR6 receptor subunit) without additional antagonists with distinct profiles. New compounds will further our understanding of kainate receptor roles in normal physiology, pathology, and clinical treatment. Finally, we anticipate that the next 10 years will see this picture refined as kainate receptors come into their own identity, likely with more surprises and puzzles, with the development of new genetic tools (e.g., conditional and restricted gene-targeted mice) and pharmacologic tools to further delineate the actions of distinct populations of kainate receptors in the mammalian brain.

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4 Delta Receptors Michisuke Yuzaki

Summary No endogenous ligands have been identified for the delta subfamily of ionotropic glutamate receptors (GluR1 and GluR2). Nevertheless, GluR2 plays indispensable roles in cerebellar functions; mice that lack the GluR2 gene display ataxia and impaired motor-related learning tasks. Recent studies of mutant mice, such as lurcher, hotfoot, and GluR2knockout mice, have provided clues to the structure and function of GluR2. In particular, morphologic and electrophysiologic analyses of hotfoot and GluR2-knockout mice have demonstrated a unique role of GluR2 in synapse formation and its maintenance. In addition, an antibody specific for GluR2‘s extracellular N-terminal indicated its direct role in controlling cerebellar long-term depression. These results suggest that GluR2 regulates distinct s pathways involved in synapse formation and synaptic plasticity. Key Words: Cerebellum; Hair cell; Ataxia, hearing; Purkinje cell; Long-term depression; Synapse formation; Cbln1; Motor learning; Orphan.

1. Introduction The delta subfamily of ionotropic glutamate receptors (iGluRs) consists of GluR1 and GluR2. Since their discovery by homology screening more than 10 years ago, the delta receptors have been regarded as “orphan receptors” because no endogenous ligands have been identified. However, recent studies on mutant mice have clearly established GluR2’s unique and crucial roles in cerebellar functions. Because historical aspects of GluR2 research (1,2) and From: The Receptors: The Glutamate Receptors Edited by: R. W. Gereau and G. T. Swanson © Humana Press, Totowa, NJ

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behavioral analyses on GluR2 mutant mice (3,4) have been reviewed in detail elsewhere, this chapter focuses on recent advances.

2. Structure 2.1. Genes In mice, the gene encoding GluR1 (Grid1) is located on chromosome 14 (14B; 13.5 cM), and the gene encoding GluR2 (Grid2) is located on chromosome 6 (6C1; 29.6 cM). GluR1 is encoded by 16 exons covering a region of ∼760 kb, and GluR2 is encoded by 16 exons covering a region of ∼1.4 Mb. Although the sizes of the encoded cDNAs (∼3 kb) are similar to those of other ionotropic glutamate receptors (iGluRs), the sizes of Grid1 and Grid2 genes are much larger than those (∼200 kb) of other iGluRs (1). In humans, the gene encoding GluR1 (GRID1) is located on chromosome 10 (q22), and the gene for GluR2 (GRID2) is located on chromosome 4 (q22). Like their mouse counterparts, both genes encompass similarly large regions. Indeed, there are only 40 human genes that span >1 Mb, and GRID2 is the thirteenth-largest known gene (5). This large size may be one of the reasons why many spontaneous mutations occur in this gene (6); at least 20 ataxic mutant mice are linked to the Grid2 locus (7,8). Although its relationship to the GRID2 locus is unclear, deletions of subregions of band 4q22 have been described in human megalencephaly (9). A high percentage of purine nucleotides and peaks of enhanced flexibility within these loci are thought to render Grid2/GRID2 susceptible to frequent spontaneous mutations (5,10). 2.2. Topology and Stoichiometry The similarity of full-length amino acid sequences between GluR1 and GluR2 is 76%. The delta family shares sequence similarity of ∼40% with the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainate receptor family and 20%–30% with the N-methyl-d-aspartate (NMDA) family (11,12). On the basis of these sequence similarities and computer-based analysis of transmembrane regions, the topology of GluR1 and GluR2 in the cell membrane has been predicted to be similar to that of other iGluRs (Fig. 1A). The accessibility of antibodies against a hemagglutinin tag attached to the N-terminal or C-terminal region of GluR2 supports this view (13). Although not directly proven, GluR1 and GluR2 are thought to assume a tetrameric stoichiometry. As described later, GluR1 and GluR2 are not coexpressed in the same neurons in most brain regions, whereas they are often coexpressed with other iGluRs. Indeed, GluR2 can form heteromers with AMPA or kainate receptors, and it modifies the channel properties of these receptors when coexpressed in heterologous cells in vitro (14). In addition, immunogold electron microscopy

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Fig. 1. Presumed membrane topology and associated intracellular molecules of GluR2. (A) Presumed membrane topology. The putative ligand-binding domain, formed by the S1 and the S2 regions, is separated by transmembrane domains 1–3. The putative ligand-binding pocket is indicated by an arrow. The most N-terminal domain (NTD) outside of the putative ligand-binding domain of GluR2 is also indicated. (B) Intracellular molecules reported to bind to the C-terminus of GluR2. The letters of the molecules that directly bind to GluR2‘s C terminus are shaded. Only representative interacting proteins are shown for clarity. For abbreviations, see the text.

revealed that GluR2 is colocalized with AMPA receptors in Purkinje cell spines (15). However, coimmunoprecipitation analysis on cerebellar lysates using an anti-GluR2 antibody indicated that the vast majority of GluR2 proteins were not coassembled with AMPA or kainate receptors in vivo (14,16). Therefore, GluR2, and, possibly, GluR1, is thought to exist as a homomeric receptor in vivo, although it is unclear whether a small proportion of GluR1 or GluR2 functions as a heteromer with other iGluRs. 2.3. Diversity Although several splicing variants of other iGluR mRNAs have been reported, there has been no evidence of alternative splicing of GluR1 and GluR2 mRNAs. Similarly, unlike GluR2 AMPA receptors, GluR2 does not

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undergo RNA editing resulting in the replacement of a glutamine with an arginine in the second transmembrane region (17). 2.4. Posttranslational Modification GluR2 is specifically phosphorylated by protein kinase C (PKC) at Ser945 within the C terminus (18). As described later, a point mutation in the third transmembrane region could cause the constitutive activation of mutant GluR2 channels (19). Phorbor ester treatment to activate PKC did not affect the constitutive channel activity of the mutant GluR2 channels (20,21). The PKC phosphorylation at Ser945 modulated the association of GluR2 and a scaffolding protein S-SCAM (22).

3. Function 3.1. In Vitro A fundamental question that remains unanswered is whether wild-type GluR1 and GluR2 serve as ion channels, like other iGluRs. Support for GluR2 channel activity has come from studies on spontaneously occurring ataxic mutant lurcher mice. As described later, GluR2 is predominantly expressed in cerebellar Purkinje cells, and a point mutation at the end of the third transmembrane region causes the constitutive activation of mutant GluR2 channels (GluR2Lc ) in lurcher, eventually leading to the death of Purkinje cells (19). It is interesting that the lurcher mutation is located in a motif that is highly conserved in all iGluRs. When a similar point mutation was introduced in AMPA and kainate receptors, these mutant iGluRs showed constitutive channel activation that reflected the corresponding wild-type properties (17). GluR2Lc displayed distinct channel properties similar to those of AMPA and kainate receptor channels: it exhibited a rectified current–voltage relationship, was sensitive to a polyamine antagonist, and showed moderate Ca2+ permeability (17,23,24). However, the current through GluR2Lc channels was reduced by pentamidine and 9-tetrahydroaminoacridine, which are antagonists that inhibit NMDA receptors but not AMPA receptors (21). These findings indicate that GluR2Lc forms an ion channel with distinct properties, although it is still unclear whether wild-type GluR2 in fact acts as a ligandgated ion channel; the ability of GluR2 to gate current may simply be a function that was lost during evolution. It is unclear whether GluR1 can also form ion channels because GluR1 with the lurcher-type mutation displayed constitutive channel activities when expressed in Xenopus oocytes in one study (20) but not in another (21). A domain transplantation approach was also used to examine the potential channel properties of GluR1 and GluR2. AMPA or kainate receptors

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containing the pore domain of GluR1 or GluR2 did not gate currents (25), suggesting that wild-type GluR1 and GluR2 may not function as channels. Alternatively, the lack of channel activity may simply reflect structural incompatibility of the pore domain of the  receptor family with the gate mechanisms employed in other iGluRs. The lack of specific pharmacologic tools has been a major obstacle in the study of the molecular mechanisms responsible for the functions of GluR1 and GluR2. Thus, the development of an antibody against the putative ligand-binding domain of GluR2 (anti-H2) revealed unexpected functions of GluR2 in vitro. The simultaneous activation of parallel fibers (PFs) and climbing fibers (CFs) induces the long-term depression (LTD) of PF–Purkinje cell transmission, which is thought to underlie motor coordination and a form of information storage in the cerebellum (26). LTD was completely blunted in GluR2-null Purkinje cells, a finding that suggests that GluR2 plays a crucial role in LTD induction. However, the reason for the LTD failure was not clear because developmental abnormalities, such as aberrant PF–Purkinje cell synapses, could also have been responsible. Several lines of evidence indicate that LTD is caused by a decrease in the number of postsynaptic GluR2 AMPA receptors (27,28). Interestingly, treatment with anti-H2 induced the endocytosis of GluR2 subunits and abrogated the subsequent induction of LTD in Purkinje cells (29). This finding indicates that a major function of GluR2 may involve active control of the AMPA receptor endocytosis, thereby modifying the synaptic plasticity at PF–Purkinje cell synapses. It is not completely clear whether anti-H2 acts as an agonist or an antagonist for GluR2. Crystallographic analysis of iGluRs indicates that the ligands must fit in the ligand-binding pocket formed by the S1 and S2 regions, inducing a clamshell-like closure in the structure of this domain (Fig. 1A) and leading to the opening of the ion channel gate. Although anti-H2 binds to the putative S1 region of the GluR2, it seems unlikely that a bulky immunoglobulin molecule could fit into the ligand-binding pocket and induce domain closure. Although the ligand-binding domain in metabotropic glutamate receptor 1 (mGluR1) forms a different type of clamshell-like domain, anti-mGluR1 antibodies to the glutamate binding sites also serve as an antagonist in mice (30) and humans (31). Therefore, GluR2 signaling may constantly suppress AMPA receptor endocytosis at the postsynaptic membrane, and anti-H2 appeared to inhibit this signal (2). Indeed, when GluR2 protein levels were decreased, the number of postsynaptic AMPA receptors was reduced in Purkinje cells (32). Conversely, postsynaptic GluR2 was upregulated in Purkinje cells of GluR2-knockout mice (33); GluR2 may be upregulated, in vain, to inhibit the endocytosis of GluR2 subunits in response to the complete loss of GluR2 in GluR2-knockout Purkinje cells.

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3.2. In Vivo Because spontaneous or induced mutant mice lacking Grid1 are unavailable, its function in vivo is not known. It is interesting that GluR1 proteins are highly expressed in the inner hair cells of the organ of Corti and in both type I and type II hair cells in the vestibular end organ in adult rats (34). Although these results suggest that GluR1 plays a role in hair cell neurotransmission, further studies using immunoelectron microscopy, as well as the generation and characterization of GluR1-knockout mice, will be necessary to clarify its role in hair cells. As described earlier, spontaneously occurring mutations in the Grid2 gene are common in mice; studies of these mice have provided great insight into the function of GluR2 in the cerebellum in vivo. Hotfoot mice are caused by a loss-of-function mutation of GluR2 and exhibit ataxia in the absence of obvious Purkinje-cell death (7). Some hotfoot mutants, like ho-Nancy, ho5J, and ho-tpr, produce no GluR2 protein. Other hotfoot mutants, like ho4J, ho-7J, ho-11J, and ho-15J, have various small in-frame deletions in the N-terminal domain of GluR2; such mutations caused retention of GluR2 in the endoplasmic reticulum (ER) (6,35,36). These findings indicate that, like other iGluRs, GluR2 must be transported to the Purkinje cell surface to function properly in vivo. Detailed morphologic analyses of GluR2-null mice (i.e., hotfoot and genetically engineered GluR2-knockout mice) have revealed that the number of PF–Purkinje cell synapses is markedly reduced in GluR2-null cerebella. It is surprising that the total spine density on GluR2-null Purkinje cells does not differ significantly from that on wild-type cells. As a result, approximately 40% of the spines are “naked” and lack presynaptic contact in GluR2-null cerebella (37,38). Although naked spines are known to appear transiently when granule cells are damaged by irradiation or other genomic mutations, such spines are eventually innervated by the remaining PFs (2). Therefore, the sustained presence of uninnervated spines is a distinctive feature of the PF–Purkinje cell synapses in GluR2-null mice. In addition, the remaining PF–Purkinje cell synapses in GluR2-null mice frequently show another specific abnormality: The length of the postsynaptic density (PSD) is disproportionally longer than that of the opposing presynaptic active zone. These findings indicate that GluR2 plays a unique role in aligning and maintaining the PSD with the presynaptic element at PF–Purkinje cell synapses (Fig. 2). GluR2 was observed in the spines of proximal dendrites in adult rats after the electrical activity of Purkinje cells was blocked by tetrodotoxin (39). Concomitantly, PF formed new synapses on these GluR2-containing proximal dendrites (40), a finding suggesting that GluR2 is instrumental in inducing PF–Purkinje cell synapses. On the other hand, Purkinje cell spines lost their contact with PFs when GluR2 proteins were decreased in the adult

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Fig. 2. In vivo function of GluR2 in the cerebellum in studies on GluR2-null mice. (A) Wild-type Purkinje cells (PC) are innervated by parallel fibers (PF) at the distal dendritic regions and by single climbing fibers (CF) at proximal sites in adult cerebellum. The length of the postsynaptic density (PSD) equals that of the presynaptic active zone. Stimulus (+stim) that evokes long-term depression (LTD) induces the endocytosis of AMPA receptors (AMPAR). (B) Purkinje cells in GluR2-null (i.e., hotfoot and GluR2 knockout) mice have abnormal PF– and CF–Purkinje cell synapses. At PF–Purkinje cell synapses, numerous free spines are not innervated by PFs (naked spines). Even at spines innervated by PFs, the length of the PSD does not equal that of the presynaptic active zone, as indicated by the arrows. LTD is completely abrogated at the PF–Purkinje cell synapses of GluR2-null mice. GluR2-null Purkinje cells remain innervated by multiple CFs that often reach the distal dendritic regions.

cerebellum of inducible GluR2-knockout mice (32). Therefore, GluR2 seems to play a crucial role in not only forming, but also in maintaining PF–Purkinje cell synapses during development and in adulthood in an activity-dependent manner. GluR2 is unlikely to be involved in normal PF–Purkinje cell synaptic transmission because the PF-evoked excitatory postsynaptic current (EPSC) was completely blocked by antagonists to conventional AMPA receptors (41). It is hard to imagine that GluR2 would be sensitive to AMPA-receptor antagonists because wild-type GluR2 does not bind to any glutamate-related agonists. The reduced amplitudes of EPSCs in response to PF stimulation observed in

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GluR2-null mice may simply reflect the reduced number of PF–Purkinje cell synapses. In addition, the reduced number of postsynaptic AMPA receptors in GluR2-null Purkinje cells and in Purkinje cells treated with anti-H2 may also contribute to the reduced amplitudes of PF-EPSC. GluR2-null mice also display abnormal synapses formed by Purkinje cells and CFs that originate from the inferior olive of the medulla. Immature Purkinje cells are normally innervated by multiple CFs. In wild-type mice, Purkinje cells become innervated by a single CF by the end of the third postnatal week (42), whereas GluR2-null Purkinje cells remain innervated by supernumerary CFs even in adulthood (43). These results are not surprising because normal PF inputs are necessary to establish a one-to-one relationship of CF–Purkinje cell synapses: The sustained innervation of Purkinje cells by multiple CFs has often been observed in mice with reduced activities of PF–Purkinje cell synapses (44,45). A distinctive feature of surplus CFs observed in GluR2-null mice is that they reach the distal dendrites of Purkinje cells, whereas surplus CFs in other mutant mice are restricted to the proximal dendrites of Purkinje cells (45). It is possible that the presence of GluR2 at the distal dendrites may inhibit innervation by CFs. Alternatively, the sustained presence of naked spines in GluR2-null mice may trigger the CF invasion. The finding that CFs can transiently innervate spines expressing GluR2 (40,46) supports the latter view. Therefore, the abnormality observed at CF–Purkinje cell synapses may be a secondary effect of the reduced number of PF–Purkinje cell synapses in GluR2-null mice. The ataxic gait and motor discoordination observed in GluR2-null mice could be caused by any of the abnormalities mentioned previously: the reduced number of PF–Purkinje cell synapses, the sustained innervation of Purkinje cells by supernumerary CFs, or the abrogated LTD at PF–Purkinje cell synapses. Acute and transient cerebellar ataxia caused by the injection of anti-H2 into the subarachnoidal supracerebellar space of adult mice (29) supports the hypothesis that GluR2 is actively involved in the maintenance of cerebellar coordination function in adults, probably by regulating LTD.

4. Expression, Trafficking, and Targeting GluR1 mRNA is continuously expressed at low levels in the pyramidal and dentate granule cell layers of the hippocampus during development and in adulthood. During the early postnatal period, GluR1 mRNA is transiently expressed at higher levels in the caudate and thalamic nuclei (12), suggesting a role in the development of neurons. As described earlier, GluR1 proteins are highly expressed in the inner hair cells in adult rats (34). GluR2 mRNA can be detected in Purkinje cells in mice as early as embryonic day 15, increases markedly during the second and third weeks of

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postnatal development, and remains high throughout adulthood (47). However, GluR2 is not completely specific to Purkinje cells; it is also expressed in several neurons in the midbrain-spinal cord region, such as the dorsal cochlear nucleus and the trigeminal motor nucleus (1,16), as well as in the pineal gland (48). GluR2 trafficking is unique in that, unlike other iGluRs, GluR2 proteins are efficiently transported to the Purkinje cell surface, leaving only small amounts of proteins in the intracellular compartments (13). In addition, GluR2 is specifically targeted at the distal dendrites of cerebellar Purkinje cells, where PF axons from granule cells form synapses, but not at the proximal dendrites, where CF forms synapses. The efficient cell-surface transport of GluR2 requires the C-terminal juxtamembrane region of 13 amino acids (13), to which unknown factors may bind. Because several isoforms of spectrin, an actin–cross-linking protein, have been localized to the Golgi complex and have been suggested to be involved in membrane trafficking (49), the association of GluR2 with spectrin (50) may be involved in the efficient cellsurface transport. Recently, mutations in -III spectrin, which is enriched in cerebellar Purkinje cells and is associated with Golgi and vesicle membranes, was reported to cause the loss of GluR2 and excitatory amino acid transporter 4 at the plasma membrane in families with spinocerebellar ataxia type 5 (51). Similarly, as discussed later, a Golgi-resident adapter protein, AP-4, which binds to the middle region of the GluR2 C-terminus (52), may also control GluR2 trafficking. Various small in-frame deletions in the N-terminal domain of GluR2 originally found in ho-4J and some other hotfoot mutant mice impair the homomeric oligomerization of GluR2 and its subsequent exit from the ER in heterologous cells (6,35). Similarly, the N-terminal domain of AMPA and kainate receptors regulates subtype-specific receptor assembly and cell-surface transport (53). These findings suggest that, like other iGluRs, the N-terminal domain of GluR2 is essential for receptor assembly and that unstable oligomers may be retained in the ER by the quality control mechanism. Immunohistochemical staining of cerebellar slices showed that GluR2 of the ho-4J homozygotes was localized to the Purkinje cell soma, where most rough ER are located. These findings indicate that GluR2 is mainly synthesized at Purkinje cell soma and transported to distal PF–Purkinje synapses by bypassing proximal CF–Purkinje cell synapses. As described earlier, GluR2 appeared in the spines of proximal dendrites when the electrical activity of Purkinje cells was blocked (39). The interaction of GluR2 and spectrin can be disrupted by physiologic concentrations of Ca2+ in vitro. Similarly, the activation of voltage-gated Ca2+ channels and a subsequent increase in intracellular Ca2+ concentration also destabilizes synaptic GluR2 clusters in Purkinje

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cells (54). It is hypothesized that the absence of GluR2 at the CF–Purkinje cell spines may be due to enormous Ca2+ spikes induced by CF activities (39).

5. Interaction Partners Because GluR2 does not seem to contribute to normal excitatory postsynaptic currents at PF–Purkinje cell synapses and is involved in postsynaptic AMPA receptor endocytosis, GluR2 may convey signals by interacting with intracellular signaling molecules via its long C-terminus. The sequences comprising the last four amino acids at the C terminus of GluR1 and GluR2 are identical (G-T-S-I) and compatible with the consensus sequence for class I PDZ ligands (-X-S/T-X-, where  indicates any hydrophobic amino acid). Indeed, yeast two-hybrid screening using the C-terminus of GluR2 as bait revealed several proteins that contained one or more PDZ domains (Fig. 1B), such as PSD-93 (55), PTPMEG (56), delphilin (57), nPIST (58), and S-SCAM (22). Although no yeast two-hybrid screenings were performed for GluR1, these proteins, except for delphilin, were explicitly or implicitly suggested to interact with the GluR1 C-terminal tail. In addition to these PDZ proteins that bind to the C-terminal end of GluR2, other PDZ proteins, such as Shank (59) and PICK1 (60), bind to somewhere in the middle of the C terminus of GluR2. PDZ proteins often contain several other domains and serve as a scaffold to organize the assembly of supramolecular complexes to modulate signaling pathways in response to synaptic transmission or to complement synaptic structure. PSD-93, a postsynaptic membrane-associated guanylate kinase (MAGUK), has three PDZ domains, to which K channels, neuronal nitric oxide synthetase, neuroligin, and TARP (Stargazin) bind, as well as an SH3 domain and a guanylate kinase domain, to which GKAP binds. PTPMEG has a band4.1 domain and a protein-tyrosine phosphatase domain. Delphilin is unique in that it is selectively expressed at PF–Purkinje cells and binds specifically to GluR2; it contains formin homology domains FH1 and FH2. Like PSD-93, S-SCAM also belongs to the MAGUK family and contains six PDZ domains, through which many molecules such as GKAP, neuroligin, -catenin, and protein tyrosine phosphatase can be brought into proximity. Shank contains an ankyrin repeat, an SH3 domain, a single PDZ domain, and a prolinerich domain. It binds to GKAP, Homer (which anchors mGluR1 and inositol triphosphate receptor 1), and cortactin. PICK1 contains a single PDZ domain, to which GluR2 AMPA receptors and activated protein kinase C (PKC) bind, and a BAR domain, to which GluR2 binds (60). Thus, AMPA receptors may be brought into proximity with GluR2 directly by PICK1 or indirectly by PSD-93–TARP interaction. Similarly, mGluR1, which is anchored to postsynaptic densities by Homer and Shank, may be associated with GluR2 directly

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via Shank or indirectly via a PSD-93–GKAP–Shank association. The plethora of these interacting molecules indicates that GluR2 is redundantly associated with many postsynaptic proteins. The role of GluR2 in forming and maintaining PF–Purkinje cell synapses suggests that it exerts a regulatory function on the cell cytoskeleton. Indeed, many of the PDZ-domain proteins mentioned earlier are associated with cytoskeleton proteins. For example, the ankyrin-repeat domain of Shank binds to spectrin. Shank can also drive the assembly of branching actin filaments via cortactin, which binds to Shank. The band-4.1 domain of PTPMEG can target GluR2 to the actin-based cytoskeleton. By means of FH1 and FH2 domains, delphilin can promote the nucleation of nonbranching actin filaments. Catenins bound to S-SCAM can also bring proteins containing FH1 and FH2 domains. EMAP, a rat homolog of a microtubule-associated protein, also binds to the GluR2’s C-terminus (61). Finally, as described earlier, GluR2 directly binds to spectrin (50). It remains to be determined which of these interacting proteins play physiologic roles in GluR2’s function and how such interactions are regulated in vivo. Although detailed behavioral analyses and LTD assays were not performed, mice lacking PSD-93 (62) or delphilin (63) displayed apparently normal gait and performance on a rotorod coordination test. Similarly, GluR2 proteins were located normally at PF–Purkinje cell postsynaptic spines, and no naked spines were observed in these knockout mice. In contrast, mice lacking PTPMEG showed attenuated LTD and poor performance in a rotorod test and eyeblink conditioning, but the overall morphology of the cerebellum was normal (64). The lack of effects on GluR2’s functions may reflect the functional redundancy of PDZ proteins that interact with GluR2. Recently, a peptide that interferes with PICK1–GluR2 binding has been shown to block the induction of LTD in cultured Purkinje cells (4). PICK1 and GRIP competitively bind to the AMPA receptor; the transfer of the receptor from GRIP to PICK1 is facilitated by PKC phosphorylation of GluR2 and has been suggested to initiate receptor endocytosis during LTD (65). It is unclear how GluR2 interferes with this pathway, but a GluR2–PICK1 interaction may play a role. The protein nPIST binds to Beclin, whose homolog in yeast induces autophagy. It is interesting that the coexpression of nPIST, Beclin, and GluR2Lc (but not wild-type GluR2) induces autophagy in HEK293 cells. Because dying Purkinje cells in lurcher mice contain the morphologic hallmarks of autophagy (58,66), the channel activities of GluR2Lc have been suggested to cause the release of nPIST and Beclin from the C terminus of GluR2Lc , thus inducing the autophagocytic death of lurcher Purkinje cells. However, as described earlier, GluR2 functions on the cell surface, whereas both nPIST and Beclin are mainly localized to the trans-Golgi network (67,68). Thus, nPIST may be involved in GluR2 trafficking in the trans-Golgi network

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(Fig. 1B). Indeed, TARP was also recently shown to associate with nPIST in the Golgi apparatus and to regulate the synaptic targeting of AMPA receptors (69).

6. Pharmacology Although the extracellular N-terminal regions of GluR1 and GluR2 contain a putative ligand-binding motif conserved in all mammalian iGluRs (Fig. 1A), no glutamate-like ligands for GluR1 or GluR2 have been identified (12,16). However, conventional radioligand-binding assays may have missed unstable or transient ligand binding. The failure to identify any ligands might also have been due to the absence of some unknown accessory protein(s) necessary for the binding of GluR2 to ligands in heterologous cells. A transgene rescue approach that does not rely on in vitro binding or functional assays was recently used to address this issue (70). An arginine residue in the ligand-binding domain is highly conserved from ancestral bacterial periplasmic amino acid–binding proteins to mammalian iGluRs. For example, a substitution of this arginine residue with lysine completely abolishes the ligand-binding or channel activities of iGluRs (71–74). It is surprising that a GluR2 transgene in which lysine replaced the conserved arginine in the putative ligand-binding motif rescued all abnormal phenotypes of GluR2-null mice (70). Although there is no direct evidence that the mutant GluR2 transgene does not bind any glutamate analogs, this finding indicates that a glutamate-like amino acid is unlikely to be required for GluR2 to function in Purkinje cells in vivo. As mentioned earlier, an anti-H2 antibody directed against the putative ligand-binding domain of GluR2 specifically abrogated LTD by disrupting the endocytosis of AMPA receptors, causing transient cerebellar ataxia. This finding indicates that GluR2 signaling may be controlled by the binding of a ligand to the putative ligand-binding domain. What is unclear is the identity of the putative endogenous ligand of GluR2. Because GluR2 is selectively expressed at PF–Purkinje cell synapses, the unknown ligand is probably supplied by PFs. On the basis of the results of the transgene rescue study, the structure of the ligand is likely to be considerably different from that of glutamate. Although glutamate is the only known amino acid neurotransmitter released from PFs, other small molecules may also be released. As mentioned earlier, a lurcher-like point mutation causes the constitutive activation of mutant GluR1 AMPA receptors. Recently, it was reported that the primary effect of the lurcher mutation was to increase the affinity of GluR1 to ambient levels of glutamate (75). If the effect of the lurcher mutation on GluR2 is similar, the leak current associated with GluR2Lc may also be caused by an increased affinity to ambient levels of some unknown endogenous ligands.

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Alternatively, an unknown secreted or membrane-attached ligand may bind to the most N-terminal domain (NTD) outside of the putative ligand-binding domain of GluR2 (Fig. 1A). In this case, the binding of anti-H2 to the putative ligand-binding domain of GluR2 may have allosterically inhibited the binding of a ligand to the NTD.

7. Modulation The phosphorylation of iGluRs by protein kinases plays a crucial role in synaptic plasticity. In the cerebellum, PKC activation is necessary and sufficient for the induction of LTD at PF-Purkinje cell synapses (76,77). It is interesting that the PKC phosphorylation at Ser945 within the C terminus of GluR2 significantly enhanced the association of GluR2 and S-SCAM (22). Although it is unclear how the PKC phosphorylation of GluR2 is related to LTD induction, the S-SCAM signaling pathway may be dynamically controlled by the PKC phosphorylation status of the GluR2 C terminus. Another signaling pathway recently identified to be intimately related to the GluR2 signaling is the Cbln1 system. Cbln1 is a member of the C1q and tumor necrosis factor families predominantly produced in cerebellar granule cells and is secreted as a hexamer (78). Remarkably, all the behavioral, physiologic, and anatomic phenotypes of GluR2-null mice—ataxia, abrogated LTD, appearance of naked and mismatched PF synapses—are shared by cbln1-null mice (79). In addition, mice that lacked both GluR2 and Cbln1 did not show an additive phenotype, but rather were similar to mice lacking only GluR2. These findings suggest that GluR2, which is localized in the PF–Purkinje cell postsynaptic densities, and Cbln1, which is expressed in granule cell presynaptic terminals, engage in a common signaling pathway or process crucial for synapse formation/maintenance and plasticity.

8. Genetic Studies Studies on spontaneous and induced mutant mice of GluR2 were described in Section 4.2.2. Behavioral analysis of GluR2 mutant mice has been reviewed in detail elsewhere (3,4).

9. Future Directions Understanding GluR2 signaling will provide key insights into normal and abnormal cerebellar functions and permit the development of novel therapeutic approaches for particular neurologic disorders. In addition, GluR1 expressed in hair cells is also likely to contribute to some aspects of hearing. Therefore, further studies are warranted to decipher the mechanisms of the signaling pathways mediated by GluR1 and GluR2.

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One unanswered fundamental question is whether GluR1 or GluR2 subunits form ion channels. Although studies on GluR2Lc currents indicated that GluR2 has the appropriate structure to act as an ion channel, it is unclear this is indeed one of the functions of wild-type GluR2. To address this question, a transgene rescue approach, which was used to analyze the putative ligand-binding domain of GluR2 (70), should be useful. If a GluR2 transgene in which its putative channel pore domain was mutated rescues the abnormal phenotypes of GluR2-null mice, GluR2 is unlikely to function as an ion channel. A key biologic process regulated by GluR2 is the endocytosis pathway of postsynaptic AMPA receptors, a pathway that is responsible for LTD. Although it is unclear at which step of this pathway GluR2 is involved, intracellular molecules interacting with the C-terminus are likely to play a role. For example, GluR2 may control the interaction of GluR2 with GRIP and PICK1. Alternatively, GluR2 may interact directly or indirectly with molecules in the classic clathrin-mediated endocytosis pathways. Because several interacting molecules are redundantly involved in similar signaling pathways, a conventional gene knockout approach may not work to identify the role of a specific molecule. An RNAi-based gene knockdown approach or the use of peptides that inhibit GluR2‘s interaction with a specific molecule may help to clarify the role of each molecule in GluR2 signaling. Another key process controlled by GluR2 is the alignment and maintenance of PF–Purkinje cell synaptic contact. This function is reminiscent of synaptic adhesion molecules, such as cadherins, protocadherins, NCAM, SynCAM, neuroligin-neurexin, and eprinB-EphB2. Thus, the N-terminal domain of GluR2 may interact with a molecule expressed on PF terminals. Alternatively, GluR2 may be indirectly involved in such functions by interacting with other postsynaptic adhesion molecules; an analogous situation is the interaction of NMDA receptors with EphB2, which binds to presynaptically expressed ephrinB. Indeed, the NTD outside of the ligand-binding region of the GluR2 subunit of AMPA receptors controls dendritic spine formation in cultured hippocampus neurons (80). The two major functions of GluR2—the stabilization of PF–Purkinje cell synaptic contact and control of postsynaptic AMPA receptor endocytosis— may be mediated by two separate signaling pathways, but they might also be controlled by a common mechanism. For example, most synaptic adhesion molecules, such as EphB and NCAM, are not simple glues; they can also act as signaling molecules. Therefore, an attractive hypothesis is that GluR2 stabilizes PF–Purkinje cell synapses by interacting directly or indirectly with a molecule expressed on PF terminals and that this interaction also modulates an intracellular signaling pathway involved in the endocytosis of AMPA receptors via the GluR2 C-terminal intracellular domain. Because Cbln1 is likely to

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share the intracellular signaling pathway with GluR2, studies on the Cbln1 system may provide insights into GluR2 signaling. Two important discoveries have been made during preparation of this manuscript. First, the structure of the ligand-binding core of GluR2 was determined by x-ray crystallography. (Proc Natl Acad Sci USA 2007;104:14116–14121). Surprisingly, D-serine and glycine were shown to bind to the ligand-binding core by interacting with the conserved arginine. Nevertheless, because a GluR2 transgene in which lysine replaced the conserved arginine in the ligand-binding core rescued all abnormal phenotypes of GluR2-null mice (70), functions achieved by binding to these ligands remain unclear. Second, GluR2 transgenes, in which its putative channel pore domain was mutated, were shown to rescue the abnormal phenotypes of GluR2-null mice (J Physiol 2007; 579.3:729–735; J Physiol 2007;584:89–96). Thus, GluR2 is unlikely to function as an ion channel.

Acknowledgments This work was supported by a Keio University Special Grant-in-Aid for Innovative Collaborative Research Projects.

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5 Ionotropic Glutamate Receptors in Synaptic Plasticity Kenneth A. Pelkey and Chris J. McBain

Summary More than 30 years have elapsed since the publication of the first reports of long-term potentiation by Bliss and Lomo (1973) and Bliss and Gardner-Medwin (1973). These two reports ushered in an exciting new era in which measurable persistent changes in synaptic strength were posited as substrates for learning and memory. Since that time, the study of long-term mechanisms of synaptic plasticity have arguably been one of the most intensely and perhaps the most rewarding fields of the neurosciences, casting important light on the nature of synaptic transmission and the events associated with the strengthening or weakening of synapses. Indeed, although once principally studied at excitatory glutamatergic synapses within the hippocampus and cortical formations, mechanisms of synaptic plasticity are observed at a myriad of synaptic connections throughout the mammalian central nervous system. Moreover, although many of these synapses share common mechanisms of plasticity, the last decade has seen an explosion in our understanding of plasticities peculiar to one synapse or another. This chapter does not attempt to cover all of these divergent mechanisms but instead focuses on those mechanisms of long-term potentiation (LTP) and depression (LTD) most commonly found within the hippocampal formation. A large portion of this review covers N-methyl-D-aspartate–receptor-dependent LTP and LTD, the two most commonly studied forms of cortical plasticity; however, it also addresses plasticity mechanisms at other hippocampal synapses that have not enjoyed the same intensity of investigation but are worthy of attention.

From: The Receptors: The Glutamate Receptors Edited by: R. W. Gereau and G. T. Swanson © Humana Press, Totowa, NJ

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Key Words: Hippocampus; Synapse; LTP; LTD; AMPA; NMDA; Plasticity; Calcium; Presynaptic; Postsynaptic; Phosphorylation.

1. NMDAR-Dependent LTP Since its first formal reports (1,2), the phenomenon of N-methyl-d-aspartate– receptor (NMDAR)-dependent long-term potentiation (LTP) has captured the fascination of neuroscientists across subdisciplines for more than 30 years (Fig. 1). The observation that brief intense synaptic activation could induce a persistent enhancement in the efficacy of neuronal communication immediately suggested a cellular mechanism by which neuronal circuits could encode memories. Since that time considerable effort has been devoted to debating whether LTP indeed corresponds to higher-order learning and memory formation. For the purposes of the present discussion we will not further

Fig. 1. N-methyl-d-aspartate (NMDAR)–dependent long-term potentiation (LTP) and long-term depression (LTD) at hippocampal Schaffer collateral–CA1 pyramidal cell synapses. A. Schematic diagram of the rodent hippocampal slice preparation, showing the main excitatory pathways (AC, associational/commissural; MF, mossy fiber; PP, perforant path; SC, Schaffer collateral). Typical electrode placements for studying synaptic plasticity at Schaffer collateral–commissural synapses are indicated. The traces are field excitatory postsynaptic potentials (EPSPs) recorded before (1) and during (2) LTP. B. Time-course plots showing alterations in field EPSP (rising slope normalized to baseline) against time during LTP (induced by a 100-Hz stimulation, 1 sec, baseline intensity) or after the induction of de novo LTD (long-term depression) (induced by 1-Hz stimulation, 15 min, baseline intensity). The black bar represents the time of the stimulus, and the numbers 1 and 2 indicate the time points illustrated in panel A. From Collingridge GL, Isaac JT, Wang YT. Receptor trafficking and synaptic plasticity. Nat Rev Neurosci 2004;5:952–962; with permission from the Nature publishing group.

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debate this issue but instead direct readers to a number of excellent reviews on the subject (3–5). Regardless of one’s opinion on the connection between LTP and higher-order brain function, it is clear that studies into the cellular and molecular mechanisms responsible for LTP have provided great insight into the basic characteristics of synaptic transmission on both sides of the cleft. In keeping with the subject of this chapter, we focus on the features of LTP directly relevant to ionotropic glutamate receptor function/signaling. With this in mind we also refrain from analysis of the controversial issue of pre- versus postsynaptic NMDAR-dependent LTP expression that dominated much of the research in the field within the first two decades of LTP investigations. Instead we concede that in all likelihood expression of NMDARdependent LTP resides to some extent on both sides of the cleft, with differential contributions throughout development and at diverse locations within the central nervous system (CNS). Because the current focus is limited to ionotropic glutamate receptor function in synaptic plasticity, the discussion will of necessity be biased toward the locus of these receptors and thus postsynaptic mechanisms. Finally, although NMDAR-dependent LTP has been described to occur at various locations throughout the CNS with subtle variations, the hippocampal CA1 area remains the prototypical LTP model and thus will dominate this survey. 1.1. Induction 1.1.1. NMDARs as Synaptic Coincidence Detectors The defining feature of NMDAR-dependent LTP is, of course, the requirement for NMDAR activation during induction as first revealed by Collingridge and colleagues (6) a full decade after Bliss and Lomo’s initial published report of LTP (1). The privileged role of NMDARs in LTP induction stems directly from their ability to detect coincident pre- and postsynaptic activity by virtue of voltage-dependent Mg2+ inhibition of the channel pore (7–9). At negative resting membrane potentials Mg2+ ions rapidly enter the NMDA channel pore and block permeation by other ions, whereas at depolarized potentials the block by Mg2+ is relieved (Fig. 2). This basic biophysical property ensures that NMDARs participate in synaptic transmission only under conditions in which the dual requirements of ligand binding and postsynaptic depolarization are met, thus enabling NMDARs to function as effective synaptic coincidence detectors. The discovery of the voltage dependence of NMDAR activation provided an immediate explanation for the selective disruption of LTP by the NMDAR antagonist APV despite no apparent influence on basal synaptic transmission (6) and provided a basic molecular framework to explain the induction of LTP for the first time. At resting membrane potentials Mg2+

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Fig. 2. Typical excitatory synapse with glutamate (Glu) being released from the presynaptic terminal to act on postsynaptic ionotropic glutamate receptors (-amino3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors [AMPARs] and N-methyld-aspartate receptors [NMDARs]. AMPARs are active at both hyperpolarized and depolarized membrane potentials, allowing for Na+ ion influx, whereas NMDARs conduct both Na+ and Ca2+ ions only at membrane potentials sufficiently depolarized to relieve Mg2+ blockade of the NMDAR channel pore. Influx of Ca2+ through NMDARs engages various postsynaptic intracellular signaling cascades to trigger the induction of long-term potentiation (LTP). CaMKII, calcium/calmodulin-dependent protein kinase II; NOS, nitric oxide synthase; PKA protein kinase A; PKC, protein kinase C. Adapted with permission from Malenka RC, Nicoll RA. Long-term potentiation–a decade of progress? Science 1999;285:1870–1874.

blockade prevents NMDARs from significantly contributing to low-frequency afferent input (test stimuli); however, during tetanization (conditioning stimuli) the summed postsynaptic depolarization produced by -amino-3-hydroxy5-methyl-4-isoxazolepropionic acid receptor (AMPAR) activation sufficiently relieves Mg2+ blockade of NMDARs to allow for activation of the liganded receptors, which trips the biochemical cascades necessary for LTP induction (10) (Fig. 2). The dual requirement of ligand binding and postsynaptic depolarization to efficiently recruit NMDAR participation in synaptic transmission also neatly explained the basic LTP properties of cooperativity, associativity, and input specificity that had been elucidated in the decade following its discovery (Fig. 3). Cooperativity describes the requirement for some threshold

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Fig. 3. Cooperativity, associativity, and input specificity of long-term potentiation (LTP). A. A relatively weak input (left) does not undergo LTP following highfrequency stimulation (HFS), whereas a strong input (right) generated by recruiting more afferent fibers does yield LTP following HFS (traces below represent synaptic events before and after HFS). The recruitment of more fibers results in cooperativity of the afferent input to depolarize the postsynaptic cell sufficiently to relieve Mg2+ block of N-methyl-d-aspartate receptors (NMDARs), allowing them to trigger LTP induction. B. Only synapses that are active during conditioning stimulation (HFS)

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stimulation to be met such that weak tetani activating very few afferent fibers fail to induce LTP (11). This cooperativity has an associative quality in that strong activation of one set of synapses can facilitate LTP induction at an independent set of adjacent active synapses on the same cell if both sets of convergent synapses are activated within a finite temporal window (11,12). Both of these properties result from the requirement that sufficient postsynaptic depolarization occurs to relieve Mg2+ block of the NMDARs and trigger LTP induction. Consistent with this proposal, the cooperativity requirement of LTP induction can be circumvented by pairing low-frequency afferent stimulation with direct postsynaptic depolarization via current injection through an intracellular recording electrode (13–15); conversely, hyperpolarizing current injection during tetanic stimulation prevents high-frequency-stimulation (HFS)–induced LTP (16). Input specificity refers to the observation that LTP is elicited only at the synapses stimulated by afferent activity but not at adjacent synapses on the same cell and is ensured by the fact that only those synapses releasing glutamate during the conditioning stimulation will efficiently activate NMDARs during the depolarizing envelope of the tetanus. 1.1.2. Increased Postsynaptic Ca2+ Is Required At roughly the same time that NMDARs were determined to be essential for LTP induction, Lynch and colleagues (17) proposed that the generation of LTP also required an increase in postsynaptic Ca2+ , because postsynaptic loading of the Ca2+ chelator EGTA prevented HFS-induced LTP at Schaffer collateralCA1 synapses. Conversely, elevation of intracellular Ca2+ by photolysis of caged Ca2+ can mimic LTP, suggesting that Ca2+ provides a sufficient trigger for LTP induction (18). Thus, the demonstration that NMDARs are in fact permeable to Ca2+ (19,20) provided a potentially satisfactory explanation of

 Fig. 3. (Continued) are potentiated, producing input specificity of LTP. In this case the inactive input (I) did not participate in conditioning stimulation, and therefore synaptic events recorded before and after HFS of the active group of inputs (A) are not different. When LTP is induced at one set of synapses on a postsynaptic cell, inactive synapses do not potentiate because NMDARs at these inactive inputs were not activated by glutamate during depolarization of the postsynaptic cell. C. If baseline weak (W) stimulation incapable of eliciting LTP on its own occurs at one set of synapses concurrent with HFS of an adjacent strong input (S), both sets of inputs will undergo LTP induction. This associativity of LTP results from activation of NMDARs at the weak input during the postsynaptic depolarization elicited by HFS of the strong input. Adapted with permission from Malenka RC. The long-term potential of LTP. Nat Rev Neurosci 2003;4:923–926.

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how their activation triggers LTP induction. Indeed, it is frequently presumed that activation of the NMDARs during HFS (or during pairing) yields a direct influx of Ca2+ through the NMDARs themselves that triggers subsequent biochemical events leading to the persistent enhancement of basal AMPARmediated synaptic transmission. Consistent with this hypothesis, postsynaptic Ca2+ transients mediated solely by NMDAR activation can be measured in response to presynaptic stimulation (21–23). However, it has been difficult to exclude contributions of alternate Ca2+ sources to Ca2+ transients evoked in dendritic spines during LTP conditioning stimulation (reviewed in refs. 24–26). Potential contributing sources of Ca2+ that could be activated in concert with NMDARs during conditioning stimulation include voltage-gated Ca2+ channels, Ca2+ -induced Ca2+ release from intracellular stores, and postsynaptic mGluR activation, all of which have been implicated to some degree in synaptic plasticity (e.g., refs. 27–29; reviewed in ref. 26). Regardless of the source, it appears that the Ca2+ signal necessary for triggering tetanus-induced LTP need only be very transient, because LTP is effectively induced when the posttetanic rise in intracellular Ca2+ is restricted to 1 hr postinduction) potentiation of NMDARs has been observed

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in other models of plasticity (131). In the extreme case of “silent synapses,” which initially generate only NMDAR-mediated responses and hence do not yield detectable synaptic responses at hyperpolarized resting membrane potentials, LTP induction leads to the sudden wholesale appearance of an AMPARmediated response without any change in the synaptic NMDAR component (127,128,132) (Fig. 4). These observations are central to the argument that LTP is expressed postsynaptically, because a presynaptic enhancement of release should affect both receptor subtypes equally (but see refs. 133 and 134). It is now generally agreed that the acute upregulation of postsynaptic AMPAR function, either by recruitment of AMPAR complexes to the postsynaptic membrane from nonsynaptic sites or via modification of the biophysical properties of existing synaptic AMPARs, represents a significant mechanism for expression of LTP (reviewed in refs. 75 and 135–137). 1.2.1. AMPAR Trafficking and LTP Expression The simple hypothesis that LTP resulted from an increase in the number of synaptic AMPARs was first proposed by Lynch and Baudry in 1984 (138), but this proposal remained largely ignored for more than a decade during the frenzied and protracted debate over pre- versus postsynaptic expression mechanisms (recounted in ref. 139). Subsequent physiologic and anatomic evidence, which revealed silent synapses that could rapidly be converted to active synapses with functional AMPARs (see Fig. 4), then rejuvenated interest in postsynaptic AMPAR insertion as a plausible mechanism for LTP expression (75,127,128,132,140–145). An important early study suggested a critical role for postsynaptic membrane fusion machinery in LTP (146). Thus, postsynaptic loading of botulinum toxin, N-ethylmaleimide (NEM), or peptide inhibitors of NSF-SNAP interactions, all of which disrupt SNARE-dependent membrane fusion events, prevented LTP (Fig. 5A, B). Conversely, postsynaptic loading of recombinant SNAP to promote SNARE-dependent membrane fusion enhanced basal synaptic transmission, leading to occlusion of LTP. These findings, combined with the demonstration that the GluR2 subunit of AMPARs directly interacts with NSF (147–149) (see Chapter 2, AMPA Receptors), immediately suggested that LTP results from postsynaptic membrane fusion of vesicles containing AMPARs, which convert silent to active synapses or increase the number of AMPARs at active synapses. Consistent with this model, studies in cultured neurons provided convincing evidence for dendritic exocytosis that was dependent on CaMKII function, thereby linking dendritic fusion events to an important mediator of LTP induction (150). Subsequently, a number of laboratories demonstrated that native AMPARs were rapidly inserted into postsynaptic membranes of dissociated cultured neurons in an activitydependent fashion (69,151–154) (Fig. 5C, D). The membrane insertion of AMPARs required the activation of NMDARs and was sensitive to disruption

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of membrane fusion machinery by tetanus toxin (Fig. 5C, D), providing a direct correlation between the culture dish model and hippocampal LTP, and thereby strengthening the argument that NMDAR-dependent LTP is expressed at least in part by the regulated insertion of new AMPARs to conditioned synapses. One avenue of investigation that has been particularly instructive in elucidating the trafficking of AMPARs during LTP is the introduction of optically and electrophysiologically tagged recombinant AMPARs into hippocampal neurons (67,68,78,155–159) (Fig. 6). Optical tagging is accomplished by the fusion of GFP or another fluorophore to recombinant receptor subunits, whereas electrophysiologic tagging results from the introduction of recombinant receptor subunits with distinct rectification properties from native AMPARs in hippocampal slice cultures (Fig. 6A, B). Such overexpressed recombinant receptor subunits do not appear to assemble with native subunits and therefore develop into functional homomeric channels. For subunits other than GluR2, these homomeric channels will exhibit an inwardly rectifying current–voltage relationship because they are sensitive to blockade of the channel pore by intracellular polyamines at depolarized membrane potentials (160–162) (see Chapter 2, AMPA Receptors); for recombinant GluR2 subunits, a mutation is made in the channel pore–encoding region of the construct that renders these homomeric receptors inwardly rectifying as well  Fig. 4. Silent synapses and long-term potentiation (LTP). A, B. Example of an experiment demonstrating the existence of silent synapses. A cell was held at a membrane potential of –60 mV, and after obtaining a small excitatory postsynaptic current (EPSC), the stimulus intensity was decreased so that no EPSCs were detected for 100 consecutive stimuli. The cell was then depolarized to +30 mV, and stimulation now evoked responses that were completely blocked by the N-methyl-d-aspartate receptor (NMDAR) antagonist D-APV. The cell was then returned to –60 mV, where again no EPSCs could be detected. Panel B shows sample consecutive sweeps (8 events, upper traces) and averages (100 events, lower traces) from each of the conditions illustrated in panel A. An average of 10 EPSCs obtained at the beginning of the experiment (panel B, far left, (60 mV higher stimulus) is also shown. C, D. Example of an experiment revealing the conversion of a silent to active synapse by LTP-inducing stimulation. In panel C the EPSC amplitude measured at a holding potential of –60 mV is plotted over time. Note the rapid appearance of detectable EPSCs following LTP induction via a pairing protocol (pairing) prior to which none were evident. Panel D shows sample consecutive sweeps (10 events, upper traces) or averages (100 events, lower traces) obtained from the experiment in panel C at the times indicated. Before pairing, stronger stimulation revealed active synapses (High Stim. average trace at left). A–D: Data adapted with permission from Isaac JT, Nicoll RA, Malenka RC. Evidence for silent synapses: implications for the expression of LTP. Neuron 1995;15:427–434.

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Fig. 5. Long-term potentiation (LTP) expression requires postsynaptic membrane fusion. A. Intracellular injection of botulinum toxin (BoTx) during sharp electrode recordings prevents LTP expression (upper panel) without affecting LTP recorded extracellularly from the surrounding noninjected cells (lower panel). B. Heatinactivated (Inact.) BoTx does not block LTP. C, D. Blockade of glycine-induced surface -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) insertion (gray signal, anti-GluR1) in cultured neurons injected with (b) tetanus toxin (TeTx) (white signal due to coinjection with Lucifer yellow) but not (c) heat-inactivated TeTx. A, B: adapted with permission from Lledo PM, Zhang X, Sudhof TC, et al. Postsynaptic membrane fusion and long-term potentiation. Science 1998;279:399–403. C, D: Adapted with permission from Lu W, Man H, Ju W, et al. Activation of synaptic NMDA receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons. Neuron 2001;29:243–254.

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(68). Because the majority of native AMPARs in principal neurons contain edited GluR2 subunits (163) and hence yield linear current–voltage relationships (see Chapter 2, AMPA Receptors), synaptic incorporation of the tagged recombinant receptors can be assayed electrophysiologically by probing for changes in the rectification properties of synaptic currents, provided the intracellular solution is supplemented with exogenous polyamines like spermine. In examining the behavior of such overexpressed receptors in CA1 pyramids of hippocampal slice cultures (Fig. 6C), Malinow and colleagues revealed that recombinant GluR1 homomeric AMPARs are excluded from synaptic sites under basal conditions but can be driven into synapses by LTP-inducing protocols in an NMDAR-dependent fashion or by coexpression of active CaMKII (67,68,155,159,164) (Fig. 6D-F). In contrast, recombinant GluR2 homomeric AMPARs readily enter active (but not silent) synapses independent of neuronal activity or conditioning stimulation (68,164) (Fig. 6D). It is interesting that the mode of recombinant AMPAR delivery to synapses— activity-dependent regulated (GluR1) versus constitutive (GluR2)—is determined by the intracellular carboxy-terminal tails of the receptor subunits. Thus, chimeric GluR1 subunits that have had their C-terminus replaced with that of GluR2 yield receptors that behave as GluR2 homomers and vice versa (68,164), suggesting that divergent intracellular protein–protein interactions of receptor subunits dictate the trafficking properties of assembled AMPARs (see Chapter 2, AMPA Receptors). Indeed, recombinant AMPARs composed of GluR1 subunits containing a mutation that disrupts binding to PDZ-domain– containing proteins such as SAP97 cannot be driven into synapses by CaMKII or LTP-inducing stimuli [155; but see 165]. Similarly, overexpressed AMPARs composed of GluR2 that have been mutated to disrupt either NSF binding or PDZ interactions (with ABP/GRIP and PICK) are not constitutively delivered to synapses (68). Based on these observations, a two-step model describing subunit-specific rules of AMPAR trafficking during LTP has emerged (68,136,166–168). It postulates that two AMPAR species exist in principal neurons: One participates in activity-dependent delivery, and the other participates in continuous replacement of receptors. AMPARs composed of GluR1 in complex with GluR2 (GluR1/2 heteromers) participate in regulated delivery and are excluded from synaptic sites in the absence of conditioning stimulation. LTP-inducing stimuli lead to the rapid incorporation of these GluR1/2 heteromers to increase the receptor complement at active synapses or to convert silent to active synapses. In contrast, AMPARs composed of GluR2 in combination with GluR3 (GluR2/3 heteromers) continuously replace synaptic receptors in a manner that maintains transmission at active and recently potentiated or unsilenced synapses. This means, of course, that the GluR1/2 heteromeric AMPARs inserted following stimulation at conditioned synapses

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 Fig. 6. Subunit specific rules governing synaptic -amino-3-hydroxy-5-methyl4-isoxazolepropionic acid receptor (AMPAR) insertion. A. Schematic of expected membrane topology of GFP-tagged GluR1 subunit. B. Current–voltage relations of glutamate-evoked whole-cell currents elicited in HEK cells expressing GFP-tagged GuR1 homomeric receptors (solid symbols, upper traces) or GluR2 subunits in combination with GluR1 (or any other GluR AMPAR subunit, GluRX, with GluR2) (open symbols, lower traces). Note the linear current–voltage relation of GluR2-containing AMPARs and inward rectification of GluR2-lacking AMPARs. C. Fluorescence (upper and middle) and differential interference contrast (DIC) (lower) images of hippocampal slices infected with tagged proteins (e.g., GFP-GluR1) allowing for their identification and recording in slice cultures. Note that fluorescent cells can readily be distinguished from neighboring uninfected cells for comparison recordings (visible electrode in lower two panels). D. Fluorescent images of CA1 pyramid dendrites infected with GFP-GluR1 (upper left) or GFP-tagged mutant GluR2 [GFPGluR(2R586Q)] that is inwardly rectifying (lower left) and rectification indices (RIs) of synaptic events in infected cells compared to uninfected neighbors. RIs calculated as the ratio of the absolute amplitude of synaptic responses obtained at –60 and +40 mV holding potentials (see traces at right). Note that GluR1 appears limited to CA1 dendritic shafts and is largely excluded from spines, whereas GluR2 is readily observed in spines. Consistent with this localization, GluR1 overexpression does not influence RIs of synaptic responses, whereas GluR(2R586Q) overexpression does, indicating that under basal conditions, recombinant GluR1 is excluded from synapses, whereas GluR2 readily enters the synaptic population. E, F. GFP-GluR1 can be driven into synapses by LTP-inducing stimulation or coexpression of active calcium/calmodulin-dependent protein kinase II. In panel E, GluR1 movement to synaptic sites is illustrated visually, whereas in panel F, GluR1 synaptic incorporation is monitored electrophysiologically. G. Effects of overexpressing C-terminal tails of GluR1 or GluR2 on LTP. A, B: Modified with permission from Shi SH, Hayashi Y, Petralia RS, et al. Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation. Science 1999;284:1811–1816. C: Adapted with permission from Hayashi Y, Shi SH, Esteban JA, et al. Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction. Science 2000;287:2262–2267. D: From Shi S, Hayashi Y, Esteban JA, et al. Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons. Cell 2001;105: 331–343; and Hayashi Y, Shi SH, Esteban JA, et al. Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction. Science 2000;287:2262–2267; with permission. E: From Shi SH, Hayashi Y, Petralia RS, et al. Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation. Science 1999;284:1811–1816; and Hayashi Y, Shi SH, Esteban JA, et al. Driving AMPA receptors into synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction. Science 2000;287:2262–2267; with permission. G: From Shi S, Hayashi Y, Esteban JA, et al. Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons. Cell 2001;105:331–343; with permission.

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will ultimately be replaced by GluR2/3 heteromers. Although the formulation of this scheme has relied heavily on observations with overexpressed receptors, attempts to interfere with the trafficking of endogenous receptors largely support the crucial role of GluR1 in activity-induced membrane insertion during LTP and of GluR2 in constitutive cycling to maintain synaptic transmission (Fig. 6G). Thus, infection of CA1 pyramidal neurons with a peptide corresponding to the carboxy-terminus of GluR1 to disrupt native GluR1-containing AMPAR protein–protein interactions and their regulated trafficking prevents LTP without any apparent effect on basal transmission (68). Conversely, overexpression of the C-terminal tail of GluR2 markedly depresses basal transmission and actually increases the magnitude of LTP (68). Furthermore, acute disruption of GluR2-NSF/AP2 interactions with peptide inhibitors introduced through the recording electrode rapidly depresses basal synaptic responses (68,147,149,169,170), although the effects of such peptides on conventional LTP have not been tested. Despite the obvious preferential insertion of recombinant GluR1 homomeric AMPARs following LTP induction, the model outlined here suggests that in uninfected neurons GluR1/2 heteromers are the dominant native AMPAR species inserted following conditioning stimulation. Consistent with this suggestion, overexpressed GluR1/2 heteromeric receptors seem to behave as GluR1 homomers and are excluded from synaptic sites unless driven by coexpression of active CaMKII (68,164). The original proposal that native GluR1/2 heteromers, rather than GluR1 homomers, are the principal AMPAR species initially inserted during LTP is largely based on the premise that all principal-neuron AMPARs contain GluR2. However, a number of recent reports clearly indicate that principal neurons contain substantial reserve pools of GluR2-lacking AMPARs (171–177), likely in the form of GluR1 homomers (163). Based on these reports, Plant and colleagues (178) recently investigated whether native GluR2-lacking AMPARs participate in LTP expression (Fig. 7). Surprisingly, LTP induction was observed to result in the rapid incorporation of native GluR2-lacking AMPARs at conditioned synapses, as indicated by both changes in spermine-dependent rectification and philanthotoxin (a GluR2-lacking AMPAR-specific antagonist) susceptibility of potentiated responses. It is interesting that, despite continued potentiation of conditioned synapses, the rectification changes and philanthotoxin sensitivity were transient, lasting approximately 20 min postinduction, indicating that the GluR2-lacking AMPARs initially inserted were replaced with GluR2containing AMPARs as proposed in the two-step model described earlier. This time course of exchange coincides with the period during which the GluR1 carboxy-terminal tail peptide manifests its block of LTP (68), suggesting that GluR1 tail interactions participate in the process of exchanging GluR2lacking for GluR2-containing AMPARs. In addition to inward rectification,

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Fig. 7. Transient incorporation of native GluR2-lacking -amino-3-hydroxy-5methyl-4-isoxazolepropionic acid receptors (AMPARs) during long-term potentiation (LTP). A, B. Two-pathway experiments (closed symbols are paired pathways and open symbols are control pathways) indicating that the LTP-induced increase in excitatory postsynaptic current (EPSC) amplitude measured at negative holding potentials (–60/–70 mV) is not mirrored by potentiation of EPSCs measured at positive holding potentials (+40 mV) when monitored within 15 min postinduction. This situation

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GluR2-lacking AMPARs differ from their GluR2-containing counterparts in having increased conductance and calcium permeability (see Chapter 1, AMPA Receptors). Thus, it is likely that the frequently observed decay of early LTP over the first 10–20 min after induction partially reflects turnover of the initially inserted GluR2-lacking, large-conductance, calcium-permeable AMPARs (CPAMPARs) for GluR2-containing, lower-conductance, calcium-impermeable AMPARs (CI-AMPARs). Moreover, the CP-AMPARs inserted initially could contribute a significant Ca2+ signal at potentiated synapses for the first 20 min postinduction, which may be necessary to “tag” recently potentiated synapses (179b) and to engage signaling cascades necessary for consolidation of LTP expression (but see ref. 30). Indeed, activation of the newly inserted CPAMPARs following LTP induction by paired stimulation appears necessary for stable LTP expression, because removal of philanthotoxin did not recover LTP and cessation of stimulation for the period during which CP-AMPARs were present prevented LTP expression (178) (but see refs. 124, 179a, and 179b for potential differences in tetanus-induced LTP assayed with field recordings). An intriguing possibility is that Ca2+ influx through the CP-AMPARs drives their replacement by CI-AMPARs in a fashion analogous to plasticity at cerebellar mossy fiber–stellate cell synapses (180–182). Whether the native CP-AMPARs are replaced by GluR1/2 or GluR2/3 CI-AMPARs remains to be resolved. The crucial role for the GluR1 subunit in LTP expression is further supported by observed deficits of conventional CA1 LTP in adult mice lacking GluR1 subunits (183,184). Even the initial potentiation following a 100-Hz tetanus, which is typically attributed to posttetanic potentiation, is largely absent in adult GluR1-knockout mice (183–185), consistent with the rapid incorporation of GluR1 homomeric CP-AMPARs following conditioning stimulation. Similarly, adult GluR1-knockout mice display absolutely no immediate potentiation of synaptic responses following a typical pairing induction protocol (184). It  Fig. 7. (Continued) yields a large change in rectification of potentiated synaptic events (panel B, EPSC−70 /EPSC+40 ), indicative of the incorporation of GluR2-lacking AMPARs during LTP. NMDA, N-methyl-d-aspartate. C. Despite continued potentiation, the change in rectification associated with LTP is transient, lasting roughly 20 min postinduction. D–F. The GluR2-lacking AMPAR specific antagonist philanthotoxin (PhTx) blocks LTP expression when applied early (D, F) but not late (E, F) after induction, consistent with the transient expression of CP-AMPARs following LTP induction. Note that PhTx does not affect control path responses or reduce potentiated responses below baseline values, consistent with a lack of CP-AMPARs at synapses prior to LTP induction. Modified with permission from Plant K, Pelkey KA, Bortolotto ZA, et al. Transient incorporation of native GluR2-lacking AMPA receptors during hippocampal long-term potentiation. Nat Neurosci 2006;9:602–604.

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should be noted, however, that typical NMDAR-dependent LTP is not absent in all brain regions nor at all ages in these mice (158,183,184,186). The resolution of this apparent discrepancy with the central role of GluR1 in LTP is the functional substitution for GluR1 by other AMPAR subunits early in development and at various locations throughout the CNS. Most notably, GluR4 and an alternatively spliced variant of GluR2 with a long cytoplasmic tail appear to be capable of sustaining CA1 LTP in young and juvenile animals, respectively (156,158). Indeed in young mice (900 residues) (Fig. 1). The iGluR subunits can be divided into four main subfamilies based on compatibility of subunit assembly, sequence similarity, and certain pharmacologic and

From: The Receptors: The Glutamate Receptors Edited by: R. W. Gereau and G. T. Swanson © Humana Press, Totowa, NJ

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Fig. 1. A. Functional ionotropic glutamate receptors (iGluRs) are tetrameric assemblies between subunits from three major subfamilies. N-methyl-d-aspartate receptors (NMDARs) differ from -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and kainate receptors (KARs) by the requirement of glycine as coagonist for channel activation. B. Placement of four GluR subunits into a low-resolution structure (gray contour) of the AMPAR obtained by single-particle electron microscopy of purified neuronal AMPARs using structures for the aminoterminal domain (NTD) (dark blue, dark green), the agonist-binding domain (ABD)

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functional features: the N-methyl-d-aspartate (NMDA) type (subunits NR1, NR2A–D, NR3A, B), the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type (subunits GluR1–4), the kainate type (subunits GluR5–7 and KA1, 2). and the orphan receptors (subunits 1 and 2). In general, a marked degree of sequence identity among all known iGluR subunits suggests that they fold into the same overall tertiary structure, and thus all known iGluR subunits share a similar architecture. Like most ligand-gated ion channels, purified preparations of iGluRs are notoriously difficult to obtain in quantities that allow for the initiation of crystallization trials, and no crystal structures are available for any intact iGluR or subunit. However, numerous biochemical, functional, and bioinformatic studies have established iGluRs as highly modular structures comprising in large part four discrete domains: two large extracellular domains (the amino-terminal and agonist-binding domains), a transmembrane domain, and an intracellular C-terminal domain (Fig. 1). Apart from the latter domain, each of the individual domains exhibits significant sequence homology to bacterial proteins with known structure and, in some instances, a related function. This similarity has provided an excellent starting point for studies of the structural as well as functional roles of the individual domains. During the past decade, structural models and discrete functional roles have been assigned to most of the subunit domains and have provided the basis for creation of mechanistic models for many of the functional and pharmacologic  Fig. 1. (Continued) (light blue, light green), and the transmembrane domains (TMDs) (red) from high-resolution structures of homologous segments found in the metabotropic glutamate receptor mGluR1, the ABD of GluR2, and the potassium channel KscA, respectively. C. Topology of the iGluR subunit. Upper: A linear representation of the domain organization of the iGluR polypeptide chain. Lower: A schematic representation of the subunit membrane topology. Each subunit folds into a modular structure of two large extracellular domains (the amino-terminal domain followed by the agonist-binding domain) connected to a transmembrane domain composed of three membrane-spanning segments (M1–3) and a membrane reentry loop (M2), and ends in an intracellular C-terminus. D: The iGluR ABD can be expressed by recombinant methods as soluble proteins that adopt a bilobed, clamshell-like fold. Shown is the crystal structure of the agonist-binding domain from the AMPA receptor subunit GluR2 without bound ligand (apo form) (Protein Data Base code 1FTO). The site of N-terminal truncation is located at the top. and the linker replacing the transmembrane domain is located at the bottom. Glutamate binds in the cleft between domain 1 (pale green) and domain 2 (light orange). B: Reprinted with permission from Nakagawa T, Cheng Y, Ramm E, et al. Structure and different conformational states of native AMPA receptor complexes. Nature 2005;433:545–549.

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features of the receptor such as partial agonism, subunit-specific agonist selectivity, receptor desensitization, and allosteric modulation. This chapter summarizes these recent advances, with focus on the correlations between iGluR structure and function. 1.1. Domain Structure 1.1.1. The Amino-Terminal Domain (NTD) Beginning at the extracellular N-terminus, all iGluRs contain a short signal peptide (10–33 residues) that target the protein to the membrane and is removed by proteolysis after membrane insertion (1). Subsequent to the signal sequence, the first ∼400–450 residues of the amino terminal fold into an autonomous domain, here denoted the N-terminal domain (NTD) (2). The iGluR NTDs have sequence homology with a group of soluble periplasmatic bacterial amino acid–binding proteins (PBPs) and with the agonist-binding domain of the metabotropic glutamate receptor mGluR1. All known structures of PBPs suggest that these proteins fold into clamshell-like structures with the ligandbinding site contained in the cleft formed between the two globular subdomains. A similar structure and location for the ligand-binding site are found for the sequence-related agonist-binding domain from mGluR1, suggesting that the function of the NTD could be to bind endogenous ligands in a putative pocket located between the lobes. In several studies, mutant subunits have been created in which the NTD has been partly or completely removed (2–8). All of these truncated subunits appear to assemble into functional receptors, in some cases functionally indistinguishable from the wild-type receptors. The nonessential nature of the NTD for the core function of the iGluRs is consistent with a regulatory role for this domain. Truncations of the NTD have been found to influence several key features of receptor function, such as desensitization and regulation of subunitspecific assembly (3,4,7,9,10). In addition, the NTD may contain binding sites for extracellular proteins involved in positioning of the receptors during synaptogenesis (11). The NR2A NMDA receptor (NMDAR) subunit constitutes the bestunderstood example of NTD regulation of receptor function. The NR2A NTD appears to constitute a high-affinity Zn2+ -binding site with an affinity in the range of 30–120 nM (5,12,13). Moreover, molecular studies support coordination of Zn2+ by a number of histidine residues, and biochemical studies further suggest that this domain can bind Zn2+ (14). Occupancy at the Zn2+ site appears to be involved in a positive allosteric interaction with glutamate binding and enhances the affinity of NR1/NR2A receptors to protons, thereby enhancing inhibition of the receptor at physiologic pH (12,13,15). Recently NR2B has been suggested to also contain a Zn2+ -binding site within the NTD

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domain (8). In addition, a number of studies suggest that the NTD is the site of action for NR2B-selective noncompetitive modulators typified by the phenolethanolamines such as ifenprodil (16–19). In contrast to NMDARs, the NTD in AMPA receptors (AMPARs) and kainate receptors (KARs) does not appear to contain inhibitory sites. 1.1.2. The Agonist-Binding Domain (ABD) The second extracellular domain within all iGluR subunits contains the agonist-binding site and is formed by two extracellular regions historically referred to as S1 and S2 (Fig. 1) (20). These regions, like the NTD, share significant sequence homology to certain periplasmatic bacterial amino acid binding–proteins (21,22). The domain, hereafter denoted the agonist-binding domain (ABD), is structurally and functionally the best-characterized part of the iGluR subunits. Protein-engineering efforts have succeeded in creating recombinant constructs that consist of the excised S1 and S2 gene sequences with an intervening artificial sequence encoding a short polypeptide (23,24). Recombinant expression of these ABD constructs from representative subunits from AMPARs, KARs, and NMDARs have successfully generated watersoluble proteins with ligand-binding activity comparable to that in full-length subunits, indicating structural identity between the binding pockets of isolated ABDs and the corresponding intact subunit. Most important, crystal structures of ABDs from the NMDAR, AMPAR, and KAR subclasses have been solved with bound agonists and antagonists (Table 1). These structures show this domain to adopt clamshell-like structure in which the S1 and S2 segments individually comprise most of each clamshell, with the agonist-binding site located deep within the cleft between the two lobes (Fig. 1); the details are further elaborated in Section 2.1. Apart from containing the agonist-binding site, the ABDs are also involved in formation of binding sites for various allosteric modulators of receptor function such as desensitization and deactivation, as is further elaborated in Section 2. 1.1.3. The Transmembrane Domain In all iGluRs, the ABD is connected to the transmembrane domain (TMD) through three short linker segments (Fig. 1). The TMD comprises the M1, M2, M3, and M4 segments. M1, M3, and M4 are presumably membranespanning -helices, whereas M2 is thought to form a non–membrane-spanning reentrant pore loop (Fig. 1). M1, M2, and M3 from each of the four subunits contribute to the formation of the core of the ion channel and have a small but significant sequence homology with the ion channel domain of K+ channels, a group of ion channels for which several high-resolution structures have become available in recent years (25,26). In iGluRs, however, the overall membrane topology of the ion channel is inverted in the membrane compared to K+ channels. Strengthening the idea of this homology is the existence of a

252

Kainate

Glycine

l-Glutamate

Apo

Ligand

HO

HO

O

HO

—

O

O

NH2

NH

O HO

NH2

O

HO

Chemical structure

AMPAR partial agonist KA-R partial agonist

Endogenous agonist

Endogenous agonist

—

Pharmacologic activity

GluR2 GluR2 GluR2-N754D GluR2-L650T GluR2-Y450W GluR6

GluR2 GluR0 (prokaryotic) NR2A NR1/NR2A dimer GluR2 GluR5 GluR5 GluR5 GluR6 GluR0 (prokaryotic) NR1 NR1/NR2A dimer

Crystallized with agonist-binding domain

PDB

1GR2 1FW0 1LBB 1P1N 2ANJ 1TT1

1FTO 1IIW 2A5S 2A5T 1FTJ 1YCJ 1TXF 2F36 1S7Y 1II5 1PB7 2A5T

Table 1 Structures of Ionotropic Glutamate Receptor Ligands and Binding Domains

61 64 104 79 191 72

64 28 80 80 64 75 72 73 72 28 85 80

Reference

253

O

HO

H N

d-Cycloserine

NH2

O

NH2

O

O

NH2

O

O

NH2

HO

HO

NH2

Cycloleucine

NH2

O

O

O

N

HO

O

HO

N

HO

HN

O

ACBC

ACPC

AMPA

Quisqualate

NR1 partial agonist

NR1 antagonist

NR1 partial agonist

NR1 partial agonist

AMPA-R full agonist KA-R full agonist (GluR5 selective)

KA-R agonist

AMPAR full agonist

NR1

NR1

NR1

NR1

GluR2 GluR2-L483Y GluR2-L483Y/L650T GluR2-L650T

GluR2-L650T GluR6

GluR2

1PB9

1Y1M

1Y1Z

1Y20

1FTM 1LB8 1P1W 1P1Q

1MM7 1P1O 1S9T

(Continued)

85

86

86

86

64 104 85 85

79 72

192

254

Iodo-Willardiine

DCKA

L-Serine

D-serine

Ligand

O

HN

O

Cl

HO

HO

N

O

I

O

NH2

N H

O

HO

NH2

O

NH2

Cl

HO

HO

O

OH

Chemical structure

AMPAR partial agonist KAR agonist (GluR5 selective)

NR1 antagonist

GluR0 agonist

NR1 partial agonist

Pharmacologic activity

Table 1 (Continued)

GluR2

NR1

GluR0 (prokaryotic)

NR1

Crystallized with agonist-binding domain

1MQG

1PBQ

1IIT

1PB8

PDB

100

85

28

85

Reference

255

CPW399

Willardiine

Fluoro-Willardiine

Bromo-Willardiine

O

HN

O

O

HN

O

O

HN

O

O

HN

O

N

N

N

N

F

HO

HO

HO

Br

HO

NH2

O

NH2

O

NH2

O

NH2

O

AMPAR partial agonist

AMPAR partial agonist KAR agonist

AMPAR partial agonist KAR agonist

AMPAR partial agonist KAR agonist

GluR2 GluR2-Y702F

GluR2

GluR2

GluR2

1SYH 1SYI

1MQJ

1MQI

1MQH

(Continued)

70 70

100

100

100

256

ATPA

Cyclothiazide (+ glutamate)

Aniracetam (+ fluorowillardiine)

CX614 (+ quisqualate)

Ligand

N

HO

O

NH2

O

N H

O

O

Cl

HO

N

O

NH

O

O

S

O

O

H2NO2S

O

N

O

Chemical structure

AMPA-R partial agonist KA-R agonist (GluR5 selective)

AMPA-R positive modulator

AMPA-R positive modulator

AMPA-R positive modulator

Pharmacologic activity

Table 1 (Continued)

GluR2

GluR2-N755S

GluR2

GluR2

Crystallized with agonist-binding domain

1NNK

1LBC

2AL5

2AL4

PDB

193

104

171

171

Reference

257

DNQX

Des-Me-AMPA

4-AHCP

Thio-ATPA

N

O2N

O2N

N

HO

O

N

HO

O

OH

S

HO

O

N H

H N

NH2

O

O

O

O

NH3

NH2

HO

HO

AMPA-R antagonist

AMPA-R agonist

AMPA-R partial agonist KA-R partial agonist (GluR5-selective)

AMPA-R partial agonist KAR agonist (GluR5 selective)

GluR2 GluR2-L483Y

GluR2

GluR2

GluR2

1FTL 1LB9

1MQD

1WVJ

2AIX

(Continued)

64 104

195

194

112

258

UBP302

UBP310

Ligand

O

O

HO

N

N

O

O

HO

HO

N

N

HO

O

O

NH3

O

S

NH3

O

Chemical structure

KA-R antagonist (GluR5 selective)

KA-R antagonist (GluR5 selective)

Pharmacologic activity

Table 1 (Continued)

GluR5

GluR5

Crystallized with agonist-binding domain

2F35

2F34

PDB

73

73

Reference

259

ACPA

2-Me-Tet-AMPA

ATPO

NS1209

N

HO

N

HO

N

O

O

N

O

H2O3P

O

O

(CH3)2NO2S

H3C

NH2 N N

O

HO

O

NH2

O

N

O

NH2

N N

HO

HO

H N

O

OH

O OH

AMPAR full agonist KAR agonist

AMPA-R agonist (GluR3/4 selective)

AMPA-R antagonist

AMPA-R antagonist

GluR2 GluR2-Y702F

GluR2

GluR2

GluR2

15ME 15MF

15MB

1N0T

1PWR

(Continued)

68 68

68

87

95

260

Domoate

2S,4R-4Methylglutamate

Br-HIBO

Ligand

HO

O

HO

HO

O

Br

O

HO

NH2

HO

N

O

NH2

O

NH

O HO

HO

Chemical structure

O

KAR partial agonist

KAR agonist

AMPA-R agonist (GluR1/2 selective) KA-R agonist (GluR5 selective)

Pharmacologic activity

Table 1 (Continued)

GluR6

GluR6

GluR2 GluR2-Y702F

Crystallized with agonist-binding domain

1YAE

1SD3

15MC 15MD

PDB

74

72

68 68

Reference

261

CNQX

Homoquinolinate

CPP

AP5

NMDA

O2N

NC

HO

O

H2O3P

H2O3P

HO

O

N H

H N

N

N

O

O

O

HO

NH

O

NH2

O

HO

N H

HO

HO

O

AMPAR antagonist

NMDAR partial agonist

NMDAR antagonist

NMDAR antagonist

NMDAR partial agonist

Not done

Not done

Not done

Not done

Not done

–

–

–

–

–

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bacterial iGluR, GluR0, that shares strong functional and structural homology with the mammalian iGluRs (27). GluR0, however, contains the signature motif of all known prokaryotic and eukaryotic K+ channels in the form of six highly conserved consecutive residues located in the reentry loop (27,28). This idea is further supported by recent studies showing that NMDA receptors are functional when M4 region alone is coexpressed as an independent polypeptide with an NR2 subunit truncated after M3 (29); this arrangement converts the topology of the NMDA receptor to one analogous to the inward rectifier K+ channel, with one notable difference being the requirement by NMDA receptors of M4 for function. According to the K+ -channel model, the iGluR ion channel is formed by equivalent contributions of M1, M2, and M3 from each subunit to formation of a symmetric pore structure that spans the membrane (20). The ion channel can be divided into three subdomains: an intracellular vestibule, a selectivity filter, and an extracellular vestibule. The selectivity filter in the K+ channel is defined by a narrow constriction formed by carbonyl groups on the peptide backbone of the reentry loop (25). Ion selectivity in iGluRs is controlled by a single residue, denoted the Q/N/R site, which controls the ion permeability of the channel by forming a cyclic motif at the tip of the selectivity filter (30). In GluR2, GluR5, and GluR6, this position is subject to RNA editing, in which a codon for glutamine is changed to arginine, thereby creating receptors with extremely low permeability for Ca2+ and a reduced single-channel conductance. In heteromeric receptors this effect is dominant; for example, AMPARs containing a single GluR2(R) subunit are essentially Ca2+ impermeable, thereby providing a developmentally and regionally control mechanism of Ca2+ permeability of AMPARs by selective RNA editing (31–34). 1.1.4. The Intracellular C-Terminal Domain (CTD) The C-terminal domain (CTD) is the most diverse domain in terms of primary structure, varying greatly both in sequence and in length among the iGluR subunits. It also shows no sequence homology to any other known proteins. Although some studies have expressed CTDs in isolation, no structural details exist for this domain (35). Adding further diversity, the CTD is alternatively spliced in selected iGluRs (1). For some iGluR subunits (e.g., NR1, NR2A), deletion of this domain does not block function, but alter regulation (36–39). Within the AMPAR subfamily, the insertion of an alternative exon is observed for GluR2 and GluR4 in certain neuronal subpopulations, generating an alternative stop codon in both subunits (40,41). These C-terminal splice variants of GluR2 and GluR4 are often referred to as “long” and “short,” respectively. The direct functional consequence of alternative splicing within the CTD is largely unknown because no differences in pharmacology

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or molecular function have been reported between CTD isoforms of any iGluR subunit. However, CTD splice variants may contain different binding sites for intracellular protein partners important for regulation of membrane trafficking or, in some cases, receptor function (42). For example, the NMDAR subunit NR1 undergoes extensive splicing within the CTD to generate four different CTDs that confer different trafficking properties on the NMDARs (43–45). In addition, the alternative CTD splicing can affect a binding site for calmodulin. Because calmodulin controls NMDAR function in terms of reducing the channel open probability fourfold on binding, alternative splicing in the CTD can indirectly control NMDAR function (46). Despite the lack of structural information of the CTD, the functional role of this domain is well characterized for many of the iGluR subunits. Here, the CTDs serve as platforms for several posttranslational modifications as well as being the major anchoring domain for protein–protein interactions with a variety of intracellular proteins involved in targeting, trafficking, and anchoring of the receptors to subcellular positions (47). In terms of posttranslational modifications, the CTD contains several functional phosphorylation sites for serine threonine kinases such as protein kinase C (PKC), protein kinase A (PKA), and calcium/calmodulin-dependent protein kinase II (CaMKII) (48). For example, AMPA receptors are known to be phosphorylated over a range of serine or threonine residues (49–54). Phosphorylation of the AMPA and kainate receptor CTD is often associated with physiologically important changes in receptor function (55–59) and for cellular mechanisms for regulation of receptor number and cellular location (42). Similarly, nonreceptor tyrosine kinases are also known to regulate iGluR function, with notable examples being upregulation of NMDAR function by Src-family tyrosine kinases (60).

2. Binding Domains: Activation and the Difference Between Agonists and Antagonists Recent advances in our understanding of the atomic structure of the iGluR agonist-binding sites have provided new opportunities to consider the molecular determinants of agonist selectivity among the different subclasses of iGluRs. These studies have turned up a number of important ideas about the molecular determinants of full agonists and partial agonists, as well as provided insight into the mechanism of action for competitive antagonists. 2.1. The iGluR Agonist-Binding Site The initial step in iGluR activation is binding of the agonist to the agonistbinding domain of each subunit. As mentioned previously, S1S2 constructs representing the ABD from multiple subunits within the NMDAR, AMPAR, and KAR subfamily have been successfully expressed as soluble proteins and

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purified in milligram quantities from insect cells, bacteria, or mammalian cells (24,61–63). Most important, these isolated ABDs have formed the basis for X-ray crystallographic experiments that have provided atomic-level threedimensional structures for GluR2, GluR5, GluR6, NR1, and NR2A, as well as the bacterial GluR0 (Table 1). All ABD structures adopt a clamshell-like conformation (Fig. 1) in which the polypeptide segment located on the N-terminal side of M1 forms most of one shell (domain 1; D1) and the segment between M3 and M4 form most of the opposite shell (domain 2; D2) (61). The binding pocket for the agonist is located in the cleft between D1 and D2, which are held together by a highly flexible “hinge” region (Figs. 1 and 2). Several structural features of agonist binding appear completely conserved in all iGluR subunits. Most important, ligands with iGluR agonistic activity contain a chemical moiety equivalent of the backbone portion of glutamate, namely the -amino and -carboxyl groups (also referred to as the amino acid moiety). The region of the binding pocket that harbors this moiety is quite similar in all ABD structures independent of subunit or agonist type and is primarily made up of residues from D1. Using the structure of glutamate bound to the ABD from the AMPAR subunit GluR2 as an example (64) (Fig. 2B), one can see that the -amino group of glutamate forms a tetrahedral network of interactions with the backbone carbonyl oxygen of P478, the side-chain hydroxyl of T480, and the carboxylate group of E705. The -carboxyl group of glutamate forms a bidentate interaction with the guanidinium group of R485 and receives hydrogen bonds from the backbone NH of T480 and S654. This binding mode of the amino acid moiety is almost identical for all agonists so far crystallized in complex with the GluR2 ABD as well as with ABDs from NMDAR and KAR subunits (Fig. 2). In contrast, greater variation is observed for the binding mode of the -positioned groups among iGluR agonists. For glutamate bound to GluR2, the -carboxyl group forms interactions with the side-chain hydroxyl group and backbone NH of T655 (Fig. 2B), whereas the interaction between the isoxazole hydroxyl group of AMPA and the NH of T655 is mediated via a water molecule. Furthermore, the side chain of Y450 forms an electron-dense ring structure above the glutamate - and -carbon atoms, resembling a lid that restrains the space available in the agonist binding pocket (Fig. 2B). Several lines of experimental work have validated that the agonist-binding site in the artificially soluble ABDs needed for crystallization faithfully resemble the binding sites in intact receptors. Most important, all engineered ABDs so far characterized display ligand-binding affinities that closely mirror those for the parent full-length subunit. Furthermore, ultraviolet (UV) absorption spectra have been used to compare the molecular configuration of the AMPAR antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) bound to full-length GluR2 with the CNQX configuration in the corresponding isolated

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Fig. 2. A. Alignments of agonist-binding residues (yellow) identified in the agonistbinding domain (ABD) crystal structures of GluR2, GluR5, GluR6, NR2A, and NR1. Analogous residues in the other iGluR subunits are included for comparison. Residue numbering is according to the total protein including the signal peptide. For reference, the predicted size of the signal peptide is included in parenthesis at the end of the alignment (SP). B. Binding of glutamate in the agonist-binding pocket of the GluR2ABD crystal structure (Protein Data Base [PDB] code 1FTJ). The protein backbone

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ABD and have found similar spectra, thereby indicating that the structure of the ABD constructs resembles the ABD of the full-length receptor (65). 2.2. Subtype Selectivity of Agonist Binding The growing list of crystal structures for ABDs (Table 1) from all three major subfamilies in complex with different agonists provides the means for understanding the structural basis of the selectivity of prototypic agonists such as NMDA, AMPA, and kainate as well as endogenous excitatory amino acids such as glutamate, glycine, aspartate, and d-serine. Furthermore, agonists that are selective for single or a subset of subunits exist within each iGluR subfamily (Table 1). For several of these ligands, structural studies in combination with site-directed mutagenesis and homology modeling have provided models for the determinants within the binding pocket that guide subunit selectivity at a level of detail unprecedented among most other receptor classes. In the following subsection, we provide a brief overview of some these structureselectivity relationships. 2.2.1. The Agonist-Binding Site of AMPAR Subunits In the four AMPA receptor subunits GluR1 to GluR4, homology modeling shows that the residues that are found directly to interact with agonists such as glutamate, AMPA, and kainate in the GluR2-ABD are fully conserved,  Fig. 2. (Continued) of D1 is shown as pale green ribbon, the protein backbone of D2 is shown as light orange ribbon, and the carbon atoms of glutamate are colored yellow. Important ligand-binding residues are shown as sticks, and dashed lines represent hydrogen bonds or salt bridges. Numbering of GluR2 residues is according to the mature protein without the signal peptide. C. Binding of glutamate in the ligandbinding pocket of the GluR6-ABD crystal structure (PDB code 1S7Y). Compared to the glutamate-bound ligand-binding pocket of GluR2, there is a loss of a direct hydrogen bond to the -amino group of glutamate at position A518 in GluR6, which is the site equivalent to T480 in GluR2. This loss is compensated by an additional water molecule that forms a hydrogen bond to the -amino group of glutamate. Numbering of GluR6 residues is according to the total protein including the signal peptide. D. Binding of glutamate in the ligand-binding pocket of the NR2A-ABD crystal structure (PDB code 2A5S). Compared to glutamate bound in the GluR2-ABD, the salt bridge between D731 and the positively charged -amino group of glutamate is absent. Instead, the -amino group of glutamate forms water-mediated hydrogen bonds to E413 and Y761. Numbering of NR2A residues is according to the total protein including the signal peptide. E. Binding of glycine in the ligand-binding pocket of the NR1-ABD crystal structure (PDB code 1PB7). Specificity of NR1 for glycine can be explained by the hydrophobic environment created by V689 and the steric barrier formed by W731. Numbering of NR1 residues is according to the total protein including the signal peptide.

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suggesting little opportunity for subunit-selective agonist binding. However, several agonists have been identified with more than 10-fold subunit selectivity among GluR1 to GluR4 (Table 1) (66,67). The structural basis of selectivity for these agonists is related to the arrangement of water molecules trapped in the agonist-bound ligand-binding cavity (68). In GluR2, four water molecules are present, and three of these participate in water-bridged interactions between side chains of GluR2 and glutamate (Fig. 2B). Although all residues that directly interact with glutamate are conserved among the AMPAR subunits (Fig. 2A), the arrangement of water molecules in the binding cavity is believed to be different, which may impose functional differences in agonist affinity and receptor activation, thereby shaping the ligand specificities among the AMPAR subunits (68–71). 2.2.2. The Agonist-Binding Site of KAR Subunits The KAR subunits display much more pronounced differences in intersubunit agonist pharmacology than AMPAR subunits. The recent structures of the ABDs from the KAR subunits GluR5 and GluR6 have demonstrated the basis for ligand discrimination at GluR5 versus GluR6, as well as revealed the structural features that determine the different pharmacology of KARs versus AMPARs (Table 1) (72–75). For glutamate, the overall binding modes to GluR5 and GluR6 closely resemble that of GluR2 (Fig. 2B, C); there are, however, some significant differences that enable the subunits to discriminate among agonists on the basis of steric occlusion. First, the binding cavities of glutamate-bound GluR5 and GluR6 are 40% and 16% larger than in the GluR2 ABD, respectively (72). Accordingly, six and five water molecules are trapped in the ligandbinding cavities of GluR5 and GluR6, respectively, as opposed to the four water molecules trapped in GluR2. One of the additional water molecules in GluR5 and GluR6 forms a hydrogen bond with the -amino group of glutamate that is not observed in GluR2 (72,75). Of particular note, there is a loss of a direct hydrogen bond to the -amino group of glutamate at position A518 in GluR6, which is the site equivalent to T480 in GluR2 and T533 in GluR5 (Fig. 2C). Consequently, GluR6 binds glutamate with lower affinity than does GluR5 (72). Furthermore, the side chains of three residues that line the agonistbinding pocket are smaller in GluR5 than in GluR6 (S736, L750, and S756 in GluR5; N721, F735, and T741 in GluR6). and the increased binding cavity in GluR5 allows room for an additional water molecule. GluR5-selective ligands displace this water molecule and are unable to bind GluR6 as a result of steric occlusion at this position (72). Mutagenesis studies have demonstrated that the exchange from S736 in GluR5 to N721 in GluR6 plays an important role in specifying the selectivity of AMPA, iodo-willardiine, and ATPA toward GluR5 (76–78). Similarly, the higher affinities of SYM2081 and kainate for

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binding KAR over AMPAR subunits result from steric occlusion at position L650 in GluR2 (61,72,79). The smaller V700 and V685 side chains in GluR5 and GluR6 replace the L650 in GluR2, and steric clash with the methyl group of SYM2081 and the isopropenyl group of kainate is therefore reduced at this position in KAR subunits. 2.2.3. The Agonist-Binding Site of NMDAR Subunits Comparison of the binding pocket of glutamate-bound NR2A with the corresponding pocket of GluR2 has revealed unexpected differences (80). Because the D731 present in NR2A is one methylene group shorter than the E705 present in GluR2, there is no salt bridge between D731 in NR2A and the positively charged -amino group of glutamate (Fig. 2). Instead, the -amino group of glutamate forms water-mediated hydrogen bonds to E413 and Y761 in NR2A. Furthermore, there is a van der Waals contact between the carboxylate of glutamate and Y730 in NR2A. Y730 of D2 is conserved among all NR2 subunits and forms an interdomain hydrogen bond with E413 of D1. The finding that D731 in NR2A does not directly bind the -amino group of glutamate was surprising because the charge-conserving substitution of the aspartate with glutamate at this position in NR2A (D731) and NR2B (D732) renders the receptor nonfunctional (81–84). The effect of the D731E (NR2A) and D732E (NR2B) mutations is therefore more likely a result of interference with the water-mediated interactions at the -amino group of glutamate and/or a disruption of the agonist-binding pocket. Modeling of NMDA into the crystal structure of NR2A-ABD suggested that the N-methyl group of NMDA is accommodated in the binding pocket by displacement of the water molecule that binds the -amino group of glutamate (80). This feature of ligand binding is likely only achieved in NR2 subunits because the aspartate side chain at this site (D731 in NR2A and D732 in NR2B) is one methylene group shorter than the glutamate side chain present in AMPAR subunits (E705 present in GluR2) (Fig. 2B). Other studies using mutagenesis and homology modeling of the agonist-binding pockets in NR2A and NR2B have suggested that NMDA is obstructed from binding AMPAR subunits because of steric clash between the N-methyl group of NMDA and M708 in GluR2, which is conserved among all AMPAR subunits (81,83). In NR2 subunits, replacement of the methionine side chain by a smaller valine (V734 in NR2A and V735 in NR2B) is believed to relieve steric occlusion of NMDA from the agonist-binding pocket (81,83). Nonetheless, the precise mechanism by which NR2 subunits selectively bind NMDA is unresolved, and the crystal structure of NR2-ABD in complex with NMDA would be highly informative. Crystal structures of the ABD from NR1 have been solved in complex with several different ligands, including the endogenous agonist glycine (Table 1)

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(80,85,86). As expected from sequence alignments, the agonist-binding pocket of NR1 is similar to that of GluR2 and NR2A, with only a few important differences that explain how NR1 discriminates glutamate and selectively binds glycine (Fig. 2E). In NR1, glutamate and other large agonists are prevented from binding by a steric barrier formed by W731. In GluR2, the equivalent site is occupied by L704, which faces away from the binding pocket (Fig. 2B), and in NR2A, the smaller side chain of Y730 is in van der Waals contact with the -carboxylate of glutamate (Fig. 2C). Furthermore, by replacement of T655 (GluR2) by V689 (NR1), there is loss of a hydrogen bond donor that is necessary to stabilize the -carboxyl group of glutamate. 2.3. Binding of Competitive Antagonists Crystal structures of iGluR ABDs in complex with compounds acting as competitive antagonists have been determined for eight different ligands (Table 1). In all of the antagonist-bound ABD crystal structures, the proteins are stabilized in a conformation that is very similar to the empty GluR2 apo conformation with little (10 degrees) is related to receptor activation (see Section 6.2.4). The interactions between the antagonists and residues of the binding pocket are sufficient to stabilize the antagonist in the binding pocket and prevent binding of agonists. The competitive antagonists 6,7-dinitro-quinoxaline-2,3-dione (DNQX) and 5,7dichlorokynurenic acid (DCKA), which bind to GluR2 and NR1, respectively, prevent agonist binding in what appears to be a similar fashion. These planar molecules interact with residues, mainly within D1, perhaps thereby depriving the amino acid moiety of the agonist of its initial contact sites with the open cleft of the binding pocket. In contrast to DNQX and DCKA, the structurally different AMPAR antagonist -ATPO- and NR1 antagonist -cycloleucine- possess amino acid moieties that bind similarly to those of agonists (86,87). However, the bulky -substituents of ATPO and cycloleucine impose steric hindrance of cleft closure, and the resulting expansion of the binding pocket allows recruitment of additional water molecules that stabilize the apo-like conformation. Recently, crystal structures of the ABD of the KAR subunit GluR5 in complex with the willardiine derivatives UBP302 and UBP310 have demonstrated a third mechanism of competitive antagonist binding (73). UBP302 and UBP310 are GluR5-selective antagonists that posses amino acid moieties and bulky -substituents (Table 1). Similar to the AMPAR antagonist ATPO and the NR1 antagonist cycloleucine, the bulky -substituents force the ABD of GluR5 to adopt an open conformation. However, unlike in all other crystal structures of AMPAR and KAR ABDs in complex with agonists and antagonists, the -amino group of the ligands surprisingly does not form direct

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interactions to the carboxyl group of E753 (E705 in GluR2; Fig. 2). Instead, the side chain of E753 adopts a conformation with striking resemblance to that of E705 in the GluR2-ABD apo structure (64). This observation led Mayer et al. (73) to suggest that UBP302 and UBP310 stabilize the GluR5-ABD in a conformation closer to the resting state as compared to the structures of GluR2-ABDs in complex with ATPO and CNQX (Table 1). In summary, competitive antagonists appear to inhibit agonist interaction by selective shielding of agonist binding to D1 residues or by additionally stabilizing ABDs in an open or partially closed inactive conformations, as will be further elaborated in Section 6.2.4. 2.4. Agonist Binding: The Cleft Closure Mechanism The activation mechanisms for ligand-gated ion channels, including the iGluRs, have been proposed to involve a sequence of discrete conformational changes that takes the receptor from an initially nonliganded closedchannel conformation to an agonist-bound closed channel, and subsequently to an agonist-bound open-channel conformation, as first proposed for nicotinic receptors (88). Agonist binding is the energy-generating process that enables subsequent conformational changes. Accordingly, the first step in the iGluR activation mechanism is likely to involve a conformational change of the ABD on binding of the agonist. Direct structural evidence for such a process was first observed when GluR2-ABD structures obtained in the absence and presence of bound agonist were compared (64). In the unbound conformation, denoted the apo state, the D1 and D2 domains are more separated than in the agonist-bound conformation, denoted the holo state, in which the clamshell structure adopts a “closed” conformation (89). This mechanism seems to be conserved in all iGluR subunits because all ABDs so far crystallized appear to be capable of adopting conformations that are closed to different degrees relative to the apo structure in the presence of agonist. Small-angle X-ray scattering (SAXS) is an important tool in probing electron density correlations on nanometer length scales and has been used to assess whether the apo and holo conformations observed for crystallized S1S2 proteins also are preferred conformations in solution in the absence or presence of agonists. Madden et al. (90) found that the volumes of ABD in the presence and absence of glutamate were in agreement with the volume of crystallized GluR2ABD in the apo and the glutamate-bound holo conformations, respectively, indicating that these conformations indeed are favorable under physiologic conditions. Taken together, these data suggest that in the absence of agonist, the ABD is at its lowest energy in the apo confirmation. Moreover, agonist binding involves a conformational change of the protein to the holo conformation, with a reduction in energy accompanying this change.

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Time-resolved monitoring of agonist binding to ABDs in solution has been achieved using time-resolved UV and infrared (IR) spectrometric measurements of shifts in the spectra for specific functional groups on ultrafast application of agonists to ABDs derived from GluR2 and GluR4, respectively (91,92). These methodologies can provide data on the kinetics of ligand binding, the formation of ligand–protein interactions, and conformational changes within the ABD. For example, Abele et al. (91) used time-resolved UV spectrometry of GluR4-ABD during glutamate binding to show that binding proceeds via a two-step mechanism in which the ligand initially makes contacts with residues on D1 followed by a relatively slower second step in which further ligand–protein and D1–D2 interactions occur. Specifically, E403 and Y451 (E402 and Y450 in GluR2) (Fig. 2) on the D1 domain were found to contribute to the rapid initial binding step, and E706 (E705 in GluR2) (Fig. 2) on the D2 domain contributes to the second binding step. The formation of the important interaction between R486 (R485 in GluR2) (Fig. 2) and the carboxyl group of glutamate could not be analyzed in these experiments. This arginine is conserved in all iGluRs (Fig. 2), and its side chain guanidinium group is thought to serve as the initial contact point for the ligand by attracting the negative -carboxylate (64). The two-step ligand binding mechanism was recently corroborated by Cheng et al. (92) in a study on the GluR2-ABD in which time-resolved Fouriertransformed infrared (FTIR) spectroscopy was used to track the formation of glutamate–ABD interactions on microsecond time scale. The FTIR methodology allowed the authors to discriminate between binding of the - and -carboxylate groups of glutamate to the protein by measuring the shift in vibrational mode of these groups on binding. Besides verifying the two-step binding mechanism suggested by Abele et al. (91), the results provided further important insights into the binding mechanism. First, when E705 (Fig. 2B) is mutated to aspartate, the time period between the first and second steps in the binding mechanism is increased, highlighting the importance of this interaction in the second, slower binding step. Second, vibrations assigned to the -carboxylate of glutamate remain unchanged during the rapid first binding step but change during the slower, second binding step, thereby indicating that interactions between this part of the ligand and the protein are formed late in the binding mechanism. The results from spectroscopic studies in combination with the crystallographic data on, in particular, the GluR2-ABD structure provide the basis for the current mechanistic and structural model for ligand binding to iGluRs. In this model, ligand binding to the agonist-binding domain proceeds via an at least a two-step mechanism in which the ligand initially makes contacts to residues on D1, inducing a relatively slower second step in which D2 undergoes a transition in which further ligand–protein and D1–D2 interactions

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occur, leading to closure of the D1 and D2 shells and locking of the ligand into the holo conformation observed in crystal structure (64,91,92). Similar “dockand-lock” mechanisms are well described for the structurally homologous PBPs (93,94), strongly indicating that D1/D2 cleft closure is an evolutionary conserved mechanism for ligand binding in this type of protein. In contrast to agonist binding, crystal structures of ABDs from all iGluR subfamilies have shown that binding of ligands with antagonistic properties to the agonist-binding site only induce minor cleft closure, or, as in the cases of a recent GluR2 structure with the antagonist NS1209 and the GluR5 structures with the antagonists UBP302 and UBP310, in a hyperextended conformation (64,68,73,85,95). In intact iGluRs, a transition similar to agonist-induced cleft closure in isolated ABDs is a compelling candidate for the first conformational change leading toward channel activation. However, in the absence of structures for a full-length iGluR, it is impossible to assign ABD conformations to functional states of the receptor. It has therefore been of great importance to verify that the ABD within functional iGluRs undergoes similar transitions during agonist binding and channel activation. A recent study by Du et al. (96) used fluorescence resonance energy transfer (FRET) to track the distance between two artificial fluorophores, one of which can absorb light and emit a photon that, if close enough, can excite the second fluorophore. Engineering of two fluorophores on D1 and D2 in functional iGluRs allowed the use of FRET to confirm that D1 and D2 move on glutamate binding. The distance of the apparent movement correlates with the difference between the locations of the domains in the apo- and glutamate-bound ABD structures, a result that provides strong evidence that cleft closure indeed occurs in the ABD in intact iGluRs on agonist binding. 2.5. Cleft Closure as the Driving Force Behind Channel Activation The number and order of discrete conformational changes that the subunit complex undergoes during the channel-opening step of the activation mechanism define the gating mechanism of the iGluRs. Decades of biophysical studies on the functional behavior of the iGluRs have established that distinct, multistate activation mechanisms can be assigned to receptors in each subfamily (97,98). AMPARs and NMDARs activate ion channels using mechanisms that differ fundamentally. NMDARs require binding of both glycine and glutamate before the ion channel is activated by a concerted rearrangement involving all four subunits into a single open state; for example, single-channel recording of NMDAR currents usually show a single conductance level (98). In contrast, each subunit within the tetrameric AMPARs can autonomously activate the ion channel; for example, binding of two subunits by glutamate can promote opening of the channel (99). Furthermore, AMPARs can activate into

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multiple open conformations with different conductance levels that correlate with the number of subunits occupied by agonists (99–101). As described in the previous section, cleft closure within the ABD is a likely candidate for the early conformational event that triggers the subsequent transition of the ion channel domain into an open conformation. The opening of the channel ultimately implies that a subdomain, the gate, of the ion channel undergoes a conformational change that relieves steric hindrance of ion flux through the ion channel. The first structural hints for a mechanism that couples cleft closure in the ABD to a conformational change within the ion channel was provided by the early GluR2-ABD structures (61,64). These structures show the molecules to be arranged as twofold-symmetric pairs in a backto-back fashion with the dimer interface formed almost exclusively between hydrophobic surface regions on the D1 domain (Fig. 3B) (64). The inter-D1 contacts formed across the dimer interface are widely believed also to be present in full-length GluR2 receptors, thereby imposing constraints on D1 movement (72,80,102–104). In contrast, the D2 domains appear to be relatively free to move. This apparent mobility of D2 is of particular interest because this domain contains the anchor points for the short segments that link the ion-channel transmembrane domain to the ABD (Fig. 1C). In all engineered ABDs, the short artificial polypeptide linker that connects the S1 and S2 segments marks the position of these anchor points (Fig. 1D). Of great interest, Armstrong and Gouaux (64) first noted that superposition of structures of apo and agonist-bound dimers displays a striking difference in the relative position of the linker in each ABD (Fig. 3B). Specifically, the distance between the linkers is increased by several angstroms in agonist-bound dimers (Fig. 3B). These results lead Armstrong and Gouaux (64) to propose a structural model for AMPA receptor activation in which cleft closure mainly involves movement of D2, whereas D1 and the dimer interface remain relatively fixed. The D2 transition leads to displacement of the linker regions, which, in the intact subunit, would subject the M1 and M3 helices to conformational strain and possibly drive the subsequent transitions toward channel opening. As will be described in further detail in Section 2.7, it has been suggested that the aforementioned strain created by D2 displacement during cleft closure specifically is relieved by a M3 transition, which then constitutes the actual gating event (62,105–108). This “D2–M3” strain model is supported by several studies in which mutations in M3 or the D2–M3 linker strongly alter channel activation rates (109,110). Furthermore, cysteine scanning mutagenesis has revealed that reactivity of engineered cysteine residues in the outer section of M3 in the NR1 and NR2A–D subunits toward sulfhydryl-modifying reagents depends on activation of the receptors. In other words, the outer section of M3 becomes more accessible to extracellular sulfhydryl reagent on agonist

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Fig. 3. A. Ligand binding to the agonist-binding domain (ABD) of the amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid receptor subunit GluR2 induces the protein to adopt distinct conformations. An “open cleft” conformation is observed in complexes with the antagonist 6,7-dinitro-quinoxaline-2,3-dione (DNQX) (Protein Data Base [PDB] code 1FTL) (upper left) or in the absence of ligand (PDB code 1FTO) (upper middle). In the presence of the agonist glutamate (PDB code 1FTJ) (upper right) the domain adopts a “close cleft” conformation.

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binding, indicating that M3 indeed is coupled to agonist-induced conformational changes during activation (105,111). The foregoing cleft closure model for iGluR activation was initially developed on the basis of structural studies on the GluR2 agonist-binding domain. It is appealing for both its simplicity and ability to describe a wide range of data for AMPARs, including the mechanism underlying partial agonism. For example, Jin et al. (100) showed that a series of partial agonists— the 5-substituted willardiines (Table 1)—varying by a single atom induce a differential degree of cleft closure that correlates with their efficacy of channel activation. This finding, along with important earlier studies (99,101), suggests that tetrameric AMPA receptors can ratchet open their channel as a function of the probability of activation for each contributing subunit, and that this probability increases with the degree of agonist-induced cleft closure, thereby providing the structural mechanism underlying partial agonism at AMPARs (Fig. 3). Structural data for the NMDAR and KAR agonist-binding domains show that agonist-binding domains from subunits belonging to these iGluR subfamilies fold in a similar manner to GluR2 and that these all assume closed cleft conformations in the presence of agonist. Consequently, it might seem reasonable to expect the structural concepts for ABD control of gating developed for AMPARs to transfer to the other iGluRs. Indeed, the first structures available for the ABD of the NR1 subunit complexed with ligands exhibited cleft closure that paralleled agonist- or antagonist-bound GluR2 structures (80,86). However, in contrast to GluR2, no substantial difference in the degree of cleft closure exists between the full agonist glycine, a partial agonist

 Fig. 3. (Continued) Lower: Structural views of the apo conformation with the antagonist and agonist-bound structures, respectively, to illustrate the difference between the conformations. B. Isolated ABDs from iGluR subunits assemble as dimers in most crystal structures, with the interface between each monomer formed by interactions between the back of domain 1 (pale green). Binding of agonist (-amino-3-hydroxy5-methyl-4-isoxazolepropionic acid [AMPA]; PDB code IFTM) induces a transition of the domain 2 (light orange) that leads to separation of the linker segments that replaces the transmembrane domains in the full-length subunits. C. Pharmacological or mutational manipulation of the stability of the dimer interface of GluR2-ABDs influences desensitization. Crystal structure of the dimer formed between monomers of the L483Y-mutated GluR2-ABD (PDB code 1LB8) shown perpendicular (left) to the molecular twofold axis or viewed from the top parallel to the twofold axis (right). Mutation of residue 483 (blue) located on domain 1 from leucine to tyrosine attenuates desensitization and stabilizes the dimer interface by interactions with Leu748 and Lys752 on the opposing ABD monomer.

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d-cycloserine, and a series of structurally related partial agonists (86). Thus, the isolated ABD of the NR1 subunit shows no relationship between the degree of agonist-induced cleft closure and agonist efficacy. This finding suggests that important differences must exist between NMDAR and AMPAR subunits with respect to how intraprotein changes transmit motions from the agonistbinding pocket to the gating domain. Inanobe et al. (86) suggested that NR1 agonists induce an incremental local rearrangement of a few specific contact residues within the NR1 binding pocket rather than changing the degree of cleft closure. The degree of local rearrangement appears to be related to the agonist efficacy, suggesting that locally reorganized residues convey conformational changes at the binding site to secondary structure elements, perhaps at the highly flexible hinge region holding D1 and D2 together. Because the dynamic behavior of the hinge region is likely to be a key determinant for cleft closure, one might speculate that binding of the NR1 glycine-site partial agonist shifts the equilibrium between “open” and “closed” cleft conformations of the ABD, with only the “closed” cleft conformation being able to activate the channel. Thus, despite the structural similarities between the ABDs from AMPA and NR1 subunits, different pictures of how agonists can influence protein structure and channel activation have emerged for the AMPAR and NMDAR subfamilies. Comparison of the crystal structures of the GluR6 KAR subunit in complex with full agonists (glutamate, 2S,4R-4-methylglutamate, and quisqualate) and a partial agonist (kainate) reveals that the latter induces a lesser degree of cleft closure (Table 1), consistent with the idea of agonist-induced cleft closure as a main determinant of efficacy for KARs (72). However, the number of structures of kainate receptor ABDs in complex with partial agonists is still too low fully to validate this conclusion. 2.6. Intradomain Dynamics During Agonist Gating The crystallographic studies described so far have revolutionized our thinking about glutamate receptor function. However, crystal structures are inherently static in nature and do not provide details of permitted motions that underlie the protein transitions under physiologic conditions. Therefore, obvious questions can be raised regarding the relevance of the functional models based on crystal structures of isolated ABDs. In addition, until the arrival of a structure of an intact iGluR, it will be essentially unknown which functional state a specific conformation of an isolated ABD represents and, for example, whether the idea of degree of cleft closure as the decisive structural determinant for agonist efficacy is valid, a paradox highlighted by the recent findings that certain partial agonists with different efficacies induce similar degrees of cleft closure in GluR2-ABD (112). Furthermore, the next challenge is to identify the peptide moieties and the motions that are

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responsible for relating the conformational transitions within the ABD to the rearrangement of the transmembrane helices taking place during gating. For these purposes, methodologies for studying protein dynamics are important tools in iGluR research. Computer-aided molecular dynamics (MD) simulations based on the growing number of ABD structures are emerging as powerful tools to monitor protein fluctuations in real time. These can provide insight into the principal intraprotein motions within the ABD that occur during the cleft closure transition (113,114). In addition, MD simulations can assess the stability of the ligand–protein interactions in the binding pocket, adding further detail to our understanding of the determinants of ligand selectivity and efficacy (114–118). As an example, results from MD simulations on the NR2A ABD docked with glutamate and the partial agonist homoquinolinate identified a subset of agonist contacts as important elements in defining the differential ability of these agonists to activate NR1/NR2A receptors (115). Specifically, analysis of binding-pocket motions during the MD simulations showed that homoquinolinate in comparison to glutamate introduced increased motion around a subset of residues on D2, leading to loss of key interactions between helix F and other sections of the protein. Analysis of singlechannel currents evoked by homoquinolinate or glutamate from NR1/NR2A receptors revealed that the reduced efficacy of homoquinolinate is due to a reduced rate of the channel activation, indicating that the homoquinolinatecomplexed NR2A-ABDs have reduced ability to translate binding energy from the binding pocket to opening of the channel. Combined with the structural models, this observation has led to the hypothesis that helix F may play a role in translation of binding energy to displacement of gating elements, an idea that is supported by both spectroscopic and crystallographic data (80,86,119). Experimental techniques that can be used to verify predictions regarding protein motions obtained through MD simulations or derived from crystal structures include nuclear magnetic resonance (NMR), ultraviolet, and infrared spectroscopy and fluorescence resonance energy transfer (35,80,86,90,92,96). Such methodologies offer the possibility for time-resolved monitoring of conformational behavior of ABDs in solution and have been used to verify that ligand binding indeed induces transitions in the ABD in full-length receptors similar to those observed in ABDs derived from S1S2 constructs (96). For example, studies employing 15 N NMR spectroscopy to monitor ligand binding to GluR2-ABD in solution have been able to detect and track changes in protein conformation and dynamics on agonist binding to specific structural elements that previously had been proposed to be prime candidates for transmission of motion from the agonist-binding site to the putative gating elements within the ion channel (119,120).

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2.7. Molecular Determinants of Gating As detailed in the previous sections, the high-resolution structures and dynamic studies of ABDs from different iGluR subunits have provided tremendous insights into conformational changes underlying gating at the level of the ABD. In terms of gating in the intact iGluR, however, this represents only one of the initial steps in a complex sequence of transitions that lead to channel opening. Indeed, it is less clear how conformational changes in the ABD are transformed into opening and closing of the ion channel itself. For iGluRs, all three transmembrane segments—M1, M3, and M4—are directly coupled to the agonist-binding domain (Fig. 1). Not surprisingly, then, point mutations in all of these segments or in the linkers coupling them to the ABD as well as the M2 loop can affect gating (38,121–124). A surprising property of mammalian iGluRs is the requirement for an additional transmembrane segment, the M4 segment, to function (125). M4 is absent in the prokaryotic iGluR, GluR0, and the distantly related K+ channel does not contain an equivalent transmembrane domain. In addition, certain noncompetitive antagonists of AMPARs act via the D1–M1 and D2–M4 linkers (126), with these same elements showing strong state-dependent rates of reactivity to cysteine-modifying reagents, indicating that they undergo molecular rearrangements with channel gating (127). Hence, many structural elements contribute to the energetics of state-dependent conformations, and integrating this information will be a great challenge even when high-resolution structures of intact iGluRs are available. Although many domains have been suggested to contribute to channel gating, the most intriguing regarding the gating domain in iGluRs appears to be the M3 segment. Indeed, a number of detailed studies have focused on the M3 segment itself or the linker region coupling it to the agonist-binding domain (D2–M3 linker) (97,111,128). In part, the interest in M3 as a gating domain reflects the dramatic effects point mutations here have on channel function (124). In addition, the homologous domain in K+ channels, the inner helix or M2, represents the major structural element lining the intracellular vestibule and defining gating (129). Considerable evidence supports the idea that the M3 segment represents the major pore-lining element in the extracellular vestibule. A notable feature of the M3 segment is the SYTANLAAF motif, the most highly conserved element in mammalian iGluR subunits (106). Substitutions of the alanines in this motif frequently yield channels with abnormal gating behavior (105,107,109–111). In addition, cysteine substitutions in M3 and in particular in SYTANLAAF show a strong state-dependent accessibility (105,107,127). Nevertheless, the mechanistic contribution of M3 is unknown despite its critical role in channel gating. A key gating feature of any channel is the activation gate—the structure that occludes the flux of ions in the closed state. The location of the activation

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gate in iGluRs is uncertain, possibly being positioned externally at the bundle helical crossing made by M3 or internally in the M2 loop (128,130). Resolving the location of the activation gate is critical to defining the mechanism of gating in iGluRs as well as to understanding how various open channel blockers with therapeutic potential interact with the pore. 2.8. Molecular Determinants of Ion Permeation and Block The ion channel associated with the iGluR, like all other ion channels, consists of a water-filled pore divided into intracellular and extracellular vestibules by a narrow constriction. Structural determinants of these various regions, based on functional experiments and the structural homology to an inverted K+ channels, are defined in general: The reentrant M2 pore loop lines the inner vestibule, with the channel’s narrow constriction located at or near the tip of this loop (like the P loop in K+ channels) and with the extracellular vestibule lined primarily by M3 and to a lesser extent by M1 (128). However, given the lack of a high-resolution structure and the asymmetric contribution of iGluR subunits to pore structure (in contrast to K+ channel subunits), many detailed structural features of the permeation pathway remain to be resolved. Furthermore, whereas key structural determinants of ion permeation and channel block are known, the mechanisms of these processes are incomplete at the structural and kinetic levels. A key determinant of the conductance and permeation properties of all iGluRs is the residue occupying a functionally critical position at or near the apex of the reentrant M2 loop, the Q/R/N site (1). All non-NMDAR subunits, except for edited ones, contain a glutamine (Q) at the Q/R site. RNA editing of the Q/R site in the AMPA receptor GluR2 or the KAR GluR5 and 6 subunits results in the glutamine being replaced by a positively charged arginine (R) in the mature protein (131). The homologous position in NMDA receptor subunits, except for NR3, is occupied by an asparagine (N). The Q/R/N site is located at or near the tip of the M2 loop and therefore is closely associated with the channel’s narrow constriction (106,132,133). Not surprisingly, then, given this key position, the residue occupying the Q/R/N site influences numerous functional properties, including single-channel conductance, Ca2+ permeability, channel block by polyamines and Mg2+ and numerous organic compounds, and assembly into heteromeric complexes (1,134,135). In addition, homomeric R-forms of KAR channels are no longer cation selective, being permeable to Cl− (136). Although the Q/R/N site strongly influences both permeation and block properties of the channel, additional structural elements also influence these properties. Notable here are two key functional properties of NMDARs: their high Ca2+ permeability and the strong voltage-dependent block of their channel by extracellular Mg2+ . These two properties confer on NMDARs key and

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distinctive roles in synaptic physiology. The block by extracellular Mg2+ is particularly distinctive because the channel is gated not only by glutamate (and glycine) but also by the membrane potential, allowing the receptor to act as a coincidence detector of pre- and postsynaptic activity (137). At a structural level, Ca2+ permeability and Mg2+ block are both strongly influenced by the channel’s narrow constriction (1). The magnitude of Ca2+ influx mediated under physiologic conditions, however, is also influenced by a cluster of charged residues, the DRPEER motif, located in the extracellular vestibule (138). This motif is unique to the NR1 subunit and is positioned in the linker between the M3 segment and the D2 lobe. It functions in part because of its net negativity: Three negative charges (one aspartate and two glutamate residues) but only one positive charge (the first arginine residues) are exposed to the water interface. Nevertheless, how the DRPEER motif, the channel’s narrow constriction, and other potential determinants of Ca2+ influx can control the process of Ca2+ influx (as well as Na+ and K+ influx) is unknown. The voltage-dependent block of NMDARs by extracellular Mg2+ is greatly influenced by structural elements located at or near the channel’s narrow constriction, mainly as contributed by the NR2 subunit (1). The mechanism of this process, however, depends on additional sites in the pore (132). Notably, the strong voltage dependence of the block—the hallmark of the block process—depends on how monovalent cations interact with the pore (139). The identity of these monovalent interaction sites will help to resolve the molecular and structural basis of one of the most intriguing biophysical properties of ion channels. One surprising feature of iGluRs, in contrast to K+ channels, is that the various subunits do not appear to contribute equally to the pore structure. This asymmetry was noted in the original identification of the N sites in NMDARs, where substitutions of the N-site asparagine in NR1 had strong effects on Ca2+ permeability but only weak effects on Mg2+ block, whereas equivalent substitutions of the N site in NR2 produced opposite effects (140). Furthermore, the channel’s narrow constriction in NMDARs is formed by nonhomologous asparagines, the NR1 N site and one adjacent to the NR2 N site, the N + 1 asparagine (133). This means that the M3 segments from the different subunits do not appear to be perfectly aligned in the pore (141). Because subunits in AMPARs may possess a twofold symmetry (108,142), they too may show a structural asymmetry. Nevertheless, the full extent of both the structural and functional asymmetry between subunits is unknown—in particular, whether the asymmetry exists only at the atomic level or on a larger scale in receptor structure as well. Understanding the structural and functional basis of permeation will also enhance our understanding of mechanisms of channel block. NMDAR

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channel blockers may become important therapeutic agents in a number of clinical settings. Excitotoxicity mediated by iGluRs, notably the NMDAR, has been implicated in the cell death associated with numerous acute and chronic brain diseases such as hypoxia/ischemia, epilepsy, and Parkinson and Alzheimer disease (143,144). NMDAR antagonists, although neuroprotective, have not been useful in the clinic because of extensive detrimental side effects. On the other hand, low-affinity NMDAR channel blockers such as memantine are approved for clinical use, with most of their therapeutic value presumably arising from their low-affinity interaction with NMDARs (144,145). Memantine and related blockers act in part via an open channel block mechanism, and the major site of action for this is near the channel’s narrow constriction. However, they also act at other sites mainly in the extracellular vestibule (127,146,147). The manner and effect of memantine and related blockers interacting with these more external sites are unknown but may be related to channel gating because memantine facilitates channel closure (148). Indeed, clarifying the structural basis for these gating effects represent a major and critical challenge, given the potential clinical usefulness.

3. Subunit Interfaces: Modeling Desensitization and Deactivation Desensitization appears to be a near-universal feature of ligand-gated ion channels and is classically defined as the diminution of response in the continued presence of an activating stimulus, for example, the ion channel is closed and the agonist is still bound tightly. Desensitization of iGluRs, especially that of the AMPARs and KARs, could serve important in vivo functions, which may contribute to shaping of the excitatory postsynaptic current (149,150), as well as provide a protective mechanism against receptor overactivation under circumstances in which malfunctioning of transmission leads to abnormal periods of exposure to high levels of glutamate (151). For most types of native AMPARs and KARs, almost complete desensitization occurs within 1000 μM) receptors or iGlu receptors. The most common mGlu1 receptor antagonist is CPCCOEt [7(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester], a selective reversible and noncompetitive antagonist of the human mGlu1 receptors (halfmaximal inhibition constant [IC50 ] 6.5 μM) without agonist or antagonist activity at human group II or III mGlu receptors (IC50 >100 μM) or iGlu receptors (159,160). CPCCOEt exhibits the greatest antagonist activity and selectivity for mGlu1 over mGlu5 receptors (161). It represents a novel class of GPCR antagonists that inhibit receptor signaling without affecting ligand binding (162). CPCCOEt noncompetitively decreases the efficacy of glutamate-stimulated PI hydrolysis without affecting the EC50 value or the Hill coefficient of glutamate, and it does not displace [3 H]glutamate binding to mGlu1a-expressing membranes. However, the interaction between mGlu1 and CPCCOEt is disrupted by mutagenesis of residues located at the extracellular surface of TM seven, suggesting that CPCCOEt inhibition is mediated by an intramolecular interaction between the agonist-bound extracellular domain and the transmembrane domain. YM-298198 (6-amino-N-cyclohexylN,3-dimethylthiszolo[3,2-a]benzimidazole-2-carboxamide) is also a noncompetitive antagonist with high affinity (IC50 = 16 nM) and selectivity for mGlu1 receptors that is inactive at other mGlu receptor subtypes, iGlu receptors, and glutamate transporters (163). YM-298198 can be administered orally and is available in radiolabeled form as [3 H]YM-298198, making it a useful tool for both in vivo and in vitro experiments (163).

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The most widely used mGlu5 receptor antagonist is MPEP [2-methyl6-(phenylethynyl)-pyridine], a potent and highly selective noncompetitive antagonist at the mGlu5 receptor subtype (IC50 =36 nM) (164–166) (for review, see ref. 167). In addition, another selective mGlu5 antagonist, MTEP (3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine), has similar potency to MPEP but superior selectivity and solubility (168). MTEP also has anxiolytic (168,169), antidepressant (170), and anti-Parkinsonian (171) properties. Finally, fenobam [N-(3-chlorophenyl)-N-(4,5-dihydro-1-methyl-4oxo-1H-imidazole-2-yl)urea] sulfate is a potent and selective noncompetitive mGlu5 receptor antagonist. Fenobam is an inverse agonist that blocks constitutive activity of mGlu5 in vitro at an allosteric modulatory site shared with MPEP and displays anxiolytic activity following oral administration in vivo (172). 3.1. Allosteric Modulators Recent studies have identified a family of highly selective allosteric modulators of the mGlu5 subtype (173). This family of closely related analogs exerts a spectrum of effects, ranging from positive to negative allosteric modulation, and includes compounds that are not direct agonists or antagonists of mGlu5 receptors but that modulate agonist and antagonist effects at mGlu5 receptors. For example, DFB (3,3’-difluorobenzaldazine) has no agonist activity but acts as a selective positive allosteric modulator of human and rat mGlu5 and potentiates threshold responses to glutamate, quisqualate, and DHPG (EC50 2–5 μM). However, at higher concentrations (10– 100 μM), DFB shifts mGlu5 agonist concentration–response curves approximately twofold to the left. CPPHA (N-[4-chloro-2-[(1,3-dioxo-1,3-dihydro2H-isoindol-2-yl)methyl]phenyl]-2-hydroxybenzamide) is also a potent and selective positive allosteric modulator of human and rat mGlu5 that alone has no agonist activity on these receptors (174). In contrast, the analog DMeOB (3,3’-dimethoxybenzaldazine) acts as a negative modulator of mGlu5 agonist activity, with an IC50 of 3 μM, whereas the analog DCB (3,3’dichlorobenzaldazine) does not exert any apparent modulatory effect on mGlu5 activity. However, DCB can act as an allosteric ligand with neutral coperativity, in that it prevents the positive allosteric modulation of mGlu receptors by DFB and the negative modulatory effect of DMeOB. Furthermore, whereas none of these analogs affects agonist binding, they do inhibit binding to the MPEP site. These studies show that related allosteric ligands induce a range of pharmacologic activities from positive to negative modulation, including neutral modulation. Studies show that an allosteric modulator can have differential effects on independent signaling pathways mediated by activation of mGlu5 in cultured rat cortical astrocytes (175). In these studies both DFB and CPPHA did

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not alter baseline calcium levels but did induce parallel leftward shifts in the concentration–response curve to DHPG and glutamate, identical to their effects on mGlu5 in heterologous expression systems. DFB also induced a similar shift of the concentration–response curve of DHPG-induced phosphorylation of ERK1/2. It is interesting that CPPHA induced an increase in basal mGlu5-mediated ERK1/2 phosphorylation and potentiated the effect of low concentrations of agonists. In contrast, CPPHA significantly decreased ERK1/2 phosphorylation induced by high concentrations of agonists. Thus, CPPHA had qualitatively different effects on mGlu5-mediated calcium responses and ERK1/2 phosphorylation. These data provide evidence that distinct allosteric potentiators can modulate coupling of a single receptor to multiple signaling pathways. These findings also provide insights into the functional role of mGlu homodimers or heterodimers when coupling to different pathways or signaling partners is mediated by distinct monomers, such as shown for mGlu1a (29). In summary, there has been substantial emphasis on identifying pharmacologic tools that can selectively activate individual mGlu receptor subtypes in the central nervous system (CNS). Many of the available pharmacologic tools show nonspecific effects on other mGlu receptor subtypes or on iGlu receptors but nonetheless have been used to obtain an enormous amount of information regarding mGlu receptor function over the years. However, until subtypeselective compounds are developed, the most useful pharmacologic tools for studying group I mGlu receptors in native cells are the group I–selective agonist, DHPG, the mGlu1-selective antagonists CPCCOEt and YM-298198, and the mGlu5-selective antagonists MPEP, MTEP, and fenobam.

4. Interacting Partners The multitude of group I mGlu receptor–interacting proteins highlights the complexity underlying mGlu receptor signaling in neurons and suggests that the functional role of individual mGlu receptors within a cell likely reflects their unique interactions with regulatory proteins within a signaling complex. For example, data suggest that mGlu1 and mGlu5 are both expressed in hippocampal CA1 pyramidal neurons, but each subtype plays distinct roles in the regulation of these cells (176). In addition, mGlu1 and mGlu5 are both expressed in interneurons but play differential roles in modeled cerebral ischemia, and only mGlu1 is implicated in pathways leading to postischemic neuronal injury (177,178). These data suggest that protein localization and protein–protein interactions in mGlu receptor signaling complexes determine the physiologic effects of receptor activation. Several studies have defined a major site for group I mGlu receptor/protein interactions at the intracellular CTD that together with the second intracellular domain comprise the G protein–binding site (179) (Fig. 8). Proteins

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Fig. 8. Several group I metabotropic glutamate (mGlu) receptor–interacting proteins have been identified, including Homer and postsynaptic density (PSD)-95, that link the receptors to the endoplasmic reticulum (ER) and the actin cytoskeleton, respectively. More recently, the group I mGlu receptors have been shown to interact with phosphofurin acidic cluster sorting proteins (PACS) that likely regulate subcellular localization. GKAP, guanylate kinase–associated protein; IP3 R, inositol 1,4,5-trisphosphate receptor.

that bind directly to the CTD of group I mGlu receptors include Homer proteins (180,181), Gq (6) and Gi/Go (182) alpha proteins, calmodulin (183), -tubulin (184,185), Siah1A (186), and protein phosphatase 1C (187). In addition, data support an indirect interaction between mGlu5 and the IP3 receptor (181,188), PLC  (189), Shank (190), arrestin (191), and Src-family protein tyrosine kinases (192) and calcineurin (193). Studies also support indirect interactions between cytoskeletal proteins and mGlu receptors: actin binds to Cupidin (Homer-2a), which in turn binds to mGlu1a (194), and actin polymerization/depolymerization regulates the movement of mGlu5 in the plasma membrane (195). 4.1. Targeting and Trafficking The best-studied mGlu receptor–interacting proteins are the Homer/Vesl (VASP/Ena-related protein induced during seizure and LTP) protein family, which localize to postsynaptic densities of excitatory synapses in the mammalian brain (194,196–198). The Homer protein family includes the inducible immediate-early gene short form of Homer-1a and the constitutively expressed long forms of Homer (1b/1c/2/3), all of which can directly interact with IP3 receptors, ryanodine receptors, type 1 and type 2 C-type TRP channels, Shank proteins, and dynamin 3 (for review, see ref. 199) Homer proteins bind directly to the CTD of mGlu1a and mGlu5 via a proline-rich consensus sequence (PPxxF) in the CTD (200) and regulate the cellular distribution of

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group I mGlu receptors. For example, expression of mGlu1a or mGlu5 alone in heterologous cells results in diffuse localization of these receptors to the plasma membrane. Cotransfection of mGlu1a or mGlu5 with Homer-1a does not change this distribution pattern (201–204). However, mGlu1a or mGlu5 cotransfected with Homer-1b/c results in receptor clustering at the plasma membrane (204,205) or decreased surface expression due to receptor retention in the endoplasmic reticulum (185,203). Similarly mGlu5 transfected alone into cerebellar granule cells causes receptor localization to cell bodies, whereas cotransfection with Homer-1b/c results in receptor redistribution to dendrites, and cotransfection with Homer-1a causes receptor redistribution to axons and dendrites (206,207), consistent with the upregulation of Homer-1a by neuronal depolarization in native cells and subsequent redistribution of mGlu5 into neurites. Homer proteins also regulate functional responses elicited by group I mGlu receptors. Expression of Homer-1b/1c/2/3 leads to decreased group I mGlu– mediated inhibition of N-type calcium currents in superior cervical ganglion neurons that is reversed with expression of Homer-1a. Homer-1b/1c/2/3 also reduces mGlu1a modulation of M-type potassium channels (188). Overexpression of exogenous Homer-1a, induction of endogenous Homer-1a, or suppression of endogenous Homer-3 in cultured cerebellar granule cells leads to a group I mGlu receptor–mediated increase in Ca2+ -activated BK channel currents, even in the presence of competitive mGlu1 and mGlu5 antagonists. These results suggest that Homer-1b/1c/2/3 proteins suppress agonistindependent activity of group I mGlu receptors, whereas Homer-1a increases agonist-independent activity (208). Proteomic studies also reveal several novel mGlu5 receptor–interacting proteins in rat brain, including the phosphofurin acidic cluster sorting protein (PACS)-1 and PACS-2 (209). The PACS are a novel gene family that bind to acidic clusters within cargo proteins and regulate trafficking of proteins such as Furin (210,211), TRPP2 (212), and Bid (213). Recent studies show that the subcellular localization and function of the TRPP2 cation channel is directed by PACS-1 and PACS-2 (212), which represents a novel molecular mechanism for trafficking of acidic cluster–containing ion channels and receptors to distinct subcellular compartments. The group I mGlu receptors are the only mGlu receptors with acidic clusters in their CTD, and depletion of PACS-1 and PACS-2 leads to differential distribution of mGlu5 in cells (214). These data suggest that the PACS proteins play a role in the trafficking, and possibly the regulation, of group I mGlu receptors. In addition, proteomic and immunoblot studies show that ischemic preconditioning alters the protein composition of a group I mGlu receptor signaling complex in rat brains (215). mGlu1 specifically interacts with neuronal nitric oxide synthase (nNOS) in ischemic

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preconditioned rat brain, although nNOS protein levels are not altered. These results suggest that ischemic preconditioning leads to altered protein complexes independent of changes in protein expression levels. In summary, the multitude of group I mGlu receptor–interacting proteins highlights the complexity underlying mGlu receptor signaling in neurons and suggests that many group I mGlu receptor–protein interactions are synapse specific. It is clear that the Homer family of proteins not only determines the localization or trafficking of group I mGlu receptors, but also can also modulate the activity of mGlu receptors in the absence of agonist. These findings suggest that the complement of proteins in each synapse may determine which signaling pathways will be used to regulate neuronal output. Thus, it will be very important to identify all mGlu receptor–interacting proteins and elucidate their role in the regulation of mGlu receptor signaling.

5. Modulation by Signaling Systems In addition to modulation of group I mGlu receptors by interacting proteins, there is both homologous and heterologous regulation of group I mGlu receptor signaling. Desensitization of mGlu1 and mGlu5 occurs similarly to other GPCRs, through phosphorylation of intracellular domains, the recruitment of G protein–coupled receptor kinases (GRKs) and  -arrestin, and the subsequent uncoupling of the receptor from G proteins. Regulators of G protein signaling (RGS) proteins are also involved in modulating group I mGlu receptor function. In addition, heterologous regulation of mGlu receptors in response to simultaneous stimulation of multiple receptor pathways is an important type of regulation involved in the integration of multiple signaling pathways. The group I mGlu receptors are largely modulated by phosphorylation at the CTD, where the majority of homologous and heterologous regulation of mGlu receptor function occurs. Many studies provide evidence for PKC-dependent desensitization and modulation of group I mGlu receptor signaling (216–221), with less evidence for modulation of group I mGlu receptors by PKA (218) or tyrosine kinases (222). Finally, there is no evidence for modulation of group I mGlu receptors by casein kinase I or II, although group I mGlu receptors do activate these kinases (71,72). Other posttranslational modifications that may modulate mGlu receptors include sumoylation, palmitoylation, and ubiquitination. Although sumoylation modulates mGlu8 (223) and palmitoylation modulates mGlu4 (224,225), there is no evidence for modulation of group I mGlu receptors by these modifications. On the other hand, Siah1A binds to the CTD of mGlu1a and mGlu5 (226) and serves as a selective ubiquitin ligase that mediates ubiquitination-dependent degradation of mGlu1 and mGlu5, thus contributing to the posttranslational downregulation of group I mGlu receptors (227).

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5.1. Modulation by PKC Glutamate elicits desensitization of the group I mGlu receptors by stimulating PKC-dependent phosphorylation of the CTD (219,228). In cultured cortical astrocytes, PKC-mediated desensitization of mGlu5 receptors attenuates phosphoinositide hydrolysis and calcium signaling but does not affect mGlu5-mediated phosphorylation of ERK2 (229), suggesting that regulation at the receptor level does not necessarily affect all signaling pathways. Direct phosphorylation of mGlu5a by PKC results in Ca2+ oscillations in mGlu5aexpressing cells, providing the first evidence that PKC phosphorylation of GPCRs is important in generating intracellular Ca2+ oscillations (230). There is also evidence for differential regulation of two distinct mGlu1a-dependent signaling pathways by PKC and PKA. Activation of PKC selectively inhibits agonist-dependent stimulation of the IP3 signaling pathway but does not affect receptor signaling via cAMP; in contrast, PKA activation potentiates agonistindependent signaling of mGlu1a via IP3 . Molecular studies demonstrate that selectivity for PKC phosphorylation occurs at a residue within the G protein– interacting domain of mGlu1 that selectively disrupts mGlu1a–Gq/11 interactions without affecting signaling through Gs (218). Heterologous modulation of group I mGlu receptors has been demonstrated through an important reciprocal feedback modulation between mGlu5 and NMDA receptors. Low concentrations of NMDA significantly potentiate mGlu5 responses via activation of the protein phosphatase, calcineurin, and reversal of phosphorylation-dependent desensitization (193,231). In addition, NMDA receptor activation induces a long-lasting potentiation of group I mGlu responses in hippocampal CA3 pyramidal cells (232), whereas higher concentrations of NMDA inhibit mGlu5 by direct phosphorylation of PKC sites in the CTD (233). Group I mGlu receptors also potentiate NMDA receptor currents (140) and NMDA-mediated cognitive behaviors (234); however, the mechanisms underlying the upregulation of NMDA receptor function by group I mGlu receptors are not well understood. Studies in CA3 pyramidal cells show that activation of mGlu1 potentiates NMDA currents via a G protein–independent mechanism involving Src kinase activation (235), whereas mGlu5-mediated enhancement of NMDA currents requires G protein activation and signaling via PKC and Src (139,141,236–238). 5.2. Modulation by G Protein-Coupled Receptor Kinases G protein–coupled receptor kinases (GRKs) are regulatory molecules that are involved in the homologous desensitization of GPCRs. GRKs recognize and phosphorylate agonist-bound, activated GPCRs (for review, see ref. 239). Arrestins then bind to GRK-phosphorylated receptors and initiate uncoupling of the receptors from heterotrimeric G proteins, resulting in desensitization

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and internalization of the receptors. The GRK family comprises six subtypes (GRK1–GRK6), which differ in their tissue distribution and structural features. Heterologous expression of mGlu1a with GRK2 and GRK5, but not GRK4 and GRK6, reduces both the constitutive and agonist-stimulated mGlu1a activity (240). However, GRK4 coexpressed with mGlu1 in cultured cerebellar Purkinje cells regulates signaling by homologous desensitization of mGlu1 (241). Thus, GRK4 likely contributes to motor learning by regulating functional responses of mGlu1 receptors in Purkinje cells. Although the accepted model for GRK-dependent desensitization involves GRK-mediated receptor phosphorylation and binding of arrestin proteins, phosphorylation-independent, GRK-mediated desensitization has also been demonstrated for mGlu1, as well as for GABAB receptors (242). These studies show that attenuation of mGlu1 signaling by GRK2 involves an interaction between the ATD of GRK2 with mGlu1 that decreases mGlu1 G protein coupling (243). The mechanism underlying GRK2 phosphorylationindependent attenuation of mGlu1a signaling requires binding of GRK2 to both Gq/11 and mGlu1a, suggesting that GRK2 can regulate receptor/G protein interactions in addition to its traditional role as a receptor kinase (242). Mutations of residues in the long mGlu1a CTD abolish GRK2-mediated inhibition of mGlu1a signaling, indicating that direct kinase binding to the G protein–coupling domain of mGlu1 is essential for the phosphorylationindependent attenuation of signaling by GRK2 (244). Finally, other studies show that the GRK2, but not GRK4, is involved in desensitization of mGlu5, but that the GRK2-mediated regulation of mGlu5 is phosphorylation dependent and requires, at least in part, a threonine 840 in the CTD of mGlu5 (245). 5.3. Modulation by Regulators of G Protein Signaling The regulators of G protein signaling (RGS) family of proteins regulate GPCR signaling pathway by directly interacting with G proteins to modulate G protein function (for review, see ref. 246). More than 20 RGS proteins have been isolated, ranging in size from 17 to 140 kDa, and specific RGS proteins regulate specific GPCR pathways (247). A conserved domain in RGS proteins encodes a GTPase-activating protein that accelerates G -catalyzed GTP hydrolysis to negatively regulate Gq and Gi-signaling proteins. However, there is little homology between the RGS family members outside of the RGS domain, and other protein domains likely control cell- and tissue-specific expression, intracellular localization, and posttranslational modification. The role of RGS proteins in the CNS has not been extensively characterized, but studies have provided evidence that RGS proteins regulate mGlu receptor function. For example, purified RGS4 blocks mGlu1a and mGlu5-mediated responses in Xenopus oocytes and also block group I mGlu receptor–mediated responses in hippocampal pyramidal cells (248). In addition, group I mGlu

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receptors expressed in sympathetic neurons inhibit IM -type potassium currents and calcium currents, but coexpression of RGS2 occluded mGlu1a inhibition of the IM and the voltage-dependent calcium current (249), presumably by blocking the activation of Gq/11. In summary, there is both homologous and heterologous regulation of group I mGlu receptor signaling, largely through phosphorylation of intracellular domains, the recruitment of GRKs and  -arrestin, and the subsequent uncoupling of the receptor from G proteins. In addition, there is a role for RGS proteins in group I mGlu receptor modulation. The group I mGlu receptors are largely modulated by PKC phosphorylation at the CTD, yet there is no evidence for posttranslational modulation of group I mGlu receptors by sumoylation and palmitoylation. However, there is a role for Siah1a ubiquitin-mediated degradation of mGlu1 and mGlu5 that contributes to the posttranslational downregulation of group I mGlu receptors (227).

6. Modulation of Synaptic Transmission Group I mGlu receptors modulate synaptic transmission by acute modulation of neurotransmitter release and by long-term modulation of synaptic activity. Although the receptors are predominately postsynaptic, they can regulate presynaptic neurotransmitter release at glutamatergic and GABAergic synapses and several signaling pathways that are involved in long-term potentiation and long-term depression. 6.1. Acute Presynaptic Modulation of Neurotransmitter Release Although group I mGlu receptors are mainly localized to postsynaptic neurons, there is evidence that these receptors both inhibit and facilitate presynaptic release of neurotransmitter in the hippocampus (125). Inhibition of presynaptic glutamate release by these receptors occurs in the hippocampus (250) and subthalamic nucleus (251) and in dopaminergic areas (252,253). The presynaptic inhibition occurs through several potential mechanisms, including inhibition of presynaptic Ca2+ channels (105,254,255) and activation of K+ channels (256). A Ca2+ -independent mechanism has also been described that is probably due to direct modulation of release machinery (257). Facilitation of presynaptic glutamate release by mGlu1 receptors has been observed in the parietal cortex (258) and in the lamprey spinal cord via group I mGlu receptor– mediated increases in intracellular Ca2+ release (256). Inhibitory or facilitatory effects of mGlu receptors on presynaptic glutamate release are dependent on the mGlu receptor subtype that is present, as well as on the rate and duration of the stimulation (146). The mGlu receptors also modulate presynaptic release of neurotransmitters other than glutamate. Group I mGlu receptors appear to play a bidirectional role in the modulation of GABAergic interneurons. Activation

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of these receptors located on the soma increases the excitability of interneurons in several areas (259–261), but group I mGlu receptors on axonal terminals inhibit the release of GABA from interneurons (45,252,260). 6.2. Long-Term Modulation of Synaptic Transmission Long-term modulation of synaptic transmission, LTP and LTD, are experimental phenomena that are expressed at excitatory synapses throughout the brain (262). Although the processes of LTP and LTD are not directly linked to memory, it is likely that synaptic restructuring is involved in memory formation. It is also clear that mGlu receptors play a role in modulating cognitive behaviors in vivo and in the induction and expression of LTP and LTD in vitro in many areas of the brain (reviewed in refs. 146 and 262– 265). In general, NMDA receptor–dependent LTP has been the most widely studied form of LTP, and in some synapses activation of mGlu receptors can modulate NMDA-dependent LTP (266,267). However, activation of either NMDA receptors or mGlu receptors is sufficient to induce LTD, depending on the synapse (262). Several second messenger systems have been implicated in the induction of LTP (reviewed in refs. 262 and 268), including PKC (269–271), PKA (272), CaMKII (273,274), and MAPK/ERK pathways (75,83). All of these second messenger pathways can be activated by group I mGlu receptors, indicating that modulation of LTP by mGlu receptor activation can occur at multiple steps within the induction pathway. Activation of mGlu receptors in the hippocampus elicits LTP (275,276) that is dependent on depolarization of the postsynaptic neuron (277–279) or activation of NMDA receptors (278–280). Group I mGlu receptors facilitate NMDA-dependent LTP by increasing MAPK activation (81) and CREB phosphorylation (81,281). LTP in the hippocampus is also facilitated by prior exposure to group I mGlu receptor agonists (282) in a PLCdependent manner (283). This “priming” of LTP transforms a small, reversible potentiation into a long-lasting enhancement by either an agonist or a synaptic stimulation protocol and can be blocked with protein synthesis inhibitors (284). The rapid priming effect (280-fold selectivity over other mGlu subtypes (EC50 > 3.5 μM on mGlu1–7)(28). For mGlu4 and 7, positive allosteric activators and modulators have been identified. The orally active compound N, N  -dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082) is an mGlu7-selective agonist that fully activates receptor signaling via an allosteric site in the transmembrane domain (35,36). In transfected mammalian cells expressing mGlu7, AMN082 potently inhibited cAMP accumulation (EC50 = 64–290 nM) with agonist efficacies comparable with those of LAP4 (35). In this study, AMN082 (up to 10 μM) had no effect on the other mGlu subtypes and N-methyl-d-aspartate (NMDA) (NR1/2A and NR1/2B) and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (GluR3) receptors. N-Phenyl-7-(hydoxylimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) has been identified as an allosteric potentiator of mGlu4 (10,37). PHCCC showed no intrinsic agonist activity on its own but increased the potency and efficacy of the orthosteric agonist L-AP4. The binding site of the active enantiomer (-)-PHCCC is localized in the transmembrane region (37). (-)-PHCCC does not potentiate or activate mGlu2, 3, 5a, 6, 7b, and 8a but has weak partial antagonist effects at some mGluRs, including mGlu1b, 2, 5, and 8 (10,37,38). Relatively few antagonists are available for group III mGluRs. MAP4 and MSOP, -methyl derivatives of the group III receptor agonists L-AP4 and L-SOP, are competitive antagonists of these receptors (IC50 = 25–190 μM), although they may have agonist activity at mGlu2 (MAP4, MSOP) and mGlu4 and 6 (MAP4) in some tissues (1,4).

4. Interacting Partners (Trafficking and Targeting) Little is known about the interaction of group III mGluRs with cytoskeletal partners involved in trafficking and subcellular targeting. The central domain

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of the ∼60-amino acid CT sequence, amino acids 883–912, has emerged as a key determinant of axon targeting versus axon exclusion and for cell surface expression of mGlu7 (31,39,40). It has been suggested that the mGlu7 CT targeting signal acts through protein sorting into specific vesicles and vesicle transport (39). It is interesting that the cytoskeletal protein -tubulin binding site is also located in this region of the mGlu7 CT and may play a role in the subcellular targeting (17). The distal region of the mGlu7 CT contains a PDZ-binding motif that, unlike mGlu1 and mGlu5, does not interact with PDZ/EVH domain proteins of the Homer/Vesl family (39) but binds the protein kinase C interacting protein (PICK1) that might be involved in the spatial organization of synaptic proteins and mGlu7 targeting (31,41). There is evidence to suggest that the interaction of the CT domains with PICK1 can be found in several members of group III mGluRs (mGlu4a, 7b, 8a, and 8b but not mGlu4b) (31).

5. Modulation of Synaptic Transmission The activation of group III mGluRs generally produces depression of glutamatergic and GABAergic synaptic transmission (2,3,5,6,29,41). Group III mGluRs can modulate the release of transmitters by acting as autoreceptors (glutamate) or heteroreceptors (-aminobutyric acid [GABA], substance P, serotonin, dopamine, and acetylcholine) (21). Decreased release of excitatory transmitters by group III mGlu activation results in the presynaptic inhibition of transmission. On the other hand, the decrease of release of GABA would indirectly have excitatory effects on synaptic transmission and neuronal excitability (disinhibition). Therefore, the overall effect of group III mGlu activation is a balance between facilitatory and inhibitory actions. Inhibition of excitatory synaptic transmission by group III mGlu activation (mainly with L-AP4) is well established and has been shown at numerous synapses in central nervous system areas. These include neocortex and olfactory cortex (lateral olfactory tract), olfactory bulb (mitral/tufted cells), cerebellum (parallel fiber to Purkinje cells), hippocampal formation CA1 (Schaffer collateral), CA3 (mossy fiber) and dentate gyrus (medial and lateral perforant path), amygdala basolateral nucleus (lateral to basolateral amygdala) and central nucleus (basolateral amygdala to central amygdala and pontine parabrachial nucleus to central amygdala), thalamus (corticothalamic), hypothalamus (supraoptic nucleus), striatum (corticostriatal), nucleus accumbens (prefrontal cortex to medial dorsal accumbens), substantia nigra pars compacta and ventral tegmental area, superior colliculus (retinocollicular), solitary tract nucleus, locus coeruleus, spinal cord dorsal horn, and motoneurons (dorsal root evoked) (29,41–47). Activation of group III mGluRs can also result in long-term depression (LTD) and inhibit long-term potentiation (LTP) in the

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hippocampus (48,49) and amygdala (50). However, LTP following group III mGluR activation by L-AP4 was also observed in the amygdala (51). In the peripheral nervous system and peripheral tissues, functional group I and II mGluRs have been demonstrated, but information about group III mGluRs is lacking, although anatomic evidence suggests their presence in primary afferent terminals (52). Furthermore, activation of presynaptic mGlu8 on primary afferent terminals in the spinal cord inhibited dorsal root–evoked transmission (28). Some evidence suggests that group III mGluRs are activated by endogenously released transmitter (glutamate). Group III antagonists increased excitatory synaptic transmission in the hypothalamus, superior colliculus, and spinal cord motoneurons (29,53,54) but had no significant effect on their own in other brain areas such as thalamus, amygdala, and periaqueductal gray (PAG) (44,45,55,56). In the retina, postsynaptic mGlu6 mediates the hyperpolarization of ON-bipolar cells in response to glutamate released from photoreceptors (3). Inhibition of GABAergic transmission by group III mGluRs has also been shown in different areas of the central nervous system, including thalamus (reticular nucleus to ventrobasal), hypothalamus (supraoptic nucleus), hippocampal CA1 interneurons and CA3 pyramidal cells, basal ganglia (striatopallidal), substantia nigra and ventral tegmental area, and spinal motoneurons (29,38,41,42,57). Furthermore, group III mGluRs can inhibit the synaptic activation of inhibitory hilar border interneurons in the dentate gyrus, thus decreasing inhibitory output (58). Activation of group III mGluRs (studied mainly with L-AP4) inhibits the release not only of glutamate, aspartate, and GABA, but also of dopamine, acetylcholine, and substance P, whereas increased release of serotonin and glutamate has been reported in the PAG (21,59). The contribution of individual group III mGlu subtypes to presynaptic inhibition is only beginning to emerge with the availability of subtype-selective agents in recent years (see Section 3). mGlu4 appears to be important for the regulation of transmission in cerebellar parallel fibers, hippocampal formation, and basal ganglia (striatopallidal synapse); mGlu7 serves as a classical autoreceptor throughout various cortical and limbic areas, basal ganglia (corticostriatal synapse), brainstem, and spinal cord; mGlu8 has been linked to the regulation of synapses in the forebrain, including hippocampal formation (particularly perforant path to dentate gyrus) and amygdala, lateral olfactory tract, and primary afferents; and mGlu6 acts postsynaptically to hyperpolarize ON-bipolar cells in the retina (1,3,41,60). Mechanisms of presynaptic inhibition by group III mGluRs involve a PTXsensitive G protein and include inhibition of presynaptic calcium currents, possibly downstream of calcium channel activation, activation of presynaptic potassium channels, and direct effects on transmitter release proteins (29).

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6. Modulation by Signaling Systems The activity of mGluRs, including group III mGluRs, is regulated not only by receptor agonists and antagonists, but also through receptor phosphorylation and dephosphorylation and modulation of GTPase-activation and G protein signaling. Receptor phosphorylation at the carboxy terminal by protein kinases A and C (PKA and PKC) mediates agonist-independent (heterologous) desensitization of group III mGluRs by uncoupling the receptor from GTP-binding proteins (6,61). Inhibition of presynaptic mGlu function by PKC is widespread and has been demonstrated for group III mGluRs at the medial and lateral perforant path-to-dentate gyrus synapses (probably mGlu8) and the Schaffer collateral-to-CA1 synapse (probably mGlu7) (6,61,62). Activation of PKA also disrupts the coupling of mGluRs to Gi and inhibits the function of several group III mGluRs (mGlu4a, 7a, and 8a) at the medial perforant path-to-dentate gyrus, mossy fiber-to-CA3, and Schaffer collateral-to-CA1 synapses (6,63). Phosphorylation of the CT domain of mGlu7 by PKC and PKA as well as by cGMP-dependent protein kinase (PKG) can inhibit calmodulin (CaM) interactions with the CT domain (64). CaM binding is required for the dissociation of G protein  subunits from the CT domain of group III mGlu7 to mediate glutamatergic autoinhibition (65). PKC phosphorylation can be inhibited by CaM and PICK1, which have been shown to bind to the CT domain of mGlu7 (6,17,31,41). CaM binding to mGlu7 overlaps the PKC phosphorylation site, thus inhibiting PKC phosphorylation of mGlu7 (17,64,65). Furthermore, PICK1, a PDZ domain–containing protein and PKC substrate, interacts with the most distal regions of the CT domain of mGlu7a and inhibits the PKC phosphorylation of mGlu7a through steric hindrance of the PKC phosphorylation site (31). PICK1 binding to the CT domain has be found in mGlu4a, mGlu7b, mGlu8a, and mGlu8b but not mGlu4b (31). Agonist-dependent (homologous) desensitization of mGluRs is mediated by G protein–coupled receptor kinases (GRKs), which form a family of isoforms (GRK1–GRK6) with different distribution and structural features (6,66). Phosphorylation of the agonist-occupied receptor at the CT domain by GRKs results in desensitization and receptor internalization through mechanisms that involve cofactors such as -arrestins (6,66). Although the regulation of group I mGlu1 and mGlu5 functions by different GRKs has been shown, little is known about the role of GRKs in group III mGluR signaling. GRK2, but not GRK4, can phosphorylate mGlu4 and desensitize the stimulation of the MAPK pathway without affecting the inhibition of cAMP formation or the receptor internalization (see Section 2) (66). Group III mGlu function can also be regulated by regulator of G protein signaling (RGS) proteins, such as RGS2 and RGS4, which facilitate G-catalyzed GTP hydrolysis, thus blocking G protein (including Gi) functions (6,17). However, it remains to be determined whether and how RGS proteins also modulate group III signaling and which mGlu subtypes are targeted.

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7. Function Group III mGluRs have emerged as drug targets for a variety of neurologic and psychiatric disorders, including epilepsies, neurodegenerative disorders such as Parkinson disease and Alzheimer disease, stress and anxiety disorders, and depression. They may also be useful in the treatment of pain. Although there is relatively little pharmacologic evidence for the endogenous activation of group III mGluRs (see Section 5), mGlu4-, 7-, or 8-knockout animals show certain behaviors associated with these disorders, suggesting perhaps that group III mGluRs are required for normal neural functions. It would appear that activation of group III mGluRs or restoring their function generally is a desirable therapeutic strategy, although exceptions may exist (see discussion of stress in Section 7.4). The fact that activation of group III mGluRs can result in LTD and inhibit LTP (see Section 5) may need to be considered in terms of undesirable drug effects on learning and memory processes. 7.1. Visual System Hyperpolarization of photoreceptors by light exposure results in a reduction of intracellular cGMP, closure of the cGMP-gated ion channel, and reduced glutamate release, which in turn produces depolarization and hyperpolarization of ON- and OFF-bipolar cells, respectively. Termination of light exposure has the opposite effect. The distinct responses of ON- and OFF-bipolar cells contribute to the discrimination of visual contrasts (3). The postsynaptic response of ON-bipolar cells is mediated by mGlu6, which is exclusively expressed at the postsynaptic site of ON-bipolar cells in both rod and cone systems. mGlu6 activation results in hyperpolarization of the ON-bipolar cell (3). Glutamate release from ON-bipolar cells in turn activates the ON pathway. Accordingly, mGlu6 deficiency results in a loss of ON responses to light stimuli but unchanged OFF responses to dark stimuli (3). Therefore, mGlu6 is a critical molecule for synaptic transmission in the ON pathway. Somewhat surprisingly, mGlu6-deficient mice retained the ability to respond to visual inputs, perhaps suggesting that OFF responses also contribute to the transmission of visual information (3). 7.2. Epilepsies Activation of group III mGluRs with L-AP4 and mGlu4- or 8-selective agonists inhibited synaptic transmission and had anticonvulsant effects in different animal models of epileptogenesis (34,56,67–69). Increased seizure susceptibility was found in mice lacking mGlu7 (70) but not mGlu8 (71). Presynaptic inhibition in the hippocampus (medial perforant path) mediated by mGlu7 but not mGlu8 was reduced in mice with pharmacologically induced status epilepticus (72). mGlu4knockout mice were resistant to absence seizures induced by GABAA -receptor antagonists (73). However, mGlu4 was upregulated in surviving hippocampal

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neurons obtained from patients with temporal lobe epilepsies, suggesting that mGlu4 can reduce seizure vulnerability (74). 7.3. Neurodegeneration and Neurotoxicity Activation of group III mGluRs, including mGlu4, 7, and 8, has been shown to have neuroprotective effects in in vitro and in vivo models of neurotoxicity, including excitotoxicity induced by NMDA and other ionotropic glutamate receptor agonists, prolonged -amyloid exposure, mechanical damage, and ischemic and hypoglycemic insults (10,34,37,75). Neurons and brain areas studied include the neocortex, hippocampus, striatum, and cerebellum. In addition, group III mGluRs have been found to protect against microglial neurotoxicity and thus could play a role in neuroinflammatory diseases such as Alzheimer disease and multiple sclerosis (24,25). The role of group III mGluRs in Parkinson disease (PD) has become an area of great interest. mGlu4 and 7 regulate synaptic transmission at multiple sites of the basal ganglia circuitry (see Section 5), where enhanced transmission is found in patients with PD and in preclinical models of PD (60). Agonists or allosteric potentiators of mGlu4 in particular show anti-parkinsonian effects such as reversal of akinesia in animal models (10,38,60). 7.4. Stress, Anxiety, and Depression Group III mGluRs are believed to be modulators of the stress response (35). Activation of mGlu7 with AMN082 increased plasma levels of the stress hormones corticosterone and corticotropin (35), whereas mGlu7-knockout mice showed upregulated glucocorticoid receptor–dependent feedback suppression of the hypothalamic-pituitary-adrenal axis, indicating an impaired stress response (76). Similarly, restraint stress had essentially no effect on the openarm activity of mGlu8-knockout mice but decreased the open-arm activity of wild-type mice in the elevated plus maze (71). Genetic ablation of mGlu7 also had antidepressant- and anxiolytic-like effects on the behavior of knockout mice in a variety of stress-related paradigms, including the forcedswim and tail-suspension tests (antidepressant-like activity) and the lightdark box, elevated plus maze, staircase test, and stress-induced hyperthermia test (anxiolytic-like activity) (1,76,77). Furthermore, mGlu7-knockout mice showed deficits in two amygdala-dependent behaviors—shock-induced fear response and conditioned taste aversion (78). On the other hand, activation of mGlu4 in the amygdala with PHCCC produced anxiolytic-like effects in the Vogel conflict drinking test, whereas the mGlu8 agonist (S)-3,4-DCPG had no effect (79,80). However, mGlu8-knockout mice showed anxiety-like behavior in the elevated plus maze (1,71). Therefore, activation of mGlu4 and blockade of mGlu7 would have anxiolytic effects, whereas the role of mGlu8 appears to be more complex.

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Fig. 1. A group III metabotropic glutamate receptor (mGluR) agonist (l-2-amino-4phosphonobutanoate [L-AP4]) inhibits responses of an amygdala neuron under normal conditions and in the arthritis pain state. Extracellular recordings from one neuron in the laterocapsular division of the central nucleus of the amygdala (CeLC). A, B. Histograms show the responses to brief (15 sec) mechanical stimuli of innocuous (100 and 500 g/30 mm2 ) and noxious intensity (1500–2500 g/30 mm2 ), which were applied to the knee joint with a calibrated forceps. Panel A shows responses in the control period before arthritis. Administration of L-AP4 (100 μM; concentration in microdialysis probe; 20 min) into the CeLC inhibited the evoked responses. Panel B shows how the evoked responses and background activity of the same neuron increased 6 hr after induction of the knee joint arthritis by intraarticular injections of kaolin and carrageenan. L-AP4 inhibited the increased responses. Bin width of the histograms in panels A and B is 1 sec. The individual action potentials (insets) illustrate that spike configuration, shape, and size remained constant throughout the long-term experiment. Calibration bars are 1 V and 1 ms. The top traces show the original recordings of the force (in grams) that was applied to the knee joint with the calibrated forceps. C, D. Histologically verified sites of the microdialysis probe and (posterior) the recording electrode in the CeLC. The boundaries of the different amygdala nuclei are easily identified under the microscope. Diagrams (adapted from ref. 89) show coronal sections through the right hemisphere at different levels posterior to bregma. Next to each

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7.5. Pain The roles of group III mGluRs in nociception and pain are less well understood than those of group I mGluRs (7,52,81,82). The role of peripheral group III mGluRs remains to be determined, but L-AP4 and the mGlu8 agonist (S)3,4-DCPG depressed dorsal root–evoked ventral root potentials and excitatory postsynaptic currents in spinal motoneurons, presumably through a presynaptic action on the central terminals of primary afferents (28,53). Activation of spinal group III mGluRs with L-AP4 inhibited mechanical allodynia but not thermal hyperalgesia (83), and central sensitization of spinothalamic tract (STT) cells in the spinal dorsal horn (84) in the capsaicin model of inflammatory pain. Inhibitory effects of L-AP4 on STT cells were also observed under control conditions before capsaicin (84). In the spinal nerve ligation model of neuropathic pain, spinal application of L-AP4 attenuated mechanical allodynia and inhibited the responses of spinal dorsal horn projection neurons but had no effect on nociceptive behavior and neuronal activity in control animals (85). Activation of group III mGluRs in the PAG with L-SOP facilitated pain behavior under normal conditions and in the late phase of the formalin pain test either by inhibiting descending pain inhibition (“disinhibition”) or activating descending facilitatory systems (55,86). It is interesting that intra-PAG perfusion of L-AP4, RS-PPG, or the mGlu8 agonist (S)-3,4DCPG increased glutamate and decreased GABA extracellular concentrations in the PAG (59). Activation of group III mGluRs in the ventrobasal thalamus resulted in the disinhibition of nociceptive processing through the presynaptic reduction of GABAergic inhibitory transmission from the thalamic reticular nucleus to the ventrobasal thalamus (87). In the laterocapsular division of the central nucleus of the amygdala (CeLC), LAP4 inhibited the sensitization of neurons in a model of arthritic pain but also affected the neurons’ responses under normal conditions, albeit less potently (88) (Fig. 1). It was further shown that the inhibition of pain-related synaptic plasticity of CeLC neurons in brain slices from arthritis rats involved a presynaptic mechanism (45) (Fig. 2).

 Fig. 1. (Continued) diagram is shown in detail the medial (CeM), lateral (CeL), and laterocapsular (CeLC) divisions of the central nucleus of the amygdala. Calibration bars for diagrams are 1 mm. Reproduced with permission from Li W, Neugebauer VE. Differential changes of group II and group III mGluR function in central amygdala neurons in a model of arthritic pain. J Neurophysiol 2006;96:1803–1815. Copyright 2006 by the American Physiological Society.

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Fig. 2. Presynaptic inhibition of synaptic transmission in the amygdala by a group III metabotropic glutamate receptor (mGluR) agonist is increased in a model of arthritic pain. A. l-2-amino-4-phosphonobutanoate (L-AP4) has little effect in a neuron recorded in the laterocapsular division of the central nucleus of the amygdala (CeLC) in a brain slice from a normal rat. B. In a slice from an arthritic rat, L-AP4 clearly inhibited synaptic transmission in a CeLC neuron. Each trace in panels A and B is the average of 10 monosynaptic excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation of afferent fibers from the brainstem. C. Cumulative concentration–response relationships show that L-AP4 inhibited synaptic transmission in CeLC neurons from arthritic rats more potently (EC50 = 11.5 nM, n = 11) than in control neurons from normal rats (EC50 = 1.18 nM, n = 11). L-AP4 was applied by superfusion of the slice for 12 min. Symbols and error bars represent mean ± SE. The EC50 values were calculated from the sigmoid curves fitted to the cumulative concentration–response

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7.6. Summary In summary, the therapeutic potential of group III mGluRs lies in their ability to exert antiepileptic (mGlu4 and 7), neuroprotective (mGlu4, 7, and 8), anti-parkinsonian (mGlu4 and 7), anxiolytic (mGlu4), and analgesic effects. On the other hand, activation of group III mGluRs can interfere with normal synaptic transmission and LTP (see Section 5), which may hint at undesirable memory and cognitive drug effects. Furthermore, mGlu6 is important for normal visual transmission. The analysis of the role of individual group III subtypes has begun only recently and needs to advance to reveal any subtypespecific therapeutic indications. Broad-spectrum group III agonists would not appear to be suitable therapeutics.

8. Future Directions Until recently, group III mGluRs were somewhat overlooked as targets for the treatment of nervous system disorders, mainly due to the lack of appropriate tools with which to study the functions of individual subtypes. With the availability of subtype-selective agents, major progress can be expected in the identification of the roles of group III subtypes in different neural systems and pathways associated with different functions and disorders. A systematic analysis of the involvement of individual subtypes in functional subsystems is needed to determine the potential therapeutic value in the treatment of disorders. The potential benefits of allosteric modulators compared to orthosteric agonists need to be examined. Finally, the interaction with cytoskeletal partners and intracellular signaling molecules is an exiting and relatively new

 Fig. 2. (Continued) data by nonlinear regression using the formula y = A + (B – A)/[1 + (10C /10X )D ], where A = bottom plateau, B = top plateau, C = log(EC50 ), X = logarithm of concentration and D = slope coefficient (GraphPad Prism 3.0). D–G. Analysis of miniature EPSCs (mEPSCs) shows that L-AP4 inhibits transmission through a prerather than postsynaptic site of action. D, E. L-AP4 decreased the frequency of mEPSCs in the presence of tetrodotoxin (TTX) (1 μM). Individual examples of voltage-clamp recordings of mEPSCs in a CeLC neuron before and during L-AP4 (1 nM) application. F. Cumulative fraction plot of the intervals between mEPSCs (interevent interval distribution). L-AP4 caused a shift toward longer intervals, indicating reduced mEPSC frequency. G. LAP4 had no effect on the mEPSC amplitude distribution. mEPSC analysis was done using MiniAnalysis program 5.3 (Synaptosoft Inc.). Reproduced with permission from Han JS, Bird GC, Neugebauer V. Enhanced group III mGluR–mediated inhibition of pain-related synaptic plasticity in the amygdala. Neuropharmacology 2004;46(7):918–926. Copyright 2004 by Elsevier.

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(for group III mGluRs) avenue that might lead to the better understanding of receptor function and to novel drug discovery.

Acknowledgments Work in the author’s laboratory is supported by National Institutes of Health (NIH) grants NS38261 and NS11255.

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13 Metabotropic Glutamate Receptor-Dependent Synaptic Plasticity Stephen M. Fitzjohn and Zafar I. Bashir

Summary Long-term potentiation (LTP) and long-term depression (LTD) are important forms of synaptic plasticity thought to underlie many brain processes such as those involved in brain development, memory, and drug addiction. The metabotropic glutamate receptors (mGluRs) are capable of inducing both LTP and LTD, and also of modulating the induction of plasticity initiated by other receptor systems. Although early work focused on the role of mGluRs in LTP, the precise nature of their involvement in LTP induction remains unclear. However, there is considerable evidence that activation of mGluRs can induce LTD in numerous brain regions. This chapter reviews the evidence for mGluR involvement in LTP induction and discusses the roles of mGluRs in LTD. In particular it describes the signaling pathways and expression mechanisms of two prominent forms of LTD—those seen in the CA1 region of the hippocampus and the cerebellum. Key Words: Long-term potentiation; Long-term depression; mGluR; Hippocampus; Cerebellum.

1. Introduction Long-term synaptic plasticity is the primary model for understanding the cellular and molecular changes underlying, for example, learning and memory, brain development, and drug addiction. In this chapter we consider the roles of metabotropic glutamate receptors (mGluRs) in two prominent forms of From: The Receptors: The Glutamate Receptors Edited by: R. W. Gereau and G. T. Swanson © Humana Press, Totowa, NJ

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synaptic plasticity—long-term potentiation (LTP) and long-term depression (LTD). The role of mGluRs in LTP was investigated intensively in the 1990s. During that time there was an accumulation of evidence showing that mGluRs can play a role in the induction of LTP in different brain regions, and much work has focused on defining the conditions and different forms of LTP under which activation of mGluRs become necessary. However, the precise function of mGluRs in the expression of LTP remains unclear. In contrast, a role for mGluRs in LTD is well established, and there is now a good deal of data on the biochemical and molecular mechanisms of induction and expression of mGluR-LTD.

2. Induction of mGluR-Dependent LTP 2.1. Pharmacological Induction of LTP Early studies showed that activation of mGluRs by the use of pharmacological agonists revealed an LTP-like increase in excitatory synaptic transmission. This phenomenon was first described in the CA1 region of the hippocampus following application of the broad-spectrum mGluR agonist ACPD (1). This form of plasticity has since been described in a variety of different regions (see ref. 2 for review), but for the purposes of this review we concentrate on the CA1 region of hippocampus. In the CA1 region, 1-aminocyclopentane-1,3-dicarboxylate (ACPD)-induced potentiation occludes with tetanus-induced LTP, suggesting overlapping mechanisms of expression with synaptically induced LTP (1). ACPD-induced potentiation persists during blockade of GABAergic inhibition but is blocked by (1) postsynaptic application of 1, 2-bis(2-aminophenoxy)ethane-N,N,N  ,N  -tetraacetic acid (BAPTA), (2) removal of area CA3, (3) inhibition of protein kinase C (PKC), and (4) depletion of intracellular calcium stores (1). Furthermore, it has been shown that ACPD-induced potentiation requires the concurrent activation of N-methyl-d-aspartate (NMDA) receptors because potentiation is blocked by the NMDA receptor antagonist 2-amino-5-phosphonopentanoate (AP5) (3–5). Basal test stimulation may provide this NMDA receptor activation, because ACPD applied in the absence of test stimulation may produce long-term depression (LTD) rather than LTP (5) (but see ref. 1). ACPD-induced potentiation is also associated with excitatory postsynaptic potential (EPSP)–spike potentiation, an effect that is prevented by blockade of -aminobutyric acid (GABA) transmission (3). Therefore, because ACPD potentiation of glutamatergic transmission is unaffected by blockade of GABAergic transmission, this suggests that the increase in EPSP-spike coupling and potentiation of the EPSP probably rely on different mechanisms. Activation of group I mGluRs has also been shown to produce slow-onset potentiation of synaptic transmission in vivo (6). However, one report suggested

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that ACPD-induced potentiation is accompanied by an increase in the size of the presynaptic fiber volley, raising the possibility that this potentiation is caused by a recruitment of presynaptic fibers brought about by a blockade of potassium channels (7). Furthermore, slow-onset potentiation induced by ACPD administration in vivo may be associated with cell death (8), raising some questions about the physiological significance of LTP induced by ACPD in vivo. 2.2. Facilitation of LTP Induction by Activation of mGluRs In some studies in the CA1 region, application of ACPD enhanced the magnitude of LTP induced by weak theta burst stimulation (TBS) but did not increase the magnitude of LTP induced by strong TBS, thereby suggesting that mGluR activation alters the threshold for LTP induction rather than affecting the maximum level of LTP that can be attained (9,10). The priming effect of ACPD was not dependent on NMDA receptor activation but relied on activation of group I mGluRs and PLC stimulation (10). Similar results have been reported in other brain regions. For example, in prelimbic cortex, application of theta burst stimulation produced no lasting increase in synaptic transmission unless delivered in the presence of the group I mGluR agonist dihydroxyphenylglycine (DHPG) (11). Similar priming effects can be produced by synaptic stimulation, and such priming is homosynaptic, mGluR dependent, and, interestingly, also dependent on protein synthesis (12). This priming effect may be important for LTP induction using weak stimulation, because LTP induced by weak TBS can be inhibited by group I mGluR antagonists and is absent in mice lacking Gq and G11 (13). In addition to converting weak LTP to strong LTP, activation of mGluRs can convert short-term plasticity (STP) into LTP in a PKC-dependent manner (14). The induction of LTP and LTD is frequency dependent (15). Typically, high-frequency stimulation (HFS) results in LTP and low-frequency stimulation (LFS) results in LTD. Activation of mGluRs, in particular group I mGluRs, has been shown to shift the frequency response function of synaptic plasticity such that stimuli normally inducing LTD resulted in LTP induction (16). Because group I mGluRs are known to elicit a depolarization of postsynaptic neurons via inhibition of a leak potassium current (17,18), it is possible that facilitation of LTP induction involves, indirectly, enhanced activation of NMDA receptor–mediated responses via this depolarization. In addition, group I mGluRs are known to facilitate directly NMDA receptor–mediated responses (19,20), which may also contribute to decreasing the threshold for LTP induction. 2.3. Role of mGluRs in Synaptically Induced LTP The most direct evidence that mGluRs play a critical role in the induction of synaptically induced LTP has come through the use of mGluR antagonists. The broad-spectrum mGluR antagonist -methyl-4-carboxyphenylglycine (MCPG)

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has been shown in a variety of different regions to block the induction of LTP [21–24; also see 2]. Furthermore, it has been demonstrated that LTP is not induced in mGluR5-knockout mice in the hippocampus (25) and in the striatum is blocked completely by combined blockade of mGluR1 and mGluR5 (26). The role of mGluRs in LTP was questioned in some early studies (27–29). It is possible that the reason for the discrepancies in results is that mGluRs have different roles in LTP under different conditions. For example, MCPG blocked LTP induced by high-frequency stimulation but not LTP induced by theta stimulation (30). Conversely, other studies have suggested that the role of mGluRs in LTP is highly dependent on weak but not strong stimulus parameters (29,31). Furthermore, there is evidence that the particular stage of development plays a crucial role in determining whether mGluRs are involved in the induction of LTP (32). Although there is agreement that mGluRs are involved in LTP under different conditions, the precise identity of the mGluR subtype involved in LTP induction in the CA1 region of the hippocampus is not entirely clear. Thus, although the broad-spectrum antagonist MCPG blocked LTP induction in several studies (21,22,32,33), another broad-spectrum antagonist of mGluRs, LY341495, failed to block LTP induction (34). The reason for this is not understood. In conclusion, although there is evidence that mGluRs can play roles in induction of LTP, the precise biochemical and molecular mechanisms are unclear.

3. Induction of mGluR-Dependent LTD 3.1. Agonist-Induced LTD 3.1.1. Stimulation of Group I mGluRs One simple means of inducing mGluR-LTD is by stimulation of mGluRs with a pharmacological agonist. Initial studies demonstrated that application of the agonist ACPD produced LTD of glutamate transmission in the dentate gyrus (35) and the CA1 region of hippocampus in vitro (36). It has since been demonstrated (using the agonist DHPG) that, within the CA1 region, the most likely receptors involved in triggering this form of LTD are group I mGluRs (37,38). Of the group I mGluRs, the mGluR5 subtype is the most likely candidate for triggering the induction of LTD. This has been demonstrated by (1) the induction of LTD by bath application of the mGluR5-selective agonist CHPG (37,38) (2) the prevention of DHPG-induced LTD with the selective mGluR5 antagonist MPEP (39,40), (3) the lack of effect of the mGluR1-selective antagonist (+)-2-methyl-4-carboxyphenylglycine (LY367385) (38,39), and (4) the lack of DHPG-induced LTD in mGluR5 knockouts (41). However, a recent study suggested that both mGluR1 and mGluR5 must be blocked to prevent the induction of LTD by DHPG (42). Given the overwhelming evidence for the selective involvement of mGluR5 in the induction of DHPG-induced LTD, this

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latter finding is somewhat confusing and requires further examination. LTD induced by group I mGluR activation was shown to be absent in Gq-knockout mice but not affected in G11 knockouts. Because both of these G proteins are triggered by group I mGluRs, this result suggests that the Gq enzyme is the important G protein in the LTD signal transduction cascade (43). Group I mGluR activation can also induce depression of glutamate transmission at various other synapses, including sensory spinal synapses (44), in the ventral septum (45), in visual cortex (46–48), in dentate gyrus (49), in the bed nucleus of the stria terminalis (BNST) (50), and in ventral tegmentum (VTA) (51). In contrast to the results in the CA1 region, the induction of LTD in the VTA appears to depend on mGluR1 because LTD is not blocked by mGluR5 antagonists but is prevented by mGluR1 antagonists. 3.1.2. Stimulation of Group II mGluRs The induction of mGluR agonist–induced LTD may not necessarily rely on only one mGluR subtype in any one brain region. For example, the selective group II mGluR agonist 2’,3’-dicarboxycyclopropylglycine (DCG-IV) also produced LTD in several brain regions, including visual cortex (52), amygdala (53), prefrontal cortex (54,55), striatum (56), nucleus accumbens (57,58), and dentate gyrus (59). As noted, the group I mGluR agonist also produces LTD in some of these regions. 3.2. Activity-Dependent LTD 3.2.1. Role of mGluR5 Unfortunately, within the confines of this chapter it is not possible to do justice to all of the published data on different stimulus protocols and experimental conditions that produce activity-dependent mGluR-LTD, and the reader is referred to the excellent recent review by Roger Anwyl (60), which deals with this issue in some detail. Suffice to say that there are many different stimulus protocols that have been used to induce mGluR-LTD in different brain regions. For example, in the CA1 region, mGluR-LTD can be induced using protocols including 5-Hz stimulation for 3 min (61) and paired-pulse LFS (PP-LFS) (62). Under these conditions, a role for synaptic activation of mGluRs in the induction of LTD in CA1 was originally demonstrated using various mGluR antagonists. The results showing occlusion of DHPG-induced LTD with PP-LFS–induced LTD (41) suggest that mGluR5 may also be the key subtype for synaptically induced mGluR-LTD in the CA1. Induction of LTD by PP-LFS was shown to be absent in Gq-knockout mice, suggesting that the Gq enzyme is the important enzyme in activity-dependent mGluR-LTD (43). 3.2.2. Role of mGluR1 At the parallel fiber-to-Purkinje cell synapse in the cerebellum, LTD is commonly induced by pairing Purkinje cell depolarization with parallel fiber

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low-frequency stimulation (63). At the corticostriatal input to neostriatal neurons, LTD can be induced by pairing postsynaptic depolarization with HFS (64). Under these conditions, LTD at both the cerebellar synapse and the input to neostriatum was blocked by antagonists selective for mGluR1 such as 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt) and LY367385 (65,66) and was absent in mGluR1-knockout mice (65,67). Nevertheless, it is also possible that mGluR5 has a role in corticostriatal LTD (66). 3.2.3. Role of mGluR2 In other regions of the brain, different stimulus protocols have been employed to induce mGluR-LTD. For example, at mossy fiber-to-CA3 synapses, 1-Hz stimulation for 15 min and 100-Hz stimulation for 1 sec have both been shown to induce LTD. Synaptically induced LTD in CA3 is prevented by broad-spectrum antagonists such as MCPG (68) or is absent in mGluR2-knockout mice (69). In the nucleus accumbens, group II mGluRs are involved in the induction of a form of LTD that requires PKA-dependent modulation of presynaptic calcium channels (57). 3.3. Developmental Changes in mGluR-LTD In some brain regions a developmental shift in the expression of group I mGluRs may occur that mirrors developmental changes in plasticity expression. For example, in the first two postnatal weeks, HFS induces mGluR5 and GABAA receptor–dependent LTD at the synapse formed by primary vestibular afferents in the medial vestibular nuclei (MVN) (70). However, during the second postnatal week, coinciding with eye opening, there is a shift to HFSinduced LTP that depends on NMDA and mGluR1 activation. LTP reaches peak levels at 3 weeks, whereas LTD is absent after the end of the second postnatal week. Coinciding with this change in plasticity, mGluR5 expression levels in the MVN start high and then decrease, whereas expression levels of mGluR1 start low and then increase with age. In addition, in perirhinal cortex, 5-Hz LTD switches from mGluR to muscarinic receptor dependent at about 2 weeks postnatal age. This switch was shown to be experience dependent and was associated with a decrease in mGluR5 levels in perirhinal cortex (71).

4. Biochemical Pathways and Expression Mechanisms Underlying mGluR-LTD Multiple second messenger cascades have been shown to be involved in mGluR-LTD in various brain regions. Here we concentrate on mGluR-LTD in brain regions in which it has been extensively studied (CA1 region of the hippocampus and cerebellum), commenting on similarities to other brain

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regions where appropriate. In addition, because the most extensively characterized forms of LTD are those involving activation of group I mGluRs, we focus on these forms of LTD, mentioning other forms of LTD where relevant (60). 4.1. CA1 Region of the Hippocampus Induction of DHPG-LTD in the CA1 involves activation of protein tyrosine phosphatases (PTPs), mitogen-activated protein kinases (MAPKs), and protein synthesis. The expression of LTD involves a loss of surface AMPA receptors, probably brought about by a change in tyrosine phosphorylation of the GluR2 AMPA receptor subunit, although a change in presynaptic glutamate release may also contribute. This form of LTD, in contrast to forms of LTD dependent on NMDA receptor activation, is independent of serine/threonine protein phosphatases (PPs) and may also be independent of changes in intracellular calcium. 4.1.1. Intracellular Calcium Multiple forms of synaptic plasticity in the central nervous system (CNS) are dependent on rises in intracellular calcium. However, DHPG-induced LTD in CA1 has been demonstrated to be independent of extracellular calcium and of release from intracellular stores, and indeed it is independent of any postsynaptic rise in calcium (72,73). This is a somewhat surprising set of results, given that group I mGluRs are known to couple through mechanisms linked to increases in intracellular calcium, indicating a novel mechanism. In contrast to DHPG-induced LTD, synaptically induced group I–dependent mGluR-LTD in CA1 was blocked by postsynaptic calcium chelators (61,74). Similarly, synaptically induced LTD in dentate gyrus (75), somatosensory cortex (76), and perirhinal cortex (77) was blocked by postsynaptic calcium chelation. This is despite the fact that very many different stimulus protocols are used to induce LTD. It is possible that the postsynaptic calcium rise could result from entry from the extracellular space (via voltage-gated calcium channels [VGCCs]) in addition to the likely release of calcium from intracellular stores that follows activation of group I mGluRs. Indeed there is evidence for both of these sources being involved in mGluR-LTD. Thus, in the CA1 region, block of L-type VGCCs can prevent mGluR-LTD (74), as can the block of T-type VGCCs (61). Similar findings have been reported in dentate gyrus (75,78). In the CA1, LFS-LTD was prevented by either block of mGluRs or block of increases in postsynaptic calcium. This suggests that there is a requirement for both activation of mGluRs and an increase in intracellular calcium most likely through activation of VGCCs (79). Depletion of intracellular calcium stores has also been shown to prevent LTD in regions including dentate gyrus (78).

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In other regions there is evidence that increases in presynaptic calcium are essential for mGluR-LTD. Membrane-permeable calcium chelators applied extracellularly blocked LTD at mossy fiber–CA3 synapses (68,80) and the amygdala (53,81), forms of LTD that are mediated by group II mGluRs. However, loading these postsynaptic neurons with high concentrations of BAPTA did not block LTD at these synapses. Together these data show that at certain synapses postsynaptic calcium does not play a role in mGluR-LTD, but increases in presynaptic calcium are essential for induction of LTD. 4.1.2. Protein Phosphorylation A range of different PKC inhibitors did not have an effect on DHPG-induced LTD in CA1 (73). However, inhibitors of MAPK do block DHPG-induced LTD, although the identity of the MAPK involved is unclear. Thus in some studies inhibitors of p38 MAPK are effective (39,82), whereas Gallagher et al. (83) reported that the extracellular signal-regulated (ERK) subclass of MAPK, but not p38 MAPK, was essential for LTD induction. ERKs are also involved in mGluR5-mediated LTD in the BNST (50). Although activation of serine/threonine PPs plays a crucial role in the induction of NMDA-LTD (84), this is not the case in the induction of mGluRLTD. In contrast, PP inhibitors enhance DHPG-LTD in the CA1 (85). However, a critical role for postsynaptic protein tyrosine phosphatases (PTPs) is important for DHPG-induced LTD (40,82,86). 4.1.3. Protein Synthesis An early finding in the study of DHPG-LTD was that this form of plasticity involved rapid dendritic protein synthesis (87). This protein synthesis, which is also required for synaptically induced mGluR-LTD, depends on translation of preexisting dendritic mRNA, because it is still observed in hippocampal slices where the dendritic region of CA1 pyramidal neurons is isolated from the cell bodies (87). A recent study suggested that this dependence on protein synthesis is absent in young (postnatal day [P] 8–P15) rats but is seen in older (>P21) animals (88). Although the identity of the protein(s) synthesized during mGluR-LTD has yet to be elucidated, a possible signal transduction pathway that results in protein synthesis has been identified whereby DHPG application induces activation of the PI3K-Akt-mTOR pathway (89), which is known to control initiation of translation. Further evidence for a role of protein synthesis in group I mGluR-LTD comes from studies using mice lacking the fragile X mental retardation protein (FMRP), a protein that is thought to inhibit translation (90). Mice lacking FMRP show a selective enhancement of mGluR-LTD in area CA1 of the hippocampus (91). Furthermore, mGluR-LTD is no longer protein synthesis dependent in these mice (92).

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4.1.4. Expression Mechanisms In region CA1 of the hippocampus, the application of DHPG produced a decrease in AMPA receptors at the cell surface (40,82,88,93,94). Such changes in surface AMPA receptor expression are prevented by inhibitors of tyrosine phosphatases, and the likely target of the PTPs activated by DHPG is the GluR2 AMPA receptor subunit (40,82). A peptide (D15) reported to block interaction of dynamin with amphiphysin, and therefore prevent receptor internalization, also blocked LTD (94). Furthermore, jasplakinolide, which stabilizes actin filaments and therefore also blocks endocytosis, was found to block the induction of mGluR-LTD (40,94). mGluRLTD at other synapses also involves a change in surface AMPA receptor expression. In the VTA, LTD induced by mGluR1 activation involves a switch in surface AMPA receptor expression from GluR2-lacking to GluR2-containing receptors (51). These results suggest that DHPG-induced LTD in the CA1 is due to postsynaptic internalization of AMPA receptors. However, in CA1 there is also evidence that mGluR-LTD may, at least in part, rely on a presynaptic component. The following lines of evidence have been found that support the presynaptic hypothesis of LTD expression: There is a change in pairedpulse facilitation with DHPG-induced LTD (40,72,88,95,96), an increase in failure rate (72), and a change in coefficient of variation (40,72). Several studies have also reported a decrease in the frequency of mEPSCs (72,93,94). However, whilst these changes are consistent with a presynaptic change in glutamate release probability, changes in failure rate, coefficient of variation and miniature excitatory postsynaptic current (mEPSC) frequency could also be consistent with postsynaptic silencing of whole AMPA receptor clusters (as discussed in refs. 40 and 72). A change in paired-pulse facilitation may indicate that a presynaptic mechanism of expression may contribute, at least in part, to DHPG-induced LTD. In addition, other observations also support a role of presynaptic changes in DHPG-induced LTD, such as (1) no change in sensitivity to exogenously applied glutamate after LTD induction (97), (2) a persistent decrease in the vesicular release of zinc (98), and (3) sensitivity of DHPG-induced LTD to treatments affecting presynaptic function (95). Thus it is possible that in the CA1 region of the hippocampus both pre- and postsynaptic changes underlie LTD. One possibility is that there is a developmental change in the expression of group I mGluR–dependent LTD. For example, Nosyreva et al. (88) recently suggested that in neonatal rats (P8–P15) mGluR-induced LTD is protein synthesis independent and presynaptically expressed, whereas in older animals (P21– P35) this LTD is associated with changes in surface expression of AMPA receptors.

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4.1.5. Retrograde Messengers If a presynaptic change does mediate, at least in part, the expression of DHPG-induced LTD, then the involvement of a retrograde messenger from post- to presynaptic cell is required. Thus, group I mGluRs are located predominantly, if not exclusively, on postsynaptic CA1 pyramidal cells (99), and DHPG-induced LTD is blocked by postsynaptic application of PTP and G protein inhibitors (40,95). Metabolites of arachidonic acid produced by the enzyme 12-lipoxygenase are candidate retrograde messengers involved in LTD induction (100). Another class of retrograde messenger, the endocannabinoids, are not involved in DHPG-induced LTD but do underlie part of the initial depression seen upon DHPG application (101), which may be mediated by activation of mGluR1 rather than mGluR5 (39). Similarly, in the BNST, activation of group I mGluRs produced a transient depression of synaptic transmission dependent on cannabinoid production, which is followed by an mGluR5-dependent, cannabinoid-independent LTD (50). One brain region in which cannabinoids are involved in LTD induction rather than just a transient depression of transmission is the dorsolateral striatum, in which activation of group I mGluRs and D2 dopamine receptors induces a form of LTD in which postsynaptic release of cannabinoids results in a presynaptic form of LTD (102). Striatal LTD is also dependent on release of calcium from intracellular stores (103). In addition, synaptically induced LTD in the nucleus accumbens, which requires mGluR5 activation, is also dependent on release of calcium from intracellular stores and production of cannabinoids (104). 4.2. Cerebellum 4.2.1. Second Messenger Pathways Synaptic mGluR-LTD at the parallel fiber-to-Purkinje cell synapse in the cerebellum involves release of calcium from intracellular stores (105). In contrast to the CA1 region of the hippocampus, activators of PKC, such as phorbol esters, induce LTD in cerebellum (106,107). In addition, a range of different PKC inhibitors have been shown to block mGlu-LTD (106), and a peptide (PKC19-36) inhibitor of PKC applied directly to the postsynaptic cell blocks LTD (106). In the cerebellum, it appears that serine/threonine PP activation prevents induction of mGluR-LTD. Thus, in the cerebellum, postsynaptic application of PP inhibitors produced a lasting depression of transmission (107) that is blocked by postsynaptic calcium chelators and that occludes synaptically induced LTD. One explanation of these results is that because phosphorylation of serine 880 on GluR2 is a key step in cerebellar LTD (108,109), activation of PPs decreases phosphorylation and thereby prevents LTD. Conversely, inhibition of PPs promotes phosphorylation and therefore allows induction of

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LTD. Similar to observed effects in the hippocampus, LTD in the cerebellum also involves rapid protein synthesis, which appears to be downstream of activation of mGluR1 (110), and LTD is also enhanced in FMRP knockouts (111). 4.2.2. Expression Mechanisms Possibly the strongest evidence for postsynaptic expression of mGluR-LTD comes from studies in the cerebellum. Associated with LTD was a decrease in sensitivity to exogenously applied AMPA receptor agonists (112,113) and a decrease in surface GluR2 expression (108). Furthermore, LTD was prevented by procedures that prevent clathrin-mediated endocytosis (114). It has been known for many years that PKC activation is crucial for LTD in cerebellum and that this is most likely due to phosphorylation at serine 880 on GluR2 (108,109). It is thought that this phosphorylation event removes glutamate receptor interacting protein (GRIP) from GluR2, and this is a key trigger for internalization of GluR2 (109). There is little convincing evidence that cerebellar LTD involves any presynaptic expression mechanisms.

5. mGluR-Mediated Inhibition of Inhibitory Synaptic Transmission So far we have concentrated on plasticity at excitatory synapses, but mGluRs can also mediate plasticity of inhibitory synaptic transmission. In the CA1 region of the hippocampus, activation of group I mGluRs by DHPG or HFS produces an LTD of inhibitory GABAergic transmission (i-LTD) (115,116). ILTD is dependent on activation of PLC but is independent of changes in postsynaptic calcium levels and involves release of cannabinoids from CA1 pyramidal neurons, which act on presynaptic GABAergic terminals to produce a persistent decrease in GABA release (116). Such a reduction in inhibitory transmission may facilitate the induction of subsequent LTP at nearby excitatory synapses, because the magnitude of LTP induced by TBS is enhanced at excitatory synapses located within 40 μm of the site of i-LTD (117). This metaplastic change lasts for at least 1 hr after i-LTD induction.

6. Role of mGluR-LTD in Drug Abuse Although multiple forms of mGluR-mediated plasticity have been demonstrated in a variety of brain regions, the physiological or pathological role of these is unclear. However, a series of recent publications have shown possible links between mGluR-LTD and drug abuse. In the nucleus accumbens, mGluR5-dependent LTD is abolished in brain slices prepared from rats that received a single in vivo exposure to cocaine (118), an effect that is associated with a decrease in expression of surface mGluR5. mGluR5-dependent LTD in

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the BNST is also reduced after cocaine administration (50). It is interesting that the reinforcing and locomotor stimulant effects of cocaine are lost in mice lacking the mGluR5 receptor (119), suggesting that activation of mGluR5 may be important for mediating some of the effects of cocaine administration. In addition to some forms of mGluR-dependent plasticity being lost in animals administered cocaine, it appears that activation of mGluRs may also reverse some of the synaptic changes associated with cocaine administration. In the ventral tegmental area, the expression of surface AMPA receptors is changed following a single cocaine injection, such that calcium-permeable AMPA receptors lacking the GluR2 subunit are driven into synapses (120). This switch in subunit composition can be reversed by administration of a potentiator of mGluR1, suggesting that such drugs may represent a novel way of reversing changes in synapse function associated with cocaine use. However, the behavioral consequences of such drugs in terms of drug addiction have yet to be determined.

7. Conclusion As will have become clear, there are far more data pertaining to the mechanisms of mGluR-LTD than to the mechanisms of mGluR-LTP. There is a great deal of interest in elucidating what the roles of mGluR plasticity might be in normal and abnormal brain function and behavior. For example, recent work has shown that drug addiction may involve mGluR-dependent plastic changes. It is envisaged that future work will expand our knowledge of the repertoire of mGluR function in the central nervous system.

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67. Aiba A, Kano M, Chen C, et al. Deficient cerebellar long-term depression and impaired motor learning in mGluR1 mutant mice. Cell 1994;79(2):377–388. 68. Kobayashi K, Manabe T, Takahashi T. Presynaptic long-term depression at the hippocampal mossy fiber–CA3 synapse. Science 1996;273(5275):648–650. 69. Yokoi M, Kobayashi K, Manabe T, et al. Impairment of hippocampal mossy fiber LTD in mice lacking mGluR2. Science 1996;273(5275):645–647. 70. Puyal J, Grassi S, Dieni C, et al. Developmental shift from long-term depression to long-term potentiation in the rat medial vestibular nuclei: role of group I metabotropic glutamate receptors. J Physiol 2003;553(Pt 2):427–443. 71. Jo J, Ball SM, Seok H, et al. Experience-dependent modification of mechanisms of long-term depression. Nat Neurosci 2006;9(2):170–172. 72. Fitzjohn SM, Palmer MJ, May JE, et al. A characterisation of long-term depression induced by metabotropic glutamate receptor activation in the rat hippocampus in vitro. J Physiol 2001;537(Pt 2):421. 73. Schnabel R, Kilpatrick IC, Collingridge GL. An investigation into signal transduction mechanisms involved in DHPG-induced LTD in the CA1 region of the hippocampus. Neuropharmacology 1999;38(10):1585–1596. 74. Bolshakov VY, Siegelbaum SA. Postsynaptic induction and presynaptic expression of hippocampal long-term depression. Science 1994;264(5162): 1148–1152. 75. Wu J, Rush A, Rowan MJ, et al. NMDA receptor- and metabotropic glutamate receptor–dependent synaptic plasticity induced by high frequency stimulation in the rat dentate gyrus in vitro. J Physiol 2001;533(Pt 3):745–755. 76. Egger V, Feldmeyer D, Sakmann B. Coincidence detection and changes of synaptic efficacy in spiny stellate neurons in rat barrel cortex. Nat Neurosci 1999;2(12):1098–1105. 77. Cho K, Kemp N, Noel J, et al. A new form of long-term depression in the perirhinal cortex. Nat Neurosci 2000;3(2):150. 78. Wang Y, Rowan MJ, Anwyl R. Induction of LTD in the dentate gyrus in vitro is NMDA receptor independent, but dependent on Ca2+ influx via lowvoltage-activated Ca2+ channels and release of Ca2+ from intracellular stores. J Neurophysiol 1997;77(2):812–825. 79. Otani S, Connor JA. Requirement of rapid Ca2+ entry and synaptic activation of metabotropic glutamate receptors for the induction of long-term depression in adult rat hippocampus. J Physiol 1998;511(Pt 3):761–770. 80. Kobayashi K, Manabe T, Takahashi T. Calcium-dependent mechanisms involved in presynaptic long-term depression at the hippocampal mossy fibre–CA3 synapse. Eur J Neurosci 1999;11(5):1633–1638. 81. Rammes G, Eder M, Dodt HU, et al. Long-term depression in the basolateral amygdala of the mouse involves the activation of interneurons. Neuroscience 2001;107(1):85–97. 82. Huang CC, Hsu KS. Sustained activation of metabotropic glutamate receptor 5 and protein tyrosine phosphatases mediate the expression of (S)-3,5dihydroxyphenylglycine–induced long-term depression in the hippocampal CA1 region. J Neurochem 2006;96(1):179–194.

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83. Gallagher SM, Daly CA, Bear MF, et al. Extracellular signal-regulated protein kinase activation is required for metabotropic glutamate receptor-dependent longterm depression in hippocampal area CA1. J Neurosci 2004;24(20):4859–4864. 84. Mulkey RM, Endo S, Shenolikar S, et al. Involvement of a calcineurin/inhibitor1 phosphatase cascade in hippocampal long-term depression. Nature 1994; 369(6480):486–488. 85. Schnabel R, Kilpatrick IC, Collingridge GL. Protein phosphatase inhibitors facilitate DHPG-induced LTD in the CA1 region of the hippocampus. Br J Pharmacol 2001;132(5):1095–1101. 86. Moult PR, Schnabel R, Kilpatrick IC, et al. Tyrosine dephosphorylation underlies DHPG-induced LTD. Neuropharmacology 2002;43(2):175–180. 87. Huber KM, Kayser MS, Bear MF. Role for rapid dendritic protein synthesis in hippocampal mGluR-dependent long-term depression. Science 2000;288(5469):1254–1257. 88. Nosyreva ED, Huber KM. Developmental switch in synaptic mechanisms of hippocampal metabotropic glutamate receptor–dependent long-term depression. J Neurosci 2005;25(11):2992–3001. 89. Hou L, Klann E. Activation of the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin signaling pathway is required for metabotropic glutamate receptor-dependent long-term depression. J Neurosci 2004;24(28):6352–6361. 90. Bear MF, Huber KM, Warren ST. The mGluR theory of fragile X mental retardation. Trends Neurosci 2004;27(7):370–377. 91. Huber KM, Gallagher SM, Warren ST, et al. Altered synaptic plasticity in a mouse model of fragile X mental retardation. Proc Natl Acad Sci USA 2002;99(11):7746–7750. 92. Nosyreva ED, Huber KM. Metabotropic receptor–dependent long-term depression persists in the absence of protein synthesis in the mouse model of fragile X syndrome. J Neurophysiol 2006;95(5):3291–3295. 93. Snyder EM, Philpot BD, Huber KM, et al. Internalization of ionotropic glutamate receptors in response to mGluR activation. Nat Neurosci 2001;4(11):1079–1085. 94. Xiao MY, Zhou Q, Nicoll RA. Metabotropic glutamate receptor activation causes a rapid redistribution of AMPA receptors. Neuropharmacology 2001;41(6):664–671. 95. Watabe AM, Carlisle HJ, O’Dell TJ. Postsynaptic induction and presynaptic expression of group 1 mGluR–dependent LTD in the hippocampal CA1 region. J Neurophysiol 2002;87(3):1395–1403. 96. Tan Y, Hori N, Carpenter DO. The mechanism of presynaptic long-term depression mediated by group I metabotropic glutamate receptors. Cell Mol Neurobiol 2003;23(2):187–203. 97. Rammes G, Palmer M, Eder M, et al. Activation of mGlu receptors induces LTD without affecting postsynaptic sensitivity of CA1 neurons in rat hippocampal slices. J Physiol 2003;546(Pt 2):455–460. 98. Qian J, Noebels JL. Exocytosis of vesicular zinc reveals persistent depression of neurotransmitter release during metabotropic glutamate receptor long-term depression at the hippocampal CA3-CA1 synapse. J Neurosci 2006;26(22): 6089–6095.

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14 Metabotropic Glutamate Receptor Ligands as Novel Therapeutic Agents Ashley E. Brady and P. Jeffrey Conn

Summary Metabotropic glutamate receptors comprise a diverse family of G proteincoupled receptors that are critical for regulating normal neuronal function in the central nervous system (CNS). The heterogeneous distribution and diverse physiologic roles of the various mGluR subtypes make them highly attractive targets for the treatment of a number of neurologic and psychiatric disorders. The discovery of subtype-selective ligands for these receptors has provided the tools to support a number of preclinical studies, suggesting the tremendous therapeutic potential that lies in the ability selectively to modulate a specific mGluR subtype. In the last few years, a major milestone in the field was achieved with the first selective mGluR ligands entering into clinical development and demonstrating efficacy in the treatment of anxiety disorders. In addition to the discovery of selective, direct-acting mGluR ligands, a novel class of mGluR-selective ligands has recently emerged. These allosteric modulators, which act through nontraditional binding sites on the mGluRs, may exhibit even greater subtype selectivity than orthosteric ligands. Furthermore, because they modulate mGluRs in an activity-dependent manner, it is possible that allosteric activators of mGluRs will be less likely to induce adverse effects or promote receptor desensitization. This chapter summarizes the critical studies that have contributed to the validation of mGluRs as therapeutic targets for the treatment of a number of CNS disorders and describes progress thus far in identifying and developing novel mGluR subtype-selective compounds.

From: The Receptors: The Glutamate Receptors Edited by: R. W. Gereau and G. T. Swanson © Humana Press, Totowa, NJ

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Key Words: Metabotropic glutamate receptor; Central nervous system disorders; Negative/positive allosteric modulator; Allosteric potentiator; Therapeutic ligand; Subtype-selective ligand; Anxiety; Parkinson’s disease; Schizophrenia; Antagonist; Agonist.

1. Introduction Metabotropic glutamate receptors (mGluRs) are members of the large family of G protein-coupled receptors (GPCRs) and play an important role in mediating the effects of glutamate, the major excitatory neurotransmitter in the central nervous system (CNS). As outlined in the preceding chapters, group I mGluRs (mGluR1 and mGluR5) are often expressed postsynaptically, where they regulate neuronal excitability, whereas group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR6, mGluR7, and mGluR8) mGluRs are predominantly expressed presynaptically, where they act as autoreceptors or heteroceptors to inhibit neurotransmitter release (1). However, a number of important exceptions to these generalizations exist, and members of each of the major groups of mGluRs can play a variety of roles in regulating neuronal, as well as glial, function. Because of the heterogeneous distribution and diverse physiologic roles of mGluR subtypes, these receptors play important roles in regulating virtually all of the major functions of the CNS. Furthermore, because of the wide diversity of this family of receptors, only specific mGluR subtypes may participate in any given function. This broad range of functional roles of a diverse family of mGluR subtypes raises the exciting opportunity for developing therapeutic agents that selectively interact with mGluRs involved in only one or a limited number of CNS functions. As the mGluR field has matured, a range of mGluR subtype-selective and group-selective ligands for these receptors has been discovered. Use of these ligands as tools has generated a large body of preclinical studies suggesting that ligands for specific mGluR subtypes have tremendous potential for the treatment of a wide variety of neurologic and psychiatric disorders including anxiety disorders (2,3), Parkinson’s disease (4), schizophrenia (5,6), drug abuse (7), Alzheimer’s disease (8), epilepsy (9), depression (10), and pain (11), among others. In the last several years, these efforts have reached a major milestone in that the first mGluR ligands have entered clinical development. Clinical studies with this first wave of mGluR ligands have revealed exciting evidence of efficacy in treatment of anxiety disorders. In this chapter, we summarize the studies that have led to mGluRs now being recognized as viable therapeutic targets for a number of CNS disorders and describe the progress that has been made in developing novel compounds, acting at either orthosteric or allosteric sites, to modulate this important GPCR family.

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2. Anxiety Disorders Anxiety disorders, including generalized anxiety disorder, panic attacks, posttraumatic stress disorder, obsessive-compulsive disorder (OCD), and social phobias, are among the most commonly occurring of all mental illnesses. Nevertheless, few therapeutic options have been available to patients suffering from these conditions. Until recently, clinical treatment of these disorders has relied most heavily on the use of benzodiazepines, such as Valium and Xanax, which have a narrow therapeutic index due to numerous adverse side effects such as sedation, memory impairment, abuse potential, and physical dependence (12). Therefore, there is a significant need for the development of new therapeutic strategies and efficacious medications with fewer adverse effects for the treatment of anxiety disorders. The most exciting advances in understanding the therapeutic potential of mGluR ligands have come from studies suggesting that ligands of specific mGluR subtypes may provide a novel approach to development of anxiolytic agents that lack many of the problems associated with the benzodiazepines (13). 2.1. Group II mGluR Agonists Group II mGluRs are localized, primarily presynaptically, in the cortex, thalamus, striatum, amygdala, and hippocampus (14), where they often serve to reduce transmission at glutamatergic synapses. These areas of the brain are thought to play a critical role in anxiety disorders (15), among other CNS disorders. For instance, the amygdaloid complex has long been postulated to play a critical role in anxiety and fear learning (15,16), and the prefrontal cortex (PFC) has been implicated in anxiety and fear in both animals and humans (17). Furthermore, hyperactivity of glutamatergic transmission in these structures is thought to be associated with the pathogenesis of anxiety and fear conditioning (15,18). Because group II mGluRs are highly localized presynaptically in forebrain regions and limbic structures (14), it has been postulated that agonists at these receptors could reduce anxiety-like behaviors by decreasing glutamate transmission in these brain regions. Based on this, there has been a major effort to test this hypothesis in preclinical models of anxiety disorders. One of the most important and earliest breakthroughs in developing drugs that target the mGluRs was development of potent and highly selective agonists for the group II mGluRs by Jim Monn, Darryle Schoepp, and their coworkers at Eli Lilly (for reviews, see refs. 3 and 19). The first of these mGluR agonists, (1S,2S,5R,6S)-2-aminobicyclo[3.1.0] hexane-2,6-dicarboxylate monohydrate (LY354740), represents a conformationally constrained analog of glutamate that is orally bioavailable and exhibits nanomolar potency for mGluR2/3. Thus, this compound has made it possible to explore the functional consequences of group II mGluR activation in vivo.

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Studies using LY354740 and related compounds have revealed robust anxiolytic-like effects in a broad range of animal models (for reviews, see refs. 13 and 19). For instance, LY354740 dose-dependently reduces fear potentiated startle (15,19,20) and increases the time spent by rats in the open arms in the elevated plus maze (19,21–24). In addition, this compound shows antianxiety or antistress activity in lactate-induced panic-like responses in panic-prone rats (25), stress-induced hyperthermia (26), and the Vogel test (27–29). A recent report by Schoepp et al. (30) evaluated another related mGluR2/3 agonist, (-)(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY404039), and found it to be 30- to 100-fold more potent than LY354740 in reducing fear-potentiated startle anxiety in rats. This compound also was shown to reduce marble burying in mice while not affecting performance on the rotorod test. These data suggest that LY404039 may be particularly efficacious in the treatment of generalized anxiety disorder (GAD) and OCD (30). Unlike benzodiazepines—the most common clinically used anxiolytics— LY354740 does not impair performance in the rotorod test, enhance sleep time induced by hexobarbital, or impair retention of memory in a passive avoidance test (20), and it also does not increase punished responding at doses that significantly reduce unpunished responding (29). In addition, in contrast to the benzodiazepines, agonists of group II mGluRs do not have a profile in animal models that is predictive of abuse liability (13). This suggests that group II mGluR agonists act by a mechanism that is distinct from that of benzodiazepines and that these compounds may not share the adverse sedative effects and potential for abuse that is associated with antianxiety agents that are available for clinical use. The distribution of group II mGluR protein and mRNA in humans is similar to that observed in rodents (19,31,32). Based on this and the exciting preclinical profile of group II mGluR agonists, LY354740 has now been used in clinical studies to evaluate its safety and potential efficacy in treating anxiety disorders in humans. Consistent with the animal studies, LY354740 reduced the number and severity of CO2 -evoked panic attacks in patients suffering from panic disorder (19,33), improved anxiety symptoms in individuals with GAD (34), and showed robust efficacy in a human model of fear-potentiated startle (35). In these initial clinical trials, there were fewer side effects observed compared to placebo, including sedation (19,33–35). Most recently, Michelson and colleagues presented a clinical comparison of the efficacy of LY354740 versus lorazepam in patients suffering from GAD (36). They reported that patients in both treatment groups showed a significantly greater improvement in the Hamilton Anxiety Scale (HAMA) compared to patients receiving placebo. In this study, patients treated with LY354740 also reported fewer adverse side effects such as somnolence, dizziness, and disturbances in attention (36). These exciting clinical findings using group II mGluR agonists represent a major

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breakthrough in mGluR pharmacology, and in neuropharmacology in general, in that they provide clinical validation of a fundamentally novel approach to the treatment of anxiety disorders. Although these results are encouraging, there are limitations to the use of the first-generation mGluR2/3 agonists in the clinic. Successful treatment of one specific anxiety disorder (or reported relief of symptoms in a human model for the disease) does not necessarily predict efficacy in all clinical manifestations of the disorder. Bergink and coworkers recently reported the results of a European phase II multicenter clinical trial evaluating the safety and efficacy of LY354740 in reducing panic attacks in patients diagnosed with panic disorder. In this 9-week study, LY354740 demonstrated a lack of efficacy at the doses tested when compared to placebo. In light of the numerous preclinical studies supporting the anxiolytic properties of mGluR2/3 agonists, and specifically LY354740, additional studies are warranted to further investigate these findings. For example, it is possible that a more potent, orally bioavailable compound or a compound that can be administered at a higher dose will be necessary for successful treatment of panic disorder (37). One of the primary shortcomings of LY354740, the first mGluR2/3 agonist introduced into clinical development, is that it has very low oral bioavailability. To address this problem, workers at Lilly developed a prodrug approach as a means to improve oral bioavailability. The prodrug, (1S,2S,5R,6S)-2[(2’S)-([2’-amino) propionyl]aminobicyclo[3.1.0]hexane-2,6- dicarboxylic acid hydrochloride (LY544344), is actively transported out of the intestinal tract into the systemic circulation, where it is then metabolized to the active compound, LY354740 (38). It is estimated in animal studies that 85% oral bioavailability of LY354740 can be achieved using this approach (39), which is a significant improvement over the 10% estimated to be absorbed when LY354740 is orally administered (40). The efficacy of LY544344 was evaluated in two animal behavioral paradigms of anxiety and was found to produce anxiolytic effects in mice at significantly lower doses than were necessary for the parent compound, LY354740 (38). This prodrug has most recently been evaluated in healthy humans for efficacy in panic anxiety induced by cholecystokinin tetrapeptide (CCK-4). Although a trend toward significant anxiolytic effects was observed in this study, we await additional clinical trials to further characterize the efficacy of this molecule in the treatment of anxiety disorders (41). 2.2. Allosteric Potentiators of mGluR2 Despite the tremendous advances in development of group II mGluR agonists and in defining their therapeutic potential, problems associated with direct-acting agonists of group II mGluRs could ultimately limit their clinical utility or even prevent their clinical use. It is not yet clear whether LY354740 and related compounds will reach the market for broad clinical use. Although

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LY354740 showed promising efficacy in phase II trials, clinical studies with this compound have been placed on hold, and it is not certain whether the safety profile of this chemical series will support its advancing to Food and Drug Administration approval. Unfortunately, all systemically active group II mGluR agonists that have been developed belong to the same chemical class as LY354740. Thus, if a problem develops with this particular series of compounds that prevents further development, it will be critical to identify novel classes of compounds that are capable of activating these receptors. In addition, group II mGluR agonists activate both mGluR2 and mGluR3, and the relative contributions of these two receptor subtypes to the actions of these drugs are not known. Finally, development of tolerance to direct agonists has the potential of limiting their clinical use. Thus, there is a critical need to build on these exciting advances by determining whether a specific mGluR subtype (mGluR2 or mGluR3) is responsible for these effects and to develop novel approaches for activating these receptors. One alternative approach to receptor activation that has been highly successful for ion channels is the use of selective allosteric potentiators of the specific receptor subtypes. This concept has recently been expanded to the mGluRs, and highly selective allosteric potentiators of different mGluR subtypes have been developed. These small molecules do not activate the mGluRs directly but act at an allosteric site on the receptor to potentiate glutamate-induced activation of the receptor. Two novel classes of compounds have been described that act as allosteric potentiators of mGluR2. These include an Eli Lilly compound, N(4-(2-methoxyphenoxy)phenyl)-N-(2,2,2-trifluoroethylsulfonyl)pyrid-3-ylmethylamine (LY487379) (42), and a structurally distinct compound developed by workers at Merck that has been termed Compound A (also known as biphenyl-indanone A [BINA]), 3’-[[(2-cyclopentyl-6,7-dimethyl-1-oxo2,3-dihydro-1H-inden-5-yl)oxy]methyl]-biphenyl-4-carboxylic acid (43,44). These are novel structural classes of compounds and represent the first clear departure from glutamate analogs as group II mGluR activators. As with other allosteric potentiators, these compounds have no effect on mGluR2 alone, but they induce a leftward shift of the concentration–response curve to glutamate (42,43,45,46). These compounds are both centrally active (42,43) and are highly selective for mGluR2 relative to mGluR3 or other mGluR subtypes, which may provide an exciting new approach to development of therapeutic agents that specifically increase activity at mGluR2 (43,46). Recent reports have suggested that mGluR2 potentiators may indeed prove to be therapeutically useful. For example, electrophysiologic studies with a compound related to LY487379, 2,2,2-trifluoro-N-[3-(cyclopentyloxy) phenyl]-N-(3-pyridinylmethyl)-ethane sulfonamide (cyPPTS), showed that this

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compound suppressed glutamatergic corticostriatal synaptic transmission only following a high-frequency stimulation. This result was in contrast to that observed with the mGluR2/3 agonist LY354740, which reduced excitatory postsynaptic potential (EPSP) amplitude in response to both low- and highfrequency stimulation (47). These findings support the idea that potentiators may be able to specifically reduce excessive glutamate release without affecting basal levels. Two additional compounds, also from the LY487379 series, were tested in rodent behavioral models of anxiety: N-[4-(4-carboxamidophenoxy) phenyl]-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride monohydrate (4-APPES) was efficacious in a rat fear–potentiated startle paradigm, and N-(4’cyano-biphenyl-3-yl)-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride (CBiPES) attenuated stress-induced hyperthermia in mice (47). Studies with BINA also support the hypothesis that mGluR2 potentiators will potentiate electrophysiologic effects of group II mGluR agonists in brain regions thought to be important for the potential anxiolytic effects of these compounds (44). Furthermore, BINA also mimicked the effects of group II mGluR agonists and mGluR2 allosteric potentiators in animal models used to predict potential anxiolytic-like activity (43,44). Unlike the short-acting LY487379, BINA exerted effects that were long lasting over a duration of at least 8 hrs, indicating that this compound is suitable for further pharmacologic evaluation in vivo (i.e., chronic studies). Because it has been postulated that allosteric potentiators could have fewer adverse effects and induce less tolerance than direct-acting agonists, it will be important in future studies to investigate the effects of chronic administration of allosteric potentiators of mGluR2 and group II mGluR agonists. These studies will allow us to determine whether mGluR2 potentiators can offer advantages over traditional group II mGluR agonists. 2.3. mGluR5 Antagonists Group I mGluRs, comprising mGluR1 and mGluR5 receptors, are Gq-linked GPCRs that are predominantly localized postsynaptically. These receptors are positively coupled to the phospholipase C (PLC) signaling pathway, and their activation leads to increases in intracellular calcium release, which facilitates the excitatory effects of glutamate (1). Because excessive glutamatergic neurotransmission in limbic and forebrain circuits is thought to be one of the primary underlying pathophysiologic causes of anxiety disorders, it has long been postulated that antagonists of group I receptors may have therapeutic efficacy. In particular, mGluR5 is expressed at high levels in brain regions believed to be involved in emotional processes, namely limbic structures, including the ventral striatum, cortex, and hippocampus. This receptor has been the focus of intensive investigation as a potential target of novel anxiolytic agents. One animal model that is often used to assess stress and anxiety-like responses is

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stress-induced hyperthermia (SIH). It is interesting that mGluR5-knockout mice display a significant reduction in SIH when compared to wild-type littermate controls (48). To confirm that the observed effect was mediated by mGluR5, the selective mGluR5 antagonist (3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine) (MTEP) also was tested in wild-type verses mGluR5-knockout animals and was found to be effective at attenuating stress-induced hyperthermia only in the wild-type animals (48). In the last few years several mGluR5-selective antagonists have been identified, including 6-methyl-2-(phenylazo)-3-pyridinol (SIB-1757) and (E)2-methyl-6-(2-phenylethenyl)pyridine (SIB-1893), which provided important proof of concept of the potential of allosteric antagonists of this receptor at the molecular and cellular level. However, due to the chemical properties and pharmacokinetics of these compounds, neither proved useful for in vivo studies (49). Recent structural derivatization around these compounds led to the discovery of 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a potent, selective, and systemically active mGluR5 allosteric antagonist (50). The effects of MPEP were evaluated in a number of different rodent behavioral models predictive of anxiolytic activity. MPEP was found to exert anxiolytic effects in several unconditioned response tests including the social exploration test (51), the elevated plus maze (51–53), marble burying (51), and stress-induced hyperthermia (26,51,53). Furthermore, anxiolytic effects of MPEP also were observed using a number of conditioned response paradigms in rats such as the Geller-Seifter test (51,54), the Vogel conflict drinking test (52,55–57), conditioned lick suppression (57), fear-potentiated startle (FPS) (54), and ultrasonic vocalization (USV) (54), as well as the four-plate test in mice (52). Thus, the mGluR5 antagonist MPEP has efficacy in a broad range of animal models that have been used to predict anxiolytic activity. Efforts to increase in vivo efficacy of MPEP through improved specificity and pharmacokinetic properties soon led to the discovery of MTEP, an analog of MPEP (58). MTEP is five times more potent than MPEP in the FPS model of anxiety when injected intraperitoneally in rats (58). In a subsequent set of in vivo studies, the anxiolytic effects of MTEP were compared to those of the benzodiazepine receptor agonist diazepam following subcutaneous injection in rats (59). In the Geller-Seifter test and the fear-potentiated startle model, MTEP was found to exhibit an anxiolytic profile similar to that of diazepam. However, it is of particular interest that, unlike diazepam, the anxiolytic effects of MTEP in the Geller-Seifter test following chronic dosing were not accompanied by a disruption in motor performance (as assessed by rotorod performance) nor was an interaction with ethanol observed (59). This indicates a clear improvement over the side-effect profile commonly associated with traditional benzodiazepine anxiolytics.

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Since the discovery of MTEP, additional compounds have been derived based on its structure. For example, 5-[(2-methyl-1,3-thiazol-4-yl)ethynyl]2,3’-bipyridine has improved oral bioavailability and demonstrates efficacy in the rat fear-potentiated startle model of anxiety (60). In addition, two novel heteroarylazoles with superior brain penetration, 3-[5-pyridin-2-yl-2H-tetrazol2-yl)benzonitrile and 3-fluoro-5-(5-pyridin-2-yl-2H-tetrazol-2-yl)benzonitrile, have been identified, the first of which also has been shown to be orally active in the rat fear-potentiated startle model, albeit with tolerance following repeated dosing (61). It is interesting that recent studies have provided the first clinical validation of mGluR5 antagonists as anxiolytic agents. Porter and coworkers have now identified mGluR5 as the molecular target of fenobam, a compound previously shown to have anxiolytic properties in both rodents and humans but with an unknown mechanism of action (62–68). These investigators found that fenobam is a selective and potent mGluR5 allosteric antagonist that shares a binding site with MPEP and has inverse agonist properties (62,63). Fenobam originally was developed by McNeil Laboratories in the late 1970s as a nonbenzodiazepine anxiolytic. As with the newer mGluR5 antagonists, fenobam exhibited anxiolytic properties in the Geller-Seifter, Vogel conflict, stress-induced hyperthermia, and conditioned emotional response paradigms in rodents (62,64). Although at that time its mechanism of action was not known, this compound progressed to clinical development and was tested in several clinical settings, where it was reported to be efficacious in treating anxiety disorders in human patients (66–68). This study is particularly exciting because it provides the first direct clinical support for mGluR5 antagonists as anxiolytic agents. Nevertheless, studies were terminated after psychostimulant side effects were reported in a phase II trial (69). 2.4. Ligands at Other mGluR Subtypes Although studies with group II mGluR agonists/potentiators and mGluR5 antagonists have received the most attention, some preclinical evidence suggests that ligands at other mGluR subtypes may also have potential as anxiolytic agents. For instance, the mGluR1 antagonists (4-methoxyphenyl)-(6-methoxy-quinazolin-4-yl)-amine HCl (LY456236) and 3-ethyl2-methyl-quinolin-6-yl)-(4-methoxy-cyclohexyl)-methanone methanesulfonate (EMQMCM) have anxiolytic effects in some animal models, including fearpotentiated startle and freezing in a fear conditioning test (57). However, the mGluR1 antagonists were not as efficacious as the benzodiazepine chlordiazepoxide when directly compared, and they failed to produce any anxiolytic effects in the elevated plus maze or the Geller-Seifter conflict test (57). There also is some evidence that ligands acting at group III mGluRs expressed in the CNS (mGluR4, 7, and 8) may exert anxiolytic activity.

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For example, several studies suggest that group III mGluR ligands produce anxiolysis in rats after intrahippocampal injection (55,70–72). In addition, anxiolytic-like effects were observed in the conflict drinking test in rats following peripheral injection of the group III mGluR agonist ACPT-1 (73). However, the ligands used in these studies are nonselective and have relatively low potencies, making it difficult to know which mGluR subtypes are most likely to be involved and whether agonists or antagonists at these specific receptors would be preferred. More recently, knockout mice with a targeted deletion of the mGluR7 gene were tested in a number of behavioral models predictive of anxiety, all of which suggest that deletion of this gene has anxiolytic-like effects. Consistent with a functional role in anxiety, mGluR7 is highly expressed in brain regions critical for the expression of anxiety and stress responses such as the amygdala, hippocampus, and locus coeruleus (74). It is interesting that, in contrast to the anxiolytic phenotype observed in mGluR7knockout animals, mice in which the gene encoding mGluR8 is deleted showed increased anxiety-like behavior (75,76). Studies to fully explore the potential utility of ligands at the group III mGluRs will be facilitated greatly by the development of more subtype-selective compounds in the future. Recently, discovery of the first selective activators of each of the major group III mGluRs has been reported, including dicarboxyphenylglycine (DCPG) as a selective agonist of mGluR8 (77), N-phenyl-7-(hydroxylimino)cyclopropa[b]chromen1a-carboxamide (PHCCC) as a selective allosteric potentiator of mGluR4 (78,79), and N,N’-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082) as a selective allosteric agonist of mGluR7 (80). Although these early compounds do not have the optimal properties to facilitate clinical testing, it is hoped that they represent the first of more selective reagents that will be developed to investigate the effects of activation and inhibition of these important members of the mGluR family.

3. Parkinson’s Disease Exciting advances in our understanding of the function of mGluRs and the distribution of mGluR subtypes in the basal ganglia suggest that members of this receptor family could serve as targets for novel therapeutic agents that would be effective in treatment of Parkinson’s disease (PD). PD is a debilitating neurodegenerative disorder that afflicts >1% of adults >65 years of age. The clinical syndrome that occurs in Parkinson patients is characterized by a disabling motor impairment that includes tremor, rigidity, and bradykinesia. The primary pathophysiologic change giving rise to the symptoms of PD is a loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) that are involved in modulating the function of the striatum and other basal

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ganglia nuclei. Unfortunately, traditional therapies for treatment of PD based on dopamine-replacement strategies eventually fail in most patients (81). Because of this, a great deal of effort has been focused on developing a detailed understanding of the circuitry and function of the basal ganglia in hopes of generating novel therapeutic approaches for restoring normal basal ganglia function in patients suffering from PD. The basal ganglia are an interconnected group of subcortical nuclei that are involved in the control of motor behavior. The striatum receives the primary inputs into the basal ganglia, whereas the substantia nigra pars reticulata (SNr) and internal globus pallidus (GPi) (or entopeduncular nucleus [EPN] in rodents) serve as the primary basal ganglia output nuclei. The striatum sends signals via inhibitory GABAergic neurons to these two output nuclei either directly or indirectly via the globus pallidus external segment (GPe) (globus pallidus [GP] in rodents) and subthalamic nucleus (STN) (Fig. 1). Whereas transmission through the “direct pathway” from the striatum to the output nuclei results in decreased output activity, signaling through the parallel “indirect pathway” increases the net output from the SNr and GPi. A delicate balance between inhibition of the output nuclei through the direct pathway and excitation through the indirect pathway is essential to normal physiologic

Fig. 1. Localization of metabotropic glutamate receptor (mGluR) subtypes within the basal ganglia motor circuit. Normal physiologic function requires a precise balance between inhibition of the output nuclei (internal globus pallidus/substantia nigra pars reticulata [GPi/SNr]) via the direct pathway (striatum → GPi/SNr) and excitation via the indirect pathway (striatum → globus pallidus [GP] → subthalamic nucleus [STN] → GPi/SNr). Because of their distribution throughout the basal ganglia, mGluRs are promising targets for regulating this circuitry. Excitatory transmission is indicated by arrows; inhibitory connections are depicted by a perpendicular bar. The dashed arrow illustrates how increased activity through the indirect pathway may lead to excitotoxic damage to substantia nigra pars compacta (SNc) dopaminergic neurons. mGluR subtype expression is shown at either cell bodies or presynaptic terminals. DA, dopamine; GABA, -aminobutyric acid; GLU, glutamate.

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function. Recent studies suggest that a loss of nigrostriatal dopamine neurons in PD patients results in an increase in activity of the indirect pathway relative to the direct pathway. The resultant increase in inhibitory output from the GPi/SNr results in reduced thalamocortical activity and produces the motor symptoms associated with PD. Consequently, pharmacologic agents that selectively decrease net transmission through the indirect pathway could be useful in ameliorating the symptoms of PD. Moreover, a number of studies raise the possibility that manipulations that reduce transmission through the indirect pathway may also slow progression of PD by reducing excessive excitatory drive to dopaminergic neurons, thereby reducing the excitotoxicity component of disease progression. 3.1. mGluR4 Agonists or Allosteric Potentiators One of the most exciting recent advances in novel pharmacologic approaches to the treatment of PD is the discovery of the robust therapeutic potential of mGluR4 agonists or allosteric potentiators in animal models of this disorder. Immunohistochemical studies reveal that this receptor is abundant on presynaptic terminals of striatal projections to the GP (82,83). As outlined earlier, this is the first synapse in the indirect pathway, and extensive studies in PD patients and animal models suggests that any manipulation that reduces transmission at this synapse could have a robust antiparkinsonian effect. It is interesting that whole-cell patch-clamp recordings from rat GP neurons reveal that activation of mGluR4 induces a robust decrease in transmission at this critical synapse (84). Based on this, it is possible that agonists of mGluR4 could have antiparkinsonian effects by actions in the GP. Consistent with this hypothesis, intracerebroventricular (UCV) injection of the group III mGluR agonist (l-2-amino-4-phosphonobutanoate (L-AP4) induced robust antiparkinsonian effects in multiple rodent models (84). Unfortunately, it has been extremely difficult to develop group III mGluR agonists with high affinity for specific mGluR subtypes that also have appropriate druglike properties. However, small molecules that act as allosteric potentiators of agonist-induced responses at mGluR4 have been identified. For example, the novel mGluR4 allosteric potentiator PHCCC was recently discovered (79). As with the mGluR2 potentiators described earlier, PHCCC does not activate mGluR4 directly, but it dramatically potentiates activation of the receptor by glutamate or L-AP4. It is interesting that this compound potentiates mGluR4 function at the striatopallidal synapse and has a robust antiparkinsonian effect in rodent models when administered by ICV injection (79). It is significant that mGluR4 agonists and potentiators had antiparkinsonian activity that was comparable to that of l-DOPA and dopamine receptor agonists. These studies provide exciting proof of concept in animal models for the potential utility of mGluR4 agonists or potentiators in reducing the symptoms of PD.

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As mentioned previously, several studies suggest that in addition to their potential efficacy in symptomatic treatment of PD, mGluR4 activators also may slow the progression of the disorder (85–88). It is believed that increased activity through the indirect pathway not only plays a role in motor dysfunction, but also may contribute to the excitotoxic damage to SNc dopamine neurons because glutamatergic neurons of the STN project both to the basal ganglia output nuclei and onto the dopaminergic neurons in the SNc. Several studies suggest that excitatory drive from the STN may contribute to the loss of dopamine neurons in animal models that involve relatively slow, progressive loss of dopamine neurons (89,90). Based on this, it is possible that reduced transmission through the indirect pathway by mGluR4 activators could also reduce dopamine cell loss. Furthermore, in addition to its effect at the striatoGP synapse, activation of mGluR4 directly reduces transmission at excitatory synapses onto SNc dopaminergic neurons (91), which could further reduce excitotoxicity in these cells. Finally, several studies suggest that mGluR4 agonists or allosteric potentiators might have other neuroprotective actions on dopamine neurons. Activation of mGluR4 on glia inhibits formation of the chemokine RANTES (85), which is involved in neuroinflammation in some neurodegenerative disorders, and mGluR4 receptor activation has direct neuroprotective effects on neurons (86,87,92). These combined actions of mGluR4 receptor activation could reduce the loss of dopamine neurons in patients with PD. Consistent with this, Battaglia and colleagues recently reported that systemic injection of PHCCC decreases nigrostriatal degeneration in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a parkinsonian model (88). Although this is a tremendous advance, PHCCC is a relatively low potency compound with limited selectivity and bioavailability. Thus, it will be important to identify novel compounds that act as allosteric potentiators of mGluR4 in the hope of identifying compounds with higher potency and selectivity than PHCCC (79,84). 3.2. Group I mGluR Antagonists In addition to mGluR4 activators, some studies raise the possibility that antagonists of group I mGluRs could have efficacy in treatment of PD. Group I mGluRs are expressed throughout the basal ganglia nuclei and regulate this motor circuit at many levels. For example, mGluR1 receptors are expressed presynaptically on dopaminergic fibers in the striatum, where they inhibit dopamine release (93). Both mGluR1 and mGluR5 are expressed in the striatum, where the net effect of their activation is to counteract dopaminergic neurotransmission (94). In addition, activation of group I mGluRs increases neuronal excitability in the GP (95,96), STN (97–99), and SNr (100). In the GP

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and SNr, mGluR1 is the predominant mGluR involved in neuronal excitation (95), whereas mGluR5 plays a dominant role in STN neurons (97,98). The role of mGluR5 in STN neurons and mGluR1 in SNr neurons is particularly relevant, given that studies have indicated that an increase in neuronal activity in the STN is the primary pathophysiologic change observed in PD patients in response to loss of nigrostriatal dopamine neurons (101). Overactivation of the STN leads to increased stimulation of GABAergic neurons of the GPi/SNr, which effectively blocks synaptic transmission from the thalamus to the cortex and results in the motor impairment observed in PD (102). Together, these studies support a role for group I mGluRs in inducing an overall effect of increasing activity through the indirect pathway, which counteracts the effects of dopamine. This implies that antagonists of these receptors could have antiparkinsonian effects. Consistent with this possibility, the mGluR5 antagonist MPEP reverses a number of parkinsonian effects of the dopamine receptor antagonist haloperidol in rats, including hypolocomotion, catalepsy, and muscle rigidity (103). Furthermore, chronic (but not acute) treatment with MPEP significantly reverses the akinetic deficits induced by bilateral nigrostriatal lesioning in rats (104). However, these effects are not as robust as the effects of other antiparkinsonian agents or as the mGluR4 agonists and allosteric potentiators described earlier, thus raising questions about whether mGluR5 antagonists are likely to have robust efficacy in humans. Selective antagonists of mGluR1 have not been rigorously tested in animal models of PD (94). However, the studies outlined here suggest that simultaneous blockade of mGluR1 and mGluR5 may be a more effective treatment strategy than blockade of only mGluR5 (105). 3.3. Group II mGluR Agonists and Allosteric Potentiators The group II mGluRs also are involved in basal ganglia neurotransmission, where they reduce transmission at corticostriatal (106–109) and STN–SNr (82) synapses. Based on our understanding of the basal ganglia circuit outlined earlier, group II mGluR–induced reduction of transmission at these two key synapses by mGluR2/3 receptor agonists may be effective in treating PD. In support of this hypothesis, the mGluR2/3 agonist LY354740 reverses parkinsonian effects of haloperidol (82,110) in rats. Furthermore, direct unilateral injection of the mGluR2/3 agonists LY379268 (111) and 2’,3’dicarboxycyclopropylglycine (DCG-IV) (112) into the SNr dose-dependently reverses akinesia in the reserpine-treated rat model of PD. However, as with the effects of mGluR5 antagonists, the actions of group II agonists are relatively modest when compared to the effects of mGluR4 agonists or allosteric potentiators.

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4. Schizophrenia Schizophrenia is a chronic and debilitating psychiatric disorder that affects approximately 1% of the world population. This disease is characterized by a number of symptoms, which are classified into three distinct categories: positive symptoms (delusions, hallucinations, disordered thought, and catatonia), negative symptoms (anhedonia, apathy, and social withdrawal), and cognitive impairment (working memory and attention deficits) (for review, see ref. 6). Over the last several decades a number of therapies have emerged for treating schizophrenia, with the majority of them focused on blocking the D2 dopamine receptor (D2R). Unfortunately, the clinical utility of these so-called typical antipsychotics such as haloperidol and chlorpromazine has been limited by the resultant extrapyramidal side effects (parkinsonism and tardive dyskinesia). Furthermore, these drugs treat only the positive symptoms of schizophrenia and have no effect on the negative symptoms or cognitive deficits. With the discovery of clozapine, “atypical” antipsychotics soon emerged as an alternative therapy for schizophrenia with some efficacy in treating both negative symptoms and cognitive deficits. Unlike the typical antipsychotics, which exhibit high affinity for the D2R, these drugs are generally more potent at blocking serotonin 5-HT2 receptors. Although the extrapyramidal side effects and tardive dyskinesia often associated with typical antipsychotic treatment were absent, patients taking clozapine were found to be at a higher risk of seizures and often developed a fatal condition called agranulocytosis. Based on the current therapeutic options available to patients suffering from schizophrenia, it is clear that there is room for improved treatment strategies. Recent clinical and basic studies suggest that a dysregulation of the glutamatergic system—specifically, changes in signaling through the ionotropic N-methyl-d-aspartate (NMDA) subtype of glutamate receptor (NMDAR), may play an important role in some of the pathologic changes associated with schizophrenia (5,113,114). This theory results primarily from the observation that NMDAR antagonists such as phencyclidine (PCP) and ketamine induce psychosis and impair cognitive functioning in healthy human volunteers, reminiscent of symptoms observed in schizophrenic patients, and exacerbate these symptoms in schizophrenic patients. Furthermore, administration of agents that enhance NMDAR function, such as agonists at the glycine-binding site on the receptor, may result in a symptomatic improvement in schizophrenic patients (6). These and other studies have led to the glutamate/NMDAR hypofunction hypothesis of schizophrenia and suggest that novel compounds that potentiate the function of NMDARs may ameliorate the symptoms of schizophrenia (for review, see ref. 5). Accordingly, the research community

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has extended a significant effort to identify agents that can restore the balance to glutamatergic neurotransmission, including those acting through mGluRs, in the hope of discovering more efficacious treatments for schizophrenia. Two specific mGluR subtypes have been identified as potentially useful therapeutic targets: mGluR5 receptors, which can directly modulate NMDAR channel function, and mGluR2/3 receptors, which regulate the release of glutamate (115). 4.1. Group I mGluR Agonists and Allosteric Potentiators Activators of mGluR5 may provide a novel approach to the treatment of schizophrenia and other disorders involving disruption in cognitive function. A number of recent studies suggest that mGluR5 is a closely associated signaling partner with the NMDAR and may play an integral role in regulating and setting the tone of NMDAR function in a variety of forebrain regions (5). For example, the activation of mGluR5 receptors enhances NMDAR currents in multiple neuronal populations (98,116–123). Based on this and a number of studies suggesting that mGluR5 and NMDAR are tightly coupled signaling partners, it was suggested that activation of mGluR5 may provide a more subtle approach to increasing NMDAR activation and could have therapeutic utility in schizophrenia patients (5). Several animal behavioral studies are consistent with the hypothesis that mGluR5 activation is linked to NMDAR function in circuits that may be important for potential antipsychotic effects. For instance, mGluR5-knockout animals exhibit deficits in prepulse inhibition (PPI) relative to wild-type controls (124,125). PPI is a model of sensorimotor gating deficits that are seen in schizophrenic patients and readily modeled in multiple animal species. Furthermore, the mGluR5 antagonists, such as MPEP and MTEP, potentiate the psychotomimetic-like effects of the noncompetitive NMDAR antagonists. Thus, MPEP increases PCP- or MK801-induced hyperlocomotor activity and disruption of PPI in rats (124,126). MPEP also potentiates methamphetamineinduced hyperlocomotion and disruption of PPI (127), suggesting that mGluR5 can modulate these behavioral responses regardless of the psychotomimetic agent used. In addition, MPEP potentiates PCP-induced disruption of learning in the repeated acquisition procedure and of spatial working memory in a delayed nonmatching to position (DNMTP) radial maze task (128,129). Finally, ICV injection of the mGluR5-selective agonist (RS)-2-chloro-5hydroxyphenylglycine (CHPG) ameliorates amphetamine-induced disruption of PPI (124). Although these findings support the notion that activation of mGluR5 may be a viable means of addressing dysregulation of NMDAR function, there are a number of reasons that the use of agonists at mGluR5 is not considered an effective therapeutic strategy. First, it has been extremely difficult to

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develop highly selective agonists of mGluR5 that have suitable properties for use as drugs due to the highly conserved glutamate-binding site (1). In addition, most glutamate-site agonists are analogs of glutamate and do not possess appropriate pharmacokinetic properties and brain penetration to allow them to be useful as drugs. Based on this, efforts have been focused on the discovery of allosteric potentiators of mGluR5. These studies have been highly successful and suggest that allosteric potentiators for this receptor provide an exciting new therapeutic avenue to pursue. Several novel positive allosteric modulators of mGluR5 have now been discovered (130–134). One of the first mGluR5 potentiators identified was N-{4-chloro-2-[(1,3-dioxo1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide (CPPHA), which has activity in the nanomolar range. Consistent with the predicted actions on NMDAR currents, this compound increases mGluR5-mediated potentiation of NMDAR in hippocampal slices (131). However, CPPHA does not have the pharmacologic properties necessary for behavioral and other in vivo studies. An important advance came with the discovery of 3-cyano-N-(1,3-diphenyl-1Hpyrazol-5-yl)benzamide (CDPPB), a novel mGluR5 potentiator with properties that allow both in vitro studies in rat brain slices and in vivo studies in animal models used to predict antipsychotic-like activity. It is interesting that CDPPB has effects in two rodent models that are consistent with antipsychotic activity. These include a decrease in amphetamine-induced hyperlocomotor activity and reversal of amphetamine induced-disruption of PPI (132,133). More recently, a structurally distinct mGluR5 positive allosteric modulator, ADX47273, was investigated in animal models of psychosis and cognition (135). Consistent with studies using CDPPB, ADX47273 dose-dependently inhibits amphetamineinduced locomotor activity and reverses memory impairment in the novel object recognition model in mice. These findings suggest that positive allosteric modulators of mGluR5 may be useful in treating both the positive and negative symptoms of schizophrenia, as well as the cognitive deficits associated with this and other disorders. 4.2. Group II mGluR Agonists and Allosteric Potentiators The NMDAR hypofunction hypothesis of schizophrenia cannot be explained simply by a deficit of glutamate in the forebrain (5). On the contrary, treatment of rats with the noncompetitive NMDAR antagonist ketamine or the competitive antagonist 2-amino-5-phosphopentanoic acid (AP-5) has been found to increase the efflux of glutamate measured in the prefrontal cortex (136,137). It is believed that these NMDAR antagonists cause this increase in prefrontal cortical glutamatergic transmission by selectively decreasing excitatory transmission onto inhibitory GABAergic neurons. The resulting decrease in inhibitory transmission would result in a disinhibition of glutamatergic neurons and a subsequent increase in cortical glutamate (6,114).

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Group II mGluRs (mGluR2/3) are localized primarily presynaptically, where they act as autoreceptors to regulate the release of glutamate (i.e., activation of these receptors results in a reduction of glutamatergic activity) in brain regions believed to play an important role in psychiatric disorders (138,139) such as the cortex, thalamus, striatum, amygdala, and hippocampus (14). Thus, targeting these receptors may provide a treatment for diseases such as schizophrenia thought to result from glutamate hyperactivity (for review, see ref. 139). The first study supporting this hypothesis showed that administration of the mGluR2/3 agonist LY354740 blocked hyperlocomotor activity, reduced stereotypy, and improved working memory impairments induced by PCP in rats (140). In addition, LY354740 reduced PCP-induced glutamate release in the prefrontal cortex and nucleus accumbens without affecting basal glutamate efflux (140). Similar inhibition of ketamine-induced glutamate release in the prefrontal cortex also was observed following administration of the more potent mGluR2/3 agonist LY379268 (141,142). Since then, a number of preclinical studies have emerged also suggesting that agonists at mGluR2/3 may be efficacious in treating schizophrenia. For example, a series of reports by Cartmell and colleagues extended this observation by showing that activation of group II mGluRs by these two structurally distinct and systemically active agonists, LY354740 and LY379268, reversed PCP-induced hyperactivity with no impairment of rotorod performance and minimal effects on amphetamineevoked fine motor movements, which is indicative of the absence of extrapyramidal side effects. Furthermore, the reversal by LY379268 was completely blocked by pretreatment with the mGluR2/3 antagonist LY341495 (143–146). These studies were corroborated by Swanson and colleagues, who also reported that LY379268 exhibited effects comparable to clozapine in blocking PCPinduced effects in control and monoamine-depleted rats (147). It is interesting that, when tested for the ability to reverse enhancement of PCP-induced behaviors following chronic (10 day) PCP-administration (i.e., resulting in PCP sensitization) in rats, LY379268 was found to effectively block PCP-evoked motor behaviors such as ambulations, fine movements, and rearings; however, chronic coadministration of LY379268 along with PCP did not prevent the onset of PCP sensitization (148). Consistent with these studies indicating that mGluR2/3 agonists show efficacy in rodent models of schizophrenia, Nakazato and colleagues also reported analogous findings in studies using two other mGluR2/3 agonists, namely 5-[2-[4-(6-fluoro-1H-indole-3-yl) piperidin-1yl]ethyl]-4-(4-fluorophenyl)thiazole-2-carboxylic acid amide (MGS0008) and (1R, 2S, 5S, 6S)-2-amino-6-fluoro-4-oxobicyclo[3.1.0]hexane-2,6-dicarboxylic acid monohydrate (MGS0028) (149). Previously, it was impossible to determine whether the efficacy of group II agonists in rodent models of schizophrenia was due to activation of mGluR2, mGluR3, or both receptors because none of the available compounds

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discriminate between these two receptor subtypes. To address this question, the effect of the mGluR2/3 agonist LY314582 (a racemate of LY354740) on spontaneous and PCP-induced locomotor activity in mGluR2-knockout mice was tested (150). Whereas LY314582 inhibited both spontaneous and PCP-induced locomotor activity in wild-type control animals, this effect was completely absent in the mGluR2-knockout mice, suggesting that these effects are mediated exclusively by the mGluR2 receptor (150). In the first study to evaluate the cognitive and antipsychotic effects of a group II mGluR agonist in humans, Krystal and colleagues found that pretreatment with the mGluR2/3 agonist LY354740 reduced the cognitive deficits caused by ketamine in healthy human volunteers (151). It was surprising, however, that LY354740 demonstrated no effect on the psychotic symptoms (psychosis, negative symptoms, perceptual changes, and mood changes) induced by ketamine, in light of previous studies with lamotrigine. Lamotrigine is a sodium channel blocker clinically used as antiepileptic drug (AED), which is reported to inhibit glutamate release and has been shown to decrease the neuropsychiatric effects of ketamine (152). It is not known whether this may have been due to an inadequate statistical power in the study or whether this is specific to LY354740 or a general property of this class of group II agonists. As mentioned earlier, this compound has poor oral bioavailabilty in humans, which could have contributed to these results. Further studies will need to be performed, perhaps using higher doses of LY354740 or a different agonist with greater bioavailability, to more conclusively determine the effects of group II agonists in this human model (151). As discussed previously, allosteric potentiators of mGluR2 have recently been introduced as a potential alternative to mGluR2/3 agonists. It is interesting that multiple mGluR2 potentiators have now been shown to reduce induction of hyperlocomotor activity by NMDAR antagonists or amphetamine (42–44,47,153–157). In contrast to mGluR2/3 agonists, the mGluR2 potentiator LY487379 also was effective at reducing amphetamine-induced disruption of PPI (157), and a distinct mGluR2 potentiator, BINA, reduces PCP-induced disruption of PPI (44). These data suggest that selective stimulation of the mGluR2 subtype may have more robust effects on sensorimotor gating than simultaneous activation of both mGluR2 and mGluR3. It is significant that each of these effects of mGluR2 potentiators were blocked by the mGluR2/3 antagonist LY341495 (44,47,157).

5. Drug Abuse Increases in glutamatergic neurotransmission within brain reward circuitries is believed to contribute to the positive reinforcing properties of addictive drugs (for review, see ref. 7). Indeed, previous studies have shown that acute

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or repeated administration of cocaine leads to increased levels of glutamate in the nucleus accumbens, a key region in the brain’s reward circuitry. These studies raise the possibility that activation of presynaptic mGluRs involved in reducing glutamate release in these regions, or blockade of postsynaptic mGluRs involved in mediating or modulating the excitatory response to released glutamate, could counteract the effects of repeated cocaine administration. mGluR5 is highly expressed in the nucleus accumbens, and repeated systemic cocaine administration increases mGluR5 mRNA levels in this region (158). It is interesting that mice with a targeted deletion of the mGluR5 gene do not respond to cocaine with the typical hyperlocomotor response and do not self-administer cocaine (159), suggesting that the reinforcing properties of cocaine are absent in these mutant mice. Furthermore, the mGluR5 antagonist MPEP dose-dependently decreases cocaine self-administration (160,161) and conditioned place preference for cocaine (162) in wild-type mice. This effect may not be restricted to cocaine, because mGluR5 antagonists also reduce the acute locomotor stimulant effects of nicotine (161), self-administration of nicotine (161,163), and drug-seeking behavior in a model of nicotine-triggered relapse to nicotine seeking (161). Evidence suggests that mGluR2/3 agonists also have efficacy in animal models of addiction. For instance, group II mGluR agonists reduce withdrawal symptoms for a number of drugs of abuse (27,164–167) and have efficacy in animal models of alcohol (168), cocaine (169,170), and heroin (171,172) seeking and relapse. Collectively, these preclinical studies suggest that mGluR5 antagonists or group II mGluR agonists may provide promising novel approaches that should be explored for the treatment of drug abuse and addiction.

6. Pain Glutamate is thought to play an important role in the processing and modulation of nociception (for review, see ref. 11). Thus, ligands at mGluRs have been investigated for their potential in treating pain associated with injury and disease. Not only are mGluRs expressed throughout the pain neuraxis, but numerous electrophysiologic and behavioral studies have implicated a role for mGluRs (reviewed in refs. 11 and 173). There is a growing body of literature supporting the role of all three groups of mGluRs in acute, persistent, and neuropathic pain. 6.1. mGluR1 Antagonists Agonists at group I mGluRs induce and potentiate nociceptive behaviors in multiple rodent models of pain (for review, see ref. 11). Prior to the availability of subtype-selective ligands, either antisense or anti–rat mGluR1

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antibodies, used to specifically block the effects of mGluR1 were found to attenuate chronic pain (174,175). In addition, several studies employing moderately selective mGluR1 antagonists have implicated these receptors in acute nociception (176–178). More recently, a number of selective antagonists have been used to show that mGluR1 also has an important role in chronic pain. Chronic pain is commonly modeled by inflammatory pain induced by injection of a noxious compound, such as formalin, into the hind paw of rats. There are two nociceptive response phases observed with the formalin test; the first (early) phase, which represents acute stimulation of nociceptors, occurs immediately following injection and is short lived, whereas the second (late) phase, indicative of central sensitization, has a delayed onset and a longer duration. The two mGluR1 selective antagonists (S)-(+)--amino-a-methylbenzeneacetic acid (LY367385) and 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) both attenuated late-phase nociception in the formalin test (179,180). LY367385 also was effective in other chronic pain models including capsaicin- and carrageenan-induced hyperalgesia and mechanical allodynia, as well as in a neuropathic pain model, the L5/L6 spinal nerve ligation (Chung model) in the rat (179). Other mGluR1-selective antagonists, including a novel series of selective noncompetitive mGluR1 antagonists, the 2,4-dicarboxy-pyrroles (181) and (3aS,6aS)-6a-naphtalen-2-ylmethyl-5methyliden-hexahydro-cyclopenta[c]furan-1-on (BAY-367620) (182), have shown efficacy in neuropathic pain models. 6.2. mGluR5 Antagonists Systemic administration of the mGluR5 antagonist MPEP has antihyperalgesic effects in rat models of inflammatory pain, including Freund complete adjuvant (FCA), which models established inflammatory hyperalgesia, and the carrageenan models, which model the development of inflammatory hyperalgesia and edema. It is important that MPEP did not alter normal responses to noxious mechanical or thermal stimulation, as assessed by paw pressure and tail flick tests, indicating that the protective role of acute pain sensation was not disrupted (183). However, MPEP did not reduce carrageenan-induced edema, which is a symptom normally treated by nonsteroidal antiinflammatory drugs (NSAIDS), and had no effect on mechanical hyperalgesia or tactile allodynia in a rat model of neuropathic pain, partial ligation of the sciatic nerve (183). These results corroborated similar findings by Dogrul and colleagues, who showed that SIB-1757 also reduced tactile allodynia and completely reversed thermal hyperalgesia in the L5/L6 spinal nerve ligation model (184). It is also important to note that, unlike opioid analgesics or NSAIDS, MPEP had no effect on motor performance (rotorod performance), nor did it induce acute gastric erosion (183), suggesting a potential advantage over traditional therapies for certain states of pain.

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Walker and colleagues extended these findings by showing that mGluR5 receptors are expressed on peripheral nociceptive afferents and that peripheral, selective blockade of these receptors can reverse mechanical allodynia in both the FCA and carrageenan models (185). These findings were further supported by a report showing expression of both mGluR1 and mGluR5 on nociceptive sensory afferents and that the group I mGluR antagonists MPEP and CPCCOEt can prevent, as well as reduce, established formalin-induced pain (186). However, unpublished studies suggest that MPEP may have similar antinociceptive actions in mGluR5-knockout mice (173). This raises the possibility that these effects could be due to off-target activity. 6.3. Group II mGluR Agonists Immunolocalization and electrophysiologic studies, as well as an observed upregulation of mGluR2/3 in inflammation, have suggested a role for group II mGluRs in pain (187), but only recently have a number of reports corroborated these studies with behavioral paradigms. For example, intraplantar administration of group II mGluR agonists attenuates both carrageenan- and capsaicininduced hyperalgesia in rats while not altering the response to acute mechanical or thermal noxious stimuli (188). Several studies have lent further support to the idea that peripheral group II mGluRs may be useful therapeutic targets, not only for pain management (189,190), but also for the prevention of inflammatory pain states (190). One possible mechanism by which group II mGluRs may modulate pain sensitization is via negative regulation of tetrodotoxinresistant sodium currents, which are known to be important in nociception and nociceptive sensitization (191). In addition to their role in inflammatory pain, group II mGluRs also have demonstrated effects in models of persistent and neuropathic pain (179,187).

7. Conclusion and Perspectives For a field in which successful efforts in drug discovery were once considered unlikely, if not impossible, the mGluR field has enjoyed dramatic progress in recent years. We have witnessed tremendous advances in the development of selective, direct-acting mGluR agonists, which have given us the opportunity to more thoroughly investigate the biology and function of this important receptor family. In addition, these compounds are beginning to provide direct evidence of efficacy in treatment of CNS disorders in clinical studies, particularly with group II mGluR agonists in the treatment of anxiety. Perhaps even more exciting has been the emergence of an entirely new arsenal of tools with the discovery of allosteric modulators of mGluRs. This new class of molecules, which act through nontraditional binding sites on the mGluRs, has made it possible to identify even more subtype-selective agents, which, in

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and of themselves, will aid us in dissecting out the individual physiologic roles of each mGluR subtype. Moreover, they will allow us to fine tune receptor function by modulating mGluRs in an activity-dependent manner, which may also lead to less receptor desensitization. These advantages over traditional orthosteric agonists may ultimately translate into clinically relevant therapeutic improvements with respect to fewer adverse side effects and less tolerance observed with chronic treatment. Since this chapter was submitted for publication, a highly selective mGluR2/3 agonist, termed LY2140023 was evaluated in a randomized phase 2 clinical trial in which it was found to be efficacious at treating both the positive and negative symptoms of schizophrenic patients without causing prolactin elevation, extrapyramidal side effects, or weight gain. (Patil et al., Nat Med 2007;13:1102-1107). These findings provide compelling evidence that activation of mGluR2/3 may be efficacious in treating schizophrenia.

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Index A Activity-based internalization, 64 Activity-dependent gene, 314 Activity-dependent plasticity, 79 ADHD See Attention deficit hyperactivity disorder Adhesion proteins, 73 Adult synapse, 71, 76, 77 Agonist-Binding Domain (ABD), 249, 251, 263, 266–268, 275 subtype selectivity of, 266 Agonist-gating, 276 Agranular frontal cortex, 53 Alkyl-benzothiadiazide, 304 Allosteric modulators, 138, 405 potentiators, 137, 471, 533, 540, 542, 544, 545 Alternative splice isoforms, 103 Alzheimer disease, 1, 24, 281, 300, 328, 333, 340, 341, 347, 353, 424, 497, 498, 530 Amino acid homology, 387 neurotransmitter, 170 Amino-terminal domain (ATD), 2, 47, 250 splice variants, 390 AMPA receptors, 1–3, 5–10, 12–15, 17–28, 48, 100, 102, 107–109, 125, 128, 133–138, 141, 142, 161–163, 165–166, 168, 170, 172, 182, 189–191, 195, 198, 201, 202, 204–207, 211, 213, 216, 219, 223–226, 228, 251, 262–264, 266–269, 275, 276, 282–286, 300, 305, 306, 319, 330, 515, 517, 520

channel biophysical properties of the, 12, 13 crystallographic resolution of, 102 desensitization, 282 modulators, 300, 313 pharmacology, 22 synaptic insertion, 204, 205 trafficking, 18, 19, 28, 190, 191, 195, 202, 205–207 Ampakine, 299–301, 303–309, 311–317, 319, 320 binding, 303 categories of, 304 potential applications of, 309 selective effects of, 304 three subclasses of, 304 Amygdala, 113, 123, 126, 140, 214, 393, 402, 416, 417, 426, 491, 492, 494, 495, 498, 499, 513, 516, 531, 538, 546 Amyotrophic lateral sclerosis (ALS), 7, 329, 422 Anchoring See Trafficking Antagonism, 115, 221, 328, 337, 341, 343, 345, 347, 349, 353, 467 Antagonist, 339 cocktail of, 117 Anti-excitotoxic therapy, 336 Anti-influenza agent, 339 Antianxiety or antistress activity, 532 See also Anxiety Antibodies, 132 Antidepressant, 24, 314, 405, 498 Antiepileptic drug, 547 Antinociceptive agents, 122 Antipsychotics, 472, 543–545, 547 Anxiety, 423–426, 472, 478, 497, 499, 530–533, 535–538, 550

565

566 Anxiolytic, 405, 425, 426, 470, 472, 478, 498, 499, 502, 531–533, 535–538 Apoptotic-like neuronal cell loss, 352, 353 Artificial cerebrospinal fluid, 107 Artificial polypeptide linker, 273 Ataxia, 21, 159, 164, 166, 167, 170, 171 Atrial natriuretic factor (ANF), 392 Attention deficit hyperactivity disorder, 24, 299, 314, 320, 423 See also Schizophrenia Autophosphorylation, 79, 186–188, 397 Auxiliary subunits, 285

B Baculovirus-infected insect cells, 366 Basal transmission, 19, 25, 181, 189, 191, 198, 221 Basolateral amygdala, 122, 123, 494 BCM theory, 208 BDNF See Brain-derived neurotrophic factor (BDNF) Beclin, 169 Benzodiazepines, 426, 536 Benzothiadiazide, 304 Benzothiazides, 23, 137 Bergmann glia, 14 Biarylpropylsulfonamide, 304 Binding pocket, 102, 163, 251, 264, 266, 268, 269, 276, 277, 367–369, 490 Biochemical pathways, 514 Brain-derived neurotrophic factor (BDNF), 24, 46, 63, 300, 301 Brainstem nuclei, 13 BTB-kelch proteins, 131

C CA1 pyramidal neurons, 25, 53, 105, 114, 119, 198, 201, 406, 516 CA1 region, 515

Index CA3 pyramidal neurons, 109, 111, 114, 117, 128, 129, 133, 214, 221, 222 Calcium, 377, 515 Calcium permeability, 28, 48, 51, 54, 80, 105, 200 Calcium-sensing receptor (CaSR), 364, 368, 391 Calmodulin-dependent kinase, 107 protein phosphatase, 210 Canavalia ensiformis, 137 Carbohydrate chains, 9 Carboxy-terminal domain (CTD), 47, 389, 390 Carboxy terminus, 364, 376, 391, 474 Cardiovascular vasodilator effect, 350 Cargo proteins, 408 Carrageenan models, 549, 550 Cell cytoskeleton, 169 Central nervous system, 7, 14, 79, 104, 109, 123, 128, 129, 179, 181, 201, 205, 214, 218, 224, 226, 300, 329, 403, 406, 411, 419, 428, 494, 495, 515, 530, 531, 537, 550 Cerebellum, 11, 13–14, 20–22, 26, 47, 49, 52, 54, 63, 69, 70, 74–75, 80, 105, 113, 121, 122, 128, 159, 161–162, 163–165, 166–167, 169, 170–171, 200, 202, 205, 214, 347, 389, 393, 394, 397, 402, 408, 411, 414, 418, 421, 423, 424, 491, 492, 494, 495, 498, 513, 514, 518, 519 morphology of, 169 Cerebral cortex, 75, 420, 424, 425 glomeruli, 74 granule cells, 22, 49, 54, 63, 70, 80, 205, 402, 408, 421 ischemia, 7, 8, 15, 341, 421 neocortex, 14 parallel fiber, 121, 214, 495

Index Cerebrovascular disease, 328 Channel activation, 272 Chinese hamster ovary (CHO), 396 Cholecystokinin tetrapeptide, 533 Cleft closure, 270, 272 Clozapine, 543, 546 Coatomer protein complex, 1, 132 Cognition, 415 Cognitive impairment, 423, 543 Coimmunoprecipitation studies, 392 Competitive antagonists, binding of, 269 Computer-aided molecular dynamics, 277 Concentration-response curve, 405, 406, 534 Conditioned lick suppression, 536 Convulsions, 134, 340 Cortical pyramidal neurons, 8 Cortico-accumbens synapses, 121 Corticostriatal synaptic transmission, 535 Crystallographic analysis, 163 C-terminal domain, 262, 376 splice variants, 103 Current-voltage relationship, 12, 105, 125, 193, 195, 226, 228 Cycloleucine, 269 Cyclothiazide, 23, 102, 282, 283, 304 Cysteine-rich domain, 365, 369 Cysteine sulfinic acid (CSA), 422 Cytoplasmic domains, 9, 103, 104, 129, 218 Cytoskeletal proteins, 21 Cytoskeleton, 72, 376 D D2 dopamine receptor (D2R), 543 “D2–M3” strain model, 273 “Deactivation-plus-desensitization” variants, 320 Dementia, 328, 329, 332, 337, 340, 341, 348–350, 353, 424 Dendrite arborization, 74 Dendrite shaft, 66

567 Dentate basket cell interneurons, 223 Dentate gyrus, 62, 123, 128, 214, 218, 221, 223, 307, 308, 393, 415, 475, 476, 491, 492, 494–496, 512, 513, 515 Dephosphorylation, 13, 65, 210, 211, 378, 397, 419, 496 Depolarization, 55, 79, 113, 117, 118, 120–122, 181, 182, 184, 218, 222, 300, 312, 316, 319, 402, 408, 413, 417, 419, 467, 497, 511, 513, 514 Depression, 19, 20, 115, 117, 119, 120, 122, 139, 179, 202, 207, 208, 210, 211, 218, 221–223, 225, 299–301, 314, 348, 477, 494, 497, 498, 510, 513, 518, 530 Desensitization and deactivation, 281, 282 Developmental delay, 423 Diabetic neuropathy, 348, 349 Digenia simplex, 132 Dihydroxyphenylglycine (DHPG), 397, 511 Dimers, 283, 392 Distal synaptic signals, 112 Dizziness, 348, 532 “Dock-and-lock” mechanisms, 272 Dopamine (DA) neurons, 417 Dorsal root ganglia, 105, 122, 134, 491 Down syndrome, 105 Drosophila, 22, 59, 61, 391, 423, 466 Drug abuse, 478, 519, 530, 547, 548 addiction, 426 clinical tolerability, 353 Dynamin, 67 Dysidea herbacea, 134 Dysiherbaine, 134 E “Editing complementary sequence”, 104 Electroencephalographic (EEG), 475 Electron microscopy, 55, 284, 393

568 Electrophysiologic tagging, 193 Endocytosis clathrin-dependent, 19, 20, 65 clathrin-independent, 67, 68 clathrin-mediated, 20, 64–66, 172, 519 Endocytotic motif, 64, 65 Endoplasmic reticulum, 5, 8, 49, 72, 80, 107, 129, 164, 375, 408 Eph family, 73 Epidermal growth factor (EGF), 67 Epilepsy, 105, 127, 133, 134, 142, 281, 329, 341, 422, 428, 469, 478, 497, 498, 530 Estradiol, 400 “ETVA” sequence, 131 Excitatory neurotransmitter, 133, 329, 331, 530 Excitatory post synaptic currents (EPSCs), 12, 13, 19, 21, 52, 109, 113, 117, 131, 165, 166, 186, 216, 228, 303–305, 510 Excitatory post synaptic potentials (EPSPs), 12, 13, 53, 54, 228, 300, 313, 402, 403, 474, 510 Excitatory synaptic transmission, 12, 108, 208, 494, 495, 510 Excitotoxicity, 7, 8, 15, 67, 71, 126, 127, 281, 328–334, 336, 338, 349, 352, 353, 421–424, 468, 498, 540, 541 pathophysiology of, 334 Exocytosis, 58, 62 Expression mechanisms, 190, 514, 517, 519 Extracellular domain (ECD), 490 Extrasynaptic membrane, 58, 62, 63, 66 F Fear conditioning, 416 Fear-potentiated startle, 532, 536, 537 Fenobam, 404–406, 425, 426, 537 Flip and flop receptors, 5, 15, 301, 306

Index Fluorescence resonance energy transfer, 272, 277, 372 Focal adhesion kinase (FAK), 397 Food and Drug Administration, 348, 534 Formalin test, 549 Forskolin, 216 Fouriertransformed infrared (FTIR), 271 Fragile X mental retardation protein (FMRP), 423, 516, 517, 519 Fragile X syndrome, 423 Functional modulation, 138 Fyn kinase, 64 G G protein signaling (RGS), 114, 115, 411, 496 G protein–coupled receptors, 113, 114, 117, 219, 363–365, 387, 409, 410, 466, 490, 496, 530 GABAergic terminals, 122, 477, 490, 519 Gadolinium, 368, 392 Gastrointestinal, 476 Gating, 22, 50, 52, 100–103, 114, 134, 207, 272, 273, 275–279, 281–283, 286, 352, 544, 547 molecular determinants of, 278 Geller-Seifter conflict test, 536, 537 Gene-targeted mice, 126 Genetic manipulation, 350 Genetic polymorphisms, 105 Genetic studies, 25, 80, 139, 171 Glaucoma, 328, 329, 332, 348–350, 353, 425 Glial cells, 7, 105, 389, 394, 395, 398, 419, 420, 477, 491 Globus pallidus, 393, 491, 539 GluR0, 2, 3, 262, 264, 278 GluR1 knockout mice, 25 GluR2 expression, 7, 8, 15, 519 GluR, 2, 46, 159, 160–173, 276 knockouts, 26 Q/R editing mutants, 26 trafficking, 167, 169

Index

569

GluR5 knockouts, 121, 139, 140 receptor antagonists, 136 RNA, editing of, 105 splice isoforms of, 137 GluR6a and GluR6b, 103, 131 GluR7 knockouts, 140 Glutamate arginine-tyrosine, 371 binding, 47, 129, 282, 366, 367, 392, 466, 545 bound confirmation, 48 carboxypeptidases, 468 free bathing solutions, 103 mediated internalization, 393 overexcitation, 478 receptor interacting protein, 20, 112, 213 receptor trafficking, 68 Glutamate/aspartate transporter (GLAST), 419 Glutamatergic neurotransmission, 132 Glutamatergic signaling, 424, 427 Glutamatergic synapses, 52, 71, 73–75, 77, 120, 179, 300, 319, 531 development of, 75 Glycine receptors, 54, 77, 350 Glycosylation, 9, 49, 106 Golgi, 9, 18, 21, 49, 58, 59, 62, 113, 167, 169, 170 Granule cells, 14, 21, 47, 52, 62, 63, 75, 80, 117, 118, 121, 128, 129, 167, 214, 218, 221, 223, 491 GTP-binding protein, 389, 395 GTPase activation, modulation of, 496 GTPase activators, 71, 72, 205, 411 Guanidinium group, 264, 271 Guanylate kinase, 49, 67 Gyrus, 118, 222, 308, 496, 513

Heterologous regulation, 409, 412, 430 Heteromeric subunit diversity, 7 Heteromultimers, 334 Heterosynaptic facilitation, 119 High-affinity glycine-binding, 55 High-frequency stimulation, 114, 118, 123, 184, 185, 217, 221, 223, 225, 317, 418, 511, 512, 535 High-resolution structural analyses, 2 High-throughput screening methods, 480 Hippocampal hemisphere, 120 neurons, 26, 61, 79, 131, 133, 134, 137, 139, 206, 207, 332, 347, 400, 402, 413, 498 slice cultures, 186, 187, 193, 195, 402 synaptic plasticity, 228, 399 Hippocampus dependent spatial memory, 26 neurotransmitter in, 412 HIV-associated dementia, 329, 332, 348, 353 Homer protein family, 407, 409 characteristic of, 376 Homocysteate, 77 Homologous regulation, 430 Homosynaptic or heterosynaptic release, 118 Hotfoot mice, 164, 167 Huntington disease, 127, 329, 332, 352, 425 Hydrolytic editing, 7 Hydrophobic interactions, 365, 392 Hyperalgesia, 416, 476, 549, 550 development of, 416 Hyperphosphorylation, 333, 347 Hyperpolarization, 495, 497 Hypothalamic nuclei, 492 Hypothalamus, 123, 393, 491, 494, 495 Hypoxic–ischemic insults, 333

H

I Immunocytochemistry, 15, 427, 428 Immunoelectron microscopy, 164 Immunoglobulin molecule, 163

Hamilton Anxiety Scale (HAMA), 532 Heptahelical transmembrane, 370 Heterologous cell cultures, 50

570 Immunogold electron microscopy, 15, 160 Immunolabeling analyses, 66 Inhibitory postsynaptic potentials, 119, 306, 403 Inhibitory strychnine-sensitive glycine receptors, 77 Inhibitory synaptic transmission, 519 Interaction partners, 19, 68, 130, 168, 474 Intermediate-conductance channels, 402 Internalization, 63, 64 Interneuron depolarization of, 121 interneuron signaling, 120 somatodendritic receptors, 121 Intracellular C-terminal domain, 262 calcium, 377, 515 protein partners, 263 signaling molecules, 168, 395, 503 trafficking determinants, 103 Intracerebroventricular, 540 Intradomain dynamics, 276 Iodo-willardiine, 267 Ion permeation, molecular determinants of, 279 Ionotropic glutamate receptor (iGluRs), 2, 100, 115, 122, 136, 159–161, 181, 247, 249–251, 262–264, 266, 269, 270, 272, 275–279, 282, 284, 285, 389, 469 agonist-binding site, 263 closure model for, 275 deactivation, 282 desensitization of, 281, 282 surprising feature of, 280 Ischemia, 7, 105, 127, 281, 333, 341, 406, 469 Ischemic insult, 15, 333 penumbra, 352 J Juxtamembrane domain, 103

Index K K+ channel model, 262, 285 KA1 and KA2 knockouts See Knockout Kainate model, 133 Kainate receptor, 23, 24, 99, 100, 102–109, 111–115, 117–142, 160, 162, 167, 219, 220, 251, 276 activation, 107, 113–115, 117–125, 127, 133, 135 agonist, characterization, 135 editing mutants, 141 intracellular trafficking of, 138 localization, polarization of, 141 in network oscillations, 124 neuronal function of, 109 pharmacology, 132 selectivity of philanthotoxin for, 118 structure, 99 in synapse development, 125 trafficking and targeting, 128 Ketamine-induced glutamate release, 546 Kinase activity, 187, 202 Kinesin association, 62 motors, 61 Knockdown approach, 172 Knockout, 25, 26, 69, 80, 81, 127, 140 See also Genetic studies Knockout mice, 8, 25–27, 111, 114, 118, 119, 121, 123, 125–127, 140, 159, 164, 165, 169, 186, 200, 201, 415, 418, 423, 424, 477, 498, 499, 512–514, 536, 538, 547, 550 L Lactate-induced panic-like responses, 532 Leptomeninges, 476 Leucine-isoleucine-valine–binding protein (LIVBP), 2, 391

Index Ligand binding, 3, 9, 134, 170, 181, 182, 268, 271, 272, 277, 365–367, 369, 391, 392, 404 domain, 3, 6, 9, 47, 48, 100, 133, 163, 170–172, 334, 370, 389, 392, 395, 467 Ligand-gated ion channels, 249, 270, 400, 403 Ligand-protein interactions, 271, 277 Lipophilic leak, 345 Locomotor hyperactivity, 314 Locus coeruleus, 491, 494, 538 Long-term depression (LTD), 13, 55, 112, 159, 163, 186, 210, 223, 399, 403, 412, 494, 510 Long-term potentiation (LTP), 13, 53, 179, 180, 207, 223, 228, 299, 300, 334, 341, 403, 412, 494, 510 pharmacologic induction of, 510 “Low-affinity” kainate receptor, 99 “Low-affinity” NMDA open-channel blockers, 345 Low-frequency stimulation (LFS), 511 Lurcher mutation, 102, 162, 170 Lymphocytes, 428 Lysine-arginine-orthinine binding protein (LAOBP), 3 M Macroscopic kainate receptor, 138 Mammalian neuroendocrine system, 77 MAPK/ERK Pathway, 397 “Marble-burying” paradigm, 475 Mass spectroscopy, 132 Mature forebrain, 77 Medial vestibular nuclei (MVN), 514 Memantine, 281, 328, 329, 337, 339, 340, 342, 343, 345–348 effects of, 346, 347 Memorial Sloan-Kettering Cancer Center Computation, 429 Memory encoding, 312 Memory molecule, 187 Merck Index, 339

571 Metabotropic, 13, 46, 64, 72, 77, 79, 113–115, 117, 119, 163, 218, 219, 250, 363, 364, 387, 466, 489, 509 Metabotropic glutamate receptor (mGluR), 72, 79, 163, 218, 250, 363, 364, 387, 466, 489, 509, 530 neurons and nonneuronal cells, 427 receptor pharmacology, 401 mGluR1 antagonists, 548 mGluR2, 221, 365, 368, 378, 514, 530–535, 540, 542, 544, 546–548, 550 allosteric potentiators, 471 mGluR4, 365, 368, 369, 379, 530, 537, 538, 540–542 mGluR5, 365, 370, 371, 374–379, 490, 495, 497, 498, 511–514, 516, 518–520, 530, 531, 535–537, 541, 542, 544, 545, 548–550 antagonists, 535, 549 Microglial neurotoxicity, 498 Micromolar potency, 135, 136, 471, 493 Miniature excitatory postsynaptic current (mEPSC), 517 Mitogen-activated protein kinase, 205, 334, 476, 515 Morphologic analyses, 17, 53, 164 Mossy fiber, 52, 75, 109, 111, 112, 114, 117–119, 121, 123, 125, 129, 131, 133, 139, 142, 200, 214, 216–219, 221–223, 225, 226, 477, 494, 496, 514, 516 control of, 539 depolarization of, 118 depression of, 218 interneuron plasticity, 222 synapse, 109, 111, 112, 119, 123, 125, 129, 131, 133, 139, 142, 214, 216, 217, 221, 222 development of, 125 termination zones, 117 Motor control, 418 Motor movement, 62, 546

572 Motor performance, disruption in, 536 Mouse tail-suspension tests, 475 Multiple sclerosis, 127, 329, 332, 491, 498 N N-glycosylation, 106 N-methyl-d-aspartate (NMDA) receptor, 2, 10, 13, 23, 26, 27, 48, 49, 78, 100, 102, 109, 112, 113, 115, 123, 128, 130, 139, 160, 162, 172, 180, 208–211, 213, 214, 216, 219, 249, 250, 262, 279, 281, 300, 312, 329, 332, 334, 336, 338, 341, 342, 347, 349–353, 400, 401, 403, 410, 413–415, 418, 420, 421, 427, 493, 510, 511, 515, 543 desensitization of, 50, 281 developmental changes in, 52, 75 endocytosis of, 66 glutamate activation of the, 71 hypofunction hypothesis of schizophrenia, 545 trafficking, 81 N-terminal domain (NTD), 100, 171, 250 Negative-feedback mechanism, 478 Neocortex, 14, 313, 314, 393, 491, 492, 494, 498 Nerve growth factor (NGF), 301 Neuregulin receptors, 75 Neurodegeneration, 334, 420, 421, 424, 478, 498 Neuroligin localization, 74 Neurologic and neuropsychiatric disorders, 82, 420, 530 Neuronal activity-regulated pentraxin, 21 Neuronal dysfunction, 7 Neuronal nitric oxide synthase, 49, 71, 408 Neuropathic pain, 328, 353 model, 549 Neurophysiology, 53 Neuroprotection, 421

Index Neuroprotective, 281, 316, 328, 329, 331, 336, 338, 339, 341, 347, 349, 350, 353, 420, 421, 424, 427, 498, 499, 541 agents, 329, 341 efficacy, 347 Neuropsychiatric disorders, 300, 313, 389, 415, 420, 428 Neurotoxicity, 328, 332, 333, 338, 347, 498 Neurotransmission, 77, 99, 115, 119, 126, 132, 164, 328, 332, 337, 338, 343, 353, 403, 415, 416, 426, 429, 469, 535, 541, 542, 544, 547 Neurotransmitter, 1, 100, 118, 170, 221, 328, 329, 331, 365, 399, 412, 414, 419, 420, 467, 530 glutamate, 328 release, modulation of, 412 Neurotrophic factors, 315 Nicotinic acetylcholine, inhibition of, 346 Nigra-dopaminergic neurons, 54 Nitric oxide synthase (NOS), 332 Nitroglycerin, 349, 350 NitroMemantines, 331, 349, 350, 353 Non-rapid eye movement (NREM), 475 Nonequilibrium currents, 134 Nonstationary fluctuation, 10 Nonsteroidal antiinflammatory drugs (NSAIDS), 549 NR1, 46–51, 54–58, 62, 65, 67, 71, 73, 80, 81, 185, 263, 268, 269, 273, 275, 276, 280, 284, 329, 334, 342, 343, 350–352 NR2, 46–51, 54–59, 65, 67, 71, 76, 77, 78, 81, 189, 262, 268, 280, 342, 343, 350–352 NSF-dependent mechanism, 201 Nuclear magnetic resonance (NMR), 277

Index O Obsessive-compulsive disorder, 423, 425, 531 Odor discrimination, 75 Olfactory, 63, 391, 393, 491, 492, 494, 495 Oligodendrocytes, 127 Oligosaccharides, 9, 137 Oocytes, 50 Open-channel block, 337, 339 Optical tagging, 193 Orthosteric agonists, 468 P Pain, 71, 81, 122, 126, 140, 142, 348, 349, 353, 416, 417, 424, 468, 469, 478, 497, 499, 530, 548–550 diabetic neuropathic, 348 Palmitoylation, 9, 106 Panic attacks, 425, 531–533 Panic disorder, treatment of, 533 Parabrachial nucleus, 491 Parkinson disease, 24, 300, 320, 329, 332, 340, 341, 424, 497, 498, 530, 538 Partial trapping, 343, 345 “Pathologically activated therapeutics”, 339 PDZ-binding domain, 47, 57, 59, 64, 74, 79 Peptide moieties, 276 Periplasmatic bacterial amino acid–binding protein, 250 Perirhinal cortex, 112, 514, 515 Perisynaptic metabotropic glutamate receptors, 72 Peritoneal kainate injection, 127 Peroxynitrite, 334 Pertussis toxin (PTX), 396, 492 Pharmacologic agents, 51, 52, 78, 121, 141, 467, 477, 540

573 profiles, 5, 115, 133, 388 therapeutic target, 27 Pharmacology, 22, 77, 132, 170, 403, 467, 493 Phencyclidine (PCP), 471, 543 Phosphofurin acidic cluster sorting protein, 408 Phosphoinositide hydrolysis, 468 Phosphoprotein phosphatase interactions, 378 Phosphorylation, 2, 3, 8, 9, 13, 20, 21, 24, 25, 49, 51, 57, 64, 65, 71, 78–80, 106, 107, 139, 162, 169, 171, 186, 187, 189, 201, 202, 205–207, 210, 211, 213, 263, 373, 376, 378, 397–400, 402, 406, 409–413, 418, 419, 474, 492, 496, 515, 516, 518, 519 intracellular domains, 409 NMDARs, 78–80 serine, 10, 49, 378 tyrosine, 64 Pick disease, 425 Placebo-controlled clinical trial, 348, 425 Plasma membrane, 9, 18, 19, 22, 49, 57–59, 100, 103, 104, 106, 129, 131, 138, 167, 205, 376, 395, 408, 414 Plasticity, 75, 123, 222, 225 Polyamine-modulated receptors, 23 Polymorphism, 105, 106 Pontine nuclei, 491 Positron emission tomography (PET), 311 Postischemic neuronal injury, 406 Postnatal synapse, 76, 77 Postsynaptic, 21, 108, 113, 128, 184, 189 clathrin-coated vesicles, 65 density, 17, 49, 71, 109, 127, 141, 164, 171, 186, 395, 407, 418 kainate receptors, 108 membrane, 54, 72–74, 163, 168, 187, 190, 191, 414, 467, 468, 477 modulation, 113 spine, 62, 72, 169

574 Posttranslational modification, 8, 49, 78, 106, 138, 162, 263, 409, 411 Posttraumatic stress disorder, 425, 531 Prepulse inhibition (PPI), 544 Presynaptic, 115, 117–122, 210, 412, 498 calcium channels, 377, 514 kainate receptors, 109, 113, 115, 117–122, 125, 129, 220 membranes, 394 neurotransmitter release, 414 receptors, 115, 118, 119, 121, 122, 490 release, 190, 223, 225, 412 Protein-engineering, 251 Protein kinase, 8, 51, 72, 79, 80, 106, 107, 162, 168, 171, 186, 188, 205, 263, 376, 378, 395, 397, 398, 399, 418, 474, 492, 494, 496, 510 Protein kinase A, 8, 188, 263, 378, 399, 474, 492 Protein kinase C, 51, 79, 106, 162, 168, 188, 263, 378, 395, 474, 494, 510 Protein phosphatase, suppression of, 188 Protein phosphorylation, 516 Protein-protein interactions, 17, 19, 24, 28, 195, 198, 228, 263, 285, 363, 376, 379, 406 Protein synthesis, 516 Proximal motifs, 65 tyrosine residue, 65 Pseudonitzschia, 134 Psychiatric disorders, 127, 299, 314, 320, 475, 477, 497, 530, 546 Pterygopalatine ganglion, 492 Pull-down assays, 132, 377, 378 Purkinje cell, 13, 14, 20, 26, 70, 113, 121, 128, 162–172, 214, 402, 411, 491, 494, 513, 518 autophagocytic death, 169 innervation of, 166 synapses, 163, 164, 166–172, 214, 513, 518

Index Purkinje neurons, 13, 128, 129, 402 Pyramidal neurons, 8, 14, 53, 54, 109, 111, 114, 115, 119–122, 127, 128, 305 depression of, 120 Pyridothiadiazine, 304 Q Quisqualate receptors, 133 R Rap signaling, 72 Raphe nuclei, 491, 492 Rapid eye movement (REM), 475 Rasmussen syndrome, 9 Rat brain development, 15 Rat forced-swim, 475 Recombinant gene, infection of, 51 Redox-sensitive cysteine, 336 Regulating calcium influx, 377 Regulator of G protein signaling (RGS), 496 Reticular formation, 491, 492 Retrograde messengers, 518 Retrograde signaling mechanism, 218 RNA editing, 10, 12, 104 RNA splice variants, 5 Rotational symmetry, 284, 285 S S-nitrosylation site(s), 336 Scaffolding molecules, 69 Schaffer collateral fiber volleys, 117 synapses, 114, 129, 208, 214 Schizophrenia, 24, 81, 106, 126, 127, 299, 300, 530, 543–545, 546 Second messenger pathways, 395, 518 Sensory brainstem regions, 492 Sensory maps, formation of, 334 Sensory spinal synapses, 513 Shank proteins, 407 Short-term plasticity, 225 Signal cascades, 186

Index Signal-to-noise ratio, 415 Signaling, 395, 409, 411, 419, 467, 476, 492, 496 See also Targetting Silent synapses, 17, 53, 55, 191 Single-channel conductance, 10, 12, 22, 79, 104, 187, 207, 262 Small-angle X-ray scattering (SAXS), 270 Small ubiquitin-related modifier, 379 Solitary tract nucleus, 491, 494 Somatosensory cortex, 515 Somnolence, 532 Spasticity, 340 Spectrometric measurements, 271 Spectroscopic and crystallographic data, 277 Spermine, 107, 138, 195, 198, 225 Spinal cord, 27, 46, 54, 113, 122, 126, 167, 306, 331, 393, 394, 412, 416, 417, 422, 427, 470, 476, 491, 492, 494, 495 Spinal nerve ligation model of neuropathic, 499 Spine morphology, 3, 18, 72 Spinothalamic tract, sensitization of, 499 Stoichiometry, 2, 5, 47, 100, 104, 121, 137, 160, 284, 286 Stratum lucidum interneurons, 120, 223 Stress, 80, 332, 333, 398, 425, 472, 497, 498, 531, 532, 535–538 Stress-induced hyperthermia, 472, 498, 532, 535–537 Stria terminalis, 513 Striatum, 65, 314, 393, 394, 400, 414, 417, 418, 421, 424–426, 469, 494, 498, 512, 513, 518, 531, 535, 538, 539, 541, 546 Subcellular trafficking, 106, 129 Substantia innominata, 491 Substantia nigra, 54, 123, 393, 402, 424, 492, 494, 495, 538, 539 Substituted cysteine accessibility methods (SCAM), 51, 343 Subsynaptic localization, 490

575 Sumoylation cascade, binding of proteins, 379 Sun model, 284 Symmetric heteromer, 8 “Symmetry mismatch”, 285 Synapse maturation, 75 Synaptic adhesion molecules, 74, 172 coincidence detectors, 181 damage, initiation of, 352, 353 kainate receptor, 108, 113, 123, 126, 135, 137, 138 neurotransmission, 338 plasticity, 1, 8, 12, 13, 15, 19–21, 24–26, 53, 74, 109, 123, 159, 163, 171, 179, 181, 186, 201, 210, 211, 214, 226, 228, 300, 319, 397, 399, 402, 403, 413–419, 423, 424, 426, 427, 477, 499, 509–511, 515 investigation of, 228 properties of, 111, 195 transmission, 1, 19, 20, 26, 28, 73, 126, 165, 168, 179, 181, 182, 185, 189, 190, 198, 218, 219, 221, 222, 225, 228, 304, 309, 329, 334, 337, 341, 387, 403, 412–415, 418, 427, 474–476, 494–498, 502, 510, 511, 519, 535, 542 modulation of, 412, 413, 474, 494 Synaptogenesis, 21, 58, 75, 76, 81, 250 early stages of, 76 SynGAP regulation, 73 Syntenin, 131 T Targetting See Trafficking Thalamocortical synapses, 112, 122, 125 Thalamus, 69, 75, 77, 105, 393, 491, 492, 494, 495, 499, 531, 542, 546 Therapeutic target for psychosis, 478 Thermal hyperalgesia, 476, 499, 549

576 Theta-burst pairing protocol, 201 stimulation, 317, 320, 511 Thymocytes, 428 TMD, 251, 364–366, 369–372, 374, 375, 379, 391 Topological structure of TARP proteins, 286 Topology, 2, 47, 100, 160, 391 Trafficking, 14, 24, 28, 54, 55, 112, 128, 129, 132, 138, 166, 193, 211, 263, 376, 493 Trans-Golgi network (TGN), 58 Transgenic knockin mice, 202 Transient ischemia, 105, 421 Transient receptor potential (TRP), 330, 402, 417 Transmembrane AMPA receptor proteins, 21, 72, 205, 285 Transmembrane domain, 5, 21, 58, 65, 251, 273, 278, 286, 334, 364, 375, 391, 404, 490, 493 Traumatic brain injury, 352, 421, 422 Trigeminal motor nucleus, 167 Triheteromeric receptors, 54 Trisynaptic hippocampal network, 309 Tubulovesicular carrier, 58, 59 Tyrosine kinase function, 189 phosphatases, 516, 517 residues, 49, 65

Index U Ultrasonic vocalization, 536 Ultraviolet (UV) absorption, 264 “Uncompetitive” antagonism, 337 Unfolded protein response (UPR), 18 V van der Waals contact, 268, 269 Vascular dementia, 340, 348, 353 Ventral pallidum, 491, 492 Ventral tegmental area, 417, 491, 495, 520 Venus flytrap domain (VFD), 364, 365, 368, 490 Vesicular retrograde retrieval system, 129 Vesicular trafficking, 18, 19 Visinin-like proteins, 132 Vogel conflict drinking test, 499, 536, 537 Voltage dependence, 345 Voltage-gated calcium, 365, 467, 515 Voltage-gated ion channels, 400 X X-ray crystallography, 301, 392 experiments, 264 Xenopus, 50, 80, 106, 137, 162, 347, 389, 395, 411 laevis oocytes, 389