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...

Author: Robert W. Gereau IV

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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 <20 pS predominating (74–76). In patches excised from neurons, native AMPARs typically exhibit a mean single-channel conductance of ∼12 pS (77,78), which represents a weighted average of all the subconductance states (Fig. 4). Nonstationary fluctuation analysis, which allows singlechannel conductance to be estimated from synaptic AMPAR-mediated currents, also indicates that synaptic AMPARs in a variety of neuronal types exhibit a similar mean single-channel conductance of ∼12 pS; similar to that observed in excised patches (79–81). The single-channel properties of AMPARs can be regulated dynamically. Phosphorylation of serine 831 on GluR1 causes the homomeric GluR1 AMPARs to open to the higher-conductance states, producing an increase in the weighted mean single-channel conductance (82). The mean single-channel conductance is also proportional to the concentration of agonist (23,83): Each subunit is thought to bind ligand independently, and as more agonist molecules are bound to the receptor complex, the predominant subconductance state increases. Recent evidence also demonstrates a strong influence on channel properties of the TARP family of proteins, which includes stargazin (84–86). Interaction of the AMPAR complex with TARPs slows AMPAR desensitization and deactivation, increases open channel probability, and increases the proportion of channel openings at the higher subconductance levels. In addition, the AMPAR interaction with TARPs dramatically alters the pharmacology of the receptor such that kainate, which is only a partial agonist at recombinant AMPARs lacking TARP, is a full agonist at AMPARs coexpressed with TARPs. GluR2 is a dominant subunit in determining the biophysical properties of the AMPAR channel (74). Channels containing GluR2 subunits have a linear current–voltage relationship (87) and are impermeable to Ca2+ , whereas those lacking GluR2 are Ca2+ permeable and show inward rectification due to a voltage-dependent block by endogenous polyamines (88–90). In addition, there is evidence that AMPARs lacking edited GluR2 exhibit considerably higher single-channel conductance than their GluR2-containing counterparts (91). The

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Fig. 4. Electrophysiologic properties of native -amino-3-hydroxy-5-methyl-4isoxazolepropionic acid receptors (AMPARs). A. AMPA receptor single-channel records in an outside-out patch excised from a developing cerebellar granule cell. Currents were evoked by glutamate (2 mM) in the presence of cyclothiazide. Dotted lines indicate subconductance levels, the solid line represents zero current, and the second and fourth traces are expanded from the first and third traces, respectively. B. Amplitude histogram for experiment shown in panel A, fitted with three Gaussians to show the main subconductance levels (3, 6, and 9 pS) observed in this patch. C. Current evoked by a 1-ms application of 1 mM glutamate (gray bar) to an outside-out patch excised from a CA3 pyramidal neuron in the presence of D-AP5 (100 μM), voltage-clamped at –70 mV. The superimposed solid line is a double-exponential fit to the decay (fast = 1.3 ms, 82%; slow = 7.9 ms, 18%). D. AMPAR-mediated miniature excitatory postsynaptic current (EPSC) (averaged) recorded from a stellate cell in neonatal mouse barrel cortex (in the presence of 0 mM Ca2+ and 8 mM Sr2+ , 100 μM D-AP5, (−70 mV holding potential), showing that the time courses of synaptic AMPAR-mediated responses are very similar to those evoked by rapid glutamate application to AMPARs in an excised patch. A, B: Reproduced from Smith TC, Howe JR. Concentration-dependent substrate behavior of native AMPA receptors. Nat Neurosci 2000;3(10):992–997. C: Lauri, S. and Isaac, J.T.R. (unpublished). D: Reproduced from Bannister NJ, et al. Developmental changes in AMPA and kainate receptor–mediated quantal transmission at thalamocortical synapses in the barrel cortex. J Neurosci 2005;25(21):5259–5271.

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differences in channel properties of GluR2-containing receptors are due to the presence of arginine at residue 607 as a consequence of the RNA editing of GluR2. This residue is in the reentrant M2 membrane-spanning region that forms the AMPAR ion channel pore (Fig. 1). The additional positive charge introduced into the pore by this editing prevents the passage of divalent Ca2+ cations and prevents the polyamine block (92). The predominance of the edited form of GluR2 in native receptors in the vast majority of postnatal neurons is the reason that the majority of native AMPARs are Ca2+ impermeable. However, Ca2+ -permeable AMPARs lacking GluR2 are found in certain cell types in the brain, for example, subsets of hippocampal interneurons (93). The voltage dependence of the polyamine block of the GluR2-lacking AMPAR also produces a novel form of short-term synaptic plasticity mediated by an activity-dependent unblock of the AMPARs (94–96). 2.2. In Vivo AMPARs mediate the vast majority of fast excitatory synaptic transmission in the mammalian brain. They are expressed in all neuronal types as well as in glia. The major role of AMPARs in mediating excitatory transmission is shown by the profound effect of infusion of an AMPAR antagonist on transmission in the hippocampus in vivo (1). Activation of native AMPARs by agonist causes a rapid opening of channels permeable to Na+ and K+ , with a reversal potential around 0 mV in vivo. At synapses this produces a transient inward current rising in a few hundred microseconds and decaying within a few milliseconds. The kinetics of this AMPAR-mediated excitatory postsynaptic current (EPSC) is primarily a function of the kinetic properties of the AMPAR channels combined with the time course of transmitter release (74,97,98) (Fig. 4). In particular, the time course of decay of the EPSC is primarily mediated by the deactivation properties of the AMPARs, which in turn is influenced by subunit composition, subunit splice variants, and degree of RNA editing. In addition, AMPAR recovery from desensitization also depends on the composition of the receptor complex and can play an important role in limiting the postsynaptic response during high-frequency repetitive activity (97). The time course of the AMPAR-mediated excitatory postsynaptic potential (EPSP), however, is influenced by a number of additional factors such as the passive electrotonic properties of the particular neuronal type in question, the contributions of subthreshold voltage-gated conductances, and the degree of spontaneous synaptic input to the neuron. Therefore, the time course of the EPSP initiated by the AMPAR-mediated EPSC can vary greatly between cell types. The fast kinetics of the AMPAR-mediated EPSC produces EPSPs with rapid kinetics that provide a precise window for coincidence detection of subthreshold input and that can generate action potentials with a high degree

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of precision (99–101). These features of precise timing also depend on the electrotonic properties of neurons. In electrically compact neurons such as subtypes of cortical GABAergic interneurons or relay cells in brainstem nuclei, AMPAR-mediated EPSPs allow for very high precision timing of input and output (102–104). Since precise timing and coincidence detection are thought to be critically important features for information processing by cortical networks (104,105), the rapid kinetics of the AMPAR channel can thus been seen to be vital to the functioning of neural networks. The AMPAR is also a major target for direct modification during the expression of the predominant forms of long-term synaptic plasticity in the brain, NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) and long-term depression (LTD) (4,18,19,106). LTP expression involves an increase in AMPAR function that is mediated at least in part by increased phosphorylation of S831 on GluR1 and the rapid incorporation of GluR1containing receptors at synapses. LTD is expressed by the rapid removal of AMPARs from synapses through a mechanism involving endocytosis and requires dephosphorylation of S845 on GluR1 and GluR2-dependent trafficking. There is a very large body of work investigating such mechanisms, and this is described in appropriate detail in a subsequent chapter. However, of particular relevance here are recent findings that long-term synaptic plasticity can cause a rapid change in the GluR2 subunit composition and as a consequence the biophysical properties of the AMPAR (61,107–109). In addition to their well-established postsynaptic function, AMPARs play other roles in the brain. There is accumulating evidence for presynaptic AMPARs regulating transmission (110,111). The best-characterized presynaptic role for AMPARs is their direct inhibition of -aminobutyric acid (GABA) release from basket cell terminals onto cerebellar Purkinje neurons (112). This is a heterosynaptic presynaptic regulation, with the glutamate being released from climbing fibers onto the same Purkinje neurons, and appears to be mediated by the canonical ionotropic AMPAR mechanism. Recent work also suggests a direct metabotropic AMPAR-mediated presynaptic inhibition at the calyx of Held (113). It has also been known for several years that certain types of nonneuronal cells in the brain can express functional AMPAR ion channels (114–116). These AMPARs can be activated by glutamate released from neurons and may be involved in a diverse range of glial processes. These include modulation of astrocytic glutamate transporter function, generation of intracellular calcium transients that influence glial morphology, release of neuroactive substances, regulation of gene expression, and modulation of the extracellular ionic environment (114,117,118). The finding that certain central nervous system (CNS) precursor cells are known to express AMPARs, which may sense ambient glutamate even before the formation of neuronal synapses,

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suggests that there may also be an important role for AMPARs in very early brain development (114).

3. Expression, Trafﬁcking, and Targeting AMPARs are widely expressed in the CNS both in neurons and in glia, and are also expressed in many peripheral neurons and in several peripheral nonneuronal cell types (114,119). In the mammalian CNS there are region, development-, and cell-specific variations in AMPAR subunit expression that profoundly affect AMPAR function (7,119,120). In adult brain, there is widespread expression of GluR1 and GluR2 but much more restricted expression of GluR3 and GluR4. In forebrain, including hippocampus and cerebral neocortex, the predominantly expressed subunits are GluR1 and GluR2, with low levels of GluR3 and GluR4 (49,57,90,121,122). In contrast, the cerebellum, retina, and thalamic reticular nucleus additionally display substantial expression of GluR4 (30,123). However, there are cell type–specific patterns of expression superimposed on the regional expression profiles. The major neuronal population in the cerebral neocortex and hippocampus— pyramidal cells—primarily express GluR1 and GluR2, resulting in GluR1/2 as the major heteromeric combination in this cell type (46,49). Although GluR2/3 has been hypothesized as the other major heteromer in cortical pyramidal neurons, expression of GluR3 is low in this cell type (∼10% of GluR1 or GluR2 levels) (46,49,57,90), making it unlikely that GluR2/3 heteromers are expressed in any high level in pyramidal neurons. In the hippocampus, neocortex, retina, and cerebellum, there are populations of GABAergic interneurons that lack GluR2 subunit expression and hence have calcium-permeable AMPARs (90,124,125). These calcium-permeable AMPARs confer novel properties on synapses in these cell types (95,108,126–128). GluR4 expression is low in the great majority of cortical neuronal types. However, GluR4 expression is relatively high in certain cell types in cerebellum such as Bergmann glia and granule cells (129,130). In addition, Bergmann glia do not express GluR2 and therefore have predominantly calcium-permeable AMPARs; the activity of these calcium-permeable AMPARs has been shown to be important in controlling the structural relationship between the Bergmann glia and cerebellar Purkinje cells (131). Cerebellar granule cells express high levels of both GluR2 and GluR4, and thus AMPARs in these neurons are very likely GluR2/4 heteromers. This cell type also is a good example of developmental regulation of splicing since there is a progressive switch from flip to flop isoforms of GluR4 mRNA during the first 2 weeks of rat brain development (132). AMPAR subunit expression is differentially regulated during development. AMPAR subunit expression is initiated during embryonic development and

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rises quickly during early postnatal development to levels of mRNA that are reported to be significantly elevated compared to those in adult brains (121). There are region- and subunit-specific differences in the developmental changes observed. In particular, GluR4 is expressed early in development in the forebrain, but declines during the first postnatal week; this period is also associated with an increase in GluR2 forebrain expression. There are also cellspecific variations in expression of the flip and flop splice isoforms that are developmentally regulated (26,121). Expression of AMPAR subunits can also be altered acutely by pathologic events such as cerebral ischemia (133,134) or drug administration (135) and by tetanic stimulation of afferents in the hippocampus (136). The cellular processes that control AMPAR gene expression are not fully understood. GluR2 is the best-studied subunit because its expression is critical in determining the biophysical properties of AMPARs. Transcription of GluR2 is under control of several independent initiation sites in the promoter that are not individually essential (52). Expression is strongly biased toward neurons due to silencer elements in the GluR2 promoter that are under control of REST (a multi–zinc finger repressor) (137). Furthermore, REST expression triggered by ischemic insult has been implicated in the downregulation of GluR2 expression, which then leads to an increase in calcium-permeable AMPARs that mediate the excitotoxicity in the hippocampus (54). Recent data also suggest that GluR2 mRNA translation can be suppressed by untranslated leader sequences that may or may not be present in the mRNA molecule, depending on which site was used for initiation of transcription (50). In relation to synaptic plasticity, there has been great interest in the idea that expression of AMPARs can be rapidly and locally regulated in dendrites. This concept arose from the observation of polyribosomes in the vicinity of synapses (138), and it has been shown that new AMPAR protein, GluR1 and GluR2 subunits in particular, can be synthesized in neuronal dendrites (58). AMPAR localization at the subcellular level has been intensely studied using immunocytochemistry, biochemistry, and immunogold electron microscopy (139–141). A substantial proportion of the AMPARs are on the surface of the cell at any one time. Since the major role of AMPARs is to mediate synaptic transmission, it is not surprising to find that the neuronal surface receptors are not homogeneously distributed but have a tendency to cluster at postsynaptic sites (Fig. 5) (141–144). This clustering is likely to be ultimately mediated by protein–protein interactions that act to link AMPARs to scaffolds in the postsynaptic density (PSD). Attempts have been made to measure the extent of the clustering by using functional and morphologic methods (140,145–150). The results indicate that the number of AMPARs correlates strongly with the size of the postsynaptic density, whereas the number of NMDARs is independent of synapse size (147,150–153). These studies suggest that the

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Fig. 5. Native -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) distribution in neurons. A. Immunocytochemical localization of surface (upper left) and total (upper right) GluR1 in a cultured hippocampal neuron using a subunit-specific antibody directed to the extracellular N-terminal (the lower panel shows the superimposed image). This demonstrates that some spines lack surface GluR1 but contain intracellular GluR1. B. Electron micrographs of sodium dodecyl sulfate–digested freeze-fracture replicas from neonatal rat cerebellum labeled with a pan-AMPAR antibody (GluR1–4) and a secondary conjugated to 5-nm gold particles. Left: The extent of the postsynaptic density (PSD) is highlighted in gray, and extrasynaptic AMPARs are indicated by arrows. Right: Higher-power image of the PSD showing extensive labeling. C. Immunogold electron micrograph of juvenile hippocampal CA1 stratum radiatum labeled with GluR2/3 antibody (b, presynaptic bouton; s, spine), showing high-density AMPAR labeling of PSD (large closed arrows)

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density of AMPARs within the PSD can be >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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256. O’Neill MJ, et al. AMPA receptor potentiators for the treatment of CNS disorders. Curr Drug Targets CNS Neurol Disord 2004;3(3): 181–194. 257. Alt A, et al. A role for AMPA receptors in mood disorders. Biochem Pharmacol 2006;71(9):1273–1288; Epub 2006 January 24. 258. Banke TG, et al. Control of GluR1 AMPA receptor function by cAMP-dependent protein kinase. J Neurosci 2000;20(1):89–102. 259. Oh MC, Derkach VA. Dominant role of the GluR2 subunit in regulation of AMPA receptors by CaMKII. Nat Neurosci 2005;8:853–854. 260. Lee HK, et al. Phosphorylation of the AMPA receptor GluR1 subunit is required for synaptic plasticity and retention of spatial memory. Cell 2003;112(5): 631–643. 261. Matsuda S, Mikawa S, H. Hirai. Phosphorylation of serine-880 in GluR2 by protein kinase C prevents its C terminus from binding with glutamate receptor– interacting protein. J Neurochem 1999;73(4):1765–1768. 262. Fu J, deSouza S, Ziff EB. Intracellular membrane targeting and suppression of Ser880 phosphorylation of glutamate receptor 2 by the linker I-set II domain of AMPA receptor–binding protein. J Neurosci 2003;23(20):7592–7601. 263. Hayashi T, Huganir RL. Tyrosine phosphorylation and regulation of the AMPA receptor by SRC family tyrosine kinases. J Neurosci 2004;24(27): 6152–6160. 264. Gomes AR, et al. Metabotropic glutamate and dopamine receptors co-regulate AMPA receptor activity through PKA in cultured chick retinal neurones: effect on GluR4 phosphorylation and surface expression. J Neurochem 2004;90(3): 673–682. 265. Carvalho AL, et al. Phosphorylation of GluR4 AMPA-type glutamate receptor subunit by protein kinase C in cultured retina amacrine neurons. Eur J Neurosci 2002;15(3):465–474. 266. Correia SS, et al. Protein kinase C gamma associates directly with the GluR4 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit. Effect on receptor phosphorylation. J Biol Chem 2003;278(8):6307–6313. 267. Zamanillo D, et al. Importance of AMPA receptors for hippocampal synaptic plasticity but not for spatial learning. Science 1999;284(5421):1805–1811. 268. Jensen V, et al. A juvenile form of postsynaptic hippocampal long-term potentiation in mice deficient for the AMPA receptor subunit GluR-A. J Physiol 2003;553(Pt 3):843–856. 269. Schmitt WB, et al. The role of hippocampal glutamate receptor-A–dependent synaptic plasticity in conditional learning: the importance of spatiotemporal discontiguity. J Neurosci 2004;24(33):7277–7282. 270. Schmitt WB, et al. Spatial reference memory in GluR-A–deficient mice using a novel hippocampal-dependent paddling pool escape task. Hippocampus 2004;14(2):216–223. 271. Reisel D, et al. Spatial memory dissociations in mice lacking GluR1. Nat Neurosci 2002;5(9):868–873. 272. Schmitt WB, et al. Restoration of spatial working memory by genetic rescue of GluR-A–deficient mice. Nat Neurosci 2005;8(3):270–272; Epub 2005 February 20.

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273. Mack V, et al. Conditional restoration of hippocampal synaptic potentiation in GluR-A–deficient mice. Science 2001;292(5526):2501–2504. 274. Mead AN, Stephens DN. Selective disruption of stimulus-reward learning in glutamate receptor gria1 knock-out mice. J Neurosci 2003;23(3):1041–1048. 275. Jia Z, et al. Enhanced LTP in mice deficient in the AMPA receptor GluR2. Neuron 1996;17(5):945–956. 276. Meng Y, Zhang Y, Jia Z. Synaptic transmission and plasticity in the absence of AMPA glutamate receptor GluR2 and GluR3. Neuron 2003;39(1):163–176. 277. Shimshek DR, et al. Forebrain-specific glutamate receptor B deletion impairs spatial memory but not hippocampal field long-term potentiation. J Neurosci 2006;26(33):8428–8440. 278. Petralia RS, et al. Loss of GLUR2 alpha-amino-3-hydroxy-5-methyl-4isoxazoleproprionic acid receptor subunit differentially affects remaining synaptic glutamate receptors in cerebellum and cochlear nuclei. Eur J Neurosci 2004;19(8):2017–2029. 279. Yan J, et al. Place-cell impairment in glutamate receptor 2 mutant mice. J Neurosci 2002;22(3):RC204. 280. Gerlai R, et al. Multiple behavioral anomalies in GluR2 mutant mice exhibiting enhanced LTP. Behav Brain Res 1998;95(1):37–45. 281. Mead AN, et al. AMPA receptor GluR2, but not GluR1, subunit deletion impairs emotional response conditioning in mice. Behav Neurosci 2006;120(2):241–248. 282. Shimshek DR, et al. Impaired reproductive behavior by lack of GluR-B containing AMPA receptors but not of NMDA receptors in hypothalamic and septal neurons. Mol Endocrinol 2006;20(1):219–231. 283. Sanchis-Segura C, et al. Involvement of the AMPA receptor GluR-C subunit in alcohol-seeking behavior and relapse. J Neurosci 2006;26(4):1231–1238. 284. Hartmann B, et al. The AMPA receptor subunits GluR-A and GluR-B reciprocally modulate spinal synaptic plasticity and inflammatory pain. Neuron 2004;44(4):637–650. 285. Feldmeyer D, et al. Neurological dysfunctions in mice expressing different levels of the Q/R site-unedited AMPAR subunit GluR-B. Nat Neurosci 1999;2(1): 57–64. 286. Higuchi M, et al. Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2. Nature 2000;406(6791): 78–81.

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 <2 hours (216,217). Thus, in the CA1 region of the hippocampus at P2, many PSDs look relatively mature and label for many of the proteins found in the adult (63). Even the spatial arrangements of these proteins is similar to that found in the adult (Fig. 4, Table 1). For example, both Shank and Homer antibodies produce distinctive clusters of labeling below the PSD (in addition to some labeling within the PSD). These clusters look similar in synapses at P2, P10, and P35; presumably this preferential position for Shank and Homer below the density reflects their positions in the chains of proteins extending from NMDARs/MAGUKs/GKAP to the deeper Shank and Homer (Fig. 4, Table 1) and from there to other proteins in internal stores and on the perisynaptic membrane surrounding the synapse. Some distinctive differences are present, however, and these seem to relate mainly to the developmental switch in NMDARs from NR2B-containing to NR2A-containing forms (63). Thus, a change of major MAGUK at the synapse from SAP102 to PSD-95 parallels this change from NR2B to NR2A (63,64,218). GKAP, the central molecule that links the NMDAR/MAGUK complex to other chains of proteins, as discussed in Section 4.2, has two forms, 130 and 95 kDa. At P2, the major GKAP is the higher-molecularweight form, whereas both forms are common in the adult (63,208,218). So the same complexes of many proteins appear to be operating at glutamatergic synapses in early postnatal and adult brains. The key difference here is that the early postnatal glutamatergic synapse (of the forebrain at least) contains a unique set of NMDARs and their associated proteins—mainly NR1/NR2B NMDARs, which may be bound to SAP102, which may link specifically to the heavy form of GKAP (63,208,218). In contrast, in the adult synapse, NR2A is the most prevalent NR2 form, although NR2B is still found there. Similarly, PSD-95 is the prevalent MAGUK even though SAP102 remains. Studies in visual neurons of NR2A KO mice show that during development, the MAGUK PSD-95 does not seem to form an effective scaffold

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for the NR1/NR2B receptors that persist in the adult (based on a selective loss of spontaneous NMDAR currents in NR2A KO mice (112)). This is consistent with the model of early synaptic NR2B-containing NMDARs bound to the MAGUK SAP102 being replaced normally by NR2A-containing NMDARs that are bound to PSD-95 in the center of the synapse. Other MAGUKs, PSD-93 and SAP97, also increase with development like PSD-95 (64,219). In addition, AMPARs become more common at synapses with maturation (65), and the adult synapse has abundant CaMKII, which is rare or absent in the early postnatal synapse (although another form of CaMKII may be present at early ages, as discussed in ref. 63). This increase in CaMKII with maturation also may be related to the switch from abundant NR2B-containing NMDARs to abundant NR2A-containing ones. NR2B-containing NMDARs bind CaMKII with high affinity, whereas those containing NR2A bind CaMKII with low affinity, and switching to the latter NMDARs results in a substantial reduction in LTP; this may lead to reduced plasticity in the glutamatergic synapses of the mature forebrain (220).

6. Pharmacology The common specific agonist for NMDARs is NMDA; it is not as potent an agonist for NMDARs as glutamate, but it shows good selectivity over other glutamate receptors including non-NMDA ionotropic and metabotropic types, probably due to the special structure of the glutamate binding site of the NMDAR as described in Section 1.2. NMDA does not show much selectivity among NMDARs, but another agonist, homoquinolinate, which is a derivate of the endogenous agonist quinolinate, may show some specificity among NR2 subunits, although there is some evidence against this (221). In addition to glutamate and quinolinate, some other endogenous agonists include l-aspartate and sulfur-containing amino acids such as homocysteate (1,222) as well as NMDA, which has been described chiefly in the mammalian neuroendocrine system (223). Homocysteate may be released from glia following glutamate activation of glial glutamate receptors and may specifically activate postsynaptic NMDARs to modulate neurotransmission (224); in vivo studies in the rat thalamus indicate that it is released following stimulation of somatosensory afferents (225). There also are a number of NMDAR agonists that act on the glycine-binding site. Endogenous d-serine is a good candidate for the major agonist for this site because it is up to three times more potent an agonist at this site, it has extracellular levels comparable to or even exceeding that of glycine, and it is specific for NMDARs because it does not activate the inhibitory strychnine-sensitive glycine receptors (226). Recent studies in the hypothalamic supraoptic nucleus confirm that the endogenous ligand for the glycine site of NMDARs is d-serine and not glycine (even the strychnine-sensitive glycine receptors in this region may be activated by taurine instead of glycine (227)).

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Most studies indicate that d-serine is secreted from glia following glutamate stimulation; this release occurs preferentially at the synapse and regulates NMDAR-mediated neurotransmission. However, there also is evidence for dserine release from neurons (228). Finally, in addition to specific agonists that act on either the glutamate- or the glycine-binding sites of NMDARs, at least one compound acts on both sites. 1-aminocyclopropanecarboxylic acid (ACPC) acts as a high-affinity full agonist at the glycine site and a low-affinity competitive antagonist at the glutamate site (229). Certain pharmacologic agents can be used to distinguish among NMDARs containing the four different NR2 subunits (5,222). However, most common antagonists and channel blockers show only minor selectivity among the four types. The most important exceptions are phenylethanolamines like ifenprodil and haloperidol, and some ifenprodil derivatives including Ro 25-6981 and CP-101,606 show an even higher preference for NR1/NR2B than ifenprodil itself (47,119). These drugs selectively suppress the response of NR1/NR2B receptors with relatively little effect on receptors containing the three other NR2 subunits. They act as a noncompetitive inhibitor by enhancing the sensitivity of the proton sensor of NR1/NR2B (5,230). NR1/NR2A receptors can be identified using the zinc chelator TPEN, which removes the tonic inhibition caused by low levels of zinc. It is interesting that in the triheteromeric receptor NR1/NR2A/NR2B, the NR2A retains its selective high affinity for zinc and the NR2B retains its selective high affinity for ifenprodil, yet either ligand produces only partial inhibition (28). Maximal inhibition is obtained only with the binding of both ligands to the receptor. Similarly, NR2A in NR1/NR2A/NR2C shows high sensitivity for zinc even though the efficacy of this zinc inhibition is much reduced compared to that of NR1/NR2A receptors. The best NR1/NR2Aselective antagonists may be the related molecules (1RS,1‘S)-PEAQX and NVP-AAM077 (117,222), although there is some evidence that the latter is not specific under certain conditions (120,231,232). Other NR2-type selective drugs include conantokin-G, which acts as a highly selective competitive antagonist for NR2B-containing NMDARs, and phenanthryl piperazinyl dicarboxylic acid (PPDA), which may preferentially block NR2C- and NR2Dcontaining NMDARs.

7. Modulation NMDARs can be modulated by posttranslational modifications such as ubiquitinization or S-nitrosylation (see Sections 1.4 and 3) or via phosphorylation by a variety of kinases. 7.1. Phosphorylation of NMDARs: CaMKII NMDA receptor function is regulated by kinases that phosphorylate NMDARs. Selectivity in sites for phosphorylation often entails a physical

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attachment of the kinase to the substrate protein, in this case either by direct binding to the NMDAR or by binding to a protein that links the kinase to the NMDAR. CaMKII can associate with NR1, NR2A, and NR2B (including two binding sites in NR2B (164)). Binding of CaMKII to NR2A and NR2B, however, does not involve direct binding of CaMKII to its major phosphorylation site on the subunit, that is, serine 1289 for NR2A (233) and serine 1303 for NR2B (234). CaMKII associates with NMDARs after autophosphorylation of CaMKII due to calcium entry from activated NMDARs and induction of LTP. This association between CaMKII and NMDARs may bring CaMKII into close proximity with AMPARs (163,235,236). The subsequent phosphorylation of AMPARs causes synapse potentiation by inducing synaptic insertion and increasing single-channel conductance of AMPARs (234,235). However, binding of CaMKII to NR2B does not require autophosphorylation of threonine 286 on the CaMKII (163). Instead, following stimulation by calcium and calmodulin, CaMKII can bind to NR2B; this interaction with NR2B puts CaMKII into an autonomous, calmodulin-trapping state that cannot be reversed by phosphatases and suppresses inhibitory autophosphorylation of T305/306. This association prevents the dissociation of CaMKII from the synapse. This association also may induce autophosphorylation of neighboring CaMKII molecules. CaMKII can form stable complexes with NR1 and NR2B but not with NR2A, and stimulation of NMDARs can increase this association (234). The high affinity of NR2B-containing NMDARs for CaMKII may be crucial for activity-dependent plasticity (220,237). CaMKII and PSD-95 also have been shown to compete for binding to NR2A (in a PSD preparation from the hippocampus (233)). In addition, CaMKII regulates the casein kinase 2 phosphorylation of the serine (1480) in the C-terminal PDZ-binding domain of NR2B, controlling the binding of NR2B to SAP102 or PSD-95 at the synapse (131); see Section 3.2.5. In addition, in LTPpotentiated hippocampal slices, both CaMKII-dependent activity and CaMKII association with NR2A/B increase, with a concomitant decrease in association between PSD-95 and NR2A/B. The association of CaMKII with NMDARs also can be influenced by protein kinase C (PKC) activation of NMDARs. PKC-dependent phosphorylation (due to stimulation with phorbol ester or the metabotropic glutamate receptor-specific agonist t-ACPD) of NR2A at serine 1416 inhibits CaMKII binding, thus promoting the dissociation of the CaMKII-NR2A complex (238). Furthermore, phorbol ester activation of PKC can induce translocation of CaMKII to synapses in cultured hippocampal neurons (239). Indeed, immunogold studies show that CaMKII in the PSD increases fivefold following depolarization (240); also see discussion in ref. 164. PKC activation also can induce rapid dispersal of NMDARs from the synapse to the extrasynaptic membrane, thus perhaps downregulating synaptic NMDARs (239).

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7.2. Phosphorylation of NMDARs: PTKs The Src protein tyrosine kinase (PTK) family includes five members in the central nervous system: Src, Fyn, Lyn, Lck, and Yes (241). Tyrosine phosphorylation of NR2A by Fyn is promoted by PSD-95, perhaps because PSD-95 acts as a physical intermediate to bring Fyn close to NR2A (133,241). The role of Fyn in phosphorylation of NR2A and NR2B was discussed in detail in Section 3. Other PTKs, including Src, Yes, and Lyn, associate with PSD-95. PSD-95 is required for the Src-mediated potentiation of NR1/NR2A receptor current in Xenopus oocytes (242). In addition, other Src PTKs as well as Fyn may be important for mediating the action of ephrins and their receptors, the Eph tyrosine kinases (see Sections 3.2.2 and 4.3); these are involved in establishing axon-dendrite connections during development (243). LTP of synaptic transmission at Schaffer collateral-CA1 synapses in the hippocampus requires upregulation of NMDAR activity by Src. To accomplish this, Src must be anchored to the NMDAR-containing complex by an adaptor protein, NADH dehydrogenase subunit 2 (ND2), which otherwise acts as a part of a mitochondrial protein complex (244). Fyn is linked to NR2B via an inhibitory scaffolding protein, RACK1. RACK1 can inhibit Fyn phosphorylation of NR2B and thus decrease NMDAR-mediated currents in the CA1 region of the hippocampus in vitro (245). 7.3. Phosphorylation of NMDARs: PKAs and PKCs Cyclic AMP-dependent protein kinases (PKAs), which can increase NMDAR activity, can phosphorylate NR1, NR2A, and NR2B and can induce synaptic targeting of NMDARs (246). PKAs can overcome constitutive type 1 protein phosphatase (PP1) activity, resulting in rapid enhancement of NMDAR currents (247). This depends on a selective anchoring of PKA to NMDARs via yotiao, a protein that binds to the C1 exon cassette, which is found in the C-terminus of some NR1 splice variants, as discussed in Section 3.1. PKAs also regulate the calcium permeability of NMDARs and directly modulate the induction of NMDAR-dependent LTP in Schaffer collateral-CA1 synapses in the hippocampus (4). PKCs already have been mentioned in relation to CaMKII function, and have various influences on glutamate receptor plasticity. PKAs and PKCs can work together, as in the endoplasmic reticulum, where they suppress the retention of NMDARs (74) (see Section 3.2.1).

8. Genetic Studies (Knockout or Other Studies) Both NR2A and NR2C KO mice show a reduction in NMDAR-mediated components of EPSCs in mature cerebellar granule cells, where these subunits are normally prevalent, yet these mice have normal motor coordination (248). In contrast, KO mice that lack both NR2A and NR2C show almost a complete

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loss of spontaneous and evoked EPSCs in immature granule cells and exhibit problems in motor coordination (248). Knockout of NR2D produces mice with lower sensitivity to stress and altered monoaminergic neuronal function (249), and overexpression of NR2D produces prominent impairment of NMDAdependent LTP (250). The most essential subunits must be NR1 and NR2B, based on transgenic mouse studies (reviewed in refs. 6 and 251). Knockout of NR1 leads to death shortly after birth. Although neuroanatomy is generally normal, some of the sensory input connections are disturbed. Because NR1 appears to be required for all functional NMDARs, NMDARs must be essential for nervous system function after birth, although they are not required for the general pattern of neural development. Mice with only a partial KO of NR1, expressing about 5% of normal levels, survive to adulthood but display behavioral abnormalities resembling schizophrenia, supporting earlier studies showing that NMDAR-noncompetitive antagonists produce symptoms of schizophrenia (252). Of the four NR2 subunits, only knockout of NR2B is fatal; like NR1, it is fatal at birth (these mice lack the suckling response), indicating the importance of the NR1/NR2B NMDARs in the development of brain function. Overexpression of NR2B enhances learning and memory (253) and affects behavioral responses to inflammatory pain (254). it is interesting that mutant mice that express NR2 subunit A, B, or C with a truncated intracellular C-terminal domain show phenotypes that are similar to those of mutant mice missing that entire subunit (255). Thus, NR2BC/C mice die perinatally, and NR2AC/C and NR2CC/C mice generally show deficits resembling those described for NR2A and NR2C KOs, illustrating the importance of C-terminal interactions in NMDAR function, as noted throughout this chapter. NR3A KO mice appear normal but show enhanced NMDA responses (in vitro; P5–8) and an increased density of dendritic spines in the cerebral cortex that is most prominent during postnatal ages; this is consistent with findings showing that NR3A modulates NMDAR activity during postnatal development (256).

9. Future Directions Questions abound concerning all steps in NMDARs trafficking and development, as is evident throughout this review: (1) During synaptogenesis, what is necessary and sufficient to form a functional NMDAR synapse? (2) What regulates the assembly of NMDAR subunits into functional receptors in the RER? What controls their exit from the ER? Does this occur in dendrites? (3) What kinds of carrier structures are used for trafficking of nascent NMDARs from the TGN to the synapse? Are there intermediate steps within endosomes? Do carriers vary with the kind of NMDAR and between NMDARs and other glutamate receptors? (4) How are NMDARs regulated

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at the synapse, and how does this differ for different kinds (e.g., NR2A- vs. NR2B- vs. NR2A/NR2B-containing NMDARs)? (5) What is the significance of the extrasynaptic NMDARs? (6) What are the roles of PDZ proteins and other associated proteins in the trafficking of NMDARs and the assembly of NMDAR-containing synapses? (7) What are the roles of NMDARs in various neurologic and psychiatric disorders? NMDAR research likely will produce more questions than answers over the next few years, and many new discoveries are expected.

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3 Kainate Receptors Anis Contractor and Geoffrey T. Swanson

Summary Kainate receptors are glutamate-gated ion channels whose functional roles in the brain have been only poorly understood until recently. A picture has developed over the last decade of kainate receptors as subtle actors in neurotransmission; they modulate excitatory and inhibitory transmission and neuronal excitability and generate small but prolonged depolarizations at a subset of postsynaptic sites. This chapter reviews a variety of aspects of kainate receptor function, including their structure, biophysical function, and activities in (and out) of synapses in the mammalian brain. Key Words: Glutamate; ligand-gated ion channel; excitatory synaptic transmission; synaptic plasticity; mossy ﬁber; RNA editing.

1. Kainate Receptor Structure 1.1. Genes Kainate receptors are formed from combinations of five distinct gene products originally named GluR5, GluR6, GluR7, KA1, and KA2. All five kainate receptor subunits were cloned within a few intensive years of discovery in the early 1990s primarily by the research groups of Steve Heinemann and Peter Seeburg (1–7). The nomenclature of the KA1 and KA2 subunits diverged because these subunits exhibited higher affinity for [3 H]kainate (KD values of 5–15 nM) and showed much greater primary sequence identity to each other (68%) than to the “low-affinity” kainate receptor subunits (∼42%). The amino acid sequences of the GluR5, GluR6, and GluR7 subunits are highly From: The Receptors: The Glutamate Receptors Edited by: R. W. Gereau and G. T. Swanson © Humana Press, Totowa, NJ

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homologous (75%–80% identity), with the bulk of intersubunit variability occurring in the amino- and carboxyl-terminal domains. Thus, the kainate receptor gene family is composed in actuality of two subfamilies of proteins. Genes encoding kainate receptors are denoted as GRIK1 to GRIK5 (short for “glutamate receptor, ionotropic, kainate” subtype) in sequential order of cloning of the cDNA. The subunit genes each contain multiple exons (e.g., 17 in the human GluR6 gene (8)) and range in size from ∼67 (KA2) to ∼670 (GluR6) (8) kilobases on five distinct human chromosomes. Alternate nomenclatures for the receptors remain in limited use, including most recently a variant recommended by the International Union of Pharmacologists consisting of GLUK5 , GLUK6 , GLUK7 , GLUK1 , and GLUK2 . However, throughout this chapter we will refer to the subunits by their original and most widely used names. 1.2. Topology and Stoichiometry The topology of kainate receptor subunits in the plasma membrane is identical to that of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartate (NMDA) receptor subunits. After a period of uncertainty and conflicting results following the cloning of the receptor subunit cDNAs, domain mapping and a predicted structural similarity with K+ channels firmly established the presence of an extracellular N-terminal domain, three membrane-spanning domains (M1, M3, and M4), a re-entrant P-loop (M2), and cytoplasmic C-terminal tail (Fig. 1) (9–12). A greater degree of uncertainty existed with respect to the precise subunit stoichiometry of ionotropic glutamate receptors (iGluRs), with a pentamer being the favored model in early years to align iGluRs with the stoichiometry of other ionotropic neurotransmitter receptors such as the nicotinic acetylcholine receptor. Evidence for a tetrameric arrangement steadily accumulated and included the growing recognition that iGluRs were, in fact, more akin structurally to K+ channels than to nicotinic or -aminobutyric acid (GABA) receptors (13) and a seminal biophysical study that explored the relationship between subunit occupancy by agonist and channel gating (14). The preponderance of evidence now supports a model of iGluRs as tetramers (15). The large N-terminal domains (NTDs) of iGluR subunits are distantly related to bacterial periplasmic binding proteins and consequently are predicted to be bi-lobate in structure (16), although this remains speculative because no N-termini have been crystallized. Like AMPA receptors, the ∼400-amino acid NTDs of kainate receptor subunits are critical for restricting multimeric assembly only to other kainate receptor subunits through the specificity of intersubunit interactions (17,18). The extracellular ligand-binding domain (LBD) of kainate receptor subunits is a bi-lobate structure, with the lobes denoted D1 and D2, formed by two

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Fig. 1. A. Illustration of the domain structure of a representative kainate receptor subunit. The N-terminal domain (NTD) is involved in the assembly of tetrameric receptors. The ligand-binding domain (LBD) is extracellular and a bilobate structure composed of two distinct domains (D1 and D2), each of which is formed from amino acids from S1, the region of the protein preceding the first membrane domain (M1), and S2, the region between M3 and M4 (see text for more detail). B. Illustration of the splice isoforms of GluR5, GluR6, and GluR7 kainate receptor subunits. The cartoons are roughly to scale relative to the primary amino acid sequence. The sites of RNA editing of the membrane domains of GluR5 and GluR6 are shown as well.

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discontinuous regions of the protein—the S1 domain preceding M1 and the S2 domain between M3 and M4 (Fig. 1A) (19). Soluble forms of the GluR5 and GluR6 LBDs were crystallized in complex with a number of agonists and antagonists (20–23), revealing a secondary structure strikingly similar to that of AMPA and NMDA receptor subunits (24,25). The correlation of structure with function following crystallographic resolution of AMPA and kainate receptor LBDs facilitated an unprecedented change in our understanding of the molecular nature of agonism and channel gating. Processes that previously were defined empirically now had mechanistic, testable models. For example, mutagenesis studies demonstrated that the diverse pharmacology exhibited by kainate receptor subunits was generated by relatively few, but important, amino acid differences in the S1-S2 domain (26,27). These critical residues are found both in the microenvironment of the “binding pocket” (28), where direct interactions with ligands occur, as well as at sites of interaction between the S1 and S2 domains. That is, several amino acid pairs in the S1 and S2 domains form interdomain salt bridges and hydrogen bonds, and these contacts have a demonstrable effect on a number of channel properties, including deactivation rates and apparent affinity (29,30). The stability of these interdomain contacts likely underlies the differences between AMPA and kainate receptors in their rate of recovery from desensitization (30); AMPA receptors recover much faster and therefore are more suited for higher-frequency signaling. The S1 and S2 domains also contain residues that form the interface between subunits. The strength of intersubunit interactions is critical to the molecular mechanisms underlying desensitization of receptors in response to the prolonged agonist binding. For AMPA receptors, the strength of this interaction can be greatly enhanced by mutation of critical residues, such as leucine 507 in GluR3 to a tyrosine (31), or by application of allosteric modulators like cyclothiazide, which bind to the intersubunit interface (32). Mutation of residues along the junctional domain of kainate receptor subunits alters desensitization rates to a more modest degree than in AMPA receptors, suggesting that interactions between the subunit interface domains are similar but not identical in the two types of receptors (33). Short extracellular “linker” regions that connect the S1 and S2 domains to the membrane domains are central to gating of the channel in response to agonist binding to an LBD. This was demonstrated dramatically in studies on the lurcher mice, which carry a spontaneous mutation in a highly conserved residue in the M3-S2 linker region of the 2 receptor subunit gene (34). The 2 lurcher subunits gate a constitutive current; analogous mutations in the GluR6 kainate receptor subunit also produce apparent agonist-independent gating and markedly slow deactivation and desensitization kinetics (35). It was demonstrated more recently that AMPA receptors containing the lurcher mutation are exquisitely sensitive to glutamate and that their “constitutive”

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activity arises from gating induced by ambient (nanomolar) levels of glutamate present in nominally glutamate-free bathing solutions (36). 1.3. Diversity Diversity in kainate receptor subunit isoforms is generated by alternate splicing, editing of mRNA transcripts, and genetic polymorphisms (in humans). Different cytoplasmic splice isoforms distribute to the plasma membrane differentially in heterologous cell lines and in neurons because they contain a variety of trafficking determinants (37), which presumably interact with a distinct set of chaperone systems to control receptor distribution. RNA editing of GluR5 and GluR6 transcripts results in profound changes in receptor function similar to that observed with the GluR2 AMPA receptor subunit. 1.3.1. Alternative Splice Isoforms GluR5, GluR6, and GluR7 mRNAs are subject to alternative splicing that alters the primary sequence of the subunit cytoplasmic C-termini (Fig. 1B) (38). GluR5 mRNA also contains a splice site that inserts an additional 15 amino acids immediately preceding the S1 domain. There are no known splice isoforms of KA1 or KA2 subunits. GluR5 subunits are denoted GluR5-1 if they contain the splice cassette in the N-terminal domain and GluR5-2 if they lack these extra amino acids (1). The large majority of functional and biochemical studies have been carried out on GluR5-2 isoforms, and indeed the functional relevance of the N-terminal splice cassette in GluR5-1 is unknown. GluR5-2a, -2b, and -2c differ in their C-terminal domains (6). GluR5-2a, the shortest splice isoform, was the first to be described functionally. GluR5-2b and GluR5-2c diverge only by virtue of an additional 29-amino acid insert in the latter subunit. A fourth GluR5 isoform, GluR5-2d, has a unique C-terminal tail and has been isolated exclusively from human cDNA libraries (39,40). GluR5-2 subunit isoforms are indistinguishable functionally; however, they are expressed at differing levels on the plasma membrane as a result of the presence or absence of intracellular trafficking determinants (41,42) (as discussed in Section 3). Cytoplasmic domains may play a role in shaping the physiologic properties of the channels, in a way that might differ between splice variants, because GluR5-2a receptor currents are quite variable in their desensitization kinetics when expressed in mammalian cell lines (43). The mechanism(s) underlying this variable desensitization are unclear. Two C-terminal splice variants have been isolated for the GluR6 subunit. The GluR6a subunit cDNA was cloned initially (3), and this subunit remains the only isoform characterized physiologically to an appreciable degree. GluR6b has a distinct cytoplasmic domain with the exception of the short conserved juxtamembrane domain (8,39). GluR6a and GluR6b have very

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similar glutamate-evoked current properties, exhibit overlapping distributions, and coassemble in the mammalian central nervous system (CNS) (44). The two GluR7 subunit isoforms also differ in their cytoplasmic domains downstream of the common juxtamembrane splice site (2,45). GluR7a was thought initially to be nonfunctional as a homomeric receptor because currents were not detected on application of agonists, but later studies showed that this occurred because glutamate has an extremely low potency for activation of either GluR7 isoform (45). The C-terminal domain in the GluR7 splice variants controls their relative level of plasma membrane expression, with the GluR7a isoform expressed at much higher levels because of the presence of a forward trafficking determinant identical to that found in GluR6a subunits (46,47). 1.3.2. RNA Editing RNA editing of kainate receptor pre-mRNAs generates diversity in the stoichiometry and function of these receptors. Two receptor subunit RNAs—those encoding GluR5 and GluR6—are subject to deamination of select adenosines through mechanisms similar to those in GluR2 AMPA receptor subunits. Enzymatic alteration of the adenosine to an inosine at the “Q/R” (glutamine/arginine) site in these kainate receptor subunit mRNAs alters the codon specificity from a glutamine to an arginine (48). GluR6 has two additional editing sites, the “I/V” (isoleucine/valine) and “Y/C” (tyrosine/cysteine) sites, that result in alteration of residues in the M1 domain (49). This enzymatic process requires formation of a secondary double-stranded RNA structure composed of complementary exonic and intronic domains; for the GluR6 kainate receptor subunit the intronic “editing complementary sequence” (ECS) is quite remote from the Q/R site codon (∼1.9 kb) (50). RNA editing is carried out by a family of enzymes called “adenosine deaminases that act on RNA” (ADAR1-3). ADAR1 shows editing activity at the GluR6 Q/R site (50), and gene-targeted mice lacking ADAR2 showed modest (GluR5) or significant (GluR6) reductions in editing of RNA transcripts (51), supporting a role for these particular enzymes in kainate receptor Q/R-site editing. The amino acid residue at the Q/R site in M2 is a critical determinant of channel properties, and thus RNA editing at this site influences several important aspects of channel function. Receptors incorporating edited GluR5(R) or GluR6(R) subunits are nearly impermeable to calcium (52,53) and have very low single-channel conductances (54). Unexpectedly, edited GluR6(R) channels, which are classically thought of as cation channels, are also slightly permeable to chloride (55). In contrast, unedited GluR5(Q) and GluR6(Q) kainate receptor subunits are weakly calcium permeable (49,52,53), exhibit significantly lower chloride permeability (55), have a ∼25fold higher single-channel conductance (54), and exhibit inwardly rectifying

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current–voltage relationships resulting from voltage-dependent channel block by intracellular polyamines (56,57). Edited and unedited subunits can coassemble to form functional receptors with intermediate degrees of rectification (58). In GluR6, the additional editing sites in M1 appear to influence calcium permeability to a slight degree but otherwise have little measurable effect on channel biophysical properties (49,53). Q/R-site RNA editing of GluR5 and GluR6 is developmentally regulated and varies among neuronal populations, unlike GluR2 Q/R-site editing (which is almost complete in early gestation). Kainate receptor mRNAs remain largely unedited in rats until late in embryonic development (59), at which time editing in most brain regions increases rapidly before reaching an equilibrium level within a week (in rodents) that is generally maintained throughout adulthood (60). Editing of GluR5 RNA in the cerebellum, as an exception, continues to increase beyond early postnatal life (60,61). In addition, GluR5 and GluR6 RNAs in glial cells from the adult optic nerve are largely unedited (62). The progression in RNA editing can be correlated with changes in kainate receptor function; for example, the increase in GluR5 editing in nociceptive neurons in rat dorsal root ganglia causes a transition from calcium-permeable to calcium-impermeable kainate receptors within the first postnatal week (63). In adult animals and in humans, the majority of GluR6 transcripts are edited at the Q/R site (>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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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 <3 sec using a photoactivatable caged Ca2+ chelator (30), potentially excluding any need for sustained Ca2+ gradients in LTP induction (31a). The Ca2+ source for triggering LTP may depend strongly on the induction protocol used, suggesting that not all NMDARdependent LTP is equivalent. Thus, in assessing the role of Ca2+ entry directly through NMDARs in LTP by examining mice expressing NMDARs with reduced Ca2+ permeability Pawlak et al. [31a] observed a selective deficit in pairing-induced LTP with no effect on HFS-induced LTP. 1.1.3. NMDAR Subunit Composition in LTP Induction In addition to voltage-dependent Mg2+ blockade and Ca2+ permeability, the relatively slow kinetics of NMDARs contributes directly to their role in LTP induction. The prolonged decay times of NMDAR-mediated currents greatly outlast the cleft glutamate transient during release, allowing for optimal temporal summation during repetitive activation. On the other hand, the slow time course of the NMDAR-mediated component of synaptic events also makes them highly susceptible to concurrently activated GABAergic inhibition during synaptic stimulation, explaining the greater ease in evoking LTP in the presence of -aminobutyric acid A receptor (GABAA R) blockers (32). Because NMDAR kinetics, and hence NMDAR-mediated Ca2+ transients, are determined by subunit composition (see Chapter 2, NMDA Receptors), one might expect differences in the abilities of distinct NMDAR subtypes to induce LTP. Within hippocampal CA1 pyramids the major NMDAR subpopulations consist of heteromeric assemblies of requisite NR1 subunits in combination with NR2A or NR2B subunits (33). It is significant that NR2B-containing NMDARs yield synaptic events that are larger and decay almost twofold slower

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than NR2A-containing NMDARs, providing a much longer window for both Ca2+ influx and temporal summation. Appropriately, initial studies on specific NMDAR subtypes in synaptic plasticity revealed that overexpression of NR2B subunits results in greatly enhanced LTP in adult mice and shifts the frequency dependence of LTP induction to lower frequencies (34). The issue of NMDAR-subtype specificity to synaptic plasticity has recently become a hotbed of controversy. In examining the role of native CA1 pyramid NMDARs pharmacologically, Liu and colleagues (35) reported the highly provocative findings that activation of NR2A subunit–containing NMDARs was specifically coupled to the induction of LTP, whereas activation of NR2B subunit–containing NMDARs selectively triggered LTD. Similar findings were subsequently reported for plasticity in the sensory cortex (36). Both studies relied to a large extent on the specificity of a relatively new pharmacologic antagonist of NR2A-containing NMDARs, NVP-AAM077, which selectively blocked LTP without affecting long-term depression (LTD) (but see ref. 37). However, subsequent investigation revealed that this compound is capable of reducing NMDAR-mediated excitatory postsynaptic currents (EPSCs) in NR2A-knockout mice, and thus is clearly not NR2A specific (37,38), calling into question the hypothesis that NR2A-containing NMDARs have exclusive access to the postsynaptic machinery necessary for LTP induction. Furthermore, LTP can be induced, albeit with less efficiency, in both NR2Aknockout mice (39) and in transgenics expressing a truncated version of NR2A that lacks the carboxyl-terminal tail (40), demonstrating that NR2B-containing NMDARs indeed contribute to LTP generation. Finally, a recent investigation demonstrated a preferential role for NR2B-containing NMDARs in triggering calcium/calmodulin-dependent protein kinase II (CaMKII)–dependent LTP in hippocampal slice cultures at a developmental time point similar to those in previous studies using acute slices from adult animals (41,42). 1.1.4. Signal Cascades Triggered During Induction Of the dizzying myriad of signaling molecules proposed to be involved in the initial transduction of NMDAR-associated Ca2+ transients into synaptic potentiation (e.g., see ref. 43), a great deal of emphasis has been placed on the role of kinase cascades. Indeed a very early report (even before the recognition of NMDAR involvement) provided evidence that HFS results in the phosphorylation of synaptic proteins and that this phosphorylation depends on the presence of Ca2+ (44). Among the numerous kinases implicated, the most widely accepted, and the one with the strongest case, appears to be CaMKII. CaMKII is principal resident of the postsynaptic density (PSD) optimally positioned to be activated by synaptically driven Ca2+ influx (45) and can remain in an activated state for very long periods of time independent of Ca2+ following autophosphorylation on threonine 286 (46,47). This feature of

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CaMKII has led to the concept that it serves as a “memory molecule” providing a trace signal demarcating previous Ca2+ signals that long outlasts the initial triggering events leading to its activation (reviewed in ref. 48). Initial experiments implicating CaMKII in LTP demonstrated that postsynaptic injection of CAMKII inhibitors blocks induction of LTP (49,50), whereas constitutively active CaMKII mimics and occludes LTP when introduced postsynaptically (51,52). Thus, CaMKII appears to be both necessary and sufficient for NMDAR-dependent LTP. Furthermore, LTP-inducing stimuli lead to the NMDAR-dependent activation and autophosphorylation of CaMKII and its translocation to synaptic locations (47,53–55). The importance of autophosphorylation of CaMKII in LTP was underscored by the absence of LTP in gene-targeted mice expressing an altered CaMKII with a point mutation at Thr-286, the autophosphorylation site (56), which extended earlier observations that deletion of the gene encoding the CaMKII alpha subunit severely impaired LTP (57). The importance of persistent autonomous CaMKII kinase activity in LTP maintenance beyond the initial induction period is questionable, however, because CaMKII inhibition after induction does not prevent LTP expression [50,58,59; but see ref. 60]. A potential resolution to this conundrum may be that CaMKII autophosphorylation at Thr-286 subserves LTP maintenance functions independent of autonomous kinase activity (61); for example, CaMKII interactions with NMDARs are modulated by Thre-286 phosphorylation (62–64). Thus, it may be that CaMKII participates in the maintenance phase of LTP through structural changes that take place at synapses following CaMKII–NMDAR interactions that are independent of kinase activity. In this scheme, the initial CaMKII kinase activity induced by Ca2+ influx through NMDARs plays a pivotal role in the induction of LTP, whereas the autophosphorylation-regulated (kinase activity– independent) NMDAR interactions would participate in LTP maintenance. Consistent with this model, Barria and Malinow (41) recently demonstrated that prevention of the association between CaMKII and NMDARs blocked LTP expression with only minimal impact on initial postpairing potentiation in hippocampal slice cultures. A final important piece of evidence implicating CaMKII as an essential mediator of LTP is that the kinase directly phosphorylates AMPARs, which increases their single-channel conductance (65,66) and controls their insertion into the postsynaptic membrane (67–69), both of which are widely supported mechanisms of LTP expression (see the discussion in subsequent paragraphs). A role for CaMKII in the early phase of potentiation following conditioning stimulation is nearly ubiquitously accepted. Thus, it is surprising how variable CaMKII inhibitors have proved in their inhibition of LTP immediately following induction, even when using pairing protocols that eliminate confounding factors such as posttetanic potentiation (e.g., compare ref. 58

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with ref. 41). The answer to this puzzle may lie in the existence of multiple Ca2+ -sensitive kinases that perform overlapping roles in the initial phases of LTP induction. Indeed, Wikstrom and colleagues (70) recently reported the existence of two parallel kinase pathways that support LTP induction in slices obtained from 2-week-old animals: One pathway involved CaMKII, and the other comprised both protein kinase A (PKA) and protein kinase C (PKC) activities. Furthermore, PKA appears to be the principal mediator of LTP induction in hippocampal slices obtained from very young animals (younger than postnatal day 10 [P10]), in which CaMKII inhibitors are without effect on LTP induction or expression (42,71). A developmental profile indicates that the role of CaMKII in LTP progressively increases from approximately P10 onward, becoming the dominant kinase beyond P20 (42). In older animals PKA has been suspected of playing a primarily modulatory role in LTP induction, because postsynaptically applied PKA inhibitors typically reduce the magnitude of early LTP (e.g., ref. 72). This modulatory role may occur through an indirect prolongation of CaMKII activity resulting from PKA-mediated suppression of protein phosphatase 1 (PP1), which generally opposes CaMKII activity (73–75). Alternatively, PKA activity may regulate the availability of AMPARs for postsynaptic insertion following LTP induction (76–78) (see further discussion later). In addition to these roles of PKA in LTP induction, there is general agreement that cAMP/PKA signaling cascades are crucial for the protein synthesis–dependent late phases of LTP expression (reviewed in refs. 4 and 79). PKC was an early candidate for a Ca2+ -sensitive mediator of LTP induction because postsynaptically applied PKC inhibitors were found to block LTP (50,80–82). Like CaMKII, PKC can become constitutively active, although the mechanism relies on cleavage of the regulatory pseudosubstrate region of the kinase rather than autophosphorylation. Such cleavage leads to release of the constitutively active kinase fragment of PKC, referred to as PKM, the levels of which have been found to increase following LTP-inducing stimuli in an NMDAR-dependent fashion (83–86). Early models proposed that NMDAR-mediated Ca2+ influx leads to calpain-mediated cleavage of PKC, yielding constitutively active PKM that participated in both LTP induction and maintenance. Indeed, PKC is a preferred substrate of calpain, and inhibition of calpain has been shown to block LTP (87–89). However, subsequent investigation by Sacktor and colleagues suggests that the increased levels of PKM following LTP induction result from increased local dendritic protein synthesis of PKMzeta, an atypical PKC isoform that lacks the pseudosubstrate regulatory domain, and hence does not require proteolytic cleavage for autonomous activity (86,90,91). Thus, it may be that conventional PKC isoforms, such as PKCalpha, are transiently engaged during LTP induction, whereas persistently active PKMzeta is synthesized locally to subserve

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LTP maintenance functions. Consistent with this scheme, conventional PKC isoforms are activated transiently within seconds of LTP-inducing stimuli, whereas PKMzeta levels persistently increase within 10–30 min following conditioning stimulation (84,86). Furthermore, specific PKMzeta inhibition reversibly blocks late-phase LTP (beyond 1 hr) without altering early LTP (92,93). Oddly, however, postsynaptic infusion of purified PKMzeta enhances AMPAR-mediated synaptic transmission in a fashion that occludes LTP, suggesting that the initial potentiation of synaptic transmission following conditioning stimulation shares essential features with LTP maintenance (most likely AMPAR phosphorylation/insertion) (92). Indeed PKC, and thus presumably PKMzeta, shares with CaMKII a phosphorylation site on GluR1 subunits (Ser831) that is implicated in LTP induction (53,65,66,94–97). A role for tyrosine kinase function in the induction of LTP was first suggested based on experiments showing that bath-applied tyrosine kinase inhibitors block LTP induction without altering preestablished LTP (98). In particular, a great deal of evidence specifically implicates the nonreceptor tyrosine kinase Src in NMDAR-dependent LTP (reviewed in ref. 99). Src is a component of synaptic NMDAR complexes capable of upregulating channel function (100), perhaps by direct NR2 subunit phosphorylation (101,102). Similar to CaMKII, PKA, and PKC, Src is activated by tetanic stimulation (103), likely via a sequential signaling cascade involving Ca2+ induced activation of the focal adhesion kinase CAKbeta/Pyk2, which subsequently activates Src (104,105). Postsynaptic injection of peptides or antibodies that selectively disrupt Src-mediated regulation of NMDARs prevent tetanusinduced LTP without altering basal synaptic transmission (103). Conversely, postsynaptically applied active Src, or a small peptide activator of Src family kinases, mimics and occludes LTP (103). In contrast to the proposed role of CaMKII/PKA/PKC in transducing NMDAR-mediated Ca2+ signals into AMPAR potentiation, Src is thought to participate in LTP induction by providing a requisite enhancement of NMDARs during conditioning stimulation; this enhancement is necessary because NMDARs are subject to tonic inhibition by the tyrosine phosphatase STEP (99,103,106). Consistent with this model, Src does not appear to regulate synaptic AMPAR-mediated currents directly, but rather requires both NMDAR function and a rise in intracellular Ca2+ to mimic and occlude LTP (100,103,104,106). Thus, it seems that Src serves as an essential effector of a positive feedback loop to boost NMDARmediated Ca2+ influx during conditioning stimulation, ensuring that intracellular Ca2+ levels reach the threshold for LTP induction, an important feature given that low levels of NMDAR-mediated Ca2+ influx may trigger LTD (see later section on NMDAR-dependent LTD). In addition to triggering postsynaptic kinase cascades, large NMDARmediated Ca2+ transients produced during conditioning stimulation may lead

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to the generation of retrograde messengers that engage the presynaptic terminal in LTP expression. If one concedes, as we have, that there is likely to be a presynaptic component to NMDAR-dependent LTP, and that induction is triggered by the postsynaptic influx of Ca2+ through NMDARs, then clearly there must indeed be such a transsynaptic retrograde signal from the postsynaptic cell to modify presynaptic release. The free radical gas nitric oxide (NO) has garnered the most attention and generated the most controversy among potential retrograde messengers (reviewed in refs. 107–110). NO is produced by NO synthase (NOS), one isoform of which is brain specific (neuronal NOS [nNOS]) and associates with the NMDAR complex via an interaction with the scaffolding protein PSD-95 (111,112). Because NOS activity is regulated by Ca2+ bound to calmodulin, it is commonly assumed that the close association of nNOS with NMDARs optimally localizes the enzyme to respond to Ca2+ influx through NMDARs. An important caveat to this scheme arises from the observation that NMDAR-dependent hippocampal LTP relies to a large extent on another NOS isoform (endothelial NOS [eNOS]), which does not associate with NMDARs, calling into question the triggering mechanism for NO production during conditioning stimulation for LTP induction (113–116). Moreover, the presence of nNOS in CA1 pyramidal cells of adult rats, the most ubiquitously studied model for LTP, is questionable, and eNOS expression may be limited to blood vessels (117). Nonetheless, substantial evidence indicates that blockade of NOS activity, even when limited to postsynaptic CA1 pyramids, or scavenging of extracellular NO prevents LTP induction (118–120), although these findings have not always been replicated in different labs (121,122). Clearly, further work is needed to establish the controversial role of NO in LTP. The study of such retrograde signaling has somewhat fallen out of favor, however, in large part due to the immense interest and current high profile of postsynaptic AMPAR trafficking as the expression mechanism for LTP. A recent report indicates that NO-mediated S-nitrosylation of N-ethylmaleimide– sensitive factor (NSF) regulates the association of NSF with GluR2, thereby regulating AMPAR surface expression in the postsynaptic membrane (123). Perhaps this emergence of NO as a player in AMPAR trafficking will regenerate interest in the role of NO in LTP. 1.2. Expression Mechanisms Whereas NMDARs are the central players in LTP induction, expression is primarily achieved by an increase in AMPAR-mediated synaptic transmission. In fact, the majority of studies focusing on this issue report a selective increase in the AMPAR-mediated component of postsynaptic responses immediately following hippocampal LTP induction without any significant change in the NMDAR-mediated component (e.g., refs. 124–129; but see ref. 130), although a delayed (>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 (
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stimulation frequency that avoided excessive Ca2+ entry though the GluR2lacking AMPARs, a typical level of potentiation was observed (190) rather than the “super LTP” observed in cells infected with recombinant GluR2 carboxyterminal tails (68). Nonetheless, the fact that pairing-induced LTP expression remains stable for the first 30–40 min after induction in the presence of the GluR2 C-tail peptide, or even in the complete absence of GluR2 subunits, conflicts with the notion that GluR2-dictated AMPAR trafficking mechanisms are necessary for LTP expression during this time frame (178). It is possible that interactions on the GluR2 C-terminal tail participate in the initiation of CP-AMPAR replacement by CI-AMPARs, and therefore blockade of GluR2 C-tail interactions allows the larger-conductance CP-AMPARs to remain at potentiated synapses, yielding enhanced LTP. In support of this hypothesis, blockade of GluR2-NSF/PICK/GRIP interactions prevents the exchange of synaptic CP-AMPARs for CI-AMPARs following conditioning stimulation in cerebellar stellate cells (181,182). Given the central roles of both kinase activity and AMPAR trafficking in LTP, it is natural to try to make a logical connection between the two events. Landmark initial studies revealed that native GluR1 subunits undergo regulated phosphorylation in the intracellular C-tail region at serine 831 (Ser831) by CaMKII during de novo LTP induction (53,65,95). In addition to Ser831, which also serves as a PKC substrate, the GluR1 C-tail can be phosphorylated at Ser845 by PKA (94) (Fig. 8A,B), and this phosphorylation has primarily been observed following LTP induction at previously depressed synapses (i.e., during de-depression) (96). Fueled by these reports, a number of studies have investigated the role of GluR1 phosphorylation in AMPAR trafficking during LTP. Most revealing perhaps is a study in which LTP was greatly diminished in adult transgenic knockin mice expressing a mutant GluR1 subunit that could not be phosphorylated at Ser831 or Ser845 due to substitution of the serine residues for alanine (97) (Fig. 8C,D). The LTP deficit in these mice was developmentally regulated and only appeared in animals older than 1 month, consistent with the developmental emergence of GluR1-dependent LTP over this time period (158,184). Incomplete LTP blockade in the GluR1 phosphomutant mice, especially within the first 10–20 min following induction (97) (see Fig. 8C,D), suggests that GluR1 Ser831 and Ser845 may not be the critical kinase substrates controlling AMPAR trafficking during LTP induction. Indeed, recombinant AMPARs composed of GluR1 subunits with an alanine substitution at Ser831 are efficiently driven into synapses by coexpression of active CaMKII or by LTP-inducing stimuli (78,155,167). Recombinant GluR1 subunits with an alanine substitution of Ser845, however, cannot be driven into synapses by constitutively active CaMKII (78). Because PKA-mediated Ser845 phosphorylation by itself was insufficient to drive recombinant GluR1 homomers into synapses, it was proposed that PKA-mediated phosphorylation

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Fig. 8. GluR1 subunit phosphorylation and long-term potentiation (LTP). A, B. -Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) (A) with sequence alignment of carboxy-terminal tails (B) of different subunits, highlighting residues important for phosphorylation and various protein–protein interactions. C, D. Hippocampal slices from adult mutant mice expressing nonphosphorylatable GluR1 subunits (GluR1 S831A/S845A phosphomutant) have reduced LTP as assayed by extracellular field potential recordings (C) or by a pairing protocol in single-cell sharp electrode recordings (D). EPSP, excitatory postsynaptic potential; Wt, wild type. A, B: Modified with permission from Song I, Huganir RL. Regulation of AMPA receptors during synaptic plasticity. Trends Neurosci 2002;25:578–588. C, D: Modified with permission from Lee HK, Takamiya K, Han JS, et al. Phosphorylation of the AMPA receptor GluR1 subunit is required for synaptic plasticity and retention of spatial memory. Cell 2003;112:631–643.

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Fig. 9. Transmembrane -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) regulatory protein (TARP)–mediated AMPAR surface insertion and synaptic targeting. A. TARPs directly interact with AMPARs early in the synthetic path (1) to promote surface trafficking (2). Subsequent phosphorylation of TARPs by calcium/calmodulin-dependent protein kinase II (CaMKII)/protein kinase C (PKC) leads to the synaptic targeting of surface TARP/AMPAR complexes by an interaction with postsynaptic density (PSD)-95 (3). B. Overexpression of stargazin (Stg) (wild type [Wt]), nonphosphorylatable Stg (S9A), or phospho-mimic Stg (S9D) all promote extrasynaptic surface expression of AMPARs (left bar chart), while only phosphomimic Stg synaptically targets AMPARs under basal conditions (compare middle and right panels; wild-type Stg not shown for basal synaptic incorporation). C. Increased synaptic responses in S9D-overexpressing neurons occludes long-term potentiation (LTP), whereas overexpression of S9A blocks LTP, likely by acting as a dominant negative. Wild-type Stg (WT)–overexpressing neurons yielded LTP comparable to uninfected neurons. A: Modified with permission from Nicoll RA, Tomita S, Bredt DS. Auxiliary subunits assist AMPA-type glutamate receptors. Science 2006;311:1253–1256. B, C: Adapted with permission from Tomita S, Stein V, Stocker TJ, et al. Bidirectional synaptic plasticity regulated by phosphorylation of stargazin-like TARPs. Neuron 2005;45: 269–277.

of Ser845 controls the availability of nonsynaptic AMPARs for synaptic incorporation by CaMKII-dependent mechanisms (78,97). Alternatively, the PKA site may be important for anchoring the newly inserted AMPARs until they can be turned over for a more stable population (97,178) because there is roughly 10–15 min of potentiation after LTP induction in neurons overexpressing GluR1 subunits with the Ser845-to-alanine mutation (78). These findings leave in question the identity of critical CaMKII substrate(s) for regulated AMPAR synaptic insertion. One line of evidence suggests that

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active CaMKII may engage a signaling cascade involving the small Ras family of GTPases and mitogen-activated protein kinase (MAPK) to mediate AMPAR insertion at conditioned synapses (157). Consistent with this model, MAPK has been proposed as an important player in LTP induction (reviewed in ref. 191). Furthermore, the synaptic Ras GTPase-activating protein SynGAP can be multiply phosphorylated by CaMKII, potentially coupling CaMKII to the Ras/MAPK pathway (192–194). A critical piece of evidence implicating Ras signaling in AMPAR synaptic insertion during LTP is the observation that neurons overexpressing dominant negative Ras lack LTP (157). However, these neurons also exhibit a transient potentiation lasting roughly 20 min postinduction that does not appear different from control neurons. Although such transient potentiation may reflect short-lived enhancement of preexisting synaptic AMPARs (195) (further discussed below), most evidence indicates that AMPAR trafficking participates in LTP within minutes of induction [e.g., 127,132,146,159,178]. Thus, the temporal requirements of Ras signaling in LTP may be more suggestive of a role in early LTP stabilization following initial AMPAR insertion. The transmembrane AMPAR regulatory proteins (TARPs) represent a particularly intriguing candidate substrate group for CaMKII in AMPAR trafficking (196) (see Chapter 2, AMPA Receptors). TARPs comprise a small family of AMPAR interacting proteins consisting of gamma-2 (also known as stargazin, Stg), gamma-3, gamma-4, and gamma-8, which are differentially expressed throughout the CNS and appear to be essential for plasma membrane AMPAR expression (197,198). Indeed, absence of the prototypical TARP Stg (gamma-2) results in a complete lack of surface AMPARs in cerebellar granule cells of stargazer mutant mice (197). Stg and related TARPS interact with AMPARs at an early stage of their biosynthesis to promote surface expression; subsequently they bind to PSD-95 to direct synaptic incorporation of the associated AMPARs (198,199) (Fig. 9A). Whereas AMPAR insertion at extrasynaptic sites seems to be a constitutive function of Stg, the synaptic incorporation of AMPARs requires CaMKII/PKC–mediated phosphorylation of the intracellular carboxy tail of Stg (200) (Fig. 9B). A critical role for phosphorylation of TARPs in LTP is supported by the finding that overexpression of mutant nonphosphorylatable Stg entirely prevents LTP (200) (Fig. 9C). The specificity of TARP phosphorylation for synaptic insertion is underscored by the fact that the nonphosphorylatable Stg actually enhances basal surface levels of AMPARs at extrasynaptic sites (200) (Fig. 9B). Conversely, recombinant mutant phosphor-mimic Stg enhances surface AMPAR levels both extrasynaptically and synaptically, leading to full occlusion of LTP (200) (Fig. 9B). A similar occlusion of LTP occurs following overexpression of PSD-95 (201,202), perhaps via the synaptic recruitment of TARP-associated extrasynaptic AMPARs; however, this would require that PSD-95 overexpression also

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enhances TARP phosphorylation (200). A role for TARPs in LTP is further supported by the highly reduced levels of LTP in mice with targeted deletion of gamma-8, the principal hippocampal TARP (203). However, interpretation is confounded by overlapping expression patterns of other TARPs and by the dramatic overall decrease in AMPAR subunit content of hippocampal neurons in the gamma-8 knockout (203). The TARP-based model of LTP implies that the initial AMPAR trafficking event following LTP induction may be a lateral movement of TARP-associated extrasynaptic surface AMPARs to conditioned synapses rather than postsynaptic exocytosis of AMPAR-containing vesicles. Oddly, however, overexpression of wild-type Stg, which dramatically upregulates surface extrasynaptic receptors, making them available for lateral diffusion, does not yield increased LTP levels (200). In addition, because there is no evidence to suggest that TARPs exhibit any subunit specificity, a TARP-based mechanism of synaptic AMPAR recruitment disputes the importance of AMPAR subunit C-terminal tail–based trafficking events. Clearly, further investigation is required to resolve this discrepancy with the large body of evidence revealing AMPAR-subunit specificity in LTP. 1.2.2. Increased AMPAR Conductance and LTP In addition to an increase in the number of AMPARs at conditioned synapses, postsynaptically expressed LTP could result from increased function of AMPARs existing at synapses prior to induction. Of particular relevance to the current discussion are reports of PKA- and CaMKII-mediated enhancement of AMPAR function resulting from increased channel open probability and conductance, respectively (66,204). The effects of PKA and CaMKII on AMPAR function result from phosphorylation of Ser845 and Ser831 of the GluR1 subunit, respectively, which are the same locations found to undergo regulated phosphorylation in response to LTP-inducing stimuli (53,65,95,96). Although evidence for a change in AMPAR open probability following LTP induction is lacking, application of peak-scaled nonstationary fluctuation analysis to dendritically recorded synaptic events before and after LTP induction has revealed that LTP can often be accounted for by increased AMPAR conductance in the juvenile hippocampus (195,205,206). Thus, it is frequently presumed that a portion of LTP is expressed as an increase in the unitary conductance of preexisting synaptic AMPARs generated by CaMKII-mediated phosphorylation of Ser831 on GluR1. Indeed this aspect of LTP expression is often referenced to explain any residual components of LTP that persist following manipulations to block the AMPAR trafficking-dependent component of LTP. However, a recent report indicates that presence of the GluR2 subunit eliminates the ability of CaMKII to upregulate the function of heterologously expressed GluR1/2 heteromeric AMPARs

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(207). In such heteromeric AMPARs, CaMKII still efficiently phosphorylates Ser831 of GluR1, but the heteromeric GluR1/2 receptors do not exhibit the increased conductance observed with homomeric GluR1 receptors. In CA1 hippocampal neurons, prior to LTP induction, synapses are dominated by GluR2-containing AMPARs, and thus these findings are difficult to reconcile with the prevailing model of increased AMPAR conductance by CaMKIImediated GluR1 phosphorylation following conditioning stimulation. Perhaps a third party is responsible for the enhanced-conductance state of AMPARs at potentiated synapses, and AMPAR-interacting TARPs immediately come to mind. In addition to its roles in AMPAR trafficking, Stg (and presumably the other TARPs) also directly modulates the gating properties of AMPARs, including single-channel conductance (208–210). Of course, it remains to be determined whether the ability of Stg to modulate AMPAR conductance is itself modifiable; however the ability of TARPs to undergo regulated phosphorylation is highly provocative (200). Enhanced AMPAR conductance observed following LTP induction also could be explained by the synaptic insertion of a population of high-conductance AMPARs. This interpretation would fit well with the incorporation of CP-AMPARs at potentiated synapses (178) because GluR2-lacking AMPARs have significantly higher conductance than their GluR2-containing counterparts. However, as outlined earlier, participation of CP-AMPAR in LTP appears to be transient, whereas the change in AMPAR conductance measured by Benke and colleagues (195) was found to be persistent. Furthermore, LTP associated with an increased AMPAR conductance did not evidence a concomitant change in AMPAR number. Clearly, further work is needed to understand the role of changes in AMPAR biophysical properties for LTP expression and the extent to which these changes contribute to LTP at various stages of development (206).

2. NMDA Receptor-Dependent Long-Term Depression If mechanisms of long-term potentiation represent an electrophysiologic correlate of memory formation, and there is much evidence to suggest that this is the case, synapses would ultimately accumulate in a persistently potentiated state in the absence of a mechanism to reverse or weaken this synaptic strengthening. In addition, any mechanism that saturates synaptic strength would be intuitively problematic for cellular function and, more importantly, lead to a loss of the tuning capabilities of any network of interconnected cells. In addition, LTP per se is insufficient fully to account for the complex process of memory storage (see ref. 48 for further discussion). With this in mind, it was widely accepted that a mechanism must exist to weaken naive synapses or reverse previously potentiated synapses, even before empirical evidence emerged for such processes. It was considered likely that the processes linked

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to strengthening and weakening synapses would be intimately linked. This is an important consideration, because if potentiation and depression were to proceed simultaneously and independently within the same synapse, both processes could ultimately saturate to produce a static synapse. Ample suggestive evidence existed for such a depotentiatating process. During experience-dependent development, for example, synapse elimination results from inactivity or downregulation of synapse strength in an anti-Hebbian manner, that is, cells that do not fire together do not wire together. A theoretical framework for bidirectional plasticity was posited by Bienenstock, Cooper, and Munro—the so-called BCM theory (210,211). In essence the BCM theory stated that active synapses would be strengthened when a critical threshold of postsynaptic response exceeded the “modification threshold,” termed m . Synaptic weakening or depression, in contrast, resulted when the sum of postsynaptic activity lay somewhere between zero and m . The overall mathematical function of the BCM theory was well described by a sinusoidal function, and the great utility of this theory was that it adequately, yet simply, recapitulated experimental data (Fig. 10). Like NMDA-dependent LTP before it, LTD of excitatory synaptic transmission was first observed within the hippocampal formation, in this case at Schaffer collateral synapses onto CA1 pyramidal cells by Dudek and Bear (212,213). Using trains of stimuli of long duration (hundreds of stimuli often for many minutes) between 0.5 and 10 Hz, they found that synaptic events progressively decreased before stabilizing at a lower synaptic strength (212) (Figs. 1 and 10). This induction protocol engaged levels of presynaptic activity at low stimulus intensity that failed to evoke a strong postsynaptic response; that is, with reference to BCM theory, this protocol failed to exceed the critical m for LTP formation. It is now well established that bidirectional plasticity occurs at many divergent synapses throughout the central nervous system, and, although subtle variations in the underlying mechanisms exist, the basic phenomenon is essentially the same. Thus, it is generally accepted that any synapse that demonstrates NMDA-receptor dependent LTP will also demonstrate NMDA receptor–dependent LTD, superficially suggesting that the phenomena might be mechanistically linked. Of course, this has turned out to be too simplified a notion and, as we will discuss, mechanisms proposed to underlie synaptic weakening are as numerous and diverse as those put forward to explain LTP. One of the first remarkable observations made about CA1 Schaffer collateral synaptic depression was the absolute requirement for NMDA receptor activation and a role for postsynaptic Ca2+ elevation (212,214). This presented something of a paradox, given that these two same ingredients are the key players in NMDA-dependent LTP (see the foregoing discussion). How could two of the most ubiquitous elements of synaptic signaling be implicated

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Fig. 10. Induction of N-methyl-d-aspartate receptor (NMDAR)–dependent bidirectional plasticity of the Schaffer–collateral synapse in CA1. A. Summary of the effects of a 900-pulse tetanus delivered at different frequencies. B. Summary of the effects of a 600-pulse, 20-Hz tetanus in different concentrations of the NMDAR antagonist AP5. C. A synaptic “learning rule” based on data such as those given in panels A and B. This learning rule is formally similar to that proposed in the Bienenstock, Cooper, and Munro (BCM) theory. Reproduced with permission from Bear MF. Mechanism for a sliding synaptic modification threshold. Neuron 1995;15:1–4.

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in mechanisms for opposing plastic phenomena at the same synapse? One immediate clue was the observation that either LTD or LTP could be evoked using the same number of stimuli just delivered at different stimulation frequencies; low-frequency stimulation paradigms typically trigger LTD, whereas high-frequency paradigms generally induce LTP (212) (Fig. 10). Nevertheless, stimulation frequency per se is not the critical factor; for example, a high-frequency induction paradigm typically used to trigger LTP will reliably induce LTD when delivered in the presence of partial NMDA receptor blockade (215) (Fig. 10). Instead, the postsynaptic Ca2+ level and, more important, the Ca2+ dynamics within the postsynaptic compartment are crucial determinants of the polarity of synaptic plasticity, and these Ca2+ dynamics vary according to the strength, duration, and frequency of stimulation paradigms used to evoke plasticity. Presynaptic activity is not even necessary for LTD induction, because either uncaging of glutamate by flash photolysis or bath application of NMDA induces robust LTD (216–218). As a simple rule of thumb, modest but prolonged elevations of postsynaptic Ca2+ in the submicromolar range induce LTD, whereas a brief Ca2+ elevation to ∼10 μM is required for LTP induction (219). At least two fundamentally different forms of NMDAR-dependent LTD are thought to exist: synaptic depression of naive synapses, which have not been subjected to potentiating stimuli, and depression of recently potentiated synapses, a process more commonly referred to as depotentiation. In both cases, intracellular elevation of Ca2+ is critically important for the downstream second-messenger systems that are engaged to produce synaptic depression. The low but persistent elevation of Ca2+ during LTD induction afforded an important clue as to the nature of downstream elements that were involved in the plasticity process. Protein phosphatases PPI and PP2B (calcineurin), a Ca2+ /calmodulin-dependent protein phosphatase, were shown to be key mediators of LTD induction (220,221). Considerable evidence now suggests that long-term depression is determined in part by the dephosphorylation of the synaptic glutamate receptor subunit GluR1. Specifically, elevation of Ca2+ during the LTD induction protocol activates calcineurin, which dephosphorylates the protein inhibitor I-1. Dephosphorylation of I-1 relieves inhibition of PP1, which in turn dephosphorylates Ser845 on GluR1— the same PKA phosphorylation site that participates in NMDAR-dependent LTP (218). Dephosphorylation of Ser845 was first described in a chemically induced model of synaptic depression (218) in which entire hippocampal slice preparations were bathed in NMDA. It is interesting that this form of LTD was not associated with the degree of phosphorylation of Ser831, another site suggested to participate in NMDAR-dependent LTP (65) (see prior discussion for details). This observation underscores the important point that LTP and LTD are not precisely inverse processes. Despite being an important

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step forward, the chemical LTD experiment revealed little about the specific involvement of synaptic AMPA receptors in LTD. In subsequent experiments, Huganir and colleagues (96) found that low-frequency stimulation of Schaffer collateral afferents led to both dephosphorylation of GluR1 Ser845 and LTD. Moreover, knockin mice in which alanine residues replaced Ser831 and Ser845 in GluR1 had both reduced LTP and an absence of NMDAR-dependent LTD (97). Together these data suggest that LTD (and LTP) follows a predictable sequence of phosphorylation and dephosphorylation steps. At naive synapses, LTD leads to a dephosphorylation of Ser845, whereas at previously potentiated synapses, LTD, or depotentiation, results from the dephosphorylation of the Ser831 site. Although clearly an important mechanism for LTD expression, this model is predicated on the presence of GluR1-containing AMPA receptors at native (and naive) synaptic sites, which is incompatible with the prevailing data suggesting that the bulk of AMPA receptors in naive tissue comprise GluR2/3 heteromers (163). In contrast, only a small fraction of synaptic receptors in naive neurons comprise GluR1/2 heteromers (136,163,222). If this receptor composition and expression arrangement is correct, then it is hard to understand how a simple model involving dephosphorylation of the different sites on GluR1 could contribute a major expression mechanism of de novo LTD, given that so few GluR1-containing AMPA receptors may actually be present at naive CA1 pyramidal cell synapses. Clearly, other GluR1-independent mechanisms must participate in synaptic depression. In the last decade considerable attention has focused on the interactions of GluR2 AMPAR subunits with various proteins present at the postsynaptic density and the roles played by these interactions in synaptic plasticity (136,222) (see Chapter 2, AMPA Receptors). As our appreciation for such molecular interactions increases, it has become apparent that a primary mechanism for NMDAR-dependent plasticity involves the highly coordinated and regulated trafficking of AMPA receptors via association with a number of molecular partners (Fig. 11). Consequently, AMPA receptor internalization has emerged as a major mechanism for LTD expression. AMPA receptor internalization proceeds through a dynamin-dependent and clathrin-mediated pathway that can be triggered by NMDA receptor activation and is Ca2+ dependent. The hexameric adenosine triphosphatase NSF directly binds the C-terminal tail of GluR2 (147,148), and agents that disrupt or prevent this interaction result in a rapid rundown of EPSCs in addition to blocking LTD triggered by NMDAR activation (147,148,169,223). This suggested that the NSF– GluR2 C-terminal interaction was responsible for maintaining a stable pool of synaptic AMPA receptors that were competent for receptor internalization during NMDA receptor activation. Lee et al. (170) then demonstrated that the clathrin adaptor protein complex AP2, a key player in endocytosis, associated

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Fig. 11. Alteration in -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor distribution during N-methyl-d-aspartate receptor (NMDAR)– dependent long-term potentiation (LTP) and long-term depression (LTD). The discovery of proteins that directly bind to AMPA receptor subunits has facilitated the understanding of the molecular mechanisms of rapid AMPAR trafficking at synapses. The association between N-ethylmaleimide–sensitive factor (NSF) and glutamate receptor subunit 2 (GluR2) is important to maintain AMPARs at synapses; its inhibition leads to the rapid internalization of a mobile pool of AMPARs. The AP2 adaptor complex binds to an overlapping region of GluR2; NSF likely occupies the site to stabilize receptors at synapses, but in response to an appropriate stimulus, it is replaced by AP2, which initiates clathrin-dependent internalization. NSF, through its ATPase activity, may help to dissociate AMPARs from their tethers, the multiple PDZ proteins ABP (AMPAR-binding protein) and GRIP (glutamate receptor–interacting protein). Another molecule that might help to dissociate AMPARs from their tethers is PICK1 (protein interacting with C-kinase), which can bind the C terminus of GluR2 and target PKC (protein kinase C) to phosphorylate serine 880 of GluR2. Once phosphorylated at this residue, GluR2 can bind PICK1 but not ABP/GRIP, which provides a mechanism by which AMPARs can be freed from ABP/GRIP for internalization. Once untethered, AMPARs probably diffuse laterally in the membrane to sites of endocytosis at the periphery of synapses. Internalized AMPARs might be recycled to the membrane, held in an intracellular pool, or degraded. The intracellular pool is also probably tethered by PDZ proteins such as ABP/GRIP and can be released by PICK1/PKC. Insertion might involve the binding of GluR1 to the multiple-PDZ– domain protein SAP97 (synapse-associated protein 97), which, in turn, binds to motor

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with a region of GluR2 that overlapped with the NSF-binding site. Synthetic peptide sequences that were previously considered to interfere only with the NSF–GluR2 interaction similarly perturbed AP2–GluR2 association. These key experiments were able to dissociate the constitutive cycling/stabilization role for NSF from a role for AP2 binding in NMDA receptor–dependent internalization and LTD (170). It remains unclear how AP2 is recruited to AMPA receptors following NMDA receptor activation, given the overlap of the NSFand AP2-binding domains; however, it is likely that under basal conditions NSF occupies this site to stabilize AMPA receptor surface expression. In response to NMDA receptor activation, AP2 binding replaces the NSF occupation to initiate receptor internalization either directly from synaptic sites or following relocation of AMPARs to extrasynaptic sites (136). The C-terminal tail of GluR2 also interacts with a number of PDZdomain–containing proteins: glutamate receptor interacting protein (GRIP), AMPA receptor–binding protein (ABP), and protein interacting with C-kinase (PICK1). GRIP and ABP are rich in PDZ domains and likely serve to anchor or stabilize AMPA receptors in both the synaptic membrane and intracellular sites (136,222). A certain fraction of GRIP is palmitoylated, which may serve to differentially target receptors to spines or intracellular clusters (224). AMPA receptor endocytosis is thought to proceed following phosphorylation of the C-terminal tail of GluR2. This important phosphorylation step results in the release of GluR2 from GRIP (which probably anchors synaptic and intracellular AMPA receptors) and allows binding to PICK1 (225–227). PICK1 may act further through its association with PKC? to facilitate phosphorylation of GluR2 on Ser880 and inhibit the binding of GluR2 to GRIP (228,229).

Fig. 11. (Continued) proteins such as myosin-VI. CaMKII (calcium/calmodulindependent protein kinase II) probably helps to drive the insertion of AMPARs by phosphorylating SAP97. AMPARs are probably inserted at sites distant from the synaptic cleft, from which they diffuse laterally within the membrane. Molecules such as the transmembrane AMPAR regulatory protein (TARP) stargazin, which is concentrated at synapses by its association with the PDZ protein PSD-95 (postsynaptic density protein 95), might help to anchor AMPARs in the synaptic density. PI3K (phosphatidylinositol 3-kinase) also binds to AMPARs and is required to maintain AMPAR surface expression during LTP. Reproduced with permission from Collingridge GL, Isaac JT, Wang YT. Receptor trafficking and synaptic plasticity. Nat Rev Neurosci 2004;5:952–962.

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3. NMDA Receptor-Independent LTP Although the bulk of research on synaptic plasticity has focused its attention on NMDAR-dependent forms of long-term plasticity, best exemplified by that observed at the Schaffer collateral synapses of CA1 pyramidal cells, many other distinct forms of NMDA receptor-independent forms of synaptic long-term plasticity exist. Within the mammalian cortical formation, the most commonly studied form of NMDA receptor–independent LTP is that found between the axons of dentate gyrus granule cells, the so-called mossy fibers, and the proximal dendrites of hippocampal CA3 pyramidal neurons. Many of the features of mossy fiber LTP are found at other synapses throughout the mammalian central nervous system, such as the cerebellar parallel fiber– Purkinje cell synapse (230,231), as well as corticothalamic synapses (232) and the lateral amygdala (233). These forms of plasticity further differ from the classic NMDAR-dependent forms of plasticity in that their expression is universally agreed on as residing in the presynaptic terminal and involving adenylyl cyclase/cAMP-dependent pathways. 3.1. Mossy Fiber NMDAR-Independent LTP Before we consider the mechanisms of mossy fiber LTP, it is worthwhile briefly considering the architecture of the mossy fiber synapse. The axons of dentate gyrus granule cells, or mossy fibers (MF), innervate their principal cell and interneuron targets via anatomically distinct synapse types (234) (reviewed in refs. 235 and 236) (Fig. 12a). MF–principal cell synapses are formed by large, complex “mossy” terminals that comprise large numbers of release sites (237). Giant MF terminals typically form up to 35 active zones (237,238) apposed to a single pyramidal cell. Historically, the large number of release sites has led to the suggestion that the strength of excitation from a single mossy fiber terminal was sufficient to reliably induce action potentials in postsynaptic CA3 pyramidal cells, and this peculiar anatomy led to the concept of the mossy fiber synapse as a “teacher”- or “detonator”-type synapse (11). In contrast, interneurons of the CA3 stratum lucidum, which are also innervated by mossy fibers, are contacted primarily via small en passant or filopodial MF synapses that emerge from the parent giant MF terminal (Fig. 12a). These MF– interneuron synapses possess numerous anatomic and physiologic properties distinct from those of synapses onto principal cells (235). In particular, MF synapses onto CA3 interneurons typically form one or a few active zones through both en passant boutons or filopodial extensions radiating from the main MF terminal. Of interest for our discussion here, presynaptic mechanisms of plasticity at CA3 pyramidal cell synapses versus interneuron synapses share few commonalities despite emerging from common mossy fiber axons.

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Fig. 12. Basic anatomic elements of mossy fiber–CA3 circuitry. A. The dentate gyrus and proximal portion of the hippocampus (MFs). Granule cells (labeled 1) give rise to extensive collateral plexuses, the mossy fibers (MFs), that are distributed throughout much of the hilus (H) and make synaptic contact with both dentate gyrus basket cells (2) and mossy cells (3). As the parent MF approaches the CA3 pyramidal cell layer (PL), the large presynaptic MF expansions begin to appear and are typically 140–200 μm apart for the entire length of the axon. These expansions form complex synapses on the proximal apical dendrites of pyramidal cells (4). The small filopodial or en passant terminals make synapses onto local circuit interneurons primarily located within the stratum lucidum (5). Scale bar, 100 μm. Left lower: Camera lucida drawings of Golgi-impregnated MFs illustrate that the filopodial extensions emerging from the large complex MF terminal (large arrows). By postnatal-day (P) 14, mature MF expansions are common. At P28, there is an increase in the number of MF expansions that have reached adult shape and size and the lengths of the individual expansions have decreased to adult levels. Scale bar, 50 μm. Right lower: Electron micrographs of different terminal types along MFs in the CA3 region (A–C, E) and of a CA3 pyramidal cell terminal (D). All electron micrographs have the same magnification, for comparison of the relative size of the terminals. (A, B) A small en passant terminal establishes a single asymmetric synapse on a dendritic shaft with long, perforated postsynaptic density (arrows). (C) A filopodial extension of a mossy terminal forms a synapse (arrow) with a substance-P-receptor–immunoreactive interneuron. (D) The postsynaptic target of a pyramidal cell terminal is a simple spine of a CA1 pyramidal neuron. (E) A large, double-headed mossy terminal forms multiple contacts (arrows) with thorny excrescences of a CA3 pyramidal cell. The individual release sites are short. Scale bars, 0.5 μm (A–D) and 1 μm (E). B. High-frequency stimulation (100 Hz for 1 sec) of mossy fiber afferents onto CA3 pyramidal cells results in robust N-methyld-aspartate receptor (NMDAR)– independent long-term potentiation as evidenced by the increase in excitatory postsynaptic current (EPSC) amplitude. Left: An individual experiment with associated averaged EPSCs as indicated by numbers above. Right: Pooled data. The identical induction paradigm results in a long-term depression at filopodial/en passant mossy fiber synapses onto CA3 stratum lucidum interneurons. These data highlight that although the expression of both forms of plasticity occur within the presynaptic terminal, an opposing change in synaptic strength occurs.

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MF LTP was effectively distinguished from NMDAR-dependent plasticity in an elegant experiment by Zalutsky and Nicoll (239), in which they demonstrated that chelation of postsynaptic Ca2+ blocked LTP at associational synapses made onto the distal dendrites of CA3 pyramidal cells (which is NMDA receptor dependent) but not at mossy fiber synapses onto proximal synapses of the same cell (a similar conclusion was reached by in ref. 240). Moreover, MF LTP was accompanied by a change in both the coefficient of variance (CV) and paired-pulse relationship of the evoked EPSC and could be induced by bath application of forskolin, an activator of PKA-dependent cascades (Fig. 13A). These results established the contentious hypothesis that both the induction and expression sites of MF LTP had their origins in the presynaptic terminal. The NMDAR independence of MF LTP is not particularly surprising because Monaghan and Cotman (241) established that NMDA-receptor binding in the stratum lucidum of CA3, the termination zone of the mossy fiber-hippocampal projection, was extremely low compared to other hippocampal or cortical regions. Although NMDA receptor–mediated events can be evoked at synapses between MFs and their targets, the ratio of NMDAR-mediated to AMPAR-mediated EPSCs at MF–CA3 pyramidal cell synapses is 30% of that found at CA3 associational–commissural synapses (242). This lack of NMDAR dependence suggested that mossy fiber LTP was nonassociative, noncooperative, and non-Hebbian (243–245) (see Fig. 2 for definitions of these plasticity terms), all hallmark features of Schaffer collateral NMDAR-dependent LTP. Although there is almost universal agreement on the presynaptic expression locus of MF LTP, the locus of induction is under some debate. Although the early studies of Zalutsky and Nicoll (239) strongly supported a presynaptic Fig. 12. (Continued) A, top: Reproduced from Claiborne BJ, Amaral DG, Cowan WM. A light and electron microscopic analysis of the mossy fibers of the rat dentate gyrus. J Comp Neurol 1986;246:435–458; with permission from Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc. The stratum lucidum inhibitory interneuron (cell 5) was added to the original figure by J. J. Lawrence and C. J. McBain. A, lower left: reproduced from Amaral DG, Dent JA. Development of the mossy fibers of the dentate gyrus: I. A light and electron microscopic study of the mossy fibers and their expansions. J Comp Neurol 1981;195:51–86; with permission from Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc. A, lower right: Reproduced from Acsady L, Kamondi A, Sik A, et al. GABAergic cells are the major postsynaptic targets of mossy fibers in the rat hippocampus. J Neurosci 1998;18:3386–3403; with permission from the Society for Neuroscience. B: reproduced from Toth K, Suares G, Lawrence JJ, et al. Differential mechanisms of transmission at three types of mossy fiber synapse. J Neurosci 2000;20:8279–8289; with permission from the Society for Neuroscience.

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Fig. 13. A. Mossy fiber–CA3 pyramidal cell long-term potentiation involves a mechanism consistent with presynaptic cyclic AMP (cAMP)/protein kinase A (PKA) activation. A. Inclusion of the activator of adenylyl cyclase, forskolin, results in longterm potentiation (LTP) of mossy fiber–CA3 pyramidal cell synaptic transmission but fails to increase excitatory transmission onto CA3 stratum lucidum interneurons. A late component of the mossy fiber–interneuron excitatory postsynaptic current (EPSC) is potentiated, consistent with a potentiation of the polysynaptic feedback circuit driven by mossy fiber–principal cell synapses. B. Mossy fiber LTP is absent in Rab3Adeficient mice. Upper: Sample traces before (1) and 55–60 min after tetanization (2) and after 10 mM L-CCG1 bath application (3) in wild-type (WT, top) and knockout (KO, bottom) mice. Lower: LTP summary graphs in wild-type (open circles) and mutant mice (black circles). LTP was induced by one train lasting 5 sec at 25 Hz in the presence of 100 mM D-AP5. A: Reproduced with permission from Maccaferri G, Toth K, McBain CJ. Targetspecific expression of presynaptic mossy fiber plasticity. Science 1998;279:1368–1370. B: Reproduced with permission from Castillo PE, Janz R, Sudhof TC, et al. Rab3A is essential for mossy fibre long-term potentiation in the hippocampus. Nature 1997;388:590–593.

locus of induction that did not rely or require postsynaptic glutamate receptor activation, it remained a possibility that other sources of postsynaptic Ca2+ could contribute to induction. Consistent with this notion, two reports suggested that induction of MF LTP required elevation of postsynaptic Ca2+ and was regulated by postsynaptic holding potential (246,247) (but see refs. 239, 240, and 244). In addition, different forms of MF LTP (one non-Hebbian and one Hebbian) were also proposed (248). To tackle these issues head on, Dan Johnston and colleagues combined electrophysiologic recording with highresolution postsynaptic Ca2+ imaging of mossy fiber synapses (249). In a carefully considered series of experiments, they concluded that two forms of MF LTP, one induced by brief high-frequency stimulation (HFS) and one by a long-duration HFS, required an elevation in postsynaptic Ca2+ . Furthermore,

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in experiments that echoed those of Nicoll and colleagues, blockade of fast ionotropic postsynaptic transmission was still capable of inducing MF LTP; unlike previous results, however, this LTP correlated with an elevation of postsynaptic Ca2+ that arose via metabotropic glutamate receptor activation and release of Ca2+ from intracellular stores. Finally, inclusion of a PKA inhibitor in the recording pipette fully blocked MF LTP induction. In short, these experiments convincingly argued for a postsynaptic induction locus that involved both mGluRs and intracellular Ca2+ elevation, and postsynaptic PKA formation. However, soon after this, Nicoll and coworkers presented data that showed largely the opposite results (250). That is, using similar animal species and nearly identical recording techniques and criteria for determining mossy fibers inputs, they demonstrated that inhibition of postsynaptic mGluRs and buffering of postsynaptic intracellular Ca2+ had little impact on mossy fiber LTP, again supporting their original theory of a presynaptic locus of MF LTP induction. Perplexing? You bet! Finally, an attempt to induce MF LTP by photolysis of postsynaptic caged Ca2+ alone failed to induce any change in synaptic transmission that could be ascribed to a presynaptic expression mechanism (251). Similarly, direct depolarization of CA3 pyramidal cells, in the absence of presynaptic activity, induces a developmentally regulated, L-type Ca2+ channel– dependent, long-lasting depression of mossy fiber synaptic transmission (252). These results argue that elevation of postsynaptic Ca2+ alone is insufficient to trigger MF LTP, although the fundamental question of a requirement for postsynaptic Ca2+ remains subject to heated debate. Given the widespread acceptance of a presynaptic locus of MF-LTP expression, were induction to be postsynaptic, a retrograde signaling mechanism would be required to signal across the synaptic cleft. Of course, numerous retrograde signaling mechanisms have been posited for a number of plasticity mechanisms; however, a compelling mechanism for transsynaptic signaling during MF LTP comes from a consideration of the EphB receptor– B-ephrin interaction. EphB receptors and their ligand partners, the B-ephrins, are enriched at many synapses and are considered important for both normal development and plasticity throughout the mammalian CNS (253,254). Within dentate gyrus granule cells, mRNAs for all three B-ephrins are detected, with ephrin-B3 the most abundant (255,256), and with protein localized to mossy fiber axon terminals (257). Using peptide interference techniques, Contractor and colleagues (258) demonstrated that induction and expression of MF LTP was impaired by disrupting Eph receptor association with postsynaptic scaffolding proteins or by blocking transsynaptic EphB receptor–B-ephrin interaction. More recently, the same group reported deficits in mossy fiber LTP in ephrin-B3–mutant mice, in which the cytoplasmic carboxy-terminal signaling domain was replaced with B-galactosidase (257). These data would

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appear to strengthen a role for B-ephrin–mediated reverse signaling into the mossy fiber presynaptic terminal as a requirement for mossy fiber LTP. So what postsynaptic glutamate receptors are involved in mossy fiber LTP? As stated previously, MF LTP proceeds in the absence of NMDA receptor activation, ruling out a role for these receptors, despite their functional presence at MF synapses (albeit reduced compared to other hippocampal synapses). The bulk of evidence suggests that the primary fast ionotropic receptor in mature MF synapses is formed by GluR2-containing, Ca2+ -impermeable AMPA receptors (259,260). Artificial incorporation of AMPA receptors containing an unedited form of the GluR2(Q) subunit, which is highly Ca2+ permeable, into mossy fibers by overexpression via Sindis viral–mediated gene transfer had little effect on mossy fiber LTP, suggesting that the postsynaptic identity of the AMPA receptors expressed there is of little importance for MF LTP (261,262). Moreover, these data further strengthen the argument against a role for postsynaptic Ca2+ elevation as an essential trigger for MF LTP induction, although the additional postsynaptic Ca2+ contribution above that provided by more traditional sources, such as VGCCs and mGluRs, would be minimal. A small but pivotal role for kainate receptors in MF LTP has been discovered more recently. Kainate receptors have long been known to be present in high density at MF synapses (263), but their role in synaptic transmission and plasticity has been somewhat elusive. During development of the mossy fiber projection, kainate receptors are first detected at the end of the first postnatal week and reach their maximal expression ∼10 days postnatal (264). Kainate receptor–mediated synaptic transmission at MF synapses is of small amplitude and slow kinetics compared to AMPAR-mediated responses, which has led to the proposal that they may be primarily involved in postsynaptic temporal integration of synaptic inputs and potentially act as triggers for MF LTP. Ionotropic signaling at the postsynaptic MF–CA3 pyramidal cell synapse requires GluR6, presumably in a coassembly with KA2 (265,266). Genetic ablation of GluR6 abolishes kainate-mediated MF EPSCs (265) and reduces MF LTP (267). The observation that genetic ablation of GluR6 protein abolishes MF kainate EPSCs suggests that KA2 receptors do not form functional homomeric receptors to compensate for the absence of GluR6. it is interesting that knockout of the KA2 subunits results in functional GluR6 receptors at MF synapses, which has led to the fascinating hypothesis that one subunit of the obligate GluR6/KA2 heteromeric receptor participates in an ionotropic role (GluR6) and the other (KA2) in a G protein–mediated metabotropic role (268). Although pharmacologic experiments have indicated a role for GluR5-containing kainate receptors in MF-LTP, GluR5-knockout mice show little deficit in MF LTP, arguing against a role for these subunits (267,269). Exploration of the role of kainate receptors in MF LTP has inevitably resurrected debate over pre- versus postsynaptic sites for the induction locus.

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If MF LTP induction and expression are entirely presynaptic, then it is hard to rationalize a role for postsynaptic kainate receptors in this scenario, especially when MF LTP can be reliably induced in the presence of both kainate and AMPA receptor antagonists (270). However, kainate receptors are also expressed on the presynaptic terminals of MF–CA3 synapses, and these receptors appears to modulate the threshold for LTP induction (269). This suggested that presynaptically expressed GluR6-containing kainate receptors acted to “boost” MF LTP induction, presumably by depolarizing the presynaptic axon or terminal. Moreover, presynaptic kainate receptors also imparted a previously unappreciated cooperative and associative property to MF LTP. The role of kainate receptors in MF LTP is discussed in additional detail in Chapter 3. As stated earlier, there is almost universal agreement that expression of MF LTP is an entirely presynaptic phenomenon that manifests as an increase in the release probability of transmitter release (Fig. 14A). Rather than repeat the

Fig. 14. Mossy fiber–CA3 pyramidal cell plasticity. A. A model of hippocampal mossy fiber long-term potentiation (LTP). LTP expression expressed presynaptically as a cyclic AMP (cAMP)/protein kinase A (PKA)–dependent increase in neurotransmitter release. B. Summary diagram of the putative signal transduction cascade mediating mossy fiber long-term depression (LTD) at pyramidal cell synapses. A basal level of activity of Ca2+ /calmodulin-dependent adenylyl cyclase 1 (AC1) provides a necessary substrate after metabotropic glutamate receptor (mGluR) activation. A decrease in cyclic AMP (cAMP) levels leads to a reduction in PKA activity, which finally lowers the release probability. AMPAR, -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor; NMDAR, N-methyl-d-aspartate receptor; PKA, protein kinase. Reproduced from Nicoll RA, Schmitz D. Synaptic plasticity at hippocampal mossy fibre synapses. Nat Rev Neurosci 2005;6:863–876; with permission from the Nature group.

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contents of numerous review articles on mossy fiber LTP expression, we direct interested readers to a recent review by Nicoll and Schmitz (236), who provide a lucid account of MF LTP expression mechanisms. Expression of MF LTP at principal cell synapses is generally considered to be triggered by Ca2+ entry into the presynaptic terminal during high-frequency stimulation. Elimination of extracellular Ca2+ during MF induction prevents MF LTP (271). Mossy fiber terminals on CA3 pyramidal neurons use both N- and P/Q-type Ca2+ channels to support neurotransmitter release, with P/Q-type channels being the greatest source of Ca2+ for basal transmission (271). Surprisingly, selective pharmacologic blockade of either has little effect on the induction of MF LTP (271), nor does MF LTP alter the selectivity of either channel type to pharmacologic intervention. This was originally interpreted as suggesting that high-frequency activity is capable of engaging the downstream targets of Ca2+ entry equally efficaciously via either N- or P/Q-type Ca2+ channel. However, although N- and/or P/Q-type channels support basal low-frequency synaptic transmission, it seems highly possible that an alternative source of Ca2+ entry is engaged during high-frequency activity. A probable candidate has emerged in the shape of R-type Ca2+ channels. The Ca2+ -channel subunit 1E is considered to be the molecular correlate for R-type Ca2+ channels and is highly enriched in dentate gyrus granule cells (272). Interestingly, loss of 1E function either through pharmacologic antagonism or genetic knockout alters MF LTP induction without altering basal synaptic transmission (273). The complete sequence of events downstream of Ca2+ entry remains unclear. However, it is well established that Ca2+ directly activates the Ca2+ /calmodulin-sensitive adenylyl cyclases, AC1 and AC8, which are enriched in dentate gyrus granule cells (Figs. 13 and 14). The resulting elevation of cAMP is necessary and sufficient to trigger MF LTP by a mechanism that is thought to involve the vesicular protein Rab3A (274) and its active-zone–located binding partner RIM1alpha (275) (Fig. 13B). How this acts to trigger a persistent increase in transmitter release probability is unclear, but it does not involve a persistent increase in presynaptic Ca2+ entry (271,276). 3.2. Mossy Fiber Pyramidal Cell NMDAR-Independent, Long-Term Depression Like other central neurons, mossy fiber synapses also possess mechanisms to persistently depress synaptic transmission in response to afferent activity. Four distinct types of LTD have been observed at MF–CA3 synapses. The first and predominant mechanism of LTD is induced by low-frequency stimulation (1 Hz for 15 min) in both juvenile and adult animals (277–279). Induction of this type of LTD is dependent on neither NMDAR activity nor postsynaptic Ca2+ but requires presynaptic mGluR2 activity and a rise in presynaptic Ca2+ and is coupled to a decrease in cAMP-dependent PKA activity (278,280)

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(Fig. 14B). Both the induction and the expression of this form of LTD are presynaptic in origin, involve a reduction in transmitter release probability, and are generally considered to be a reversal of the processes responsible for conventional NMDAR-independent mossy fiber LTP (278). Second, highfrequency stimulation (100 Hz, 1 sec) of mossy fibers in rats on postnatal day 6–14 also induces LTD (279,281). Although the mechanism for this form of LTD is not clear, its induction requires neither NMDAR nor mGluR activities, but it does depend on postsynaptic Ca2+ elevation, and it has an expression locus that is presynaptic (279). Third, intracellular “tetanization” of postsynaptic CA3 pyramidal neurons induces either LTD or LTP at MF– CA3 synapses in slices from juvenile (7- to 16-day-old) rats (282). This form of LTD also has a presynaptic expression locus, and the form of plasticity evoked appears to depend largely on the initial synaptic release probability (282). Fourth and finally, another developmentally regulated form of mossy fiber LTD is triggered solely by prolonged depolarization of the postsynaptic CA3 pyramidal cell (252). This heterosynaptic LTD depends on postsynaptic Ca2+ elevation through L-type Ca2+ channels and release of Ca2+ from inositol 1,4,5-trisphosphate (InsP3) receptor–sensitive intracellular stores. This LTD is independent of NMDA, mGlu, cannabinoid, or opioid receptors and does not require coincident synaptic activity. Of particular interest, Ca2+ imaging of both proximal and distal CA3 pyramidal neuron dendrites demonstrated that the depolarizing induction paradigm differentially elevated intracellular Ca2+ levels. L-type Ca2+ -channel activation was observed principally at the most proximal locations where mossy fibers make synapses and is consistent with the known expression pattern of L-type Ca2+ channels on pyramidal cells (283). It is significant that depolarization-induced LTD did not occlude conventional 1-Hz–induced LTD or vice versa, suggesting that independent mechanisms underlie each form of plasticity. The paired-pulse ratio and coefficient of variation of synaptic transmission were unchanged after depolarizationinduced LTD, suggesting that the expression locus of LTD is entirely postsynaptic. Moreover, peak-scaled nonstationary variance analysis indicated that this depolarization-induced LTD correlated with a reduction in postsynaptic AMPA receptor numbers without a change in AMPA receptor conductance. 3.3. Mossy Fiber Interneuron Plasticity So far this discussion has focused on plasticity of mossy fiber synapses made onto CA3 pyramidal cells. However, as described earlier, mossy fiber axons also innervate local circuit inhibitory interneurons residing within the dentate gyrus, the hilar region, and the stratum lucidum of the CA3 hippocampus. Although mechanisms of synaptic depression have been extensively studied at mossy fiber–stratum lucidum interneuron synapses (see later discussion), only

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two studies have reported long-term potentiation or de-depression at mossy fiber-interneuron targets. 3.3.1. Mossy Fiber LTP at Dentate Basket Cell Interneurons An associative form of MF LTP was described by Jonas and colleagues (284) in experiments employing paired recordings between dentate gyrus granule cells and basket cells or low-frequency stimulation of afferent fibers. This form of LTP was expressed presynaptically in a mechanism reminiscent but not identical to MF–CA3 pyramidal cell LTP. Although MF– CA3 pyramidal cell LTP involves PKA-dependent cascades, MF–basket cell LTP primarily involves a PKC-dependent cascade. In addition, induction of associative LTP was blocked by postsynaptic infusion of the Ca2+ chelator bis-(aminophenoxy)ethane-tetraacetic acid (BAPTA), indicating a postsynaptic locus of MF–basket cell LTP induction. It interesting that, by using a nonassociative protocol in which the postsynaptic cell was held under voltage clamp and incapable of firing action potentials, the same authors demonstrated a long-term depression at MF–basket cell synapses consistent with LTD seen at MF synapses onto stratum lucidum interneurons in the CA3 hippocampus (260,285). In conclusion, despite the presynaptic locus of both forms of LTP (MF–CA3 pyramidal cell and MF–basket cell), there are differences in both the induction and expression mechanisms that are critically dependent on the identity of the postsynaptic target (235). 3.3.2. Mossy Fiber LTD at Stratum Lucidum Interneurons Interneurons of the stratum lucidum are primarily targeted by the filopodial extensions and en passant MF synapses and only rarely by the larger MF boutons that typically contact principal cells. Given that the filopodial extensions originate from the large MF terminal, it might be assumed that any change in presynaptic release probability arising from LTP in the large MF bouton would distribute evenly to all synapses, resulting in long-term changes at both MF–pyramidal cell and interneuron synapses. However, quite the opposite is the case. The same high-frequency, nonassociative stimulation protocol that induces LTP at principal cell synapses induces two forms of LTD at interneuron synapses (286,287) (Fig. 15). Postsynaptically, AMPARs at MF–stratum lucidum interneuron (SLIN) synapses comprise a continuum ranging from GluR2-lacking, Ca2+ -permeable channels (CP-AMPARs) to GluR2-containing, Ca2+ -impermeable channels (CI-AMPARs) (260,288). Both CI-AMPAR– and CP-AMPAR–containing MF–SLIN synapses exhibit LTD in response to high-frequency stimulation, but LTD arises from two distinct mechanisms at each synapses type (260,285–288) (Fig. 14). Induction of each is blocked by inclusion of the Ca2+ chelator BAPTA in the postsynaptic compartment, indicating a postsynaptic contribution to the induction

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Fig. 15. Two forms of mossy fiber–CA3 interneuron long term depression (LTD). A. Long-term depression at mossy fiber–interneuron synapses comprised of Ca-impermeable -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) has a postsynaptic locus of induction and expression, is N-methyld-aspartate (NMDA) receptor dependent, and involves an endocytosis of surface AMPA receptors reminiscent of NMDAR-dependent LTD observed at principal cell synapses. B. Long-term depression at mossy fiber–interneuron synapses comprised of Ca2+ -permeable AMPA receptors has a requirement for a postsynaptic increase in intracellular Ca2+ but is expressed presynaptically. This form of LTD is NMDA receptor independent and requires activation of presynaptic mGluR7 and downstream protein kinase C (PKC)–dependent cascades to reduce transmitter release probability. Reproduced from Nicoll RA, Schmitz D. Synaptic plasticity at hippocampal mossy fibre synapses. Nat Rev Neurosci 2005;6:863–876; with permission from the Nature group.

for each (see the MF–CA3 pyramidal cell LTP described earlier). Activityinduced LTD of MF–SLIN transmission at CI-AMPAR synapses is mediated by postsynaptic NMDAR activation and subsequent AMPAR internalization similar to other excitatory synapses throughout the CNS (286,288) (see earlier discussion of NMDAR-dependent LTD at principal-cell synapses). In contrast, LTD at CP-AMPAR containing MF–SLIN synapses is NMDAR independent and is expressed presynaptically as a reduction in transmitter release (286). The rise in postsynaptic Ca2+ likely comes from influx through the CP-AMPARs themselves, but the identity of any retrograde signals that control presynaptic release is unknown. It is interesting that whereas MF–CA3 pyramidal cell LTP involves PKA-dependent cascades (see earlier discussion for details), transmission and plasticity at either type of MF–interneuron synapse is largely insensitive to manipulations that elevate cAMP levels (285). This compartmentalization of the biochemical machinery highlights one potential functional

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utility of the filopodial versus large-terminal arrangement of the mossy fiber presynaptic complex. How the cAMP-dependent cascade is segregated across the interterminal compartments is not understood. Similar to LTP at MF–basket cell synapses, LTD at MF–stratum lucidum CP-AMPA receptor synapses is blocked by antagonists of PKC (287). Furthermore, activation of mGluR7, which specifically localizes to MF terminals opposing stratum lucidum interneurons and not PYRs (289,290), is an important step in the induction of presynaptic MF–SLIN LTD (287) (Fig. 15B). The joint requirement for a rise in postsynaptic Ca2+ by influx through CP-AMPARs and presynaptic mGluR7 activation imparts a Hebbian coincidence detection feature to LTD at CP-AMPAR containing MF–SLIN synapses. Blockade of mGluR7 activation during high-frequency stimulation blocks LTD induction and reveals a posttetanic potentiation. It is significant that mGluR7 appears to function as a metaplastic switch at MF–SLIN synapses, whose activation and surface expression govern the direction of plasticity. In naive slices, mGluR7 activation during high-frequency stimulation generates MF–SLIN LTD, depressing presynaptic release through a PKC-dependent mechanism. Following agonist exposure, mGluR7 undergoes internalization, unmasking the ability of MF–SLIN synapses to undergo presynaptic potentiation or de-depression in response to the same HFS that induced LTD in naive slices. Thus, the selective mGluR7 accumulation at MF terminals contacting SLINs and not PYRs provides cell-target specific plasticity and bidirectional control of feedforward inhibition (287).

4. Short-Term Plasticity at Ca2+ -Permeable AMPA Receptors Most forms of short-term plasticity of synaptic transmission have their origins within the presynaptic terminal and typically involve changes in transmitter release probability as a consequence of elevation in intracellular Ca2+ concentration (for review see ref. 291). Discussion of these mechanisms of short-term plasticity are beyond the immediate scope of this chapter because they tend not to depend on glutamate receptor activation per se. However, an entirely postsynaptic form of short-term plasticity exists at AMPA receptors lacking the GluR2 subunit. As described earlier, AMPA receptors lacking the GluR2 subunit are highly permeable to Ca2+ ions. Ca2+ permeability is controlled by a single amino acid in the second membrane-associated domain, the so-called Q/R site. In addition to controlling Ca2+ permeability, the Q/R site influences the sensitivity of the receptor complex to block by polyamine spider toxins and endogenous intracellular polyamines, such as spermine and spermidine. This voltage-dependent block by internal polyamines underlies the inward rectification observed in native AMPA receptors lacking the GluR2 subunit (160–162). Under basal conditions most principal neurons of the

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mammalian CNS are considered to express high levels of GluR2 and only transiently express Ca2+ permeable AMPA receptors during an early phase of NMDAR-dependent LTP expression (178). In contrast, some hippocampal and neocortical local circuit GABAergic inhibitory interneurons possess glutamate receptors with inwardly rectifying current–voltage relationships and appreciable Ca2+ permeability. The presence of these receptors is correlated with a low abundance of GluR2 mRNA expression (292). In addition to their important role in rapid interneuron synaptic signaling (293) and mechanisms of long-lasting plasticity in interneurons (see earlier discussion), these receptors also endow cells with a novel mechanism of short-term plasticity. Highfrequency repetitive activation of Ca2+ -permeable AMPA receptors by synaptic glutamate (260,294) or application of exogenous glutamate receptor agonists (295) results in a use-dependent unblock by internal polyamines that confers a novel mechanism of short-term synaptic plasticity with an entirely postsynaptic locus (Fig. 16). Given that the polyamine-binding site on the channel is within the intracellular channel vestibule, the presence of polyamine block interferes with current flow on channel activation. The complete unblock of polyamines requires multiple pulses or synaptic stimuli (Fig. 16A). Moreover, facilitation is frequency dependent, and the number of pulses required for maximal facilitation differs depending on the receptor subunit composition studied (295). This mechanism of short-term facilitation has been observed at synapses formed between layer II/III pyramidal cells and multipolar interneurons (294) as well as mossy fiber–hippocampal stratum lucidum interneuron synapses (260). This short-term facilitation appears not to depend on Ca2+ permeation or ion flux through the receptor per se but arises entirely from a voltage- and use-dependent relief of block by internal polyamines. Relief of block by polyamines requires that the channels open, and the rate of unblock is more rapid at more-negative potentials (Fig. 16). Removal of internal polyamines by dialysis results in a loss of the facilitatory mechanism (260). These data suggest that Ca2+ -permeable AMPA receptor synapses are “tonically” blocked by polyamines and that on repetitive activation, block is relieved. Of particular interest during current facilitation, the current–voltage relationship is temporarily transformed from inwardly rectifying to linear, consistent with a transient relief of block of the channel by polyamines (Fig. 16). Facilitation of currents lasts only for a limited time before reblock of the channel occurs. The reblocking mechanism proceeds without requiring the channel to reopen, suggesting that polyamines do not only act as classic open channel blockers as was previously thought. Polyamine block of Ca2+ -permeable AMPA receptors also confers a strong voltage dependence to the rise time of currents. Because the channel is blocked by polyamines in the closed state, significant unblock of the channel must occur before the steady-state current amplitude is reached. This has the effect of slowing the time course of current activation at more-positive voltages, a result

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Fig. 16. Calcium-permeable (CP) -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and short-term plasticity. A. Upper: The mechanism of polyamine block of AMPARs. Left: GluR2-lacking CP-AMPARs are tonically blocked by intracellular polyamines. Unblock of polyamines from the channel vestibule is use dependent and typically requires repetitive synaptic stimulation to reach maximal current amplitude (far left). Lower: At mossy fiber–interneuron CP-AMPAR synapses, normalizing the first excitatory postsynaptic currents (EPSCs) in the train evoked at both –20 and –80 mV reveals a greater degree of facilitation in EPSCs evoked at −20 mV. This voltage-dependent increase in the degree of facilitation at more-positive potentials is consistent with the greater tonic block by polyamines of AMPA receptors held at depolarized potentials. Right: Summary histogram of data from nine experiments in which, regardless of whether the synapse was facilitating or depressing, the EPSC5 /EPSC1 ratio was always larger at more-positive (–20 mV) potentials at which the intracellular polyamine block is relieved. B. High-frequency stimulation relieves AMPAR channels from polyamine block in recombinant channels. Currents in GluR2(Q) channels were recorded with 25 μM spermine added to the intracellular solution: current–voltage relationship for control (open circles) and facilitated currents (closed circles). In controls, glutamate was applied 20 ms after stepping the potential from –80 mV to various test potentials (–80 to +60 mV) at 0.2 Hz. An identical voltage protocol was used for the facilitated currents, except that a 100 Hz train (10 pulses) of glutamate was applied at –80 mV (conditioning glutamate pulses)160 ms before the step to the test potential. Inset: Example recordings at a test potential of +40 mV; arrows indicate glutamate application during the test step. For the current–voltage curve, smooth lines are fitted sixth-order (control) or eight-order polynomials, and each point represents the mean of 10 sweeps. A: Kindly provided by Derek Bowie. B: Taken with permission from Toth K, Suares G, Lawrence JJ, et al. Differential mechanisms of transmission at three types of mossy fiber synapse. J Neurosci 2000;20:8279–8289. C: Reproduced with permission from Rozov A, Zilberter Y, Wollmuth LP, et al. Facilitation of currents through rat Ca2+-permeable AMPA receptor channels by activity-dependent relief from polyamine block. J Physiol 1998;511(Pt 2):361–377.

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not seen in the absence of internal polyamines. This would suggest that the rise times of synaptic currents through native Ca2+ -permeable AMPA receptors would also be voltage dependent and slowed compared with Ca2+ -impermeable receptors, which would have a major impact on the temporal integration of synaptic transmission in cells possessing Ca2+ -permeable AMPA receptors. Of particular interest, the voltage-dependent rate of channel unblock confers a hysteresis to the current–voltage relationship during rapid voltage ramps. One can envision that excitatory postsynaptic potential (EPSP) amplitudes in cells containing these receptors will be strongly influenced not only by the frequency of synaptic transmission but also by the membrane potential “history” of the postsynaptic cell, resulting in a complex pattern of modulation. Because polyamines are an essential component of the intracellular milieu, it is likely that the free polyamine concentration and the metabolic state of the cell will be permissive for the occurrence of such a mechanism of plasticity.

5. Conclusion As can be seen from our brief discussion of a number of mechanisms of hippocampal synaptic plasticity, we have learned much in the intervening 30 years or so since the first description of synaptic long-term potentiation. However, we are a long way from truly connecting the mechanisms of hippocampal plasticity observed in vitro to the behavioral correlates of learning and memory observed in a freely moving animal. Nevertheless, the future is extremely bright for investigation of synaptic plasticity. The introduction of higher-resolution imaging and electrophysiologic and genetic tools together with a better understanding of the temporal nature of protein–protein interactions of essential pre- and postsynaptic components can only propel this field rapidly forward. Throughout this chapter we have tried to pay careful attention to the role of particular glutamate receptor subtypes involved in plasticity induction and expression; however, synaptic plasticity is not limited to glutamate receptor synapses and is found at a number of chemically distinct synapses. These synapses presumably also harbor their own complexities, and it is hoped that in the next 30 years much will be learned about their underlying mechanisms and, more important, the physiologic role long-term plasticity plays at each and every one.

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6 Structural Correlates of Ionotropic Glutamate Receptor Function Anders S. Kristensen, Kasper B. Hansen, Lonnie P. Wollmuth, Jan Egebjerg, and Stephen F. Traynelis

Summary Recent structural and functional studies of ionotropic glutamate receptors (iGluRs) have begun to offer rare insight into the structure–function relationship for an integral membrane protein. In particular, advances in our understanding of iGluR structure are providing an opportunity to interpret functional work in terms of potential conformational changes. Moreover, working hypotheses derived from structural insight offer an opportunity to enrich and guide functional studies. This chapter summarizes knowledge of glutamate receptor structure, with an emphasis on how it has shaped our functional understanding of these receptors. Key Words: Receptor structure; Efﬁcacy; Activation mechanism; NMDA receptor desensitization; AMPA receptor desensitization; Kainate receptor desensitization

1. Introduction: Domain Organization Within the iGluR Subunit Ionotropic glutamate receptors (iGluRs) are integral membrane proteins formed by the tetrameric assembly of large subunits (>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

Structural Correlates of Ionotropic GluR Function

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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

Structural Correlates of Ionotropic GluR Function

265

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.

Structural Correlates of Ionotropic GluR Function

267

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 (<6 degrees) or no cleft closure compared to the apo form, which is consistent with the hypothesis that cleft closure (>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 <20 ms. This desensitization is thought to be dominated by intraprotein conformational changes, as will be discussed in detail in Section 3.2. 3.1. Desensitization of NMDA Receptors Desensitization of NMDA receptors is slower and more complex. At least four different forms of desensitization have been functionally described. First, a decrement of current can be observed that correlates with the magnitude of the response and is voltage dependent, which has been ascribed to Ca2+ entry and inactivation of receptor function that may involve uncoupling of NMDA receptors from the cytoskeleton (46,152–154). Second, a negative allosteric

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interaction between the glutamate-binding site and the glycine-binding site can cause a decrement of current under conditions with submaximal glycine (155–158). The basis for this is a near-instantaneous reduction in affinity at the glycine site once glutamate binds, causing a reequilibration of the receptor with glycine. If the glycine site is not saturated, glycine will unbind, relaxing to a new equilibrium with a macroscopic time course reflecting the on and off rates for glycine. A third form of desensitization has recently been described by a similar mechanism. Zn2+ binding to NTD has a positive allosteric interaction with the glutamate-binding site, meaning that when glutamate binds, the receptor affinity for Zn2+ increases. This will lead to a reequilibration with ambient Zn2+ immediately after glutamate binding, with occupancy relaxing to a new equilibrium as Zn2+ binds to the higher-affinity site, which causes receptor inhibition (15,159). Lastly, a fourth form of desensitization that does not involve, Ca2+ , glycine, or Zn2+ has been described. This desensitization may proceed by similar mechanisms as sometimes observed for AMPAR and KAR and appears to accelerate its time course with dialysis (160,161). 3.2. Structural Basis for Fast iGluR Desensitization and Deactivation Like the gating mechanism, the molecular events underlying rapid iGluR desensitization appear complex, and for many years they were generally not well understood. However, recent years have yielded important new hypotheses into the structural mechanism of iGluR desensitization, again accelerated through structural studies on the GluR2-ABD in combination with several extensive functional studies on full-length iGluR (69,104,162–164). Prior to the elucidation of the ABD structure, several point mutations that altered desensitization rates provided initial clues to the location of the domains involved in adoption of the desensitized conformation (164–167). As discussed in Section 2.5, GluR2-ABDs form a dimeric complex in most crystallization studies. In AMPARs, the majority of residues identified as critical for desensitization rates reside at the interface formed between the ABDs in the dimer, implying that the conformation of the “dimer interface” is important for the stability of the desensitized receptor conformation. The recognition of the dimer interface as a potential main determinant for desensitizing drove the recent work by Sun et al. (104) on the structural basis of desensitization. In this study, crystal structures of a wild type and of a non-desensitizing mutant (L483Y) GluR2-ABD were determined in the absence or presence of the classical inhibitor of AMPA receptor desensitization, cyclothiazide. These structures were nearly identical, including the organization of the dimer interface. However, the presence of the non-desensitizing L483Y mutation or cyclothiazide seemed to stabilize the strength of the dimer interface as measured in biochemical experiments in which the kinetics of formation of

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ABD dimers were monitored, showing that blockade of desensitization greatly accelerated the rate of dimer formation in solution. L483 is located in the dimer interface, and mutation to tyrosine promotes formation of additional interactions with residues located on the opposite monomer (Fig. 3C). The dimer interface also contains two binding sites for cyclothiazide, which, when present, mediate additional stabilizing interactions across the interface. In contrast, when Sun et al. (104) examined structures of a GluR2-ABD carrying a mutation that enhances desensitization in full-length GluR2 (S754D), a dramatically different dimer interface was observed, denoted “relaxed.” As in the “non-desensitized” dimer, the relaxed interface is formed between residues on D1, although with a different set of contact points and monomer orientation. Therefore, on basis of the very similar dimer interfaces observed in structures of ABD constructs representing agonist-bound, non-desensitized receptors, Sun et al. (104) proposed that (1) the “constrained” dimer type must represent the resting or activated receptor conformations, and (2) transition into the desensitized state involves rearrangement of the constrained interface, perhaps into the “relaxed” conformation. This led the authors to propose a mechanistic scheme for activation and desensitization in AMPARs in which agonist binding can initiate two different transition pathways. Both include initial cleft closure and adoption of the holo conformation by movement of D2, generating a short-lived transition state with structural tension in the ABD. Subsequently, tension can be relieved by the gating transition in the ion channel without affecting the constrained dimer interface. However, tension also can be relieved by rearrangement of the dimer interface into the relaxed, desensitized conformation instead of movement of the gating domain. The desensitized conformation is likely more energetically favorable than the activated conformation, thereby accounting for the fact that the majority of receptors become trapped in the desensitized state during prolonged exposure to agonist. The hypothesis by Sun et al. (104) implies that the stability of the constrained dimer interface is a major determinant for transitions between the activated and the desensitized states. Corroborative evidence for this idea has subsequently been provided by studies in which introduction of mutations designed to either increase or decrease stability of the constrained dimer interface in general has been found to affect the desensitization kinetics in full-length AMPARs in the predicted manner, for example, by either increasing or decreasing desensitization (102). Furthermore, the importance of the dimer interface to desensitization is supported by the positive correlation between the degree of domain closure observed with partial AMPAR agonists and the degree of desensitization produced by those agonists (70,100,104,168). If degree of agonistinduced D1/D2 cleft closure is the primary determinant for structural tension within ABD in AMPARs, the likelihood of transitions into the desensitized

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conformation must be expected to also correlate with degree of cleft closure. Indeed, a positive correlation between degree of cleft closure and extent of desensitization has been found for most agonists subjected to parallel structural and functional characterization at GluR2 (70,100,104). The Sun model provides a unified mechanism for activation and desensitization in which both processes initially proceed through transitions within the ABD. However, other domains can also influence desensitization. For example, within the short linker segment between D2 and M3, mutation of a single arginine residue to glutamate severely reduces desensitization in AMPARs (169). Furthermore, recent work by Robert and Howe (162) suggests that desensitization is independent of the number of subunits occupied by an agonist; for example, a single occupied subunit can promote transitions into the desensitized state. Most likely, the details of AMPAR desensitization cannot be finally elucidated until structures of full-length AMPAR become available, preferably in each of the three basic conformations: resting, active, and desensitized. In contrast to AMPARs and KARs, NMDARs do not exhibit fast desensitization. The recent structure of a NR1/NR2A dimer shows an arrangement of the ABDs in which intermonomer interactions between D1 and D2 contribute to the interface (80). This structure indicates a distinct mechanistic role of the dimer interface in NMDARs. One of the unique hallmarks of NMDAR function is the slow deactivation (the transitions leading to closing of the channel and unbinding of the agonist) compared to AMPARs and KARs. In AMPARs, ligands that modulate receptor deactivation (aniracetam and CX416)(Table 1) bind to sites in the D1–D1 dimer interface and stabilize the non-desensitized constrained interface at a site in close proximity to the flexible “hinge” between D1 and D2, consequently stabilizing the closed cleft conformation and preventing cleft opening and agonist dissociation (171). If is interesting that in the NR1/NR2A dimer interface, an NR1 tyrosine (Y535) has interactions with NR2A that closely mimic the interactions made possible by aniracetam in AMPARs, thereby suggesting that the relatively slow rate of NMDAR deactivation is due to this “built-in” modulator of the NR1/NR2A interface and further emphasizing the important role of the dimer interface in iGluR function (80).

4. Quaternary iGluR Structure 4.1. Electron Microscopy Structures of AMPA Receptors The tetrameric stoichiometry for iGluRs has recently been supported by single-particle images of recombinant and native AMPARs receptors obtained through electron microscopy (142,172,173). Although these images show the receptors at low resolution, several structural features can be extracted. First, an internal two fold rotational symmetry is indicated in the receptor structure,

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consistent with the indications that iGluRs assemble as a dimer-of-dimers (64,80,162,174,175). The proposed twofold rotational symmetry for iGluRs is in contrast to the symmetry observed in structures of other ion channels such as tetrameric K+ channels and the pentameric nicotinic acetylcholine receptor, in which the quaternary subunit arrangement leads to rotational symmetries that correlate with subunit number (176,177). However, it has been suggested that the iGluRs contain “symmetry mismatch” between the ABDs on which the dimer-of-dimer model is built and the structure of the ion channel, which should adopt fourfold rotational symmetry (128), thereby implying that the K+ -channel model for the structure and function of the iGluR ion channel retains its legitimacy. Recent studies utilized antibodies to localize the ABD and NTD in low-resolution images of AMPAR surface contour (172,178). These approaches further allowed an evaluation of surface contour and thus ABD and NTD localization in the absence and presence of ligand. Nakagawa et al. (172) suggested, based on using this approach, that continued presence of ligand causes substantial rearrangement of the position of the NTD, which they interpreted as desensitization. However, heterologous expression of homomeric GluR4 receptors with truncation of the NTD still generates AMPARs with unchanged receptor function in terms of rapid desensitization and deactivation, suggesting that the NTD is not crucially involved in these mechanisms (2). Still, the electron microscopy data are intriguing, and if similar changes in shape accompany desensitization in receptors in intact membranes and synapses, it would represent an important new advance in our appreciation of receptor function. 4.2. Auxiliary Subunits The iGluRs engage in numerous protein–protein interactions with intracellular postsynaptic proteins at different time points during receptor trafficking and positioning in the synapse. The majority of these molecular interactions involve the intracellular C-terminal domains that contain recognition sites for a number of well-described postsynaptic anchoring and scaffolding proteins (42). The transmembrane AMPA receptor regulator proteins (TARPs) are the only known type of iGluR interacting proteins that are integral membrane proteins (179). TARPs selectively interact with iGluRs from the AMPAR subfamily early in the synthetic pathway and are critical for proper expression and positing of the receptor into the cell surface (180–185). Recently, TARPs have been shown not only to interact with AMPARs during trafficking, but also to be mandatory components in the majority of AMPAR complexes in the brain, suggesting that TARPs in fact are “auxiliary” subunits for the mature AMPARs (178,186). In the electron microscopic structures of native AMPARs, the presence of TARPs is clearly observed as an enlargement of the transmembrane domain of the receptor (172). Furthermore, association of the TARP

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subtype -2–also known as stargazin–with AMPARs has recently been found to influence several important functional properties of the receptors leading to enhanced response to glutamate (187–189). When expressed with recombinant AMPARs, stargazin increases channel conductance and reduces desensitization (187,188,190). The topological structure of TARP proteins shows that they have four transmembrane domains with intracellular N- and C-terminal domains. So far, the only data regarding the structural basis for TARP regulation of AMPAR function come from a study in which a series of TARP chimeras between stargazin (-2) and another TARP family member (-5) that do not enhance receptor function were used to map the first extracellular loop of stargazin to be critical for effects on receptor function, perhaps by formation of direct interactions with structure elements in the linker regions or the ABD that are involved in gating and desensitization (179,188). The discovery of the role of TARPs as functionally important auxiliary AMPAR subunits has come as a surprise, and a number basic questions regarding the role and mechanism of TARP regulation of AMPARs need to be answered, including structural questions such as the stoichiometry and orientation of TARP proteins relative to AMPAR subunits and the identity of regions and residues on the AMPAR subunits directly interacting with TARPs.

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7 Positive Modulators of AMPA-Type Glutamate Receptors Progress and Prospects Gary Lynch and Christine M. Gall

Summary Ampakines are small synthetic compounds that cross the blood–brain barrier and enhance fast excitatory synaptic responses mediated by -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)–type glutamate receptors. Their binding site has been identiﬁed and shown to be appropriately located for slowing receptor deactivation and desensitization, the two processes that terminate excitatory transmission. After reviewing these mechanisms, this paper takes up the question of why ampakines, although targeted at receptors found throughout the brain, produce surprisingly discrete behavioral effects. Three processes are hypothesized to be involved: (1) the facilitation of excitatory inputs to inhibitory interneurons balances the effects of ampakines on glutamatergic neurons; (2) the compounds have preferences for receptor subtypes and thus for particular brain regions; and (3) the potency of the ampakines scales positively with the complexity of brain networks. The discrete effects of the drugs raise the possibility of using them in the treatment of psychiatric disorders. Ampakines improve performance of complex behaviors in rodents and primates, accelerate learning across a diverse array of tests, and have positive effects in animal models of schizophrenia, attention deﬁcit hyperactivity disorder (ADHD), and depression. Improved memory scores and a reduction in ADHD symptoms are also reported in studies with human subjects. It is hypothesized that these behavioral effects reﬂect facilitation of communication within cortex, a lowered threshold for the From: The Receptors: The Glutamate Receptors Edited by: R. W. Gereau and G. T. Swanson © Humana Press, Totowa, NJ

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induction of long-term potentiation, and enhanced cortical regulation of lower brain systems. Finally, ampakines upregulate the production of brain-derived neurotrophic factor (BDNF). Experimental work prompted by this observation indicates that chronic ampakine treatments cause sizeable improvements in animal models of Parkinson disease, excitotoxic brain damage, and age-related losses of synaptic plasticity. Key Words: Ampakine; memory; long-term potentiation; brain-derived neurotrophic factor; cognitive disorders; hippocampus.

1. Introduction -Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are not an obvious target for pharmaceuticals. The receptors mediate fast excitatory transmission throughout the central nervous system, and so one could reasonably assume that positive or negative modulators would produce gross disturbances to essential brain operations. It is hard to imagine, for example, that changing the strength of excitatory input to the dorsal motor nucleus of the vagus nerve could have anything other than disastrous consequences. That the first ampakines—positive AMPA receptor modulators that cross the blood–brain barrier and enhance fast excitatory post synaptic potentials (EPSPs)—produced subtle effects thus came as something of a surprise. The drugs at physiologically effective doses did not noticeably disturb complex behaviors, alter motivation, or elicit evidence of emotional reactions (1–3). Later work with humans confirmed that moderate doses of ampakines are not experienced in any way different from placebos (4). Yet the drugs have sizeable effects on learning and in animal models of various psychiatric diseases (see later discussion). Explaining this selectivity is a primary issue for studies on ampakines; this review will survey experimental work and recent hypotheses related to the question. The reasons for developing ampakines range from the subtle to the obvious. In principle, at least, the drugs could be used to compensate for reductions in the numbers or strength of glutamatergic synapses, conditions that are thought to obtain in Alzheimer disease and possibly other neuropsychiatric disorders. It is also the case that AMPA receptors provide the depolarization needed to unblock the N-methyl-d-aspartate (NMDA) receptors that induce long-term potentiation (LTP), a likely substrate of memory; therefore, enhancing AMPA receptor currents could, in the absence of prominent side effects, accelerate learning. Other possibilities arose when it was found that ampakines have particularly large effects in the cortical telencephalon (2,5). It has long been held that cortex acts as a brake on lower brain systems that generate and/or modulate psychological states (6–10); if so, then ampakines, by increasing transmission in cortex and its outputs, could potentially normalize activity in

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ascending systems implicated in diseases such as schizophrenia and depression. The considerable work that has gone into testing these potential applications constitutes the second topic reviewed here. The trajectory of ampakine research changed following the discovery that the compounds upregulate the production of neurotrophins by adult forebrain neurons (11). Prior studies had shown that intense neuronal activity induces the neurotrophins brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in hippocampus and cortex of adult rodents (12–16), but it was not known whether modest increases in excitatory drive would produce similar effects. The demonstration that this is indeed the case prompted a successful effort to develop ampakine treatment regimens that produce chronic increases in BDNF protein levels (17). BDNF promotes survival and growth in a variety of neuronal populations (18,19), has positive effects in animal models of degenerative diseases including Parkinson (20–22) and Huntington (22–25) disease, and is argued to offset depression (26,27). A recent and rapidly growing body of experimental work indicates that the neurotrophin also facilitates the formation of LTP (28–31); in fact, it appears to be the most potent endogenous modulator of the potentiation effect so far discovered. Induction of BDNF expression by ampakines thus might have beneficial effects on a broad array of pathologic conditions, as well as producing a chronic enhancement of memory encoding. A survey of work on ampakines and neurotrophins and on the critical question of why BDNF so powerfully affects LTP forms the third topic of this review. Before taking up the foregoing material, it is necessary to first consider the mechanism of action of the ampakines and the subcategories into which the compounds fall.

2. Mechanism of Action 2.1. Ampakines and Receptor Kinetics A combination of site-directed mutagenesis and X-ray crystallography experiments have provided a detailed hypothesis about the operation of AMPA receptors and the manner in which it is affected by ampakines (32–35). Figure 1 is a highly schematic picture of the AMPA receptor, various features of which have been ignored or distorted so as to emphasize points related to drug action. The receptor is a tetramer composed of homologous subunits (GluR1–4) that can be assembled in a variety of combinations (e.g., one GluR1, two GluR2s, one GluR3). The subunits come in two RNA splice variants labeled “flip” and “flop” (36). Flip and flop receptors differ in their biophysical properties, including the rate at which they desensitize (see later discussion) and in their distribution across brain regions and cell types (36,37). Each subunit of the tetramer has two large extracellular domains (A and B in Fig. 1A) connected so as to form a “V” that encloses the transmitter binding site (32,34). It is

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Fig. 1. Ampakines and the operation of -amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA)–type glutamate receptors. A. AMPA receptors are composed of four subunits that assemble into two dimers. Each subunit has two extracellular domains that connect to form a V-shaped structure. The binding site for glutamate is contained within the interior of the V (black oval). Binding causes the domains to move together (dashed arrows), thereby creating tension on the transmembrane segment of the subunit. When this occurs across the multiple subunits, the channel formed by the transmembrane segments opens; this is the onset of the excitatory postsynaptic current (EPSC). The current terminates when the domains move away from each other (“deactivation”) or when binding disturbs the dimer

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significant that there is good evidence (34) that the subunits pair up, resulting in a receptor with two dimers and four transmitter sites. The ampakine binding pocket is located at the dimer interface, near the junction of the “V” in each member of the dimer (Fig. 1A) (32). Receptor dynamics are thought to operate as follows. Binding of the transmitter causes a closure of the extracellular “V’s”, an effect that creates tension on the transmembrane region of the subunit. When this occurs across the multiple subunits, the receptor pore, formed by the transmembrane regions of the subunits, opens, and sodium flows through the membrane. This is the rising phase of the excitatory postsynaptic current (EPSC). The synaptic current terminates when one of two events occurs. First, the extracellular domains separate, and the transmitter is released. This relaxes the tension on the transmembrane region, and the receptor channel closes, a sequence referred to as deactivation. Second, the binding event disturbs dimerization, resulting in a situation in which the transmitter remains bound but the receptor channel closes; this mode of terminating the synaptic current is called desensitization. As described later, deactivation appears to be the predominant means for ending AMPA receptormediated synaptic currents, with the effects of desensitization typically being restricted to periods of high-frequency synaptic activity. Ampakines, by binding to a site near the juncture of the two extracellular domains (32), cause the “V” formed by the domains to reopen more slowly than normal; this slows deactivation by delaying the release of the transmitter. However, the ampakine site is also situated at the interface of the dimer; binding there tends to stabilize the dimer configuration and thus reduces the likelihood that receptors will shift into the desensitized state (32,38). In all, Fig. 1. (Continued) configuration (“desensitization”). Ampakines (white oval) bind to a site in the dimer interface near the hinges of the V’s formed by the two extracellular domains of each subunit in the dimer. From this position, ampakines delay the reopening of the extracellular domains and stabilize the dimer configuration; thus they slow the two processes that terminate the synaptic current. B. There are several structurally distinct families of positive modulators, as illustrated with these physiologically effective examples. C. The manner in which ampakines affect synaptic responses; the upper records in each case were collected immediately prior to infusion of the compounds. Some ampakines have a substantially greater effect on deactivation than on desensitization; these variants (“deactivation type”) increase the amplitude of the excitatory postsynaptic potential (EPSP) but have somewhat smaller effects on its duration. Most ampakines, however, affect both deactivation and desensitization, although often to different degrees; these compounds (“deactivation + desensitization type”) increase both the amplitude and half-width of the synaptic response. A: Based on refs. 32, 34, 48, and 54.

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then, ampakines increase the time constants for the two biophysical processes that terminate the synaptic current. 2.2. Categories of Ampakines It is important to recognize that various ampakines differentially affect deactivation and desensitization. The original compounds were benzamides, but work since then has resulted in benzothiadiazide (39), pyridothiadiazine (40), biarylpropylsulfonamide (41), and alkyl-benzothiadiazide (42) derivatives that all appear to be centrally effective (Fig. 1B). Some of these agents (e.g., CX516) have much larger effects on deactivation than on desensitization, whereas others (e.g., cyclothiazide) exhibit the reverse order of potency (43). The large majority of ampakines affect both processes (43,44), although typically not to same extent. As might be expected, these distinctions have important consequences for how the various ampakines affect synaptic transmission. Versions that predominantly affect deactivation increase the amplitude of the EPSP while producing relatively smaller changes in its duration (Fig. 1C). Compounds strongly biased toward desensitization have only minor effects on single excitatory post synaptic currents (EPSCs) (43), although they do prolong the responses generated by 40- to 50-Hz afferent activity (45,46). Together, these results suggest that deactivation regulates the waveform of the EPSC under most conditions, with a contribution from desensitization appearing as responses occur rapidly enough that (1) a significant percent of the receptor pool is occupied and (2) successive inputs fall within the time course of desensitization. Finally, ampakines that slow both deactivation and desensitization increase the amplitude of the EPSC and prolong its duration (Fig. 1C). This result suggests that desensitization plays a major role in terminating the synaptic current when deactivation has been slowed. If deactivation is only slightly more rapid than desensitization, then modest slowing of the former by an ampakine would result in the latter becoming the likeliest means for closing the receptor channel. That the three subclasses of ampakines (deactivation type, desensitization type, deactivation and desensitization type) have substantially different effects on synaptic physiology, strongly implies that they will also differ in important ways in how they influence brain physiology. This expectation has been confirmed with regard to seizures, LTP, and the induction of growth factors (see later discussion).

3. The Selective Effects of Ampakines The initial behavioral experiments with ampakines found that the drugs, at doses that enhanced synaptic responses in vivo, produced no overt disturbances in tests in radial mazes and water mazes (1,2,47). Further work extended this

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selectivity to tests of reaction time, motivation, and motor performance (3). The question then arose as to why drugs that act globally on the large majority of synapses in the brain do not produce more dramatic changes in physiology and behavior. Work over the last decade indicates that three distinctly different factors are responsible for the selectivity of ampakines. 3.1. Ampakines Increase Both Excitation and Inhibition Figure 2 illustrates a conventional view of the local circuit organization in hippocampal field CA1. As shown, there are three sites at which positive modulators of AMPA receptors might act: (1) the glutamatergic inputs to the target pyramidal cells; (2) the synapses formed by these same inputs on GABAergic interneurons that provide feedforward inhibition to the target pyramidal neurons; and (3) the connections between the recurrent collaterals of the glutamatergic pyramidal cells and feedback GABAergic interneurons. Assuming that the ampakine acted equally on these three sets of AMPA receptors, then the response of the pyramidal cell would be composed of an enhanced monosynaptic EPSC followed quickly by an enhanced disynaptic

Fig. 2. Design of local circuits in the cortical telencephalon. Shown are two GABAergic neurons (gray circles), representing two broad classes of interneurons, and a single glutamatergic pyramidal neuron (white circle with an apical process). In reality, the pyramidal cells far outnumber the interneurons. Three classes of circuits are illustrated: (1) monosynaptic excitatory between the input and the pyramidal neuron; (2) disynaptic, feedforward connections between (a) the input and a GABAergic cell, which then (b) forms an inhibitory synapse with the pyramidal cell; (3) a trisynaptic feedback series in which (a) the excitatory input activates the pyramidal cell, which then (b) forms a glutamatergic (excitatory) synapse with the second interneuron, which then (c) synapses with the pyramidal neuron. -Amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptors are present on the two (-aminobutyric acid (GABA) cells as well as in the initial contact with the glutamatergic neuron; ampakines thus, in principle, will enhance both excitation and inhibition of the pyramidal cell. Experimental studies have confirmed this prediction.

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inhibitory postsynaptic current (IPSC). If the first of these is sufficiently large, then the net response of the circuit will be a brief period of enhanced spiking followed by enhanced suppression of further spiking (Fig. 2). The idea that ampakines enhance disynaptic, feedforward inhibition was confirmed in early studies on the compounds (48). More recent work demonstrated that ampakines increase EPSCs on feedforward and feedback GABAergic interneurons and made the further point that the magnitude of this effect varies across compounds and classes of interneurons (49). The flow of activity through cortical networks is mediated by excitatory transmission and maintained within acceptable limits by local inhibition. It appears that behaviorally effective doses of ampakines, by increasing both of these processes, do not cause network activity to reach pathophysiologic levels. However, at high concentrations, the compounds appear to overcome inhibitory regulation and initiate epileptiform activity. 3.2. Preferences for Receptor Subtypes As noted, AMPA receptors are made up of any of several combinations of subunits; different brain regions express particular subunits to different degrees (50–52), making it likely that the subunit combinations vary across areas. Moreover, some neurons express one splice variant, whereas others have the second. Field CA3 of hippocampus, for example, is greatly enriched in flip receptors, whereas field CA1 contains flop variants (36). Certain ampakines are known to have very different flip/flop affinities (53–57), which makes it very likely that the drugs will have correspondingly stronger effects in regions expressing their preferred receptor subtype (58). The idea that most ampakines will act in a regionally differentiated manner was confirmed in studies describing a fourfold-greater effect of one variant in hippocampus as compared to the thalamic reticular nucleus expressing a different combination of subunits (59). Much more work is needed in this area, but regional selectivity helps to explain why ampakines that increase activity in cortical networks do not produce evidence of hyperactivity in subcortical regions. 3.3. Network Complexity Unlike the case for other transmitter systems, glutamatergic neurons connect together to form networks that have serial, parallel, and positive feedback features. There are no precedents for predicting the types of functional effects that might follow from enhancing all connections within such systems. Moreover, glutamatergic networks clearly vary in complexity, from the relatively simple reflexes of spinal cord and brainstem to the incredibly elaborate organizations proposed for the cortical telencephalon. How

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complexity affects drug action is thus a question that arises in dealing with glutamate receptor modulators Figure 3 describes an experiment that tested the effects of an ampakine on the input and output stages of the three-stage (dentate gyrus, field CA3, field CA1) intrahippocampal circuit (60). A single stimulation pulse was delivered to the

Fig. 3. Effects of ampakines in complex networks. A. Design of an experiment in which the effects of an ampakine were tested simultaneously on monosynaptic

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perforant path, and recordings were made in the molecular layer of the dentate gyrus (monosynaptic) and in the apical dendrites of field CA1 (trisynaptic connection). Note that the field EPSP recorded in the latter location occurs after an approximately 10-ms delay, confirming that the response depends on transmission across multiple steps. The ampakine concentration was selected to be at threshold for the monosynaptic EPSP. As shown, the compound had much larger effects in field CA1 than it did in the dentate gyrus, suggesting that its actions scale with the number of synaptic stages involved in generating the test response. Related studies found that the threshold for the ampakine is about threefold lower when measured on polysynaptic versus monosynaptic hippocampal responses (61). The complexity results are not unexpected, given that positive modulation can cause a much larger percentage increase in the probability of a cell Fig. 3. (Continued) and trisynaptic stages of the principle intrahippocampal circuit. Single stimulation pulses were delivered to the perforant path projections from cortex to the dentate gyrus, and field excitatory postsynaptic potentials (EPSPs) were recorded in the dentate gyrus (monosynaptic) as well as at the terminus (in field CA1) of the two-stage network formed by the mossy fibers and Schaffer-commissural projections (trisynaptic). Note that the latter response had a delayed onset after the stimulus artifact, confirming its polysynaptic nature. Infusion of a low concentration of ampakine produced a <10% increase in the dentate gyrus response (asterisk) while more than doubling the trisynaptic CA1 EPSP. B. The all-or-none nature of cell spiking introduces a nonlinear element to ampakine actions. The cell on the left has several active synapses that collectively generate an EPSP that is near threshold for triggering an action potential. The right-hand graph shows the relationship of the depolarizing postsynaptic responses generated by the input to the probabilistic spike threshold; the middle of the depolarization range produced by the input falls in the low-probability portion of the discharge curve. Adding a threshold concentration of ampakine has little effect on the synaptic responses (“response”) but, because of the steep depolarization/spike probability curve, causes a marked increase in the likelihood of the target cell generating an action potential. C. Increasing spike probability can explain the amplification of ampakine effects in complex networks. Case A illustrates a balanced series of networks in which input and output magnitudes are roughly equal. Case B shows a hypothetical case in which transmission strength at individual synapses is slightly reduced from normal. Because of the nonlinearity of the depolarization/spike probability curve, a 10% decrease in input strength results in the loss of one third of the responding cells. This effect accumulates across the successive stages of the circuit, such that the third stage generates no output at all. Accordingly, EPSP size at connections between networks falls drastically from the input (monosynaptic) stage to the third (trisynaptic) stage. Adding an ampakine would restore transmission to normal levels and return the system to the condition found in case A; the drug in this case would have the disproportionate effect on the polysynaptic EPSP shown in panel A.

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spiking than it does on the EPSCs that trigger the spike (Fig. 3B). Applied to networks where large numbers of cells are brought close to action potential threshold by typical-sized inputs, the disproportionate action on spiking would have the dramatic effects observed in the trisynaptic hippocampal network (Fig. 3C). These results lead to the hypothesis that the potency of an ampakine on a given behavior will depend on the complexity of the networks engaged by that behavior. If the most complex networks used by the brain are indeed those (1) found in the cortical telencephalon and (2) activated during cognitive behaviors, then this argument predicts that ampakines will have pronounced effects on restricted classes of measures. Evidence in support of this argument is described later.

4. Potential Applications of Ampakines The parameters that describe fast excitatory transmission in cortex are the result of physical limits of the system and a long evolutionary history, much if not most of which occurred in a context unrelated to current environments. The extent to which these parameters are in any sense close to optimal for dealing with challenging cognitive problems cannot be estimated from current information about the operation of complex networks. The same can be said for the characteristics of LTP, the presumed substrate of memory within cortical systems. Ampakines adjust the parameters for both synaptic transmission and LTP, and testing how this might affect cognitive-type behaviors was a primary motive for developing the compounds. Beyond this, there are reasons to suspect that certain disease states involve impaired transmission in cortex and/or impairments to LTP. Thus, independent of their effects on normal operations, there was a real possibility that ampakines would have important therapeutic benefits. 4.1. Effects on Complex Behavior Figure 4 describes the performance of monkeys presented with a cue and then, after delays of 5–60 sec, required to pick that cue out from a group of similar stimuli (i.e., delayed-match-to-sample). The stimuli are changed between trials, so long-term memory functions are minimized. The graph describes performance accuracy as a function of the number of objects presented in the choice phase of the paradigm and the delay between presentation of the sample and the choices. This is clearly a difficult problem for monkeys, involving the application of a learned protocol to specific instances, attention, and a short-term memory system. As shown in Fig. 4A, ampakine treatment produced a marked improvement in performance, particularly on the most complex version of the test (i.e., choose from six objects) (62). Similar

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Fig. 4. Effects of an ampakine on complex behavior and cortical activity in a primate. A. Monkeys were given a visual cue and then required to select it, after delays ranging from 5 to 30 sec, from a set of two to six similar cues in order to obtain a reward (delayed-match-to-sample). The graphs show the percentage of correct choices as a function of (1) the delay between the stimulus and choice and (2) the number of items presented during the choice phase of the test. As shown, an ampakine markedly improved the accuracy of the selections, particularly on the most difficult

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results were obtained in rats performing a much simpler version of the task (63). Together these results suggest that positive modulation of fast excitatory transmission improves performance of complex behaviors. The foregoing findings provide a first (and negative) answer to the question of whether excitatory transmission in cortical networks is in any sense optimized for cognition but leave open the issue of why AMPA receptor upmodulation enhances performance. One possibility is suggested by the earlier arguments on complexity: If ampakines increase the reliability of communication within the cortical telencephalon, then they should have the effect of increasing the size of the networks that can be engaged by behavior. This could be a real advantage in dealing with difficult problems that presumably require large networks. This idea was investigated in the primate studies by mapping brain activity during performance of the delayed-match-to-sample problem in the presence and absence of the ampakine (62). Figure 4B illustrates a typical result. Performance under placebo conditions was accompanied by high levels of activity, relative to those found in an awake but inactive state, in the dorsal frontal cortex, the medial temporal lobe, and the somatosensory cortex contralateral to the hand used to make choices. The ampakine increased activity in the two associational areas but did not affect it in the primary sensory cortex. Although the observed drug effects could reflect increased firing by a fixed population of cells, a more likely explanation is that additional neurons were recruited into the networks producing the behavior. Note also that the positron emission tomography (PET) scan results provide a striking example of the degree to which ampakines act in a regionally selective fashion associated with specific behaviors.

Fig. 4. (Continued) trials (i.e., those involving six choices). B. Positron emission tomography images from a monkey performing the match-to-sample problem under the influence of a placebo or an ampakine. Left: Cortical activity during performance versus the awake state outside the test situation. The dorsal frontal cortex (DPFC), the medial temporal lobe (MTL), and the somatosensory cortex (S1) were active during the task. Right: The difference during task performance following placebo versus ampakine injections. Note that the frontal and temporal associational areas, but not the primary sensory area, engaged by the task under control conditions are enhanced by the ampakine. The compound also increased aggregate cortical activity in the precuneus, a large associational region that was not involved in the task under placebo conditions. Modified from Porrino LJ, Daunais JB, Rogers GA, et al. Facilitation of task performance and removal of the effects of sleep deprivation by an ampakine (CX717) in nonhuman primates. PLoS Biol 2005;3:e299. Available at http://plos.org/journals/ index.html.

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4.2. Memory Encoding Early work demonstrated that ampakines lower the threshold for the induction of LTP by increasing the depolarization needed to unblock the NMDA receptors that trigger the potentiation effect (47). As expected from this, the compounds improve retention scores in a diverse array of testing paradigms; it is noteworthy that the potency of the compounds in these tests corresponds to that for their physiologic effects [e.g., 64]. Positive results were obtained both for forms of memory that normally decay over hours and for long-term memory (Table 1). Ampakines do not increase arousal or produce other behavioral effects that might account for the improvements in memory. They cause a modest reduction of exploratory activity by rats in open fields, but even this effect is absent in radial mazes (64). Tests for drug-related changes in response latencies and motivation were also negative (3). Moreover, there was no evidence for side effects that might account for improved retention in human subjects. In the latter studies, the tested ampakine (CX516) produced substantial improvements in nonsense syllable recall in elderly subjects (65,66)

Table 1 Ampakines facilitate learning in various paradigms

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and, at lower doses, significantly increased retention scores in a variety of tests carried out with young adults (4). There were no detectable effects on heart rate, visual recognition, or motor performance (4). When asked, the subjects could not report whether they had taken a placebo or the ampakine. It should be noted the ampakine used in these human trials has modest potency and a short half-life; a critical issue for future research is to determine whether the same absence of psychological side effects obtains with the more powerful variants used in animal experiments. The results from the preclinical work on long-term memory suggest that the tested ampakine accelerated learning without increasing the strength of memory. Thus, for example, extinction of conditioned fear proceeded as rapidly in treated rats as in controls (67). Recent work showed that the ampakine used in these experiments (CX516) reduces the number of theta bursts needed to induce LTP but does not increase the degree of potentiation produced by an optimal theta train; that is, it lowered the threshold but did not raise the ceiling for LTP (68). This would predict the observed acceleration of learning without an increase in memory strength. CX516 belongs to the subgroup of ampakines that slow deactivation with little effect on desensitization and, as a result, increase the amplitude but not the duration of EPSPs (see prior discussion). Variants that do prolong synaptic responses (i.e., that slow deactivation and desensitization) lower the LTP threshold and greatly increase the percentage potentiation produced by conventional theta trains (68). The observed differences between the two types of ampakines accord with the hypothesis that the duration of the fast EPSP regulates the LTP ceiling (69). A question of great interest is whether, as might be expected from the LTP results, the differences will be reflected in the strength of learning. If this proves to be the case, then it will be possible to explore the extent to which two primary LTP parameters—ease of induction and magnitude—are in any sense close to optimum for complex learning. 4.3. Regulation of Lower Brain Systems Linked to Neuropsychiatric Disorders The various subcortical systems targeted by ongoing efforts to develop psychiatric medicines are regulated by descending glutamatergic projections from cortex. Thus, the advent of AMPA-receptor modulators opened the way for testing whether enhanced transmission, within both the neocortex and presumably its efferents, would help to normalize disturbances arising from aberrant activity in these target systems. Initial experiments in this area found that ampakines markedly reduce the stereotyped behavior produced in rats by methamphetamine and the excessive dopaminergic activity it produces (70). Subsequent studies found that ampakines act synergistically with typical and

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atypical neuroleptic drugs in correcting the repetitive behaviors induced by amphetamine (71,72). Other work on dopamine-related diseases showed that ampakines essentially eliminate locomotor hyperactivity in a mouse model of attention deficit hyperactivity disorder (ADHD) (73). As with learning, the potencies of the compounds in suppressing abnormal behavior and in enhancing synaptic responses were correlated. A recent clinical study of patients with adult ADHD showed that daily ampakine treatments reduced hyperactivity scores by 6 points on a conventional rating scale, a result that was highly significant relative to changes seen with placebos (9 points on this scale is the distance between the means of diagnostic categories; i.e., “severe” to “moderate” to “mild”) (H. Mansbach, Cortex Pharmaceuticals, unpublished data). There is evidence supporting the idea that ampakines produce the aforementioned effects by increasing activity within the neocortex. Early experiments using expression of the activity-dependent gene c-fos during exploration of a novel environment demonstrated that an ampakine at doses that suppress behavioral hyperactivity increased aggregate neuronal activity in cortex relative to that found in the striatum (5). Subsequent work measured c-fos expression in a portion of cortex needed to correct circling behavior induced by (1) unilateral 6-hydroxydopamine lesion of the ascending dopamine projections and (2) sensitization and then acute treatment with methamphetamine (74). Ampakines blocked methamphetamine-induced rotational behavior and increased aggregate neuronal activity in the cortical areas controlling forelimb movements as assessed by levels of c-fos expression. Moreover, the c-fos analysis indicated that the compounds had a greater effects on the dopaminedepleted side responsible for suppressing the circling behavior (72). In addition to showing that ampakines increase cortical activity related to the control of subcortical abnormalities, the bilaterally differentiated effect in this paradigm dramatically illustrates the argument that they act selectively on complex circuits engaged by behavior. Ampakines are also reported to have strong, positive effects in animal models of depression (75,76), a disease with a major serotonergic component. In one experiment, the dose–response curve for conventional antidepressant drugs was shifted fivefold to the left, indicating that compounds interact synergistically with antidepressants. These synergies support the hypothesis that various psychiatric disorders arise from an imbalance between activity in subcortical systems related to the disorder and the cortical systems that normally regulate such activity (10,77). Disturbances in both components of the feedback loop proposed by such models would tend to have multiplicative effects; similarly, drugs acting at both ends of the loop should positively interact with regard to restoring balance in the system.

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5. Ampakines and Neurotrophic Factors 5.1. Ampakines Upregulate BDNF The discovery that intense neuronal activity triggers the production of neurotrophins (12,14) was followed by studies showing that increased expression could be elicited by afferent stimulation comparable to that used to induce LTP (78) and, more recently, by engagement in specific behaviors such as wheel running (79) and learning (80,81). Tests of whether the increases in excitatory drive produced by ampakines are sufficient to upregulate neurotrophins in mature neurons were originally carried out using cultured hippocampal slices (11) and confirmed for dissociated neurons using a different structural variant of the compounds (82). Figure 5 illustrates a typical result in the slice model; a 6-hr infusion of the ampakine produced a profound increase in BDNF mRNA levels throughout the principal cells of hippocampus and retrohippocampal cortex. BDNF protein levels were elevated along with the mRNA. Electrophysiological recordings showed that the drug increased overall firing rates of the neurons but did not produce signs of pathophysiology such as epileptiform discharges. Upregulation of BDNF, although to a lesser degree than was obtained in the slices, was also found in vivo following a single

Fig. 5. Ampakine treatment increases neuronal brain-derived neurotrophic factor (BDNF) expression. Photographs of film autoradiograms show 35S-cRNA in situ hybridization labeling of BDNF mRNA in cultured hippocampal slices harvested without treatment (A) (control, Con) or after treatment with the ampakine CX614 (3 hr, 50 μM) alone (B), CX614 plus the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) (C), or CX614 plus the N-methyl-d-aspartate (NMDA) receptor antagonist 2-amino-5phosphonovaleric acid (APV). As shown, ampakine-induced increases in BDNF mRNA content are blocked by CNQX but not APV. EC, entorhinal cortex; sg, stratum granulosum; sp, stratum pyramidale. Calibration bar = 300 μm. From Lauterborn JC, Lynch G, Vanderklish P, et al. Positive modulation of AMPA receptors increases neurotrophin expression by hippocampal and cortical neurons. J Neurosci 2000;20:8–21.

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intraperitoneal injection with an ampakine that has a half-life of <40 min (11). Antagonists of L-type calcium channels depressed the in vitro effects of the ampakine in some hippocampal subdivisions, whereas AMPA-receptor blockers uniformly eliminated the increases in BDNF mRNA. Surprisingly, NMDA-receptor antagonists were ineffective. Many of the potential therapeutic applications involving BDNF require that elevated production of the neurotrophin be maintained for many days. Tests of whether ampakines can produce such chronic effects demonstrated that daily 3-hr treatments in cultured slices resulted in severalfold increases in neurotrophin mRNA and protein that stayed in place largely unchanged across 5 days of testing (17). As will be described later, there is evidence that even shorter treatment periods will suffice to produce a chronic increase in BDNF levels. One other result emerging from these studies is likely to be of critical importance in the development of an ampakine strategy for manipulating BDNF production: Compounds that slow both deactivation and desensitization were far more effective in upregulating the neurotrophin than were variants that acted on deactivation alone [11; C. Gall and J. C. Lauterborn, unpublished observations]. This indicates that prolongation of excitatory synaptic responses is needed to induce BDNF expression. This could relate to the evidence implicating voltage-sensitive calcium channels in regulatory mechanisms; that is, longer periods of depolarization may be needed to produce an influx of the cation sufficient to initiate signaling pathways to the genome. 5.2. Potential Applications of Ampakine-Induced Increases in BDNF There appear to have been no attempts to use chronic elevation of BDNF in animal models of neurodegenerative diseases, perhaps the most likely application of the technology. However, daily injections of ampakines are reported profoundly to reduce the loss of dopaminergic terminals in the caudate following partial 6-hydoxydopamine lesions of the ascending nigrostriatal projections (83). These effects could be obtained when the injections were started hours or even days after the lesions and were associated with increases in growth-associated protein-43 (GAP-43) expression, thereby suggesting that the effects were at least in part due to sprouting of undamaged fibers. Moreover, the anatomic sparing was accompanied by substantial improvements in neurologic symptoms (83). A number of studies have shown that BDNF stimulates axonal growth, and it is therefore possible that upregulation contributed to the results in the lesion study. A second experiment demonstrated that acute and chronic ampakine treatments were neuroprotective against excitotoxic brain damage in a mouse model (84). The authors showed that the drug treatment caused a pronounced increased in BDNF production in the target areas, and

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there is a significant body of work indicating that the neurotrophin can reduce cell death with excitotoxic insult (24,85). In addition to its pro-survival and growth-inducing effects, BDNF acts acutely at synapses to promote LTP (28–31,86,87). This involves both a lowering of the threshold for the potentiation effect and an increase in its maximum value (88,89). Moreover, these effects are obtained with brief applications of very low (2 nM) concentrations of BDNF (29). It is significant that the pronounced influence of BDNF is observed with theta burst stimulation but not with long trains of high-frequency stimulation (31), indicating that the neurotrophin acts as a modulator rather than an essential ingredient in the production of LTP. Two observations suggest that this modulatory influence is necessary under physiologically plausible conditions: (1) theta burst stimulation, which mimics patterns of activity seen during learning (90,91), is particularly well suited for triggering the release of BDNF (92), and (2) agents that scavenge released BDNF, and thereby competitively block association of the neurotrophin with its TrkB receptor, prevent the formation of theta-induced LTP (29,30). These results raise the possibility of chronically enhancing LTP by stably elevating BDNF levels with periodic injections of an ampakine. Tests of this idea were conducted using the recently discovered LTP deficit in the hippocampus of middle-aged rats. This deficit appears in the basal but not apical dendrites of field CA1 pyramidal cells by 7 months of age (93) and thus can be considered as a consequence of early aging. Ongoing studies led to the surprising finding that upregulation of protein levels in middle-aged rats could be achieved, and sustained for days, with twice-daily intraperitoneal injections of a drug variant with a 15-min half-life (94). Accordingly, slices were prepared from middle-aged rats 1 day after 4 days of treatment, a time point at which the ampakine would be gone from the system but BDNF concentrations were still well above normal. Under these conditions, normal LTP was restored to the basal dendrites (Fig. 6). Tests of whether the rescue of potentiation is due to increased BDNF release during theta stimulation will probably require identifying the mechanisms whereby the neurotrophin promotes LTP and then determining whether these signaling pathways are enhanced following chronic ampakine treatment. Some progress has been made along these lines. Infused BDNF enhances the postsynaptic response to theta stimulation (29), but subsequent work suggests that endogenous neurotrophin does not produce this effect (C. S. Rex et al., unpublished results). This makes it likely that the modulator acts at some aspect of LTP that lies downstream from initial induction and expression events. Continuing progress in identifying the cellular mechanisms of LTP consolidation engaged in the several minutes after theta burst stimulation (88) will point to signaling pathways that are likely to be responsive to BDNF.

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Fig. 6. Reversal of age-related loss of synaptic plasticity following chronic treatment with an ampakine. A, B. Micrographs illustrating the features of the system used to test for plasticity in middle-aged rat hippocampus. The low-power micrograph shown in panel A illustrates the distribution of the Schaffer-commissural projections from field CA3 to CA1. The marked differences in the anatomy of the treelike apical dendrites and the more bushlike basal dendrites are evident in the labeled cell shown in panel B. C. Long-term potentiation is impaired in the basal but not in the apical dendrites of field CA1 in middle-aged rats. A comparison of slices from young adult (2–3 months) and middle-aged (7–9 months) rats indicates that long-term potentiation (LTP) induced with conventional theta burst stimulation is impaired in the basal dendrites but not

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6. Summary Thanks to recent work on the structure and operation of AMPA receptors and to earlier excised patch studies, a plausible picture of how ampakines work is now available. The compounds bind to a strategic site such that they slow deactivation and desensitization, thereby stabilizing the bound receptor in the channel open configuration. Some ampakines largely affect deactivation, whereas others slow both deactivation and desensitization. The former group increases the amplitude of synaptic responses, whereas the latter group increases amplitude and duration. This proves to be a critical difference. Despite the presence of AMPA receptors in the great majority of brain synapses, ampakines have surprisingly selective actions on physiology and behavior. This appears to arise from the operation of three factors: (1) the compounds do not necessarily affect the balance of excitation and inhibition in local circuits; (2) they have preferences for AMPA-receptor subtypes, and therefore exert regionally differentiated effects on brain activity; and (3) their functional influence varies with the complexity of the networks engaged to perform a given behavior, resulting in much greater effect in cortical telencephalon than at other sites in brain. Potential applications for acute treatments with ampakines fall into three broad groups. First, by enhancing transmission within cortical networks, the drugs could potentially facilitate cognitive behaviors that presumably depend on extremely complex versions of such networks. Behavioral studies have provided support for this idea, and imaging work confirms that ampakines selectively increase aggregate neuronal activity within areas of associational cortex linked to these behaviors. Second, by increasing depolarization at glutamatergic synapses, ampakines promote LTP and thus potentially those forms of memory dependent on this form of synaptic plasticity. The predicted effect Fig. 6. (Continued) in their apical counterparts. The bar graphs to the right of each graph summarize the percentage of potentiation remaining 70 min after the delivery of the theta bursts. The evident difference between young adulthood and early middle age was highly significant for the basal dendrites. D. Daily treatment with an ampakine rescues LTP in the middle-aged hippocampus. Slices were prepared from 7- to 9month-old rats that had been treated with vehicle or a short–half-life ampakine for the preceding 4 days. Slices were prepared 18 hr after the last ampakine treatment, a time point at which brain-derived neurotrophic factor (BDNF) levels were elevated and the short–half-life drug was long removed. As is evident from the graph, the ampakine-pretreated cases had near-normal LTP in the basal dendrites of field CA1. fEPSP, field excitatory postsynaptic potential. Based on refs. 93 and 94.

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of ampakines on retention scores has been observed in numerous studies of memory types that last for hours as well as in tests of long-term memory. In the latter case, it appears that the tested compounds accelerate learning rather than increasing the strength of memory. However, the “deactivation-only” type of ampakine tested in these studies does not increase the magnitude of LTP. The “deactivation-plus-desensitization” variants that do raise the ceiling on LTP remain to be tested for their effects on long-term memory. Third, by increasing communication within cortex and its outputs, ampakines could potentially enhance descending regulation over disturbances in subcortical systems that are thought to contribute to psychiatric disorders. Evidence in favor of this has been obtained in multiple animal models of conditions involving abnormal dopaminergic activity, including a mutant mouse that expresses symptoms resembling those found in ADHD. Recent clinical trials with adult ADHD patients have also been positive. Experimental work demonstrated that ampakines upregulate the production of neurotrophins, compounds that have significant potential for treating a variety of degenerative conditions. As with LTP, there are significant differences between “deactivation-only” versus “deactivation-plus-desensitization” variants of the drugs in this regard. Various studies indicate that it will be possible to produce chronically elevated concentrations of BDNF with appropriate drug treatment regimens.. Initial attempts to use ampakines to treat neuropathology have produced intriguing results for excitotoxic damage and in animal models of Parkinson disease. In addition to its chronic actions, BDNF is released by the naturalistic theta burst stimulation pattern and acts as an acute, positive modulator of LTP. Upregulation of the neurotrophin by brief daily treatments with an ampakine was associated with a reversal of age-related losses in LTP.

Acknowledgments This work was supported in part by grants NS051823 and NS045260 from the National Institute for Neurological Disorders and Stroke and grants CP19982 and CP22357 from Cortex Pharmaceuticals, Inc.

References 1. Granger R, Staubli U, Davis M, et al. A drug that facilitates glutamatergic transmission reduces exploratory activity and improves performance in a learningdependent task. Synapse 1993;15:326–329. 2. Staubli U, Rogers G, Lynch G. Facilitation of glutamate receptors enhances memory. Proc Natl Acad Sci USA 1994;91:777–781. 3. Larson J, Lieu T, Petchpradub V, et al. Facilitation of olfactory learning by a modulator of AMPA receptors. J Neurosci 1995;15:8023–8030. 4. Ingvar M, Ambros-Ingerson J, Davis M, et al. Enhancement by an ampakine of memory encoding in humans. Exp Neurol 1997;146:553–559.

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8 Clinically Tolerated Strategies for NMDA Receptor Antagonism Huei-Sheng Vincent Chen, Dongxian Zhang, and Stuart A. Lipton

Summary Many potentially neuroprotective drugs have failed in human clinical trials because of side effects that cause normal brain function to become compromised. An important example concerns antagonists of the Nmethyl-d-aspartate type of glutamate receptor (NMDAR). Glutamate receptors are essential to the normal function of the central nervous system. However, their excessive activation by excitatory amino acids such as glutamate is thought to contribute to neuronal damage in many neurologic disorders ranging from acute hypoxic–ischemic brain injury to chronic neurodegenerative diseases such as Alzheimer disease, Parkinson disease, Huntington disease, HIV-associated dementia, multiple sclerosis, glaucoma, and amyotrophic lateral sclerosis. The dual role of NMDARs in particular for normal and abnormal functioning of the nervous system imposes important constraints on possible therapeutic strategies aimed at ameliorating neurologic diseases. Blockade of excessive NMDAR activity must therefore be achieved without interference with its normal function. In general, NMDAR antagonists can be categorized pharmacologically according to the site of action on the receptor–channel complex. These include drugs acting at the agonist (NMDA) or coagonist (glycine) sites, channel pore, and modulatory sites, such as the S-nitrosylation site, where nitric oxide (NO) reacts with critical cysteine thiol groups. Because glutamate is thought to be the major excitatory transmitter in the brain, generalized inhibition of a glutamate receptor subtype like the NMDAR causes side effects that clearly limit the potential for clinical applications. Both competitive NMDA and glycine antagonists, From: The Receptors: The Glutamate Receptors Edited by: R. W. Gereau and G. T. Swanson © Humana Press, Totowa, NJ

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even though they are effective in preventing glutamate-mediated neurotoxicity, will cause generalized inhibition of NMDAR activities and thus have failed in many clinical trials. Open-channel block, a form of uncompetitive antagonism, is the most appealing strategy for therapeutic intervention during excessive NMDAR activation because this action of blockade requires prior activation of the receptor. This property, in theory, leads to a higher degree of channel blockade in the presence of excessive levels of glutamate and little blockade at relatively lower levels, for example, during physiologic neurotransmission. As an alternative strategy, genetic manipulation of NR3 subunits can reduce glutamateinduced currents and Ca2+ inﬂux through NMDARs without completely blocking their activation. Based on this molecular strategy of action, this chapter reviews the logical process that was applied over the last decade to develop memantine as the ﬁrst clinically tolerated yet effective agent against NMDAR-mediated neurotoxicity. Phase 3 (ﬁnal) clinical trials have shown that memantine is effective in treating moderateto-severe Alzheimer’s disease while being well tolerated. Memantine is also in trials for additional neurologic disorders, including other forms of dementia, glaucoma, and severe neuropathic pain. In addition, taking advantage of memantine’s preferential binding to open channels and the fact that excessive NMDAR activity can be downregulated by S-nitrosylation, combinatorial drugs called NitroMemantine have recently been developed. These drugs use memantine as a homing signal to target NO to hyperactivated NMDARs to avoid systemic side effects of NO such as hypotension (low blood pressure). These second-generation memantine derivatives are designed as pathologically activated therapeutics, and in preliminary studies they appear to have even greater neuroprotective properties than memantine. Key Words: NMDA receptor; Neuroprotective; Memantine; Nitric oxide; S-Nitrosylation; 116: pg 110–116 Open-channel block; Uncompetitive antagonism; Fast off-rate.

1. Introduction Dementia and cerebrovascular disease (stroke) are among the leading causes of death, disability, and economic expense in the world. For example, Alzheimer disease (AD) ranks fourth as a cause of mortality in the United States. In fact, it has been estimated that as the population continues to age, treatment of patients with dementia will consume the entire U.S. gross national product by the latter decades of this century. Excitotoxic cell death, also termed excitotoxicity, is thought to contribute to neuronal cell injury and death in this and other neurodegenerative disorders. Excitotoxicity is defined as excessive exposure to the neurotransmitter glutamate or overstimulation of its

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membrane receptors, leading to neuronal injury or death. Glutamate, however, is the major excitatory neurotransmitter in the brain and mediates critical synaptic transmission for the normal functioning of the nervous system. Based on the pharmacology of agonist sites, there are three classes of glutamategated ion (or ionotropic) channels, known as -amino-3-hydroxy-5-methyl4-isoxazolepropionate (AMPA), kainate, and N-methyl-d-aspartate (NMDA) receptors. Among these, the ion channels coupled to classical NMDA receptors (NMDARs) are generally the most permeable to calcium (Ca2+ ). Excitotoxic neuronal cell death is mediated in part by overactivation of NMDARs, which results in excessive Ca2+ influx through the receptor’s associated ion channel. As a consequence of Ca2+ accumulation, excessive activation of the NMDARs leads to production of damaging free radicals and other enzymatic processes contributing to cell death (Fig. 1) (1–3). Many neurodegenerative diseases, including AD, Parkinson disease, Huntington disease, HIVassociated dementia, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and glaucoma, are caused by different mechanisms but may share a final common pathway to neuronal injury due to the overstimulation of glutamate receptors, especially of the NMDA subtype (3). Acute disorders, such as stroke, central nervous system (CNS) trauma and epilepsy, also manifest a component of excitotoxicity. Hence, NMDAR antagonists could potentially be of therapeutic benefit in a number of acute and chronic neurologic disorders manifesting excessive NMDA receptor activities (4). NMDARs are made up of different subunits: NR1 (whose presence is mandatory), NR2A–D, and, in some cases, NR3A or B subunits, which the Lipton laboratory recently cloned and characterized. The receptor is probably composed of a tetramer of these subunits (for further details, see chapter on NMDA Receptors). The subunit composition determines the pharmacology and other parameters of the receptor–ion channel complex (5). Alternative splicing of some subunits, such as NR1, further contributes to the diversity of pharmacologic properties of the receptor (6). The subunits are differentially expressed both regionally in the brain and temporally during development. Physiologic NMDAR activity is therefore essential for normal neuronal function (7). Potential neuroprotective agents that block virtually all NMDA receptor activity will therefore very likely have unacceptable clinical side effects. For this reason, many previous NMDAR antagonists have disappointingly failed advanced clinical trials for a number of neurodegenerative disorders. In contrast, studies in the Lipton laboratory have shown that the adamantane derivative memantine preferentially blocks excessive NMDA receptor activity without disrupting normal activity. Memantine does this through its action as a low-affinity, uncompetitive open-channel blocker with a relatively rapid off-rate from the channel. This chapter reviews the molecular mechanism of

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Fig. 1. Schematic illustration of the apoptotic-like cell death pathways triggered by excessive N-methyl-d-aspartate receptor (NMDAR) activity. The cascade of steps leading to neuronal cell death include (1) NMDAR hyperactivation; (2) activation of the p38 mitogen activated kinase (MAPK)–myocyte-specific enhancer factor 2C (MEF2C) (transcription factor) pathway (MEF2C is subsequently cleaved by caspases to form an endogenous dominant-interfering form that contributes to neuronal cell death (71); (3) toxic effects of free radicals such as nitric oxide (NO) and reactive oxygen species (ROS); and (4) activation of apoptosis-inducing enzymes including caspases and apoptosis-inducing factor. cyt c, cytochrome c; nNOS, nitric oxide synthase. Adapted from the Lipton website of the Burnham Institute for Medical Research, www.burnham.org.

memantine’s clinically tolerated action and also the basis for the drug’s development in treating several neurologic disorders. Of note, other calcium-permeable channels and routes of calcium entry, such as transient receptor potential (TRP) channels, acid-sensing ion channels, and calcium-permeable AMPA receptors, are also known to contribute to the excitotoxicity (8–11). In addition, there are alternative approaches to providing protection against NMDAR-mediated excitotoxicity, such as

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methods uncoupling NMDARs from their downstream effectors (e.g., nitric oxide synthase; also see ref. 4). It is not our intention here to cover all possible strategies against calcium-mediated excitotoxicity. The purpose of this review is to provide a brief and perhaps somewhat surprising primer on excitotoxicity as a promising target of neuroprotective strategies and to present a scientific and clinical overview of the excitotoxicity blocker memantine. Some preliminary information on second-generation memantine derivatives, termed NitroMemantines, is also provided. As an alternative strategy, genetic manipulation of NR3 subunits is used to reduce glutamate-induced currents and Ca2+ influx through NMDARs without completely blocking their activation.

2. Excitotoxicity 2.1. Deﬁnition and Clinical Relevance The ability of the nervous system rapidly to convey sensory information and complex motor commands from one part of the body to another and to form thoughts and memories is largely dependent on a single powerful excitatory neurotransmitter—glutamate. There are other excitatory neurotransmitters in the brain, but glutamate is the most common and widely distributed. Most neurons (and also glia) contain high concentrations of glutamate (∼10 mM) (1); after sequestration inside synaptic vesicles, glutamate is released for very brief amounts of time (milliseconds) to communicate with other neurons via synaptic endings. Because glutamate is so powerful, however, its presence in excessive amounts or for prolonged periods of time can literally excite cells to death. This phenomenon was first documented when Lucas and Newhouse (12) observed that subcutaneously injected glutamate selectively damaged the inner layer of the retina (representing primarily the retinal ganglion cells). John Olney, in seminal work, later coined the term “excitotoxicity” to describe this phenomenon (13,14). A large variety of insults can lead to the excessive release of glutamate within the nervous system and, thus, excitotoxicity. When the nervous system suffers a severe mechanical insult, as in head or spinal cord injury, large amounts of glutamate are released from injured cells. These high levels of glutamate reach thousands of nearby cells that had survived the original trauma, causing them to depolarize, swell, lyse, and die by necrosis. The lysed cells release more glutamate, leading to a cascade of autodestructive events and progressive cell death that can continue for hours or even days after the original injury. A similar phenomenon occurs in stroke; the ischemic event deprives many neurons of the energy they need to maintain ionic homeostasis, causing them to depolarize and propagate the same type of autodestructive events that are seen in traumatic injury (1,15). This acute form of cell death occurs by a necroticlike mechanism, although a slower component leading to an apoptotic-like

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death can also be present, as well as a continuum of events somewhere between the two (see later discussion). A slower, subtler form of excitotoxicity is implicated in a variety of chronic and slowly progressing neurodegenerative disorders as well as in the penumbra of stroke damage. In disorders such as AD, Huntington disease, Parkinson disease, multiple sclerosis, HIV-associated dementia, ALS, and glaucoma, it is hypothesized that chronic exposure to moderately elevated glutamate concentrations or glutamate receptor hyperactivity for longer periods of time than occur during normal neurotransmission triggers cellular processes in neurons that eventually lead to apoptotic-like cell death, a form of cell death related to the programmed cell death that occurs during normal development (Fig. 1) (2,16–19). More subtle or incipient insults can lead to synaptic and dendritic damage in the early stages of disease. This process may be reversible and so is of considerable therapeutic interest. It is significant that elevations in extracellular glutamate are not necessary to invoke an excitotoxic mechanism. Excitotoxicity can come into play even with normal levels of glutamate if NMDAR activity is increased, for example, when neurons are injured and thus become depolarized (more positively charged); this condition relieves the normal block of the ion channel by magnesium (Mg2+ ) and thus abnormally increases NMDAR activity (20). In addition, increased activity of the enzyme nitric oxide synthase (NOS) is associated with excitotoxic cell death. The neuronal isoform of the enzyme is physically tethered to the NMDA receptor and activated by Ca2+ influx via the receptor-associated ion channel, and increased levels of nitric oxide (NO) have been detected in animal models of stroke and several neurodegenerative diseases (21). Recent studies have indicated that excessive NR2B-mediated (mainly extrasynaptic) NMDAR activity may contribute to neurotoxicity, whereas NR2A-mediated (predominantly synaptic) NMDAR activity may promote survival in the face of various forms of stress (22–24). Thus, it has been proposed that NR2B-specific antagonists might be useful neuroprotectancts (4). Alternatively, as discussed here, drugs that are designed to preserve normal synaptic activity while blocking excessive extrasynaptic activity might also be effective and, most important, avoid clinical intolerability (3). 2.2. Links Between Vascular Dementia and Excitotoxic Damage The glutamate content of whole brain is approximately 10 mM. Because of the activity of glutamate transporters, most of this glutamate is intracellular. The extracellular glutamate concentration in brain has been estimated to be approximately 0.6 μM. The sensitivity to excitotoxicity of cultured cortical neurons isolated away from astrocytes or of hippocampal neurons in intact

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tissue is approximately 2–5 μM glutamate (1). Therefore, the ambient concentrations of glutamate are close to those that can cause neuronal death, and it is important that extracellular glutamate concentration and compartmentalization be exquisitely controlled to prevent excitotoxicity. On the other hand, with 10 mM glutamate present in cells, the potential for disaster is obviously great. Extracellular glutamate levels have been shown to rise in the face of hypoxic–ischemic insults. There will probably prove to be several mechanisms for the excess accumulation of glutamate even in a single disorder such as ischemia (1). Energy failure might cause abnormal accumulations of glutamate either by impairment of uptake (into neurons and especially astrocytes) mediated via glutamate transporters or by reversal of the direction of transport. This series of events would be followed by injury to some neurons and abnormal potentiation of glutamate release from others. With glutamate release from injured neurons and excess physiologic release from otherwise intact neighboring neurons, the process might then develop a self-propagating, vicious cycle that extends the area of neuronal damage. Deprivation of oxygen and glucose, for example during ischemia, causes a decrement in the production of high-energy phosphate compounds and “energy failure.” However, short-term energy failure per se is not particularly toxic to neurons. What does make energy failure highly neurotoxic is the activation of glutamate receptor–dependent mechanisms. If suitable glutamate antagonists block these mechanisms, then neurons can survive a period of oxygen and metabolic substrate deprivation (1). 2.3. Possible Links Between Excitotoxic Damage and Alzheimer Disease There are several potential links between excitotoxic damage and the primary insults of Alzheimer disease, which, based on rare familial forms of the disease, are believed to involve toxicity from misfolded mutant proteins (25). These proteins include soluble oligomers of -amyloid peptide (A) and hyperphosphorylated tau proteins (26). For example, oxidative stress and increased intracellular Ca2+ generated by A have been reported to enhance glutamatemediated neurotoxicity in vitro. Additional experiments suggest that A can increase NMDA responses and thus excitotoxicity (27–29). Another potential link comes from recent evidence that glutamate transporters are downregulated in Alzheimer disease and that A can inhibit glutamate reuptake or even enhance its release (30,31). Moreover, excessive NMDAR activity has been reported to increase the hyperphosphorylation of tau, which contributes to neurofibrillary tangles and is involved in NMDA-mediated neurotoxicity (32). The NMDAR antagonist memantine has been found to offer protection from these neurotoxic processes, as discussed later.

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2.4. Pathophysiology of Excitotoxicity: Role of the NMDA Receptor NMDA receptors are implicated in neuronal survival and maturation (33,34), neuronal migration (35), induction of long-term potentiation (LTP) (a cellular/electrophysiologic correlate of learning and memory formation (36), formation of sensory maps (37,38), and neurodegeneration (1,7,39,40). In contrast to AMPA- and kainate-type glutamate receptors, all functional NMDARs are heteromultimers (5). Conventional NMDARs composed of NR1 and NR2A–D subunits require dual agonists—glutamate and glycine— for activation (Fig. 2A). The activity of the NMDAR-associated channel is modulated by a voltage-dependent block of Mg2+ (41,42), and the channel manifests high permeability to Ca2+ (43). NMDA is generally not thought to be an endogenous substance in the body; it is an experimental tool that is highly selective for this subtype of glutamate receptor and therefore became the source of its name. Under normal conditions of synaptic transmission, the NMDAR channel is gated by extracellular Mg2+ sitting in the channel and only activated for brief periods of time. This brief opening of the NMDARs allows Ca2+ (and other cations) to move into the cell for the subsequent physiologic functions. Under pathologic conditions, however, overactivation of the receptor relieves the Mg2+ block and causes an excessive amount of Ca2+ influx into the nerve cell, which then triggers a variety of processes that can lead to necrosis, apoptosis, or dendritic/synaptic damage. These detrimental processes include Ca2+ overload of mitochondria, resulting in oxygen free radical formation, activation of caspases, and release of apoptosis-inducing factor; Ca2+ -dependent activation of neuronal NOS, leading to increased NO production and the formation of toxic peroxynitrite (ONOO− ) and Snitrosylated glyceraldehyde-3-phosphate dehydrogenase (GAPDH); and stimulation of mitogen-activated protein kinase p38 (MAPK p38), which activates transcription factors that can go into the nucleus to influence neuronal injury and apoptosis (Fig. 1) (16,44–50). In NMDARs, the following membrane topology has been proposed (Fig. 2B) (5). (1) The N-terminal domain contains the first ∼380 amino acids that are related to the bacterial periplasmic binding protein sequence designated leucine/isoleucine/valine-binding protein (LIVBP), the Zn2+ -binding site, the proton site, and other modulatory sites; (2) four transmembrane domains (M1–M4) are present, and the selectivity filter of the channel pore is formed by M2 (a P-loop region); (3) the ligand-binding domains are formed by the pre-M1 (S1) and M3–M4 linker region (S2); (4) a cytoplasmic C-terminal domain interacts with intracellular proteins (43); and (5) the pre-M1 segment, the C-terminal portion of the M3 segment, and the N-terminal region of the M4 segment form the extracellular channel vestibule (51). In general, NMDAR antagonists can be categorized pharmacologically into four major groups according to site of action on the receptor–channel complex

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Fig. 2. N-Methyl-d-aspartate receptor (NMDAR) model illustrating important binding and modulatory sites. A. Glu or NMDA, glutamate or NMDA-binding site; Gly, glycine-binding site; Zn2+ , zinc-binding site; NR1, NMDAR subunit 1; NR2, NMDAR subunit 2A; SNO, cysteine sulfhydryl group (−SH) reacting with nitric oxide species (NO); X, Mg2+ -, MK-801–. and memantine-binding sites within the ion channel pore region. B. Schematic representation of various domains of the NMDAR subunit. Top: Linear sequence. Bottom left: Proposed three-dimensional folding. Bottom right: Proposed tetrameric structure of the classical NMDAR. ATD, amino-terminal domain; S1 and S2, agonist-binding domains; M1–M4, the four transmembrane domains; CTD, carboxyl-terminal domain (61).

(52): drugs acting at (1) the NMDA (agonist) recognition site, (2) the glycine (coagonist) site, (3) the channel pore, and (4) modulatory sites, such as the redox modulatory site, the proton-sensitive site, the high-affinity Zn2+ site, and the polyamine site. The degree of NMDAR activation and consequent influx of Ca2+ and Na+ into the cell can be altered by higher levels of agonists

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and by substances binding to one of the modulatory sites on the receptor. The two modulatory sites that are most relevant to this review are open-channel blocker sites within the ion channel pore and S-nitrosylation site(s) located toward the N-terminus (and hence extracellular region) of the receptor. Note that S-nitrosylation reactions represent transfer of NO to a thiol or sulfhydryl group (−SH) of a critical cysteine residue (53). This reaction modulates protein function, in this case decreasing channel activity associated with stimulation of the NMDA receptor. Each of these sites can be considered potential targets for therapeutic intervention to block excitotoxicity, as explained later. Moreover, other modulatory sites also exist on the NMDA receptor and may in the future prove to be of therapeutic value. These include binding sites for Zn2+ , polyamines, the drug ifenprodil, and a pH (i.e., proton)-sensitive site (4). In addition, three pairs of cysteine residues at extracellular domains contribute to the redox sites and can modulate NMDAR function by virtue of their redox sensitivity (53). These redox-sensitive cysteine residues may constitute a unique “NO-reactive molecular oxygen sensor” in the brain, enhancing the degree of downregulation of NMDA receptor function by S-nitrosylation in the presence of low pO2 levels, and thus dictating the pathologic effects of hypoxia that are mediated via the receptor (53a).

3. Rational Drug Design of Clinically Tolerated NMDA Receptor Antagonists Excitotoxicity is a particularly attractive target for neuroprotective efforts because it is implicated in the pathophysiology of a wide variety of acute and chronic neurodegenerative disorders (1). The challenge facing those trying to devise strategies for combating excitotoxicity is that the same processes that, in excess, lead to excitotoxic cell death are, at lower levels, absolutely critical for normal neuronal function. To be clinically acceptable, an anti-excitotoxic therapy must block excessive activation of the NMDAR while leaving normal function relatively intact to avoid side effects. Drugs that simply compete with glutamate or glycine at the agonist-binding sites block normal function and therefore do not meet this requirement, and have thus failed in clinical trials because of side effects (drowsiness, hallucinations, and even coma) (54–56). In fact, competitive antagonists compete one for one with the agonist (glutamate or glycine) and therefore will block healthy areas of the brain (where lower, more physiologic levels of these agonists exist) before they can affect pathologic areas (where higher levels of agonist accumulate). Thus, such drugs would preferentially block normal activity and would most likely be displaced from the receptor by the high concentrations of glutamate for prolonged periods that can exist under excitotoxic conditions.

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3.1. Advantages of Uncompetitive Antagonism and Open-Channel Block The term “open-channel blocker of the NMDAR” means that the drug enters the receptor-associated ion channel only when it is open. It is important that this type of drug will be most effective in the face of excessive (pathologic) activity because statistically more channels are open and available to be blocked. This mechanism of inhibition, whose action is contingent on prior activation of the receptor by the agonist, is defined as “uncompetitive” antagonism. Open-channel block is a most appealing strategy for therapeutic intervention during excessive NMDAR activation because the action of blockade requires prior activation of the receptors. This property, in theory, leads to a higher degree of channel blockade in the presence of excessive levels of glutamate and little blockade at relatively lower levels, for example, during physiologic neurotransmission (see Memantine section for details) (1,40,57–60). In fact, an uncompetitive open-channel blocker would prevent more severe excitotoxic processes better than mild disease. One would predict, for example, that moderate-to-severe dementia, involving excessive NMDAR activity leading to neuronal injury or death, would be treated more effectively than mild dementia or other excitotoxic disorders, involving only somewhat increased physiologic firing. (Of course, these drugs will not reverse very severe disease because the neurons will already be lost.) Although preferential neuroprotection from moderate-to-severe excitotoxic processes seems counterintuitive, the drug’s uncompetitive mechanism of action readily explains this uncanny phenomenon. 3.2. The Importance of Off-Rate from Channel Block In recent years it has become clear that among open-channel blockers of the NMDAR, the details of the kinetics of blockade are important to avoid side effects (58,60). A relatively fast off-rate (and hence a short dwell time in the channel) would prevent the drug from accumulating in open channels. This avoids progressive blockade of normal synaptic transmission and relatively spares synaptic activity (3,59). In contrast, a drug with a slow off-rate would build up in the ion channels that underlie synaptic events and consequently interfere with normal neurologic function. At a given membrane potential, the macroscopic pseudo–first-order-rate constant for blocking the channel is dependent not only on diffusion and channel open probability, but also on the drug’s concentration (for a quantitative description, see refs. 3 and 60). In contrast, the off-rate is an intrinsic property of the drug–receptor complex, unaffected by drug concentration. The apparent affinity of a channel-blocking drug is related to its off-rate divided by its on-rate. Thus, a relatively fast offrate can be a major contributor to a drug’s low affinity for the channel pore.

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It has been proposed that a clinically tolerated neuroprotective drug would consist of a low-affinity, open-channel blocker with a relatively fast off-rate (61). Hence, the drug would not substantially interfere with normal synaptic neurotransmission in an accumulative fashion. As a result, the drug would be both effective and well tolerated. As a useful analogy, the NMDA receptor can be thought of as being like a television set. The agonist sites are similar to the “on/off” switch of the television. Drugs that block cut off all normal NMDAR function. What is needed is the equivalent of the “volume” control (or, in biophysical terms, the gain) of the receptor. Then, excessive Ca2+ influx through the NMDARassociated ion channel would be prevented by simply turning down the “volume” of the Ca2+ flux toward normal values. A blocker that binds at a site within the channel, similar to the action of physiologic levels of Mg2+ , could act as a sensor and provide an “automatic” volume control. It is important that the automatic volume control needs to reach an optimal level. In the case of Mg2+ itself, the block is too ephemeral, a so-called “flickery block,” and the cell continues to depolarize (become positively charged because of Ca2+ and Na+ entry) until Mg2+ is repelled and the block is totally relieved. Hence, in most cases Mg2+ does not effectively block excessive Ca2+ influx to the degree needed to prevent neurotoxicity. If, on the other hand, a channel blocker binds with too high an affinity, it will accumulate in the channels, block normal activation, and thus prove clinically unacceptable. Following the television set analogy, turning the volume all the way down is as bad as turning off the “on/off” switch in terms of normal functioning of the television. This is the case with MK-801; it is a very good blocker of excitotoxicity, but because its dwell time in the ion channel is so long (reflecting its slow off-rate and high affinity), it progressively blocks critical normal functions. MK-801 can thus produce coma. Drugs with slightly shorter but still excessive dwell times (off-rates) make patients hallucinate (e.g., phencyclidine, also known as Angel Dust), or so drowsy that they classify as anesthetics (e.g., ketamine). A clinically tolerated NMDAR antagonist would not make a patient drowsy, hallucinate, or comatose, and in fact should spare normal neurotransmission while blocking the ravages of excessive NMDA receptor activation. An uncompetitive, open-channel mechanism of blockade coupled with a longer dwell time in the channel (and consequently a slower off-rate) than Mg2+ but a substantially shorter dwell time (faster off-rate) than MK-801 would yield a drug that blocks NMDAR-operated channels only when they are excessively open while relatively sparing normal neurotransmission. 3.3. Targeting Therapeutic Agents to the Pathologically Active Area An NMDAR open-channel blocker binds increasingly well to neurons manifesting excessive channel activity; these are by definition potentially

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vulnerable neurons. Hence, other anti-apoptotic or pro-survival moieties can be attached to an open-channel blocker to form a new adduct that is targeted to vulnerable neurons, giving the second moiety specificity of action to the sick neuron. Using this concept, the Lipton laboratory and its colleagues have generated a series of second-generation drugs that will have even greater neuroprotective properties than the original. These second-generation drugs can take advantage of the fact that the NMDAR has other modulatory sites (or “volume” controls) in addition to its ion channel that offer safe but effective clinical intervention. One example of an additional modulatory site(s) on the NMDAR that can be taken therapeutic advantage of involves the action of nitric oxide (NO). Transfer of NO to thiol (−SH) groups on critical cysteine residues of the NMDAR (a reaction termed S-nitrosylation) decreases excessive receptor activity (46,62,63). However, if administered systemically, NO can cause serious side effects, including severe hypotension (low blood pressure) by virtue of its ability to produce vasodilation, and may even be toxic by nitrosylating other targets such as GAPDH or reacting with superoxide anion (O2 − ) to form peroxynitrite (ONOO− ). To avoid this problem, the NO group is tethered to an appropriate open-channel blocker to specifically target NO to the NMDAR nitrosylation sites. Such combinatorial drugs, linking the principles of open-channel block and S-nitrosylation of the NMDAR to provide two “volume controls,” show great clinical potential. This newly recognized mode of action for drugs has been designated “pathologically activated therapeutics” (PAT, meaning a gentle tap). By virtue of their relatively gentle binding, PAT drugs work best under pathologic conditions while exerting minimal effects on normal brain activity. Many believe that these simple concepts embody the future of clinically tolerated neuroprotective drug design. The Lipton laboratory was the first to show that the adamantane derivative memantine fulfills these mechanistic criteria (1,40,58,60).

4. Memantine as an NMDAR Antagonist 4.1. Uncompetitive Open-Channel Block of NMDARs Memantine (MEM; 1-amino-3,5-dimethyl-adamantane; Fig. 3) was first synthesized by Eli Lilly and Company and patented in 1968, as documented in the Merck Index. It is a derivative of amantadine, an anti-influenza agent (64). Memantine has a three-ring (adamantane) structure with a bridgehead amine (−NH2 ) that under physiologic conditions carries a positive charge (−NH3 + ). Memantine has been used clinically with an excellent safety record for more than 20 years in Europe to treat Parkinson disease, spasticity, convulsions, vascular dementia (65), and Alzheimer disease (66). The reported efficacy of amantadine and memantine in Parkinson disease, which was discovered

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Fig. 3. Structure, kinetic, and dose–response analysis of memantine (MEM). A. Left: Chemical structure of amantadine. Right: Chemical structure of memantine, which has methyl group (-CH3 ) side chains (unlike amantadine). B. Blockade of 200 μM N-methyl-d-aspartate (NMDA)–activated currents by 1 and 12 μM MEM recorded from a solitary neuron at a holding potential of -60 mV. Kinetic analysis with single-exponential fitting revealed that the on-time (time until peak blockade) of 12 μM memantine was approximately 1 sec, whereas the off-time (recovery time) from the effect was ∼5 sec. C. Dose–response curve for MEM constructed using IMEM /Icontrol (%) versus MEM concentration (58,60).

by serendipity in a patient taking amantadine for influenza, led scientists to believe that these compounds were dopaminergic or possibly anticholinergic drugs. Work at a small German company named Merz first suggested that the drug might be an NMDA-receptor inhibitor, but in these early studies it was initially described to be “potent,” which in fact it is not (67,68). The Lipton laboratory discovered that memantine acts as an open-channel blocker of the NMDAR-coupled channel pore, and that the drug blocks NMDA-evoked responses via uncompetitive antagonism with a 50% inhibition constant (IC50 ) of ∼1 μM at −60 mV (58,60). This blocking site senses <50% of the

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transmembrane potential from the outside surface and interacts with the Mg2+ blocking site in NMDA-gated channels (69). In addition (70), it was reported that the concentration of memantine in the parenchyma of a postmortem brain of a Parkinsonian patient who had received the standard dose of 20 mg memantine/d) was approximately 1–12 μM. At 1–12 μM, memantine is within the effective range of its NMDA-antagonistic action (Ki ≈ 1 μM) but is below the effective level of memantine at any other known receptor or ligand-gated channel (58). The antagonistic action of memantine on NMDARs is therefore thought to be the principal mechanism in the therapy of Parkinson disease, and possibly cerebral ischemia, dementia, and epilepsy (1,40,71). Most important, the Lipton laboratory showed why memantine could be clinically tolerated as an NMDA-receptor antagonist; namely, it was an uncompetitive open-channel blocker with a dwell time/off-rate from the channel that limited pathologic activity of the NMDA receptor while sparing normal synaptic activity (58–60). This most astonishing property of memantine is illustrated in Fig. 4 (58). In this experiment, the concentration of memantine was held constant (at a clinically achievable level of 1 μM) while the concentration of NMDA was increased over a wide range. It was found that the degree to which this fixed concentration of memantine blocked NMDA receptor activity actually increased as the NMDA concentration was increased to pathologic levels. This is classical “uncompetitive” antagonist behavior. In fact, the component of the excitatory postsynaptic current due to physiologic activation of NMDA receptors is inhibited by only 10% or less (59). During prolonged activation of the receptor, however, as occurs under excitotoxic conditions, memantine becomes a very effective blocker. In essence, memantine only acts under pathologic conditions without much affecting normal function, thus relatively sparing synaptic transmission, preserving long-term potentiation, and maintaining physiologic function on behavioral tests such as the Morris water maze (59). This notion is supported by the safety and efficacy profiles of memantine in two recent clinical trials for the treatment of Alzheimer’s disease (72,73). This strategy of selecting NMDAR antagonists of low-affinity/fast off-rate is in contrast to most drug discovery by the pharmaceutical industry, which uses high-affinity screens of the target to look for new drug therapies. Neuroprotective agents that work by high-affinity binding to the NMDAR result in blockade of virtually all receptor activity; thus, these drugs manifest unacceptable clinical side effects. Instead, several years ago, the Lipton laboratory proposed to protect the brain with drugs that do not bind very well under physiologic conditions but are nevertheless selective under pathologic conditions for a particular target, such as the NMDAR (58,74). It is important not to confuse affinity with selectivity; as long as a drug acts selectively and specifically on the target of interest and the effective concentration can be achieved, a high affinity per se is not the key issue.

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Fig. 4. Paradoxically, a fixed dose of memantine (i.e., 1 μM) blocks the effect of increasing concentrations of N-methyl-d-aspartate (NMDA) to a greater degree than lower concentrations of NMDA. This finding is characteristic of an uncompetitive antagonist (58).

4.2. Memantine Interacts with the Intracellular Mg2+ -Blocking Site in the NMDAR Channel Core Early studies indicated that memantine exerts its effect on NMDA receptor activity by binding at or near the Mg2+ site within the ion channel (58,60). Because of its interaction with external Mg2+ and based on mutational analysis of the NMDAR by others (75), the specific site of memantine action was assumed to be near the external Mg2+ -blocking site at the selectivity filter region of the NMDAR-associated channel (76). This region is formed by asparagine (N) residues at the “N-site” of NR1 and “N+1 site” of NR2 subunits (43). Compared to physiologic block by external Mg2+ , a common explanation for the safety and effectiveness of memantine has been that memantine represented a “better magnesium,” manifesting a somewhat slower unblocking rate, moderate voltage dependence, and slightly higher affinity (76). However, when applied from the intracellular versus extracellular surface, Mg2+ interacts differently on the N-site residues of NR1 and NR2 subunits (77,78). The Nsite asparagine of the NR1 subunit represents the dominant blocking site for intracellular Mg2+ , whereas the N- and N+1-site asparagines of the NR2A subunit form the critical blocking site for extracellular Mg2+ . Recently, the Lipton laboratory performed a series of experiments using point mutations and substituted cysteine accessibility methods (SCAM) to show that the N-site asparagine of the NR1 subunit, located at the selectivity filter of the NMDAR-

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associated channel, is the specific and predominant blocking site for memantine (69). The N- and N+1-sites of NR2A subunits provide the major electrostatic interaction with memantine on binding to this deep, specific site (Fig. 5). The differential contribution to memantine block by the N- and N+1-site asparagines in NR1 and NR2 subunits is reminiscent of their effects on intracellular Mg2+ blockade (69). The distinct patterns of interaction of memantine with the channel selectivity filter may confer on memantine unique kinetic features leading to the drug’s excellent clinical tolerability. In line with these results, memantine, in the absence of extracellular Mg2+ , displays minimal differences in blocking NMDARs containing various NR2 subunits (79). None of the NR1 splice variants differentially affected memantine antagonism in the absence of external Mg2+ (69). 4.3. A Second Binding Site for Memantine in NMDA-Gated Channels Several studies have also reported a second binding site for memantine in NMDA-gated channels (79–82). This second site was reported to have a much lower affinity, minimal voltage dependence, and a noncompetitive mechanism of block. There were, however, several differences among these studies, including the estimated IC50 , the precise degree of voltage dependence, and the location of this blocking site in the NMDA-gated channel. By point mutations and SCAM, the second (superficial) memantine-blocking site, located at the extracellular vestibule of the channel, appears to be nonspecific and overlaps the site occupied by the nonspecific pore blocker hexamethonium. Residues in the post-M3 segment of the NR1 subunit are not directly involved in memantine binding (69). There is an important therapeutic implication of our recent description of the location of the second memantine binding site, which concerns the uncompetitive mechanism of antagonism displayed by memantine. Uncompetitive, unlike competitive or noncompetitive, antagonists can block excessive activation of NMDARs while sparing normal neurotransmission (provided their off-rate is sufficiently rapid); this is the property most likely responsible for the clinically tolerated mechanism of action of memantine, as explained earlier. Memantine blocks NMDARs via an uncompetitive mechanism at low micromolar concentrations, yet it possesses a noncompetitive component (sometimes called “partial trapping” in the channel) at higher concentrations (60,81). Lipophilic leak of memantine from its blocking site cannot explain this noncompetitive component (83,84). Instead, the noncompetitive behavior may be explained by the second site of memantine binding, which has very low affinity and is located at the channel vestibule. Occupancy by memantine of this shallow site may allow dissociation of the drug in either the open or closed conformation, resulting in a form of noncompetitive antagonism. This

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Fig. 5. A trapping, uncompetitive scheme for memantine (MEM) action. A. Response to 200 μM N-Methyl-d-aspartate (NMDA) was first blocked by 2 μM MEM at (60 mV, and then both agonists and antagonists were washed out by rapidly exposing the cell to a solution containing only the control solution for 11.3 sec to demonstrate MEM trapping (shown at the left)). After washout with control solution, a second application of NMDA alone displayed a fast-rising phase of channel activation followed by slow relaxation, representing recovery from MEM blockade. This slow relaxation was similar to the regular recovery phase from MEM block that occurred without trapping, as shown at the right. B. Difference in predicted degrees of blockade between noncompetitive and uncompetitive antagonist action of MEM. Left: Scheme for noncompetitive antagonism, with C representing the closed channel; O, the open channel; C*-MEM, the blocked and closed channel; O-MEM; the open but blocked channel; , the microscopic on-rate; , the microscopic off-rate; [MEM], the concentration of the blocker; K, the equilibrium constant for opening from the closed state. The affinity of the blocker for the closed and open channel is the same. The open probability of the unblocked channel is the same as that of the blocked channel. Right: Scheme for uncompetitive antagonism. K/ is the equilibrium constant for opening from the C*-MEM state, and the rest of the symbols have the same meaning as before. The blocker does not bind to the closed channel in this paradigm.

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superficial site of memantine action represents nonspecific binding and may also explain the noncompetitive component of many other very low affinity open-channel blockers. Most important, a large difference in the affinity between these two sites of memantine binding is crucial for maintaining the selectivity of this type of “low-affinity” NMDAR open-channel blocker (69). Thus, it is thought that the distinct patterns of interaction and the relative degree of affinity of memantine for these two binding sites contribute to the drug’s excellent pharmacologic profile of clinical tolerability. 4.4. Voltage Dependence, Partial Trapping, and Lipophilic Leak of Memantine Many possible factors have also been suggested for memantine’s clinical tolerability, including moderate-to-low affinity, moderate voltage dependence, fast blocking and unblocking kinetics, and partial trapping in the NMDARassociated channel (25). All these macroscopic explanations are based on the assumption that memantine and other open-channel blockers bind at the same site as extracellular Mg2+ in the channel selectivity filter. Recently, it was shown that this assumption is incorrect. Instead, memantine interacts with the intracellular Mg2+ -blocking site, which is located slightly deeper than the extracellular Mg2+ -blocking site (69). In addition, a second, superficial site of memantine action represents relatively nonspecific binding and may explain the noncompetitive (or nontrapping) component of memantine at near-millimolar concentrations. For so-called “low-affinity” NMDA open-channel blockers, the apparent affinity, apparent unblocking rate, apparent voltage dependence, lipophilic leak (or closed-channel egress) (81), and degree of trapping of each open-channel blocker will represent “mixed” properties of both sites if the relative affinities are not too far apart. Therefore, despite prior reports, none of these properties at one site alone can explain the variable clinical tolerability of low-affinity NMDAR antagonists. In the case of memantine, however, the affinities of the two sites are sufficiently distinct so that the pharmacologic properties of the specific site may account for its lack of side effects (69). In our hands, memantine at therapeutic concentrations displays Fig. 5. (Continued) C. Computer-simulated degree of blockade for a noncompetitive antagonist (broken line) and uncompetitive antagonist (solid curve) with the models and parameters indicated in panels A and B. The inhibition equilibrium constant (Ki ) for the memantine blockade was assumed to be 1.2 μM, and the concentration of MEM was 6 μM. The empirical data points were very close to those predicted theoretically for pure uncompetitive antagonism (60).

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minimal closed-channel block or egress (minimal lipophilic leak), and it therefore behaves like a perfect uncompetitive blocker (Fig. 6) (60). Moreover, the relatively rapid off-rate of memantine from the inner-channel site is the predominant factor accounting for its clinical tolerability at low micromolar concentrations. 4.5. Other Possible Effects of Memantine At various concentrations, many different mechanisms of action of memantine have been reported: (1) antiviral action by inhibition of viral coat protein function (64), (2) inhibition of muscle-type nicotinic acetylcholine (ACh) receptors at the frog neuromuscular junction (85), (3) antagonistic effects on NMDARs, (4) potentiation of strychnine-sensitive, glycine-activated currents (86) (however, this result has not been substantiated by us or others), and (5) at concentrations >100 μM, memantine may block voltage-sensitive sodium channels (87) and has nonspecific effects on cell membranes (88,89). In rat brain preparations, memantine does not interact directly with dopamine, opioid, -aminobutyric acid (GABA), or 1- and 2-adrenergic receptors

Fig. 6. Atomic model showing two memantine (MEM)-binding sites in the channel permeation pathway of the N-methyl-d-aspartate receptor (NMDAR). Locations of memantine-binding sites in the channel permeation pathway are shown at the level of the channel selectivity filter (the specific site) and at the L651 residue of the NR1 subunit (the nonspecific site). Internal permeant Cs+ can compete with externally applied MEM for binding, and MEM binding interacts with the intracellular Mg2+ -binding site (60,69).

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and has no effect on the uptake of the neurotransmitters noradrenaline or serotonin (88). Memantine has been found to block serotonin (5-HT3 ) receptor channels at concentrations approaching those that block NMDA receptor channels (90,91). Memantine’s effect on 5-HT3 receptors may possibly further enhance cognitive performance. Memantine has also been reported to inhibit 7 nicotinic acetylcholine receptors in the Xenopus oocyte expression system and in rat cultured hippocampal neurons with an affinity similar to that reported for native NMDA receptors (92,93). These results, however, remain controversial, in part because desensitizing currents were evaluated rather than steady-state currents, and their significance thus needs to be confirmed (94). 4.6. Efﬁcacy of Memantine in Animal Models of Alzheimer’s Disease Concerning its use in Alzheimer’s disease (AD), in the rat, memantine at therapeutic concentrations reduced the loss of cholinergic neurons in the nucleus basalis caused by NMDA-mediated toxicity or mitochondria toxins (25). Memantine has been reported to offer protection from neurotoxicity engendered by intrahippocampal injection of A (95) and to enhance the processing of non-amyloidogenic -amyloid precursor protein (25). Memantine also improved performance on behavioral tests (T-maze and Morris water maze) in a transgenic mouse model of familial AD consisting of a mutant form of amyloid precursor protein and presenilin 1 (96). In addition, memantine was recently found to reduce tau hyperphosphorylation, at least in culture (97). Chronic infusion of memantine also attenuated neuronal loss, improved short-term memory impairment and reduced learning deficits and neurotoxicity caused by quinolinic acid–induced entorhinal cortex lesions in the rat (reviewed in ref. 98). However, the exact mechanism(s) of protection by memantine from these animal or culture models of AD remains to be elucidated, although a mechanism related to its NMDAR antagonism is favored. 4.7. Neuroprotective Efﬁcacy of Memantine in Animal Models and Clinical Results in Humans The neuroprotective properties of memantine have been studied in a large number of in vitro and in vivo animal models by several laboratories (98), although of course this remains very difficult to demonstrate directly in humans. Among the types of neurons protected by memantine both in culture and in vivo in animal models are cerebrocortical neurons, cerebellar neurons, and retinal neurons (58,59,62,99). In addition, in a rat model of stroke, memantine, given as long as 2 hr after the ischemic event, reduces the amount of brain damage by approximately 50% (58,59).

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A series of human clinical trials has recently been completed that investigated the efficacy of memantine for the treatment of AD, vascular dementia, HIV-associated dementia, diabetic neuropathic pain, depression, and glaucoma. Some of these studies have only recently been completed and remain unpublished at this time except in abstract form. One recent, high-profile publication reported the results of a U.S. phase 3 (final) clinical study showing that memantine (20 mg/d) is efficacious for moderate-to-severe AD (72). Another study reported that, in combination with Aricept, memantine treatment somewhat improves memory and function in moderate-to-severe Alzheimer’s patients (73). Both studies revealed excellent clinical tolerance and minimal side effects from therapeutic doses of memantine. These positive results of clinical studies for treating AD convinced the European Union and the U.S. Food and Drug Administration to approve memantine for the treatment of this form of dementia. Concerning other forms of dementia, one European multicenter, randomized, controlled trial reported that memantine was beneficial in severely demented patients, probably representing both AD and vascular dementia (100). Another recent publication of a randomized, placebocontrolled clinical trial also described significant benefit from memantine therapy (20 mg/d) in mild-to-moderate vascular dementia (101). These clinical trials are summarized in Table 1. Most trials have reported minimal adverse effects from memantine. The only memantine-induced side effects encountered were rare dizziness and occasional restlessness/agitation at higher doses (40 mg/d), but these effects were mild and dose related. Memantine is also under investigation as a potential treatment for other neurodegenerative disorders, including HIV-associated dementia, neuropathic pain, and glaucoma, as well as depression and movement disorders. It is significant that the uncompetitive mode of memantine action would predict that, at a fixed dose, memantine would work better for severe conditions, for example, excessive glutamate receptor activity to the point of causing cell death, than more mild conditions manifest by slightly elevated synaptic transmission. Bearing this out, recent studies support that memantine may Table 1 Clinical Trials with Memantine German/Merz phase 3 trial for vascular dementia and Alzheimer’s disease Karolinska/Italian phase 3 trial for vascular dementia Two U.S. multicenter phase 3 trials for Alzheimer’s disease U.K. phase 3 trials for vascular dementia French phase 3 trial for vascular dementia U.S. phase 2 trials for neuropathic pain and HIV-associated dementia

+ + + + + ±

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have a larger effect in moderate-to-severe dementia than in mild dementia. Another case in point is neuropathic pain, which is thought to be mediated at least in part by excessive NMDAR activity. Given the uncompetitive antagonism for memantine, more severe pain, for example, the nocturnal pain of diabetic neuropathy, might be expected to benefit from memantine to a greater extent than milder forms of neuropathic pain. In fact, a phase 2B clinical trial suggested that this is indeed the case, whereas preliminary reports indicate that milder pain conditions were not statistically benefited by memantine in phase 2/3 clinical trials. Along these same lines, one would predict that a higher concentration of memantine would be needed to combat pain than to prevent neuronal cell death because greater NMDA receptor activity is associated with cell death (a greater proportion of channels will be blocked in the face of increasing NMDAR activity). Again, clinical trials have suggested that this is indeed the case because 40 mg/d of memantine was been needed in successful pain studies but only 20 mg/d in severe dementia studies. However, further clinical trials will be necessary to prove the efficacy of memantine for severe neuropathic pain. As promising as the results with memantine are, additional modulatory sites (the “volume controls”) on the NMDA receptor appear to block excitotoxicity even more effectively and safely than memantine alone. New approaches in this regard are explored in what follows.

5. NitroMemantines NitroMemantines are second-generation memantine derivatives that were designed to have enhanced neuroprotective efficacy without sacrificing safety. As mentioned earlier, a nitrosylation site(s) is located on the N-terminus or extracellular domain of the NMDA receptor, and S-nitrosylation of this site (NO reaction with the sulfhydryl group of the cysteine residue) downregulates (but does not completely shut off) receptor activity (Fig. 2). The drug nitroglycerin, which generates NO-related species, can act at this site to limit excessive NMDA receptor activity. In fact, in rodent models, nitroglycerin can limit ischemic damage (102), and there is some evidence that patients taking nitroglycerin for other medical reasons may be resistant to glaucomatous visual field loss as well (103). Consequently, the Lipton group characterized S-nitrosylation sites on the NMDA receptor to determine whether a nitroglycerin-like drug could be designed that could be more specifically targeted to the receptor. In brief, five different cysteine residues on the NMDA receptor were found to interact with NO. One of these, located at cysteine residue 399 (C399) on the NR2A subunit of the NMDA receptor, mediates approximately 90% of the effect of NO under our experimental conditions (63). From crystal structure models and

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electrophysiologic experiments, it was shown that NO binding to the NMDAR at Cys399 apparently induces a conformational change in the receptor protein that makes glutamate and Zn2+ bind more tightly to the receptor. The enhanced binding of glutamate and Zn2+ in turn causes the receptor to desensitize and, consequently, the ion channel to close (53). Electrophysiologic studies have demonstrated this effect of NO on the NMDA channel (46,62,63). Unfortunately, nitroglycerin is not very attractive as a neuroprotective agent. The same cardiovascular vasodilator effect that makes it useful in the treatment of angina could cause dangerously large drops in blood pressure in patients with dementia, stroke, traumatic injury, or glaucoma. However, the open-channel block mechanism of memantine not only leads to a higher degree of channel blockade in the presence of excessive levels of glutamate, but it also can be used as a homing signal for targeting drugs, for example, the NO group, to hyperactivated, open NMDA-gated channels. The Lipton laboratory has been developing combinatorial drugs (NitroMemantines) that theoretically should be able to use memantine to target NO to the nitrosylation sites of the NMDAR to avoid the systemic side effects of NO. Two sites of modulation are analogous to having two volume controls on a television set for fine tuning the audio signal. Preliminary studies have shown NitroMemantines to be highly neuroprotective in both in vitro and in vivo animal models. In fact, it appears to be more effective than memantine. Moreover, because of the targeting effect of the memantine moiety, NitroMemantines appear to lack the blood pressure– lowering effect typical of nitroglycerin. More research needs to be performed on NitroMemantine drugs, but, by combining two clinically tolerated drugs (memantine and nitroglycerin), a new, improved class of PAT drugs has been created that are both clinically tolerated and neuroprotective.

6. Strategy of Genetic Manipulation to Inhibit Excessive NMDAR Activity Another method for inhibiting NMDA receptors uses a molecular approach by incorporating NR3A or 3B subunits into the NMDA receptor complex. NR3A and 3B represent the third and final group of subunits in the NMDAR family, recently cloned by our group and others (104–109). NR1 and NR2A–D subunits assemble to form conventional NMDARs, whose activation requires glycine and glutamate as coagonists (110,111). Conventional NMDARs made up of NR1 and NR2A–D subunits manifest high permeability to calcium and exhibit strong voltage-dependent magnesium block. In contrast, NR1 and NR3A, B subunits in the absence of NR2 subunits functionally assemble to form excitatory glycine receptors, because they require glycine alone for activation in the absence of glutamate or NMDA (108). Receptors made up of NR1 and NR3 subunits desensitize at high glycine concentrations, are less

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permeable to calcium, and are relatively resistant to magnesium block (108). In addition, when coexpressed with NR1 and NR2 subunits in heterologous cells, NR3 subunits modulate NMDAR activity by decreasing subunit conductance, Ca2+ permeability, and Mg2+ sensitivity (104,105,107–109,112–114,116). The fast desensitization, decreased conductance, and low Ca2+ permeability in NR3-containing receptors effectively antagonizes NMDA receptors and decreases overall Ca2+ entry into the cells on receptor activation. Consistent with these properties of NR3-containing receptors, mice genetically lacking NR3A manifest increased NMDA-induced currents and dendritic spine density (112).

Fig. 7. Schematic model for the permeation pathway of N-methyl-d-aspartate receptor (NMDAR) channels. A. Model of the permeation pathway proposed for NMDAR channels containing NR1 and NR2 subunits. The M3 segments from the two subunits are staggered relative to each other in the vertical axis of the channel. The N-site residue in the M2 segment of NR1 and the N + 1-site residue in NR2 constitute the selectivity filter of the channel pore (5). B. Proposed model for NR1/NR3A. NR3 subunits replace NR2 subunits to form a symmetric glycine-activated channel. The NR3A M3 segment forms a nonmobile, rigid structure that participates in the formation of the outer vestibule of the channel. This rigid NR3 M3 domain results in a ring of threonine residues (T) composed of NR3A threonines and the homologous NR1 threonines. Because of the lack of motion of the NR3 subunit at this critical region during channel gating, the ring of threonines forms a constriction in the outer vestibule of the NMDAR channel, external to the known selectivity filter in pore loop of the M2 region (labeled as N-site asparagines). Speculation suggests that this external constriction limits ionic flow (resulting in smaller conductance), disrupts divalent hopping relays (producing less calcium permeability), and changes the sensitivity to divalent blockade (rendering the channel less sensitive to magnesium block). The conformation and role of the M2 segment from NR3A subunits remain to be elucidated (indicated with a question mark) (118).

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Structural determinants underlying the properties of NR1/NR3 receptors have recently been elucidated by a number of studies. NR3 subunits bind glycine with very high affinity (116), and this binding is required for activation of NR1/NR3 receptors (117). Unlike conventional NMDA receptors, simultaneous glycine binding to NR1 and NR3 subunits results in significant desensitization, whereas glycine binding to NR3 alone but not NR1 evokes largely nondesensitizing currents. In addition, NR3 subunits appear to play a significant role in gating of NR1/NR2/NR3 receptors. The M3 segment of the NR3 subunit aligns symmetrically with that of the NR1 subunit to restrict externally applied sulfhydryl-specific reagents (Fig. 7). This alignment may contribute to the smaller conductance and relatively low Ca2+ permeability of NR3-containing receptors (118). Moreover, the channel outer vestibule is affected by NR3, which may explain the dominant-negative effect of the NR3 subunit on channel behavior when coexpressed with NR1 and NR2 subunits. Rather than the M2 segment of the NR3 subunit contributing to the selectivity filter of the channel, NR3 subunits appear to modify channel properties via the novel mechanism of forming a narrow constriction at the outer vestibule of the channel. In some sense the effect of NR3 on the outer vestibule of the channel resembles the antagonist effect of memantine in the channel to limit excessive current flux. Thus, these subunits represent natural “antagonists” to decrease the activity of NMDA receptors in vivo (118).

7. Conclusions and Future Prospects Necrosis- and apoptosis-mediated excitotoxic cell death is implicated in the pathophysiology of many neurologic diseases. This type of excitotoxicity is caused, at least in part, by excessive activation of NMDA-type glutamate receptors. Intense insults, such as that occurring in the ischemic core after a stroke, trigger massive stimulation of NMDA receptors, leading to neuronal cell swelling and lysis (necrosis). In contrast, more moderate NMDAR hyperactivity, such as that occurring in the ischemic penumbra of a stroke and in many slow-onset neurodegenerative diseases, results in a moderately excessive influx of calcium ions into nerve cells, which, in turn, triggers free radical formation and multiple pathways, leading to the initiation of synaptic damage and apoptotic-like neuronal cell loss (2). However, NMDA receptor activity is also required for normal neural function. Until recently, all drugs that showed promise as inhibitors of excitotoxicity also blocked normal neuronal function and consequently had severe and unacceptable side effects, and so clinical trials for stroke, traumatic brain injury, and Huntington disease all failed (119,120). In the last decade, the Lipton laboratory has shown that memantine represents a class of drugs that have relatively low-affinity and act as an

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uncompetitive, open-channel blockers. Due to its uncompetitive antagonism and relatively fast off-rate, memantine blocks excessive NMDAR activation but spares low (physiologic) levels of NMDAR activity seen during normal neurotransmission. It is significant that memantine binds at the “intracellular” Mg2+ site in the channel pore and displays differential affinity for specific and nonspecific binding sites on the NMDAR. These molecular interactions confer on memantine favorable kinetic properties that contribute to the drug’s clinical tolerability as well as its neuroprotective profile (69). The discovery that memantine, a low-affinity but still highly selective agent with a mechanism of uncompetitive antagonism, is neuroprotective yet clinically tolerated has triggered a paradigm shift in drug development by the pharmaceutical industry. Clinical studies have borne out our hypothesis that low-affinity/fast–off-rate memantine is a safe NMDA receptor antagonist in humans and beneficial in the treatment of neurologic disorders mediated, at least in part, by excitotoxicity. The NitroMemantines are second-generation NMDA receptor antagonists that may work even better than memantine. They use the memantine moiety as a homing signal for the targeted delivery of NO to a second modulatory site on the NMDAR. Work is progressing rapidly in this area of investigation. Further clinical studies of the efficacy of memantine in the treatment of AD, vascular dementia, HIV-associated dementia, glaucoma, and severe neuropathic pain are underway, and there is every reason to expect the results to be positive, although this is, of course, not yet proven except in the case of Alzheimer’s disease and possibly vascular dementia (Table 1). The efficacy of memantine in neurodegenerative diseases and its ability to protect neurons in animal models of both acute and chronic neurologic disorders suggest that memantine and drugs acting in a similar manner could become very important new weapons in the fight against neuronal damage. In addition, the latest NMDAR subunit family to be discovered, NR3A and NR3B, in some sense is reminiscent of the effect of memantine in that these subunits appear to constrict the outer vestibule of the NMDAR-associated channel and thus limit excessive Ca2+ influx.

Acknowledgments We like to thank our colleagues for their contributions to this work, which is updated here with an emphasis on Alzheimer’s disease. We are especially grateful to Drs. Joachim Bormann, Yun-Beom Choi, Nobuki Nakanishi, and Jonathan S. Stamler for their discussions or collaborations. This work was supported in part by the American Heart Association and NIH grants P01 HD29587, R01 EY50477, and R01 EY09024.

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85. Masuo K, Enomoto K, Maeno T. Effects of memantine on the frog neuromuscular junction. Eur J Pharmacol 1986;130:187–195. 86. Lampe H, Bigalke H. Modulation of glycine-activated membrane current by adamantane derivatives. Neuroreport 1991;2:373–376. 87. Netzer R, Bigalke H. Memantine reduces repetitive action potential firing in spinal cord nerve cell cultures. Eur J Pharmacol 1990;186:149–155. 88. Osborne NN, Beale R, Golombiowska-Nikitin K, et al. The effect of memantine on various neurobiological processes. Arzneimittelforschung 1982;32: 1246–1255. 89. Wesemann W, Sontag KH, Maj J. Pharmacodynamics and pharmacokinetics of memantine [in German]. Arzneimittelforschung 1983;33:1122–1134. 90. Rammes G, Rupprecht R, Ferrari U, et al. The N-methyl-d-aspartate receptor channel blockers memantine, MRZ 2/579 and other amino-alkyl-cyclohexanes antagonise 5-HT(3) receptor currents in cultured HEK-293 and N1E-115 cell systems in a non-competitive manner. Neurosci Lett 2001;306:81–84. 91. Reiser G, Binmoller FJ, Koch R. Memantine (1-amino-3,5-dimethyladamantane) blocks the serotonin-induced depolarization response in a neuronal cell line. Brain Res 1988;443:338–344. 92. Maskell PD, Speder P, Newberry NR, et al. Inhibition of human alpha 7 nicotinic acetylcholine receptors by open channel blockers of N-methyl-daspartate receptors. Br J Pharmacol 2003;140:1313–1319. 93. Aracava Y, Pereira EF, Maelicke A, et al. Memantine blocks alpha7* nicotinic acetylcholine receptors more potently than N-methyl-d-aspartate receptors in rat hippocampal neurons. J Pharmacol Exp Ther 2005;312:1195–1205. Epub 2004 November 2. 94. Banerjee P, Samoriski G, Gupta S. Comments on “Memantine blocks alpha7* nicotinic acetylcholine receptors more potently than N-methyl-d-aspartate receptors in rat hippocampal neurons.” J Pharmacol Exp Ther 2005;313:928–9; author reply, 30–33. 95. Miguel-Hidalgo JJ, Alvarez XA, Cacabelos R, et al. Neuroprotection by memantine against neurodegeneration induced by beta-amyloid(1–40). Brain Res 2002;958:210–221. 96. Tanila H, Minkevicine R, Banjeree P. Behavioral effects of subchronic memantine treatment in APP/PS1 double mutant mice modeling Alzheimer’s disease. J Neurochem 2003;85(Suppl 1):42. 97. Iqbal K, Li L, Sengupta A, et al. Memantine restores okadaic acid-induced changes in protein phosphatase-2A., CAMKII and tau hyperphosphorylation in rat. J Neurochem 2003;85(Suppl 1):42. 98. Parsons CG, Danysz W, Quack G. Memantine is a clinically well tolerated Nmethyl-d-aspartate (NMDA) receptor antagonist—a review of preclinical data. Neuropharmacology 1999;38:735–767. 99. Osborne NN. Memantine reduces alterations to the mammalian retina, in situ, induced by ischemia. Vis Neurosci 1999;16:45–52. 100. Winblad B, Poritis N. Memantine in severe dementia: results of the 9M-Best Study (Benefit and Efficacy in Severely Demented Patients During Treatment with Memantine). Int J Geriatr Psychiatry 1999;14:135–146.

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9 The Structures of Metabotropic Glutamate Receptors David R. Hampson, Erin M. Rose, and Jordan E. Antﬂick

Summary Interest in the structures of the metabotropic glutamate receptors continues to increase for a variety of reasons, including the fact that they are now established drug targets and are linked to a wide spectrum of physiologic processes both within and outside the central nervous system. This chapter summarizes our knowledge of the structures of the eight receptor subtypes, including the alternatively spliced forms, and the experimental approaches that have been used to study them. The large size and multiple domains of these proteins are conducive to further advances in the development of drugs for potential therapeutic use, and for basic research directed toward elucidating the intrinsic signaling mechanisms of these complex molecules. Increasingly detailed analyses of protein–protein interactions between the metabotropic glutamate receptors and other signaling molecules will also contribute to a deeper understanding of how this class of receptors and related G protein-coupled receptors, function at the molecular level within biologic membranes in vivo. Key Words: G protein-coupled receptor; Venus ﬂytrap; Heptahelical domain; Class C; Cysteine-rich domain; Dimer interface; Allosteric modulator.

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

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1. General Structural Features of Metabotropic Glutamate Receptors and Class C G Protein-Coupled Receptors The metabotropic glutamate receptors (mGluRs) are members of the class C subclass of receptors within the G protein-coupled receptor (GPCR) superfamily. Class C receptors are also referred to as the glutamate group in the GRAFS classification system of GPCRs outlined by Fredriksson et al. (1). Class C receptors encompass four main groups of receptors: Group I consists of the calcium-sensing receptor (CaSR), the V2R pheromone receptors, the T1R taste receptors, and the amino acid-activated GPRC6A; group II includes the mGluRs; group III consists of the -aminobutyric acid B (GABAB ) receptor; and group IV consists of the orphan receptor GPRC5 (2). With the exception of GPRC5, all members of this family possess a large extracellular ligandbinding domain, also referred to as the Venus flytrap domain (VFD), a prototypical heptahelical transmembrane domain (TMD), and an intracellular carboxy terminus (Fig. 1). With the exception of the GABAB receptors, class

Fig. 1. A metabotropic glutamate receptor (mGluR) dimer, including the ligandbinding domain, the cysteine-rich domain (CRD), and the seven-transmembrane domain (7TMD); the intracellular carboxyl tail of the receptor is not shown. The ligand-binding domain is a representation of the crystal structure, whereas the cysteinerich and 7TMD domains are homology models of mGluR1 based on the structures of the extracellular domain of the type I tumor necrosis factor receptor and rhodopsin, respectively. The actual orientation of the Venus flytrap domain and Cysteine-rich domain relative to the YTMD domain is not known; the figure depicts one possible arrangement. VFD, Venus flytrap domain.

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C receptors also possess a relatively short cysteine-rich domain (CRD) situated between the VFD and the TMD regions. The eight mammalian mGluRs are subclassified into three groups based on sequence similarity and pharmacologic properties. Group I receptors include mGluR1 and mGluR5; group II receptors include mGluR2 and mGluR3; and group III receptors are composed of mGluR4, mGluR6, mGluR7, and mGluR8. In cell lines, group I receptors couple to Gq and the Gq-like family of G proteins and the stimulation of phospholipase C and release of intracellular calcium, whereas the group II and III receptors couple to the Gi and Go family of G proteins and the inhibition of adenylyl cyclase. However, in the nervous system, the mGluRs couple to multiple signal transduction pathways, including the modulation of voltage-gated calcium and potassium channels. The CRD and TMD regions of the mGluRs do not appear to have counterparts in prokaryotes; the TMD also has very low sequence similarity to other G protein-coupled receptors, including rhodopsin and members of the rhodopsin family of receptors. However, O’Hara et al. (3) first reported that the VFD of the mGluRs show low sequence similarity to the periplasmic binding proteins in bacteria that transport amino acids and other nutrients into the cell. Thus, the VFDs of the mGluRs and other class C receptors may have evolved from the prokaryotic periplasmic binding proteins (4). It is clear that the mGluRs, like other neurotransmitter receptors, are oligomeric protein complexes. Most of the evidence indicates that the mGluRs are homodimers, although some groups have reported the presence of heterodimers composed of one mGluR subunit partnered with a subunit of another class C GPCR. The existence of higher-order structures in vivo, such as tetramers formed from dimers of dimers, has not been ruled out. The protomers within a dimeric mGluR complex are linked together at several points, including a covalent disulfide bond and additional noncovalent bonds in the VFD (5,6), hydrophobic interactions in the TMDs, and probably additional associations in the carboxy termini. The dimeric configuration of the VFD and ligand binding both remain intact when this domain is expressed as an isolated protein (7,8).

2. The Venus Flytrap Domain The VFD of the mGluRs is a large bilobed structure composed of approximately 600 amino acids. The crystal structures of the VFD of rat mGluR1 with and without bound glutamate (9–11), mGluR3, and mGluR7 (12) show that the two VFDs within each of the protomers face away from each other. Lobe 1 is situated on top of lobe 2, which in turn presumably sits on top of the CRD. A molecule of glutamate binds within a cleft formed between lobes 1 and 2 and induces a large 31-degree conformational shift resulting in the closure of the

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two lobes. Based on data obtained from the crystal structures of mGluR1 with bound glutamate or the antagonist -methyl-4-carboxyphenylglycine (MCPG), two primary configurations have been observed: an open/open form and a closed/open form. The open/open form is seen with the antagonist while the closed/open form was observed with glutamate. In the absence of ligands, both conformations were observed suggesting that the VFD maintains a dynamic equilibrium whereby agonists stabilize the closed/open configuration. Some competitive antagonists, such as MCPG and (S)-2-amino-2-methyl-4phosphobutanoic acid (MAP4) jam the VFD in the open configuration by the actions of a methyl group in the molecule that causes a steric clash with a residue situated on lobe 2. Thus mutation of tyrosine 227 to alanine in mGluR8 creates space into which this methyl group can fit into upon closure of the lobes; this mutation therefore converts MAP4 from an antagonist into an agonist capable of activating the receptor (13). In all mGluRs, the conformational change initiated in the VFD is then transmitted to the TMD (via the CRD) and ultimately results in the activation or inhibition of an effector protein. In the heteromeric GABAB receptor, only the GABAB -R1 subunit contains a GABA-binding site, and therefore only one molecule of GABA is needed to fully activate the receptor. In homodimeric mGluRs, a single molecule of glutamate bound to only one of the two protomers results in activation of the receptor complex. However, it has been suggested that full receptor activation may require agonist bound to both protomers (14,15), whereas others have speculated that agonist bound to both protomers may induce an “insulated state” whereby the receptor becomes desensitized (12). The extracellular VFD contains several sites of asparagine-linked glycosylation that are required for folding and cell surface expression but not for ligand binding (8,16,17). In the crystal structure of mGluR1 generated from protein purified from baculovirus-infected insect cells, two N-linked carbohydrates at asparagines 98 and 223 were present; neither glycosylation site is fully conserved in all eight mGluRs. Additional roles of N-linked carbohydrates beyond folding and surface expression have not been identified. The crystal structure of mGluR1 also revealed the presence of a bound divalent metal ion coordinated by asparagine 90, aspartate 92, and leucines 95 and 96. These amino acids are located far from the glutamate-binding site, and, based on its hexavalent coordination, the bound ion is most likely magnesium (9). This cation might be involved in receptor folding. We note, however, that unlike calcium, which can activate mGluR1 directly (18,19), magnesium ions do not directly activate this receptor, but they do potentiate the responses to glutamate (19). The site of action of the activating and potentiating effects of divalent cations on the mGluRs might be localized in or near the glutamate-binding pocket because they are obligatory for agonist binding to the isolated VFDs of group I and group II mGluRs (20).

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2.1. The Glutamate-Binding Pocket From an evolutionary perspective, the glutamate-binding pocket buried within the VFD has ancient origins. The primordial class C receptor likely arose during early metazoan evolution and may have been preadapted as a glutamate receptor for its later use at excitatory synapses (21). The glutamatebinding pocket in the mGluRs occupies a relatively small cavity within the crevice of the VFD compared to other class C receptors (22) and is highly selective for glutamate over other amino acids (23,24). From the crystal structure it appears that there are approximately 12 residues in the binding pocket that establish bonds with the bound glutamate molecule. As described later, additional bonding interactions occur with more complex agonists and antagonists. Within the pocket, several residues are highly conserved in the mGluRs; these conserved residues interact with the -carboxy and -amino groups of the glutamate ligand. Of particular importance are the four residues that establish bonds with the -amino group of the bound ligand; these residues are essential for ligand binding and are a signature feature of all mGluRs and most, if not all, class C amino acid–binding receptors (4,25,26). In contrast to the conserved amino acids that interact with the -carboxy and -amino groups of the glutamate ligand, the residues that establish bonds with the side chain of the glutamate ligand are not conserved and are the principal determinants of receptor subtype selectivity for orthosteric agonists and antagonists (27). The analogous set of residues in other members of class C receptors mediate amino acid ligand selectivity—for example, the preference for basic amino acids for the 5.24 and GPRC6A receptors (25,28–30) and the preference for large hydrophobic amino acids in the CaSR (31). Orthosteric mGluR agonists and antagonists can be divided into three groups: nonselective compounds that show relatively little selectivity among the eight subtypes, compounds that display selectivity towards group I, group II, or group III receptors, and drugs that show substantial selectivity toward a single receptor subtype. Most mGluR ligands fall in the group-selective class, and, in many cases, the molecular basis for this selectivity has been determined. The highly potent group I selective agonist quisqualate binds in the glutamate pocket in an orientation that allows additional interactions not present in the glutamate molecule. This was demonstrated by the observation that mutations in residues in the mGluR1 pocket that interact with the side chain -carboxyl group of the bound glutamate eliminate responsiveness to glutamate but not to the larger and more complex structure of quisqualate (32). DCG-IV and LY354470 are two examples of potent group II–selective orthosteric agonists. The molecular basis for the high affinity and selectivity of DCG-IV binding for group II receptors is mediated in part by an interaction between a third carboxylic acid group on DCG-IV not present in the glutamate molecule and a tyrosine and arginine present in the binding pocket of group II

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receptors (33). The selectivity of LY354740 to group II receptors is mediated by several residues, in particular, arginine 57 in mGluR2 and aspartate 146, which is located outside of the pocket and appears to mediate the higher affinity of LY354740 for mGluR2 versus mGluR3 (34). A salient characteristic of group III receptors is their selective activation by phosphonate- or phosphate-containing compounds such as l-amino-4phosphonobutyric acid (L-AP4), l-serine-O-phosphate (L-SOP), and RSphosphonophenylglycine (PPG). This selectivity is mediated by a cluster of amino acids in the pocket, primarily composed of the side chains of lysines and arginines, that converge to form a positively charged microenvironment; these residues establish bonding interactions with the negatively charged phosphonate group on L-SOP and L-AP4 (35). In some cases agonist selectivity across mGluR groups is mediated by only two residues in the binding pocket. For example, mutating lysine 74 together with lysine 317 or lysine 74 and glutamate 287 to the equivalent amino acids in mGluR1 resulted in complete switching of its pharmacologic profile whereby the double mutants displayed affinities for quisqualate that were similar to mGluR1 (36). The role of the amino acid at position 74 in the group III receptors is also highlighted by the work of Rosemond et al. (37). In this case, a molecular modeling and mutagenesis strategy was used to elucidate the molecular basis for the approximate 1000-fold difference in agonist affinity between the high-affinity mGluR4 receptor and the low-affinity mGluR7 receptor. Mutating asparagine 74 in the pocket of mGluR7 to lysine as in mGluR4 increased agonist affinity by 12-fold, and significantly, molecular models of the two receptors were crucial in revealing the identity and location of additional residues outside the pocket that also contribute to the differences in affinity of group III receptors. 2.2. The VFD Dimer Interface The dimer interface between the two VFDs likely participates in initiating the receptor activation process. A hydrophobic patch of residues at the interface appears to be involved in receptor activation; replacement of isoleucine 120 with alanine in this region in mGluR1 resulted in the elimination of signaling, likely by disrupting bonding interactions within this hydrophobic region and impairing the conformational change required to initiate receptor activation (32). Cocrystallization of the mGluR1 VFD with both the trivalent element gadolinium and glutamate, together with mutagenesis experiments, have demonstrated that the ion is bound to a site nested within several acidic residues located at the lobe 2–lobe 2 interface (10,38). Gadolinium acts by alleviating the mutual repulsion of the acidic residues projecting into the dimer interface region between the two protomers. Gadolinium is also a potent agonist of the calcium-sensing receptor (CaSR), but its site of action has not been identified. In mGluR1 this cation both potentiates the sensitivity to glutamate

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and, like calcium, acts as an agonist in the absence of glutamate whereby it may induce a closed–closed state of the two VFDs (39). Together, these studies and others provide examples of how the molecular space within the binding pocket can be probed to elucidate the molecular basis for orthosteric drug actions. Moreover, exploration of the dimer interface has revealed further insight into the mechanisms of receptor activation, and this region may prove to be a fruitful target for future drug development (40).

3. The Cysteine-Rich Domain The cysteine-rich domain (CRD), which links the VFD to the TMD, is composed of approximately 60–70 amino acids, 9 of which are cysteines. With the exception of the GABAB receptor, which lacks this domain, these 9 residues are highly conserved throughout class C receptors. With the CaSR, deletion of the CRD resulted in a protein that was expressed on the cell surface but was nonfunctional (41). Truncated forms of group II (mGluR3) and group III mGluRs (mGluR4 and mGluR8), which included all of the VFD plus part or all of the CRD, were retained in intracellular compartments when transfected into mammalian cells (17,42). However, group II and group III mGluR expression constructs encompassing the VFD but completely devoid of the CRD were secreted and retained their ligand-binding capabilities. The expression constructs used for X-ray crystallographic analysis of the VFD of mGluR1 and mGluR7 also excluded the CRD, whereas the mGluR3 structure included the CRD (6,9,12). Together, these findings suggest that the VFD and the CRD fold independent of one another and that the CRD is not essential for ligand binding. Despite the conservation of the CRD throughout the mGluRs and its importance in signaling from the VFD to the TMD, little is known about the structure of this region prior to the availability of the mGluR3 structure containing both the VFD and the CRD (12). Previously, several groups had formulated computer-generated models of the CRD structure. One group (43) used a “threading” approach whereby the CRD sequences from all eight mGluR subtypes were “fed” through a protein–folding template to calculate the most energetically favorable arrangement. Several possible structures were generated, which all share common secondary structural elements, namely two sets of antiparallel -strands that form a compact structure. It is interesting that it was also proposed that four of the nine cysteine residues within the CRD coordinate a Zn2+ ion. An alternative model of the CRD was constructed using a section of the tumor necrosis factor receptor as the template (44). This model also features four -strands and, in addition, includes three intramolecular disulfide bridges. This model further predicted the presence of two copies of a module termed A1, which also exists as a repeat motif in the tumor necrosis

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factor receptor and in epidermal growth factor–like domains. A consensus sequence for the mGluR A1 module was established as Cys-Xaa2 -Gly-Xaa-bXaa-Xaa 4−9 -Cys, where b indicates any amino acid with a large side chain. Of interest was the speculation that two conserved hydrophobic amino acids (a phenylalanine and a valine) and one of the cysteines could potentially participate in dimer formation. Muto et al. reported the structure of mGluR3 that encompasses both the VFD and the CRD (12). The structure of the mGluR3 CRD contains three -sheets, each composed of two short, antiparallel -strands. All of the cysteine residues are fully oxidized; four intradomain disulfide bridges contribute to stabilizing the internal architecture of the domain. In contrast, the remaining cysteine residue (C527 in mGluR3) forms a disulfide bond with C240 in lobe 2 (12,45). In addition, the short connection between the C terminus of the structure (E567) and the N terminus of the predicted TMD (A577) contains nine residues, which likely restrict the relative positions of the two domains (12). Thus, it is most likely that the CRD plays a major role in transmitting the conformational change in the ligand-binding domain to the TMD region. Furthermore, this domain prevents a direct interaction between the ligandbinding and TMD regions because the distance between the lobe 2 domain and the C-terminus of the CRD domain is too great. In summary, the structure of mGluR3 reveals that the CRD is tightly linked to the VFD by a disulfide bridge, suggesting a potential role in transmitting ligand-induced conformational changes to the downstream TMD region. The structure also indicates that potential lateral interactions between the two CRDs of the two protomers may exist that could facilitate clustering of the dimeric receptors on the cell surface.

4. The Heptahelical Transmembrane Since the initial cloning of the mGluRs in the early 1990s, it has been assumed, based on hydropathy plots, that the mGluRs possess the prototypical heptahelical TMD topology; some experimental evidence now exists supporting this topology. A study by Bhave et al. (46) used native and engineered glycosylation sites in mGluR5 to examine TMD topology. Mutation of only one of six possible N–glycosylation consensus sequences produced a mobility shift compared with a mutant in which all native N-glycosylation sites had been mutated, demonstrating that this site (asparagine 444) in the VFD accounts for all of the detectable native N-glycosylation in mGluR5. To analyze the structure further, asparagine N-glycosylation protection assays of truncated mGluR5a fusion proteins were conducted, in which the C-terminal domain of mGluR5a was fused to mGluR5a proteins truncated at each of the hydrophilic loops separating the seven TMDs. An HA epitope tag was inserted into the

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N-terminal domain and used in western blots following asparagine protease treatment of vesicles prepared from COS7 cells transfected with the mGluR5a fusion constructs. Fusion constructs in which the mGluR5a C-terminal tail lies in a “trans” configuration to the extracellularly located N-terminal HA tag will be proteolyzed, whereas those lying in a “cis” conformation will be protected along with the tag. These experiments confirmed an alternating pattern of cytoplasmic to extracellular tags beginning with intracellular loop 1 through to extracellular loop 3 (46). The sequence identity shared between bovine rhodopsin (the prototypical class A receptor) and the TMDs of the mGluR subtypes is very low (6%–15% amino acid sequence identity). The TMD helices in the mGluRs are separated by short intra- and extracellular loops (4). The second intracellular loop in the mGluRs is the largest, with a maximum of 27 residues, whereas the third intracellular loop in rhodopsin is the largest. The primary features of the mGluR third intracellular loop are conserved in class C receptors (except retinoic acid–induced receptor), with a basic residue at its N-terminal end and the motif (F/I/L)N(E/D)xK at its C-terminal end (4). Intracellular loops 2 and 3 form a cavity within which the C-terminus of the G protein alpha subunit interacts, and these two loops are thought to determine the receptors’ G protein coupling selectivity (47). The third intracellular loop in class C receptors is thought to play a role equivalent to that of the second intracellular loop in rhodopsin. When the intracellular loops of rhodopsin were replaced by those of mGluR6, only the constructs in which the third intracellular loop was replaced by the second intracellular loop of mGluR6 were functional (48). An intracellular amphipathic helix 8 occurs in rhodopsin parallel to the plane of the lipid bilayer just after TM7 and is thought to also play a role in G protein coupling. It has been suggested that a similar amphipathic helix may be present in the mGluRs (49). Within the TMD helices of class C receptors, only 19 residues are conserved. A highly conserved arginine at the end of TM3 of rhodopsin-like receptors is part of the DRY motif (aspartate-arginine-tyrosine). This motif plays a pivotal role in the equilibrium between active and inactive confirmations of the receptor. This arginine residue is conserved in the mGluRs (4). A disulfide bond that links the top of TM3 with the second extracellular loop in all GPCRs is also conserved in class C members (except retinoic acid–induced receptors). A tryptophan residue in TM6 is conserved between class A and class C (except GABAB ) receptors and is part of a hydrophobic pocket that constitutes the ligand-binding site in some family A receptors. This residue is also thought be critical in the transformation from the active to the inactive state of the receptor. Finally, the NPxxY motif in TM7 of rhodopsin is also conserved in class C receptors (as xPKxY); the proline residue in this motif causes a kink

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in the TM7 helix and is important for the correct coupling of the receptor to G protein (50). 4.1. The Role of Intracellular Loops 2 and 3 in Receptor Activation Tateyama et al. (51) used fluorescence resonance energy transfer (FRET) technology to study activation-induced structural changes in the TMD of the mGluRs. Two fluorophores, cyan fluorescent protein and yellow fluorescent protein, were inserted into the second or third intracellular loop or at various positions in the C-terminal tail. Insertion of the fluorophores into the third intracellular loop prevented membrane trafficking of mGluR1, suggesting that the third intracellular loop is a key determinant in the formation of a functional receptor. Insertion into the first and second intracellular loops caused a decrease and an increase in the FRET signal, respectively, on glutamate binding. Tateyama et al. (51) proposed a model whereby agonist binding leads to a shift in the intracellular domains such that the second intracellular loops of each monomer move closer together, hence the increase in the FRET signal. Notably, a dose–response effect was observed in which increasing concentrations of extracellular glutamate correlated with both increased FRET in the second intracellular loop dimer and increased release of intracellular calcium. Constitutively active mutations have been identified in bovine rhodopsin between the third intracellular loop and TM6, a region important for G protein coupling and receptor activation. A constitutively active mutation has been identified in mGluR8 at glutamine 695 in the second intracellular loop, which is highly conserved among all mGluR subtypes and other family C GPCRs, such as the CaSR (52). The activity of the mutant increases when this glutamine is substituted with a nonpolar or small, uncharged, polar amino acid (e.g., alanine, cysteine, isoleucine, methionine, valine, serine, or threonine) and decreases with ringed or acidic amino acids (phenylalanine, proline, tryptophan, tyrosine, aspartate, or glutamate). These results suggest that a conformational change around glutamine 695 occurs during G protein activation, perhaps favoring the active state of the receptor. Further analysis of the second intracellular loop of mGluR8 led to the identification of specific residues responsible for the G protein selectivity. Five of these residues (arginine 672, isoleucine 673, arginine 675, isoleucine 676, and proline 687) are conserved in all mGluR subtypes, and two residues (phenylalanine 677 and isoleucine 690) are conserved among the Gi/o-coupled mGluR subtypes (52). The latter two residues could potentially be important determinants of Go-subtype coupling in mGluRs. Several intracellular proteins have been shown to modulate G protein coupling and receptor activation. GPCR kinases (GRKs) phosphorylate rhodopsin-like GPCRs and promote the binding of arrestins, resulting in the

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desensitization and internalization of the targeted receptor. GRK-mediated inhibition of mGluR1, however, is phosphorylation and -arrestin independent (53). Dhami et al. (53) discovered that GRK2 inhibits mGluR1 by binding to a region in the second intracellular loop, presumably preventing the binding of the G protein. Together these findings confirm that second intracellular loop in the mGluRs is an important determinant of G protein selectivity and is essential for activation of the receptor. 4.2. Structural Determinants of Allosteric Modulation The study of the structure–activity relationships of allosteric modulators of the mGluRs has provided important information regarding the mechanism of receptor activation. Positive allosteric modulators do not directly activate the receptor, but instead potentiate the response of an agonist binding to the orthosteric site. A growing number of positive and negative allosteric modulators have been discovered, some of which are listed in Tables 1 and 2, respectively (54–70). An interesting observation is that when the extracellular domain of an

Table 1 Positive Allosteric Modulators of Metabotropic Glutamate Receptors

BBB, blood–brain barrier; mGluR, metabotropic glutamate receptor; MPEP, 2-methyl-6phenylethynylpyridine; NMDA, N-methyl-D-aspartate; PHCCC, N-phenyl-7-(hydroxylimino) cyclopropa[b]chromen-la-carboxamide; TM, transmembrane; TMD, transmembrane domain.

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Table 2 Negative Allosteric Modulators of Metabotropic Glutamate Receptors

CPCCOEt, 7-Hydroxyiminocyclopropan[b]chromen-la-carboxylic acid ethyl ester; DHPG, (R,S)3,5-dihydroxyphenylglycine; EM-TBPC, 1-ethyl-2-methyl-6-oxo-4 (1,2,4,5-tetrahydrobenzo[d]azepin-3yl)-1,6-dihydro-pyrimidine-5-carbonitrile; IC50 half-maximal inhibition constant; mGluR, metabotropic glutamate receptor; MPEP, 2-methyl-6-phenylethynylpyridine; MTEP, 3[(2-methyl-1,3-thiazol-4yl)ethynyl]-pyridine; Noncomp., noncompetitive; TM transmembrane; TMD, transmembrane domain.

mGluR is deleted, positive allosteric modulators can act as agonists by inducing responses in the absence of an orthosteric agonist. Goudet et al. (71) examined this phenomenon by comparing the activity of the positive allosteric modulator 3,3’-difluorobenzaldazine (DFB) on wild-type mGluR5 and truncated mGluR5 (with the extracellular domain and/or C-terminus removed). They observed that only glutamate activated the wild-type receptor and that DFB directly activated the truncated mGluR5 constructs (71). The results from this study suggested that the TMD is involved in the transition from an inactive to an active state of the receptor. 7-Hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt) is a noncompetitive antagonist of mGluR1 that binds within TM7 at threonine 815 and alanine 818. Conformational changes within the TMD induced by glutamate binding to the orthosteric site is inhibited by CPCCOEt, as observed by inhibition of the FRET signal induced by 1 mM l-glutamate (51). Thus, within the TMD several residues are required for transformation

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of the receptor from the inactive to the active state, and these residues are blocked by the binding of negative allosteric modulators (52). Three aromatic residues—tryptophan 798, phenylalanine 801, and tyrosine 805—are thought to be essential for binding and inhibitory activity of the another mGluR1 negative allosteric modulator, 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydrobenzo[d]azepin-3-yl)-1,6-dihydro-pyrimidine-5-carbonitrile (EM-TBPC) (72). EM-TBPC may bind within this aromatic cluster in TM6 and block the conformational change centered around tryptophan 798, an amino acid critical for transition to the active state of the receptor. It therefore appears that negative and positive allosteric modulation of mGluRs involves interruption or enhancement, respectively, of conformational changes within the TMD. Allosteric modulators have also provided insight into how each protomer of an mGluR homodimer contributes to activation of the receptor. Truncated mGluR dimers consisting of the isolated TMDs appear to function asymmetrically such that activation of only one of the TMD protomers is required for full receptor activity. One molecule of DFB, a selective positive allosteric modulator of mGluR5, is required to elicit full agonist activity in a chimeric mGluR1/mGluR5 TMD heterodimer (73). The phenomenon of asymmetric activation of a symmetric mGluR homodimer may be explained by steric hindrance, which prevents conformational changes within the TMD from occurring in both protomers simultaneously. Hlavackova et al. (74) assessed the contribution of the two protomers to receptor output by creating two chimeric mGluR1 receptors, R1c1 and R1c2, by replacing the C-terminal tail of mGluR1 with the tails of GABAB R1 and GABAB R2, respectively. The GABAB R1 subunit does not reach the cell surface alone due to the presence of an endoplasmic reticulum retention signal in its C-terminal tail; this signal is masked by the presence of the C-terminal tail of GABAB R2 (74). Thus, these chimeras provided control of dimer composition at the cell surface. In addition, each chimera was tagged with a fluorophore (EuCryptate or Alexa647) and an epitope (HA or c-myc) to measure FRET and to track expression. By producing a point mutation in the third intracellular loop of one chimera, it was determined that both TMDs are required to activate G proteins for full receptor activity. However, this mutation did not inhibit the ability of the TMD to reach an active conformation. For a receptor dimer combination in which only one TMD is maintained in the inactive state with the negative modulator (inverse agonist) 2-methyl-6-phenylethynylpyridine (MPEP), the associated subunit is still able to generate the full response of the receptor to l-glutamate. The interpretation of these findings was that the mutation in intracellular loop 3 impairs G protein activation but not the ability of the transmembrane domain to reach an active conformation; therefore the TMD of a single protomer is turned on per dimer.

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5. The Carboxy Terminus 5.1. Homer Proteins The C-terminal domain (CTD) of the mGluRs is the least conserved region within the family and is also the domain encompassing the alternative splice forms. The CTD is a substrate for palmitoylation (75) and for protein kinases and phosphatases (76). One of the more intensely studied protein–protein interactions is with the clustering protein Homer (77). All long forms of Homer proteins (Homer1b/c/d, Homer 2a/b, and nearly all of the Homer 3 splice variants) have coiled-coil C-termini, which allow them to homoand heterodimerize with themselves (78). Another characteristic of Homer proteins is that they all contain an Ena/vasodilator-stimulated phosphoprotein homology-1 (EVH1) domain, which is responsible for protein–protein interactions, including those seen with the mGluRs (79). The sequence of this domain is related to the vasodilator-stimulated phosphoprotein (VASP) and PSD/Discs/ZO-1 (PDZ) domains (78). Homer proteins recognize and bind the proline-rich sequence PPxxF in the C-terminal tail of group I mGluR long splice variants mGluR1a and 5a/b (80). Competition of Homer proteins for this site results in differential receptor trafficking and signaling. For example, neurons at rest constitutively express the long isoform Homer1c, which promotes intracellular retention of mGluR5. Upon neuronal stimulation, Homer1a expression is upregulated and results in targeting of mGluR5 to the plasma membrane (80). Similarly, coexpression of mGluR1a with Homer1c results in decreased cell surface expression and noncovalent association with the IP3 receptor. In this way Homer proteins are able to interact with mGluRs and regulate clustering, scaffolding, and signal transmission (81). Homer proteins also allow the mGluRs to form unique signaling complexes. Under some conditions, quisqualate has been shown to promote neuronal survival and prevent apoptosis (82,83), but the mechanism remained unknown until a novel signaling complex was identified (84). PIKEL, a long version of the homologous nuclear protein PIKE, binds Homer1c to form a unique signaling complex with mGluR5. Following agonist stimulation of mGluR5, PI3K is activated by association with the mGluR5-Homer1c-PIKEL complex and transmits antiapoptotic signals (84). Another signaling cascade mediated by Homer binding to the CTD of mGluR5 promotes phosphorylation of ERK1/2 of the MAP kinase pathway. mGluR5 contains a PPxxF motif between amino acids 1154 and 1161 that facilitates binding of Homer1b/c. Mutation of LTPPSPFR to LTPLSPRR in this region abolishes Homer1b/c binding and prevents the subsequent phosphorylation of ERK1/2 (85). 5.2. Interactions with the Cytoskeleton In addition to interactions with intracellular proteins to create signaling complexes, the CTD is also important for mGluR interactions with various

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components of the cytoskeleton. mGluR5 has been shown associate with -tubulin, and this interaction, which anchors the receptor to the cytoskeleton, depends on the CTD region binding directly to tubulin or binding via a microtubule-associated protein (86). This interaction was clearly demonstrated in neuronal growth cones using a single-particle tracking assay of a CTD deletion mutant of mGluR5 (N887stop) whereby the truncated receptor diffused freely in the membrane compared to the anchored wild-type receptor. The significance of this interaction is not clear, although it was proposed that it may function to anchor the receptor to a specific location either during growth cone navigation or for proper synaptic communication at mature synapses. Another interaction of mGluRs with the cytoskeleton is through noncovalent linkage to filamin. An alanine scan was used to map regions of the mGluR7b splice variant that bind filamin A, and three additional interacting proteins were identified via yeast two-hybrid and GST-pull-down assays (87). Filamin A binding occurs over a 10-residue span between valine 909 and isoleucine 918, PP11 binds over five residues flanking serine 912, and the binding of syntenin and PICK1 is mediated by three amino acids—tryptophan 915, tyrosine 916, and valine 922. It is interesting that mutation of tryptophan 915 to alanine abolished interaction of mGluR7b with all of these proteins. Although this overlap initially suggested that these proteins may compete for binding at this residue, competition experiments concluded that this was not the case (87). Instead, a trimeric protein complex was proposed whereby mGluR7b and PP11 bound to either PICK1 or syntenin. A structural model proposed for this tail region of mGluR7b suggested that the tail depends on a hydrophobic interaction of tryptophan 915 with another, unidentified section of the C-terminus; the interaction is thought to form a structure that coordinates the spatial location of these proteins in a ternary complex. 5.3. The Role of the CTD in Regulating Calcium Inﬂux and Intracellular Calcium Release Group III mGluRs attenuate calcium influx and glutamate release by inhibition of presynaptic calcium channels through G subunits. It was proposed that activated calcium-calmodulin displaces prebound G from the CTD of mGluR7 (88). The “released” G subunit is then available for downstream signaling, such as inhibition of the N-type Ca2+ channels (89), and subsequent reduction in glutamate release. This specific interaction is most likely mediated by an association with PICK-1, a protein that is important in receptor clustering (90) and function (91,92) in the active zone of presynaptic terminals (89). In contrast, mGluR8, also inhibited N-type channels, although the calcium-calmodulin binding region in the CTD was not necessary for this interaction. Furthermore, the entire CTD was not necessary for cell surface expression or G protein coupling, and the degree of calcium channel inhibition

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was identical between mGluR8a and 8b splice variants (88). This indicates that unlike mGluR7, the C-terminus of mGluR8 is not needed for cell surface expression or interaction with N-type calcium channels. Both mGluR1 and mGluR5 stimulate the release of calcium from intracellular stores due to their association with the IP3 receptor via the Homer proteins. However, different response profiles have been observed between the two receptors. Whereas mGluR1 promotes fast, transient calcium release, mGluR5 induces calcium oscillations (93,94). This phenomenon has been determined to be a consequence of protein kinase C (PKC) phosphorylation (95). Phosphorylation of serine 839 by PKC occurs at the proximal portion of the C-terminal tail of mGluR5 and is responsible for calcium oscillations (95). Whereas mGluR5 isoforms have a threonine at position 840 that is permissive in allowing PKC phosphorylation at serine 839, the C-terminal tail of mGluR1a has a nonpermissive aspartate at the homologous position (aspartate 854), which precludes phosphorylation of serine 843 by PKC. 5.4. Phosphoprotein Phosphatase Interactions in the CTD Protein phosphatase 2C has been shown to interact with the short, 50-amino acid CTD of mGluR3; this binding motif is not conserved in mGluR2, and therefore PPC2 lacks the ability to bind the CTD of mGluR2 (96). Serine 845 in mGluR3 is the site for phosphorylation by protein kinase A and dephosphorylation by PP2C. It was also found that phosphorylation at this site decreases the binding of PP2C (96). The exact role of this protein–protein interaction is unknown, although several possibilities have been proposed, including the modulation of receptor trafficking, signaling, and desensitization. Using both yeast two-hybrid and GST-pull-down assays, the phosphatases PP11C and PP12C were identified as proteins interacting with mGluR1a and mGluR5a and 5b (97). The PP11 binding motif was localized to a 20-amino acid span in the distal section of the CTD and contained partially conserved sequences in mGluR1a (KSVSW) and mGluR5a/b and mGluR7b (KSVTW). An alanine scan of this region of the CTD of the mGluRs revealed that, except for the serine in the second position (serine 892 for mGluR1a, serine 885 for mGluR5, and serine 912 for mGluR7b), all other residues were necessary for PP11 binding. Competition experiments with CTD constructs showed that the C-terminal tails of mGluR1a, mGluR5a/b, and mGluR7b can compete with one another in a concentration-dependent manner for binding of PP11. In another study, it was established that mGluR5 but not mGluR1 activation results in PP2A inhibition and an increase in ERK1/2 stimulation (98). This study builds on previous work by this group showing that mGluR5 signals through Homer1b/c (85) and supports the idea that PP2A is involved in the mGluR5 signaling pathway to ERK by a novel mechanism of action. Overall the results demonstrate that an additive effect on phosphorylation of ERK1/2 is

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seen when inhibition of PP2A is paired with mGluR5 activation, and this effect ensures that the signal from mGluR5 is efficiently transmitted to downstream targets. 5.5. Binding of Proteins from the Sumoylation Cascade A yeast two-hybrid screen using the CTDs of mGluR8a and 8b as bait identified the protein sumo1 (small ubiquitin-related modifier) and several of its downstream targets in the sumoylation cascade as potential interacting proteins, but only Pias1 was confirmed by a GST pull-down assay (99). Pias1 (protein inhibitor of activated STAT) is an E3 protein ligase that facilitates sumo1 conjugation to proteins (100). Pias1 was shown to interact with all group III mGluRs, displaying the highest binding affinity for mGluR8a, followed by mGluR7a, mGluR6, and mGluR4. A minimal binding sequence of DRPNGE was identified between amino acids 875 and 880 in the group III receptors but not the group II receptors, which explains the absence of this interaction with mGluR3 (99). The significance of sumoylation is unknown, although it has been proposed that it might antagonize ubiquination of mGluRs by another mGluR interacting protein, seven in absentia homolog 1 (siah1). Siah1A binds to a specific siah-interacting domain in the C-terminal of the long splice variants of the group I mGluRs and promotes receptor degradation by ubiquination of lysine residues (101). It was also shown that multiple lysine residues throughout the CTD and TMD are ubiquinated and these events promote degradation by the proteosomal complex. This protein–protein interaction therefore has the ability to posttranslationally regulate the amount of mGluR protein at the cell surface.

Conclusion The in-depth understanding of the structures of the mGluRs that now exists is just beginning to bear fruit in terms of providing a more complete picture of the conformational changes and associated receptor activation mechanisms that occur subsequent to agonist binding. We also have at this point, a reasonably detailed, albeit incomplete, view of the diverse complement of proteins that associate with the mGluRs to propagate signals within cells both pre- and postsynapically. In light of the pharmaceutical industry’s focus on the development of allosteric modulators, more detailed structural information on the TMD regions of the mGluRs will likely benefit drug discovery and development efforts. As an added bonus, this structural knowledge may also translate into a more refined comprehension of receptor blocking and activation mechanisms that may have implications that extend beyond the mGluRs to other members of the G-protein coupled receptor superfamily.

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78. Kato A, Ozawa F, Saitoh Y, et al. Novel members of the Vesl/Homer family of PDZ proteins that bind metabotropic glutamate receptors. J Biol Chem 1998;273(37):23969–23975. 79. Beneken J, Tu JC, Xiao B, et al. Structure of the Homer EVH1 domain–peptide complex reveals a new twist in polyproline recognition. Neuron 2000;26(1): 143–154. 80. Abe H, Misaka T, Tateyama M, et al. Effects of coexpression with Homer isoforms on the function of metabotropic glutamate receptor 1alpha. Mol Cell Neurosci 2003;23(2):157–168. 81. Duncan RS, Hwang SY, Koulen P. Effects of Vesl/Homer proteins on intracellular signaling. Exp Biol Med (Maywood) 2005;230(8):527–535. 82. Flor PJ, Battaglia G, Nicoletti F, et al. Neuroprotective activity of metabotropic glutamate receptor ligands. Adv Exp Med Biol 2002;513:197–223. 83. Copani A, Bruno VM, Barresi V, et al. Activation of metabotropic glutamate receptors prevents neuronal apoptosis in culture. J Neurochem 1995;64(1):101–108. 84. Rong R, Ahn JY, Huang H, et al. PI3 kinase enhancer–Homer complex couples mGluRI to PI3 kinase, preventing neuronal apoptosis. Nat Neurosci 2003;6(11):1153–1161. 85. Mao L, Yang L, Tang Q, et al. The scaffold protein Homer1b/c links metabotropic glutamate receptor 5 to extracellular signal–regulated protein kinase cascades in neurons. J Neurosci 2005;25(10):2741–2752. 86. Serge A, Fourgeaud L, Hemar A, et al. Active surface transport of metabotropic glutamate receptors through binding to microtubules and actin flow. J Cell Sci 2003;116(Pt 24):5015–5022. 87. Enz R, Croci C. Different binding motifs in metabotropic glutamate receptor type 7b for filamin A, protein phosphatase 1C, protein interacting with protein kinase C (PICK) 1 and syntenin allow the formation of multimeric protein complexes. Biochem J 2003;372(Pt 1):183–191. 88. Guo J, Ikeda SR. Coupling of metabotropic glutamate receptor 8 to N-type Ca2+ channels in rat sympathetic neurons. Mol Pharmacol 2005;67(6):1840–1851. 89. Millan C, Castro E, Torres M, et al. Co-expression of metabotropic glutamate receptor 7 and N-type Ca(2+) channels in single cerebrocortical nerve terminals of adult rats. J Biol Chem 2003;278(26):23955–23962. 90. Boudin H, Doan A, Xia J, et al. Presynaptic clustering of mGluR7a requires the PICK1 PDZ domain binding site. Neuron 2000;28(2):485–497. 91. Dev KK, Nakajima Y, Kitano J, et al. PICK1 interacts with and regulates PKC phosphorylation of mGLUR7. J Neurosci 2000;20(19):7252–7257. 92. Perroy J, El FO, Bertaso F, et al. PICK1 is required for the control of synaptic transmission by the metabotropic glutamate receptor 7. EMBO J 2002;21(12):2990–2999. 93. Kawabata S, Kohara A, Tsutsumi R, et al. Diversity of calcium signaling by metabotropic glutamate receptors. J Biol Chem 1998;273(28):17381–17385. 94. Kawabata S, Tsutsumi R, Kohara A, et al. Control of calcium oscillations by phosphorylation of metabotropic glutamate receptors. Nature 1996; 383(6595):89–92.

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9 The Structures of Metabotropic Glutamate Receptors David R. Hampson, Erin M. Rose, and Jordan E. Antﬂick

Summary Interest in the structures of the metabotropic glutamate receptors continues to increase for a variety of reasons, including the fact that they are now established drug targets and are linked to a wide spectrum of physiologic processes both within and outside the central nervous system. This chapter summarizes our knowledge of the structures of the eight receptor subtypes, including the alternatively spliced forms, and the experimental approaches that have been used to study them. The large size and multiple domains of these proteins are conducive to further advances in the development of drugs for potential therapeutic use, and for basic research directed toward elucidating the intrinsic signaling mechanisms of these complex molecules. Increasingly detailed analyses of protein–protein interactions between the metabotropic glutamate receptors and other signaling molecules will also contribute to a deeper understanding of how this class of receptors and related G protein-coupled receptors, function at the molecular level within biologic membranes in vivo. Key Words: G protein-coupled receptor; Venus ﬂytrap; Heptahelical domain; Class C; Cysteine-rich domain; Dimer interface; Allosteric modulator.

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

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1. General Structural Features of Metabotropic Glutamate Receptors and Class C G Protein-Coupled Receptors The metabotropic glutamate receptors (mGluRs) are members of the class C subclass of receptors within the G protein-coupled receptor (GPCR) superfamily. Class C receptors are also referred to as the glutamate group in the GRAFS classification system of GPCRs outlined by Fredriksson et al. (1). Class C receptors encompass four main groups of receptors: Group I consists of the calcium-sensing receptor (CaSR), the V2R pheromone receptors, the T1R taste receptors, and the amino acid-activated GPRC6A; group II includes the mGluRs; group III consists of the -aminobutyric acid B (GABAB ) receptor; and group IV consists of the orphan receptor GPRC5 (2). With the exception of GPRC5, all members of this family possess a large extracellular ligandbinding domain, also referred to as the Venus flytrap domain (VFD), a prototypical heptahelical transmembrane domain (TMD), and an intracellular carboxy terminus (Fig. 1). With the exception of the GABAB receptors, class

Fig. 1. A metabotropic glutamate receptor (mGluR) dimer, including the ligandbinding domain, the cysteine-rich domain (CRD), and the seven-transmembrane domain (7TMD); the intracellular carboxyl tail of the receptor is not shown. The ligand-binding domain is a representation of the crystal structure, whereas the cysteinerich and 7TMD domains are homology models of mGluR1 based on the structures of the extracellular domain of the type I tumor necrosis factor receptor and rhodopsin, respectively. The actual orientation of the Venus flytrap domain and Cysteine-rich domain relative to the YTMD domain is not known; the figure depicts one possible arrangement. VFD, Venus flytrap domain.

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C receptors also possess a relatively short cysteine-rich domain (CRD) situated between the VFD and the TMD regions. The eight mammalian mGluRs are subclassified into three groups based on sequence similarity and pharmacologic properties. Group I receptors include mGluR1 and mGluR5; group II receptors include mGluR2 and mGluR3; and group III receptors are composed of mGluR4, mGluR6, mGluR7, and mGluR8. In cell lines, group I receptors couple to Gq and the Gq-like family of G proteins and the stimulation of phospholipase C and release of intracellular calcium, whereas the group II and III receptors couple to the Gi and Go family of G proteins and the inhibition of adenylyl cyclase. However, in the nervous system, the mGluRs couple to multiple signal transduction pathways, including the modulation of voltage-gated calcium and potassium channels. The CRD and TMD regions of the mGluRs do not appear to have counterparts in prokaryotes; the TMD also has very low sequence similarity to other G protein-coupled receptors, including rhodopsin and members of the rhodopsin family of receptors. However, O’Hara et al. (3) first reported that the VFD of the mGluRs show low sequence similarity to the periplasmic binding proteins in bacteria that transport amino acids and other nutrients into the cell. Thus, the VFDs of the mGluRs and other class C receptors may have evolved from the prokaryotic periplasmic binding proteins (4). It is clear that the mGluRs, like other neurotransmitter receptors, are oligomeric protein complexes. Most of the evidence indicates that the mGluRs are homodimers, although some groups have reported the presence of heterodimers composed of one mGluR subunit partnered with a subunit of another class C GPCR. The existence of higher-order structures in vivo, such as tetramers formed from dimers of dimers, has not been ruled out. The protomers within a dimeric mGluR complex are linked together at several points, including a covalent disulfide bond and additional noncovalent bonds in the VFD (5,6), hydrophobic interactions in the TMDs, and probably additional associations in the carboxy termini. The dimeric configuration of the VFD and ligand binding both remain intact when this domain is expressed as an isolated protein (7,8).

2. The Venus Flytrap Domain The VFD of the mGluRs is a large bilobed structure composed of approximately 600 amino acids. The crystal structures of the VFD of rat mGluR1 with and without bound glutamate (9–11), mGluR3, and mGluR7 (12) show that the two VFDs within each of the protomers face away from each other. Lobe 1 is situated on top of lobe 2, which in turn presumably sits on top of the CRD. A molecule of glutamate binds within a cleft formed between lobes 1 and 2 and induces a large 31-degree conformational shift resulting in the closure of the

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two lobes. Based on data obtained from the crystal structures of mGluR1 with bound glutamate or the antagonist -methyl-4-carboxyphenylglycine (MCPG), two primary configurations have been observed: an open/open form and a closed/open form. The open/open form is seen with the antagonist while the closed/open form was observed with glutamate. In the absence of ligands, both conformations were observed suggesting that the VFD maintains a dynamic equilibrium whereby agonists stabilize the closed/open configuration. Some competitive antagonists, such as MCPG and (S)-2-amino-2-methyl-4phosphobutanoic acid (MAP4) jam the VFD in the open configuration by the actions of a methyl group in the molecule that causes a steric clash with a residue situated on lobe 2. Thus mutation of tyrosine 227 to alanine in mGluR8 creates space into which this methyl group can fit into upon closure of the lobes; this mutation therefore converts MAP4 from an antagonist into an agonist capable of activating the receptor (13). In all mGluRs, the conformational change initiated in the VFD is then transmitted to the TMD (via the CRD) and ultimately results in the activation or inhibition of an effector protein. In the heteromeric GABAB receptor, only the GABAB -R1 subunit contains a GABA-binding site, and therefore only one molecule of GABA is needed to fully activate the receptor. In homodimeric mGluRs, a single molecule of glutamate bound to only one of the two protomers results in activation of the receptor complex. However, it has been suggested that full receptor activation may require agonist bound to both protomers (14,15), whereas others have speculated that agonist bound to both protomers may induce an “insulated state” whereby the receptor becomes desensitized (12). The extracellular VFD contains several sites of asparagine-linked glycosylation that are required for folding and cell surface expression but not for ligand binding (8,16,17). In the crystal structure of mGluR1 generated from protein purified from baculovirus-infected insect cells, two N-linked carbohydrates at asparagines 98 and 223 were present; neither glycosylation site is fully conserved in all eight mGluRs. Additional roles of N-linked carbohydrates beyond folding and surface expression have not been identified. The crystal structure of mGluR1 also revealed the presence of a bound divalent metal ion coordinated by asparagine 90, aspartate 92, and leucines 95 and 96. These amino acids are located far from the glutamate-binding site, and, based on its hexavalent coordination, the bound ion is most likely magnesium (9). This cation might be involved in receptor folding. We note, however, that unlike calcium, which can activate mGluR1 directly (18,19), magnesium ions do not directly activate this receptor, but they do potentiate the responses to glutamate (19). The site of action of the activating and potentiating effects of divalent cations on the mGluRs might be localized in or near the glutamate-binding pocket because they are obligatory for agonist binding to the isolated VFDs of group I and group II mGluRs (20).

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2.1. The Glutamate-Binding Pocket From an evolutionary perspective, the glutamate-binding pocket buried within the VFD has ancient origins. The primordial class C receptor likely arose during early metazoan evolution and may have been preadapted as a glutamate receptor for its later use at excitatory synapses (21). The glutamatebinding pocket in the mGluRs occupies a relatively small cavity within the crevice of the VFD compared to other class C receptors (22) and is highly selective for glutamate over other amino acids (23,24). From the crystal structure it appears that there are approximately 12 residues in the binding pocket that establish bonds with the bound glutamate molecule. As described later, additional bonding interactions occur with more complex agonists and antagonists. Within the pocket, several residues are highly conserved in the mGluRs; these conserved residues interact with the -carboxy and -amino groups of the glutamate ligand. Of particular importance are the four residues that establish bonds with the -amino group of the bound ligand; these residues are essential for ligand binding and are a signature feature of all mGluRs and most, if not all, class C amino acid–binding receptors (4,25,26). In contrast to the conserved amino acids that interact with the -carboxy and -amino groups of the glutamate ligand, the residues that establish bonds with the side chain of the glutamate ligand are not conserved and are the principal determinants of receptor subtype selectivity for orthosteric agonists and antagonists (27). The analogous set of residues in other members of class C receptors mediate amino acid ligand selectivity—for example, the preference for basic amino acids for the 5.24 and GPRC6A receptors (25,28–30) and the preference for large hydrophobic amino acids in the CaSR (31). Orthosteric mGluR agonists and antagonists can be divided into three groups: nonselective compounds that show relatively little selectivity among the eight subtypes, compounds that display selectivity towards group I, group II, or group III receptors, and drugs that show substantial selectivity toward a single receptor subtype. Most mGluR ligands fall in the group-selective class, and, in many cases, the molecular basis for this selectivity has been determined. The highly potent group I selective agonist quisqualate binds in the glutamate pocket in an orientation that allows additional interactions not present in the glutamate molecule. This was demonstrated by the observation that mutations in residues in the mGluR1 pocket that interact with the side chain -carboxyl group of the bound glutamate eliminate responsiveness to glutamate but not to the larger and more complex structure of quisqualate (32). DCG-IV and LY354470 are two examples of potent group II–selective orthosteric agonists. The molecular basis for the high affinity and selectivity of DCG-IV binding for group II receptors is mediated in part by an interaction between a third carboxylic acid group on DCG-IV not present in the glutamate molecule and a tyrosine and arginine present in the binding pocket of group II

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receptors (33). The selectivity of LY354740 to group II receptors is mediated by several residues, in particular, arginine 57 in mGluR2 and aspartate 146, which is located outside of the pocket and appears to mediate the higher affinity of LY354740 for mGluR2 versus mGluR3 (34). A salient characteristic of group III receptors is their selective activation by phosphonate- or phosphate-containing compounds such as l-amino-4phosphonobutyric acid (L-AP4), l-serine-O-phosphate (L-SOP), and RSphosphonophenylglycine (PPG). This selectivity is mediated by a cluster of amino acids in the pocket, primarily composed of the side chains of lysines and arginines, that converge to form a positively charged microenvironment; these residues establish bonding interactions with the negatively charged phosphonate group on L-SOP and L-AP4 (35). In some cases agonist selectivity across mGluR groups is mediated by only two residues in the binding pocket. For example, mutating lysine 74 together with lysine 317 or lysine 74 and glutamate 287 to the equivalent amino acids in mGluR1 resulted in complete switching of its pharmacologic profile whereby the double mutants displayed affinities for quisqualate that were similar to mGluR1 (36). The role of the amino acid at position 74 in the group III receptors is also highlighted by the work of Rosemond et al. (37). In this case, a molecular modeling and mutagenesis strategy was used to elucidate the molecular basis for the approximate 1000-fold difference in agonist affinity between the high-affinity mGluR4 receptor and the low-affinity mGluR7 receptor. Mutating asparagine 74 in the pocket of mGluR7 to lysine as in mGluR4 increased agonist affinity by 12-fold, and significantly, molecular models of the two receptors were crucial in revealing the identity and location of additional residues outside the pocket that also contribute to the differences in affinity of group III receptors. 2.2. The VFD Dimer Interface The dimer interface between the two VFDs likely participates in initiating the receptor activation process. A hydrophobic patch of residues at the interface appears to be involved in receptor activation; replacement of isoleucine 120 with alanine in this region in mGluR1 resulted in the elimination of signaling, likely by disrupting bonding interactions within this hydrophobic region and impairing the conformational change required to initiate receptor activation (32). Cocrystallization of the mGluR1 VFD with both the trivalent element gadolinium and glutamate, together with mutagenesis experiments, have demonstrated that the ion is bound to a site nested within several acidic residues located at the lobe 2–lobe 2 interface (10,38). Gadolinium acts by alleviating the mutual repulsion of the acidic residues projecting into the dimer interface region between the two protomers. Gadolinium is also a potent agonist of the calcium-sensing receptor (CaSR), but its site of action has not been identified. In mGluR1 this cation both potentiates the sensitivity to glutamate

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and, like calcium, acts as an agonist in the absence of glutamate whereby it may induce a closed–closed state of the two VFDs (39). Together, these studies and others provide examples of how the molecular space within the binding pocket can be probed to elucidate the molecular basis for orthosteric drug actions. Moreover, exploration of the dimer interface has revealed further insight into the mechanisms of receptor activation, and this region may prove to be a fruitful target for future drug development (40).

3. The Cysteine-Rich Domain The cysteine-rich domain (CRD), which links the VFD to the TMD, is composed of approximately 60–70 amino acids, 9 of which are cysteines. With the exception of the GABAB receptor, which lacks this domain, these 9 residues are highly conserved throughout class C receptors. With the CaSR, deletion of the CRD resulted in a protein that was expressed on the cell surface but was nonfunctional (41). Truncated forms of group II (mGluR3) and group III mGluRs (mGluR4 and mGluR8), which included all of the VFD plus part or all of the CRD, were retained in intracellular compartments when transfected into mammalian cells (17,42). However, group II and group III mGluR expression constructs encompassing the VFD but completely devoid of the CRD were secreted and retained their ligand-binding capabilities. The expression constructs used for X-ray crystallographic analysis of the VFD of mGluR1 and mGluR7 also excluded the CRD, whereas the mGluR3 structure included the CRD (6,9,12). Together, these findings suggest that the VFD and the CRD fold independent of one another and that the CRD is not essential for ligand binding. Despite the conservation of the CRD throughout the mGluRs and its importance in signaling from the VFD to the TMD, little is known about the structure of this region prior to the availability of the mGluR3 structure containing both the VFD and the CRD (12). Previously, several groups had formulated computer-generated models of the CRD structure. One group (43) used a “threading” approach whereby the CRD sequences from all eight mGluR subtypes were “fed” through a protein–folding template to calculate the most energetically favorable arrangement. Several possible structures were generated, which all share common secondary structural elements, namely two sets of antiparallel -strands that form a compact structure. It is interesting that it was also proposed that four of the nine cysteine residues within the CRD coordinate a Zn2+ ion. An alternative model of the CRD was constructed using a section of the tumor necrosis factor receptor as the template (44). This model also features four -strands and, in addition, includes three intramolecular disulfide bridges. This model further predicted the presence of two copies of a module termed A1, which also exists as a repeat motif in the tumor necrosis

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factor receptor and in epidermal growth factor–like domains. A consensus sequence for the mGluR A1 module was established as Cys-Xaa2 -Gly-Xaa-bXaa-Xaa 4−9 -Cys, where b indicates any amino acid with a large side chain. Of interest was the speculation that two conserved hydrophobic amino acids (a phenylalanine and a valine) and one of the cysteines could potentially participate in dimer formation. Muto et al. reported the structure of mGluR3 that encompasses both the VFD and the CRD (12). The structure of the mGluR3 CRD contains three -sheets, each composed of two short, antiparallel -strands. All of the cysteine residues are fully oxidized; four intradomain disulfide bridges contribute to stabilizing the internal architecture of the domain. In contrast, the remaining cysteine residue (C527 in mGluR3) forms a disulfide bond with C240 in lobe 2 (12,45). In addition, the short connection between the C terminus of the structure (E567) and the N terminus of the predicted TMD (A577) contains nine residues, which likely restrict the relative positions of the two domains (12). Thus, it is most likely that the CRD plays a major role in transmitting the conformational change in the ligand-binding domain to the TMD region. Furthermore, this domain prevents a direct interaction between the ligandbinding and TMD regions because the distance between the lobe 2 domain and the C-terminus of the CRD domain is too great. In summary, the structure of mGluR3 reveals that the CRD is tightly linked to the VFD by a disulfide bridge, suggesting a potential role in transmitting ligand-induced conformational changes to the downstream TMD region. The structure also indicates that potential lateral interactions between the two CRDs of the two protomers may exist that could facilitate clustering of the dimeric receptors on the cell surface.

4. The Heptahelical Transmembrane Since the initial cloning of the mGluRs in the early 1990s, it has been assumed, based on hydropathy plots, that the mGluRs possess the prototypical heptahelical TMD topology; some experimental evidence now exists supporting this topology. A study by Bhave et al. (46) used native and engineered glycosylation sites in mGluR5 to examine TMD topology. Mutation of only one of six possible N–glycosylation consensus sequences produced a mobility shift compared with a mutant in which all native N-glycosylation sites had been mutated, demonstrating that this site (asparagine 444) in the VFD accounts for all of the detectable native N-glycosylation in mGluR5. To analyze the structure further, asparagine N-glycosylation protection assays of truncated mGluR5a fusion proteins were conducted, in which the C-terminal domain of mGluR5a was fused to mGluR5a proteins truncated at each of the hydrophilic loops separating the seven TMDs. An HA epitope tag was inserted into the

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N-terminal domain and used in western blots following asparagine protease treatment of vesicles prepared from COS7 cells transfected with the mGluR5a fusion constructs. Fusion constructs in which the mGluR5a C-terminal tail lies in a “trans” configuration to the extracellularly located N-terminal HA tag will be proteolyzed, whereas those lying in a “cis” conformation will be protected along with the tag. These experiments confirmed an alternating pattern of cytoplasmic to extracellular tags beginning with intracellular loop 1 through to extracellular loop 3 (46). The sequence identity shared between bovine rhodopsin (the prototypical class A receptor) and the TMDs of the mGluR subtypes is very low (6%–15% amino acid sequence identity). The TMD helices in the mGluRs are separated by short intra- and extracellular loops (4). The second intracellular loop in the mGluRs is the largest, with a maximum of 27 residues, whereas the third intracellular loop in rhodopsin is the largest. The primary features of the mGluR third intracellular loop are conserved in class C receptors (except retinoic acid–induced receptor), with a basic residue at its N-terminal end and the motif (F/I/L)N(E/D)xK at its C-terminal end (4). Intracellular loops 2 and 3 form a cavity within which the C-terminus of the G protein alpha subunit interacts, and these two loops are thought to determine the receptors’ G protein coupling selectivity (47). The third intracellular loop in class C receptors is thought to play a role equivalent to that of the second intracellular loop in rhodopsin. When the intracellular loops of rhodopsin were replaced by those of mGluR6, only the constructs in which the third intracellular loop was replaced by the second intracellular loop of mGluR6 were functional (48). An intracellular amphipathic helix 8 occurs in rhodopsin parallel to the plane of the lipid bilayer just after TM7 and is thought to also play a role in G protein coupling. It has been suggested that a similar amphipathic helix may be present in the mGluRs (49). Within the TMD helices of class C receptors, only 19 residues are conserved. A highly conserved arginine at the end of TM3 of rhodopsin-like receptors is part of the DRY motif (aspartate-arginine-tyrosine). This motif plays a pivotal role in the equilibrium between active and inactive confirmations of the receptor. This arginine residue is conserved in the mGluRs (4). A disulfide bond that links the top of TM3 with the second extracellular loop in all GPCRs is also conserved in class C members (except retinoic acid–induced receptors). A tryptophan residue in TM6 is conserved between class A and class C (except GABAB ) receptors and is part of a hydrophobic pocket that constitutes the ligand-binding site in some family A receptors. This residue is also thought be critical in the transformation from the active to the inactive state of the receptor. Finally, the NPxxY motif in TM7 of rhodopsin is also conserved in class C receptors (as xPKxY); the proline residue in this motif causes a kink

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in the TM7 helix and is important for the correct coupling of the receptor to G protein (50). 4.1. The Role of Intracellular Loops 2 and 3 in Receptor Activation Tateyama et al. (51) used fluorescence resonance energy transfer (FRET) technology to study activation-induced structural changes in the TMD of the mGluRs. Two fluorophores, cyan fluorescent protein and yellow fluorescent protein, were inserted into the second or third intracellular loop or at various positions in the C-terminal tail. Insertion of the fluorophores into the third intracellular loop prevented membrane trafficking of mGluR1, suggesting that the third intracellular loop is a key determinant in the formation of a functional receptor. Insertion into the first and second intracellular loops caused a decrease and an increase in the FRET signal, respectively, on glutamate binding. Tateyama et al. (51) proposed a model whereby agonist binding leads to a shift in the intracellular domains such that the second intracellular loops of each monomer move closer together, hence the increase in the FRET signal. Notably, a dose–response effect was observed in which increasing concentrations of extracellular glutamate correlated with both increased FRET in the second intracellular loop dimer and increased release of intracellular calcium. Constitutively active mutations have been identified in bovine rhodopsin between the third intracellular loop and TM6, a region important for G protein coupling and receptor activation. A constitutively active mutation has been identified in mGluR8 at glutamine 695 in the second intracellular loop, which is highly conserved among all mGluR subtypes and other family C GPCRs, such as the CaSR (52). The activity of the mutant increases when this glutamine is substituted with a nonpolar or small, uncharged, polar amino acid (e.g., alanine, cysteine, isoleucine, methionine, valine, serine, or threonine) and decreases with ringed or acidic amino acids (phenylalanine, proline, tryptophan, tyrosine, aspartate, or glutamate). These results suggest that a conformational change around glutamine 695 occurs during G protein activation, perhaps favoring the active state of the receptor. Further analysis of the second intracellular loop of mGluR8 led to the identification of specific residues responsible for the G protein selectivity. Five of these residues (arginine 672, isoleucine 673, arginine 675, isoleucine 676, and proline 687) are conserved in all mGluR subtypes, and two residues (phenylalanine 677 and isoleucine 690) are conserved among the Gi/o-coupled mGluR subtypes (52). The latter two residues could potentially be important determinants of Go-subtype coupling in mGluRs. Several intracellular proteins have been shown to modulate G protein coupling and receptor activation. GPCR kinases (GRKs) phosphorylate rhodopsin-like GPCRs and promote the binding of arrestins, resulting in the

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desensitization and internalization of the targeted receptor. GRK-mediated inhibition of mGluR1, however, is phosphorylation and -arrestin independent (53). Dhami et al. (53) discovered that GRK2 inhibits mGluR1 by binding to a region in the second intracellular loop, presumably preventing the binding of the G protein. Together these findings confirm that second intracellular loop in the mGluRs is an important determinant of G protein selectivity and is essential for activation of the receptor. 4.2. Structural Determinants of Allosteric Modulation The study of the structure–activity relationships of allosteric modulators of the mGluRs has provided important information regarding the mechanism of receptor activation. Positive allosteric modulators do not directly activate the receptor, but instead potentiate the response of an agonist binding to the orthosteric site. A growing number of positive and negative allosteric modulators have been discovered, some of which are listed in Tables 1 and 2, respectively (54–70). An interesting observation is that when the extracellular domain of an

Table 1 Positive Allosteric Modulators of Metabotropic Glutamate Receptors

BBB, blood–brain barrier; mGluR, metabotropic glutamate receptor; MPEP, 2-methyl-6phenylethynylpyridine; NMDA, N-methyl-D-aspartate; PHCCC, N-phenyl-7-(hydroxylimino) cyclopropa[b]chromen-la-carboxamide; TM, transmembrane; TMD, transmembrane domain.

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Table 2 Negative Allosteric Modulators of Metabotropic Glutamate Receptors

CPCCOEt, 7-Hydroxyiminocyclopropan[b]chromen-la-carboxylic acid ethyl ester; DHPG, (R,S)3,5-dihydroxyphenylglycine; EM-TBPC, 1-ethyl-2-methyl-6-oxo-4 (1,2,4,5-tetrahydrobenzo[d]azepin-3yl)-1,6-dihydro-pyrimidine-5-carbonitrile; IC50 half-maximal inhibition constant; mGluR, metabotropic glutamate receptor; MPEP, 2-methyl-6-phenylethynylpyridine; MTEP, 3[(2-methyl-1,3-thiazol-4yl)ethynyl]-pyridine; Noncomp., noncompetitive; TM transmembrane; TMD, transmembrane domain.

mGluR is deleted, positive allosteric modulators can act as agonists by inducing responses in the absence of an orthosteric agonist. Goudet et al. (71) examined this phenomenon by comparing the activity of the positive allosteric modulator 3,3’-difluorobenzaldazine (DFB) on wild-type mGluR5 and truncated mGluR5 (with the extracellular domain and/or C-terminus removed). They observed that only glutamate activated the wild-type receptor and that DFB directly activated the truncated mGluR5 constructs (71). The results from this study suggested that the TMD is involved in the transition from an inactive to an active state of the receptor. 7-Hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt) is a noncompetitive antagonist of mGluR1 that binds within TM7 at threonine 815 and alanine 818. Conformational changes within the TMD induced by glutamate binding to the orthosteric site is inhibited by CPCCOEt, as observed by inhibition of the FRET signal induced by 1 mM l-glutamate (51). Thus, within the TMD several residues are required for transformation

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of the receptor from the inactive to the active state, and these residues are blocked by the binding of negative allosteric modulators (52). Three aromatic residues—tryptophan 798, phenylalanine 801, and tyrosine 805—are thought to be essential for binding and inhibitory activity of the another mGluR1 negative allosteric modulator, 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydrobenzo[d]azepin-3-yl)-1,6-dihydro-pyrimidine-5-carbonitrile (EM-TBPC) (72). EM-TBPC may bind within this aromatic cluster in TM6 and block the conformational change centered around tryptophan 798, an amino acid critical for transition to the active state of the receptor. It therefore appears that negative and positive allosteric modulation of mGluRs involves interruption or enhancement, respectively, of conformational changes within the TMD. Allosteric modulators have also provided insight into how each protomer of an mGluR homodimer contributes to activation of the receptor. Truncated mGluR dimers consisting of the isolated TMDs appear to function asymmetrically such that activation of only one of the TMD protomers is required for full receptor activity. One molecule of DFB, a selective positive allosteric modulator of mGluR5, is required to elicit full agonist activity in a chimeric mGluR1/mGluR5 TMD heterodimer (73). The phenomenon of asymmetric activation of a symmetric mGluR homodimer may be explained by steric hindrance, which prevents conformational changes within the TMD from occurring in both protomers simultaneously. Hlavackova et al. (74) assessed the contribution of the two protomers to receptor output by creating two chimeric mGluR1 receptors, R1c1 and R1c2, by replacing the C-terminal tail of mGluR1 with the tails of GABAB R1 and GABAB R2, respectively. The GABAB R1 subunit does not reach the cell surface alone due to the presence of an endoplasmic reticulum retention signal in its C-terminal tail; this signal is masked by the presence of the C-terminal tail of GABAB R2 (74). Thus, these chimeras provided control of dimer composition at the cell surface. In addition, each chimera was tagged with a fluorophore (EuCryptate or Alexa647) and an epitope (HA or c-myc) to measure FRET and to track expression. By producing a point mutation in the third intracellular loop of one chimera, it was determined that both TMDs are required to activate G proteins for full receptor activity. However, this mutation did not inhibit the ability of the TMD to reach an active conformation. For a receptor dimer combination in which only one TMD is maintained in the inactive state with the negative modulator (inverse agonist) 2-methyl-6-phenylethynylpyridine (MPEP), the associated subunit is still able to generate the full response of the receptor to l-glutamate. The interpretation of these findings was that the mutation in intracellular loop 3 impairs G protein activation but not the ability of the transmembrane domain to reach an active conformation; therefore the TMD of a single protomer is turned on per dimer.

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5. The Carboxy Terminus 5.1. Homer Proteins The C-terminal domain (CTD) of the mGluRs is the least conserved region within the family and is also the domain encompassing the alternative splice forms. The CTD is a substrate for palmitoylation (75) and for protein kinases and phosphatases (76). One of the more intensely studied protein–protein interactions is with the clustering protein Homer (77). All long forms of Homer proteins (Homer1b/c/d, Homer 2a/b, and nearly all of the Homer 3 splice variants) have coiled-coil C-termini, which allow them to homoand heterodimerize with themselves (78). Another characteristic of Homer proteins is that they all contain an Ena/vasodilator-stimulated phosphoprotein homology-1 (EVH1) domain, which is responsible for protein–protein interactions, including those seen with the mGluRs (79). The sequence of this domain is related to the vasodilator-stimulated phosphoprotein (VASP) and PSD/Discs/ZO-1 (PDZ) domains (78). Homer proteins recognize and bind the proline-rich sequence PPxxF in the C-terminal tail of group I mGluR long splice variants mGluR1a and 5a/b (80). Competition of Homer proteins for this site results in differential receptor trafficking and signaling. For example, neurons at rest constitutively express the long isoform Homer1c, which promotes intracellular retention of mGluR5. Upon neuronal stimulation, Homer1a expression is upregulated and results in targeting of mGluR5 to the plasma membrane (80). Similarly, coexpression of mGluR1a with Homer1c results in decreased cell surface expression and noncovalent association with the IP3 receptor. In this way Homer proteins are able to interact with mGluRs and regulate clustering, scaffolding, and signal transmission (81). Homer proteins also allow the mGluRs to form unique signaling complexes. Under some conditions, quisqualate has been shown to promote neuronal survival and prevent apoptosis (82,83), but the mechanism remained unknown until a novel signaling complex was identified (84). PIKEL, a long version of the homologous nuclear protein PIKE, binds Homer1c to form a unique signaling complex with mGluR5. Following agonist stimulation of mGluR5, PI3K is activated by association with the mGluR5-Homer1c-PIKEL complex and transmits antiapoptotic signals (84). Another signaling cascade mediated by Homer binding to the CTD of mGluR5 promotes phosphorylation of ERK1/2 of the MAP kinase pathway. mGluR5 contains a PPxxF motif between amino acids 1154 and 1161 that facilitates binding of Homer1b/c. Mutation of LTPPSPFR to LTPLSPRR in this region abolishes Homer1b/c binding and prevents the subsequent phosphorylation of ERK1/2 (85). 5.2. Interactions with the Cytoskeleton In addition to interactions with intracellular proteins to create signaling complexes, the CTD is also important for mGluR interactions with various

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components of the cytoskeleton. mGluR5 has been shown associate with -tubulin, and this interaction, which anchors the receptor to the cytoskeleton, depends on the CTD region binding directly to tubulin or binding via a microtubule-associated protein (86). This interaction was clearly demonstrated in neuronal growth cones using a single-particle tracking assay of a CTD deletion mutant of mGluR5 (N887stop) whereby the truncated receptor diffused freely in the membrane compared to the anchored wild-type receptor. The significance of this interaction is not clear, although it was proposed that it may function to anchor the receptor to a specific location either during growth cone navigation or for proper synaptic communication at mature synapses. Another interaction of mGluRs with the cytoskeleton is through noncovalent linkage to filamin. An alanine scan was used to map regions of the mGluR7b splice variant that bind filamin A, and three additional interacting proteins were identified via yeast two-hybrid and GST-pull-down assays (87). Filamin A binding occurs over a 10-residue span between valine 909 and isoleucine 918, PP11 binds over five residues flanking serine 912, and the binding of syntenin and PICK1 is mediated by three amino acids—tryptophan 915, tyrosine 916, and valine 922. It is interesting that mutation of tryptophan 915 to alanine abolished interaction of mGluR7b with all of these proteins. Although this overlap initially suggested that these proteins may compete for binding at this residue, competition experiments concluded that this was not the case (87). Instead, a trimeric protein complex was proposed whereby mGluR7b and PP11 bound to either PICK1 or syntenin. A structural model proposed for this tail region of mGluR7b suggested that the tail depends on a hydrophobic interaction of tryptophan 915 with another, unidentified section of the C-terminus; the interaction is thought to form a structure that coordinates the spatial location of these proteins in a ternary complex. 5.3. The Role of the CTD in Regulating Calcium Inﬂux and Intracellular Calcium Release Group III mGluRs attenuate calcium influx and glutamate release by inhibition of presynaptic calcium channels through G subunits. It was proposed that activated calcium-calmodulin displaces prebound G from the CTD of mGluR7 (88). The “released” G subunit is then available for downstream signaling, such as inhibition of the N-type Ca2+ channels (89), and subsequent reduction in glutamate release. This specific interaction is most likely mediated by an association with PICK-1, a protein that is important in receptor clustering (90) and function (91,92) in the active zone of presynaptic terminals (89). In contrast, mGluR8, also inhibited N-type channels, although the calcium-calmodulin binding region in the CTD was not necessary for this interaction. Furthermore, the entire CTD was not necessary for cell surface expression or G protein coupling, and the degree of calcium channel inhibition

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was identical between mGluR8a and 8b splice variants (88). This indicates that unlike mGluR7, the C-terminus of mGluR8 is not needed for cell surface expression or interaction with N-type calcium channels. Both mGluR1 and mGluR5 stimulate the release of calcium from intracellular stores due to their association with the IP3 receptor via the Homer proteins. However, different response profiles have been observed between the two receptors. Whereas mGluR1 promotes fast, transient calcium release, mGluR5 induces calcium oscillations (93,94). This phenomenon has been determined to be a consequence of protein kinase C (PKC) phosphorylation (95). Phosphorylation of serine 839 by PKC occurs at the proximal portion of the C-terminal tail of mGluR5 and is responsible for calcium oscillations (95). Whereas mGluR5 isoforms have a threonine at position 840 that is permissive in allowing PKC phosphorylation at serine 839, the C-terminal tail of mGluR1a has a nonpermissive aspartate at the homologous position (aspartate 854), which precludes phosphorylation of serine 843 by PKC. 5.4. Phosphoprotein Phosphatase Interactions in the CTD Protein phosphatase 2C has been shown to interact with the short, 50-amino acid CTD of mGluR3; this binding motif is not conserved in mGluR2, and therefore PPC2 lacks the ability to bind the CTD of mGluR2 (96). Serine 845 in mGluR3 is the site for phosphorylation by protein kinase A and dephosphorylation by PP2C. It was also found that phosphorylation at this site decreases the binding of PP2C (96). The exact role of this protein–protein interaction is unknown, although several possibilities have been proposed, including the modulation of receptor trafficking, signaling, and desensitization. Using both yeast two-hybrid and GST-pull-down assays, the phosphatases PP11C and PP12C were identified as proteins interacting with mGluR1a and mGluR5a and 5b (97). The PP11 binding motif was localized to a 20-amino acid span in the distal section of the CTD and contained partially conserved sequences in mGluR1a (KSVSW) and mGluR5a/b and mGluR7b (KSVTW). An alanine scan of this region of the CTD of the mGluRs revealed that, except for the serine in the second position (serine 892 for mGluR1a, serine 885 for mGluR5, and serine 912 for mGluR7b), all other residues were necessary for PP11 binding. Competition experiments with CTD constructs showed that the C-terminal tails of mGluR1a, mGluR5a/b, and mGluR7b can compete with one another in a concentration-dependent manner for binding of PP11. In another study, it was established that mGluR5 but not mGluR1 activation results in PP2A inhibition and an increase in ERK1/2 stimulation (98). This study builds on previous work by this group showing that mGluR5 signals through Homer1b/c (85) and supports the idea that PP2A is involved in the mGluR5 signaling pathway to ERK by a novel mechanism of action. Overall the results demonstrate that an additive effect on phosphorylation of ERK1/2 is

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seen when inhibition of PP2A is paired with mGluR5 activation, and this effect ensures that the signal from mGluR5 is efficiently transmitted to downstream targets. 5.5. Binding of Proteins from the Sumoylation Cascade A yeast two-hybrid screen using the CTDs of mGluR8a and 8b as bait identified the protein sumo1 (small ubiquitin-related modifier) and several of its downstream targets in the sumoylation cascade as potential interacting proteins, but only Pias1 was confirmed by a GST pull-down assay (99). Pias1 (protein inhibitor of activated STAT) is an E3 protein ligase that facilitates sumo1 conjugation to proteins (100). Pias1 was shown to interact with all group III mGluRs, displaying the highest binding affinity for mGluR8a, followed by mGluR7a, mGluR6, and mGluR4. A minimal binding sequence of DRPNGE was identified between amino acids 875 and 880 in the group III receptors but not the group II receptors, which explains the absence of this interaction with mGluR3 (99). The significance of sumoylation is unknown, although it has been proposed that it might antagonize ubiquination of mGluRs by another mGluR interacting protein, seven in absentia homolog 1 (siah1). Siah1A binds to a specific siah-interacting domain in the C-terminal of the long splice variants of the group I mGluRs and promotes receptor degradation by ubiquination of lysine residues (101). It was also shown that multiple lysine residues throughout the CTD and TMD are ubiquinated and these events promote degradation by the proteosomal complex. This protein–protein interaction therefore has the ability to posttranslationally regulate the amount of mGluR protein at the cell surface.

Conclusion The in-depth understanding of the structures of the mGluRs that now exists is just beginning to bear fruit in terms of providing a more complete picture of the conformational changes and associated receptor activation mechanisms that occur subsequent to agonist binding. We also have at this point, a reasonably detailed, albeit incomplete, view of the diverse complement of proteins that associate with the mGluRs to propagate signals within cells both pre- and postsynapically. In light of the pharmaceutical industry’s focus on the development of allosteric modulators, more detailed structural information on the TMD regions of the mGluRs will likely benefit drug discovery and development efforts. As an added bonus, this structural knowledge may also translate into a more refined comprehension of receptor blocking and activation mechanisms that may have implications that extend beyond the mGluRs to other members of the G-protein coupled receptor superfamily.

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78. Kato A, Ozawa F, Saitoh Y, et al. Novel members of the Vesl/Homer family of PDZ proteins that bind metabotropic glutamate receptors. J Biol Chem 1998;273(37):23969–23975. 79. Beneken J, Tu JC, Xiao B, et al. Structure of the Homer EVH1 domain–peptide complex reveals a new twist in polyproline recognition. Neuron 2000;26(1): 143–154. 80. Abe H, Misaka T, Tateyama M, et al. Effects of coexpression with Homer isoforms on the function of metabotropic glutamate receptor 1alpha. Mol Cell Neurosci 2003;23(2):157–168. 81. Duncan RS, Hwang SY, Koulen P. Effects of Vesl/Homer proteins on intracellular signaling. Exp Biol Med (Maywood) 2005;230(8):527–535. 82. Flor PJ, Battaglia G, Nicoletti F, et al. Neuroprotective activity of metabotropic glutamate receptor ligands. Adv Exp Med Biol 2002;513:197–223. 83. Copani A, Bruno VM, Barresi V, et al. Activation of metabotropic glutamate receptors prevents neuronal apoptosis in culture. J Neurochem 1995;64(1):101–108. 84. Rong R, Ahn JY, Huang H, et al. PI3 kinase enhancer–Homer complex couples mGluRI to PI3 kinase, preventing neuronal apoptosis. Nat Neurosci 2003;6(11):1153–1161. 85. Mao L, Yang L, Tang Q, et al. The scaffold protein Homer1b/c links metabotropic glutamate receptor 5 to extracellular signal–regulated protein kinase cascades in neurons. J Neurosci 2005;25(10):2741–2752. 86. Serge A, Fourgeaud L, Hemar A, et al. Active surface transport of metabotropic glutamate receptors through binding to microtubules and actin flow. J Cell Sci 2003;116(Pt 24):5015–5022. 87. Enz R, Croci C. Different binding motifs in metabotropic glutamate receptor type 7b for filamin A, protein phosphatase 1C, protein interacting with protein kinase C (PICK) 1 and syntenin allow the formation of multimeric protein complexes. Biochem J 2003;372(Pt 1):183–191. 88. Guo J, Ikeda SR. Coupling of metabotropic glutamate receptor 8 to N-type Ca2+ channels in rat sympathetic neurons. Mol Pharmacol 2005;67(6):1840–1851. 89. Millan C, Castro E, Torres M, et al. Co-expression of metabotropic glutamate receptor 7 and N-type Ca(2+) channels in single cerebrocortical nerve terminals of adult rats. J Biol Chem 2003;278(26):23955–23962. 90. Boudin H, Doan A, Xia J, et al. Presynaptic clustering of mGluR7a requires the PICK1 PDZ domain binding site. Neuron 2000;28(2):485–497. 91. Dev KK, Nakajima Y, Kitano J, et al. PICK1 interacts with and regulates PKC phosphorylation of mGLUR7. J Neurosci 2000;20(19):7252–7257. 92. Perroy J, El FO, Bertaso F, et al. PICK1 is required for the control of synaptic transmission by the metabotropic glutamate receptor 7. EMBO J 2002;21(12):2990–2999. 93. Kawabata S, Kohara A, Tsutsumi R, et al. Diversity of calcium signaling by metabotropic glutamate receptors. J Biol Chem 1998;273(28):17381–17385. 94. Kawabata S, Tsutsumi R, Kohara A, et al. Control of calcium oscillations by phosphorylation of metabotropic glutamate receptors. Nature 1996; 383(6595):89–92.

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10 Group I Metabotropic Glutamate Receptors (mGlu1 and mGlu5) Julie Anne Saugstad and Susan Lynn Ingram

Summary The metabotropic glutamate (mGlu) receptors are a family of eight G protein-coupled receptors that modulate cell excitability and synaptic transmission in the nervous system. Group I mGlu receptors are generally coupled to Gq stimulation of phospholipase C and the release of intracellular calcium. The physiologic effects of group I mGlu receptors in the brain are diverse and highlight the importance of these receptors in normal brain function. This chapter describes what is known about the structure, signaling, regulation, and function of the group I mGlu receptors in the nervous system, as well as the roles of these receptors in disease. It also discusses emerging roles in both central and peripheral nonneural tissues. Because the group I mGlu receptors modulate rather than mediate excitatory neurotransmission, they are exciting targets for new therapeutic strategies. However, further understanding of the complex regulatory mechanisms associated with these receptors is required to design more beneﬁcial therapies for neurodegenerative and neuropsychiatric disorders. Key Words: Group I metabotropic glutamate receptors; Gq; Synaptic plasticity; Anxiety; Addiction; Pain; Neurodegeneration; Neuropsychiatric disorder; Fragile X; MicroRNA.

The metabotropic glutamate (mGlu) receptors are a family of eight G protein–coupled receptors (GPCRs) that modulate cell excitability and synaptic transmission in the nervous system. The mGlu receptor family is divided into three groups based on amino acid homology, signal transduction From: The Receptors: The Glutamate Receptors Edited by: R. W. Gereau and G. T. Swanson © Humana Press, Totowa, NJ

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pathways, and pharmacologic profiles (Fig. 1). The group I mGlu receptors (mGlu1 and mGlu5) couple to the Gq signaling pathway, are predominantly postsynaptic, and in general potentiate cell excitability. The group II (mGlu2 and 3) and group III mGlu (mGlu4, 6, 7, and 8) receptors couple to Gi/Go signaling pathways, are predominantly presynaptic, and in general decrease cell excitability by inhibiting glutamate release from presynaptic terminals (1). In this chapter we describe what is known about the structure, signaling, regulation, and function of the group I mGlu receptors in the central nervous system. We also discuss emerging roles for these receptors both within and outside the brain. However, the divergent coupling of GPCRs to second messenger pathways paints a typical picture that is frustrating for those trying to understand all of the functions of GPCR activation: There appears to be no set rule for coupling between receptor subtypes and second messenger pathways, and one can find examples of inhibition and potentiation for practically any type of ion channel studied. This frustration extends to studies on the molecular mechanisms that underlie group I mGlu receptor function in that they couple to several signaling pathways and modulate cell excitability by a variety of methods that vary based on the cell type, the synapse, and

Fig. 1. The eight cloned metabotropic glutamate (mGlu) receptor subtypes are classified into three groups based on their amino acid identity and their predominant G protein signaling pathway used in cells, as depicted, as well as on their pharmacologic profile.

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the proteins that comprise the signaling complex. The recent development of pharmacologic tools, such as allosteric modulators that can tease apart the complexity of the group I mGlu receptor signaling pathways, will only add to this depth and breadth of information, but will certainly present exciting new avenues for future research. Thus, although we have tried to present much of what is known about the group I mGlu receptors, their effects in the brain are diverse and highlight the importance of these receptors in normal brain function, as well as their potential as therapeutic targets for neurologic and neuropsychiatric disorders.

1. Structure The mGlu1 and mGlu5 proteins contain seven hydrophobic regions with a large extracellular ligand-binding domain and an intracellular carboxy-terminal domain (CTD) and are members of the family C GPCRs, which are structurally distinct from the other GPCR families. The mGlu receptors form homodimers that can couple to distinct signaling pathways, and they can form heterodimers with other members of the family C GPCRs. The group I mGlu receptors are abundantly expressed throughout the brain, and in some regions they show very distinct, nonoverlapping expression patterns. In addition, although it is generally accepted that both mGlu1 and mGlu5 are located on the postsynaptic density, perisynaptic to ionotropic glutamate receptors (iGluRs), the mGlu5 subtype is also localized to presynaptic and nuclear membranes and is the predominant subtype in glial cells. 1.1. Genes Experiments in the mid-1980s showed that application of excitatory amino acids to rat hippocampal slices (2) and cerebellar cultures (3) enhanced the hydrolysis of membrane inositol phospholipids. These studies provided the rationale for the existence of a second messenger–coupled glutamate receptor in rat brain, and soon thereafter the first Rattus norvegicus heterotrimeric GTP-binding protein (G protein)–coupled glutamate receptor was isolated with the use of expression cloning. Distinct pools of rat cerebellar complementary DNA (cDNA) clones were used as templates for the in vitro transcription of complementary RNA (cRNA), and cRNA pools were injected into Xenopus laevis oocytes. The presence of a glutamate-activated GPCR was assessed by measuring the calcium-dependent chloride current, and positive cRNA pools were successively fractionated into smaller pools until the cDNA encoding mGlu1a was ultimately isolated (4,5). The cDNA encoding mGlu5 was subsequently isolated by homology cloning (6). The mGlu1 and mGlu5 receptor subtypes comprise the group I mGlu receptors (Fig. 1). The rat mGlu1 gene (Grm1) is located on chromosome

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1:5318616–5744590 and aligns with the mouse mGlu1 gene located on chromosome 10:7248561–21359563 and the human mGlu1 gene located on chromosome 6:135263257–150277927. The rat mGlu5 gene (Grm5) is located on chromosome 1:143935631–144555825 and aligns with the mouse mGlu5 gene located on chromosome 7:5251451–139739826 and the human mGlu5 gene located on chromosome 11:58029473–88990550 (7–9). There are four carboxy-terminal and one amino-terminal splice variants for rat mGlu1 and one carboxy-terminal splice variant for rat mGlu5 (Fig. 2). The mGlule (10) is an amino-terminal splice variant that results in the expression of a soluble protein that comprises only the extracellular amino-terminal domain (ATD). The carboxy-terminal splice variants arise due to the insertion of a stop codon

Fig. 2. Schematic of the rat group I metabotropic glutamate (mGlu) receptor splice variants. In addition to mGlu1a and mGlu5a, there are five splice variants for the mGlu1 receptor and one splice variant for the mGlu5 receptor. The proteins comprise a large extracellular amino-terminal domain that, except for mGlu1e, is followed by a seven-transmembrane domain and carboxy-terminal domains of varying lengths. Most studies have focused on the predominant splice variants mGlu1a, mGlu1b, mGlu5a, and mGlu5b. ANF, atrial natriuretic factor. Adapted from Hermans E, Challiss RA. Structural, signalling and regulatory properties of the group I metabotropic glutamate receptors: prototypic family C G-protein-coupled receptors. Biochem J 2001:359(pt 3): 465–484.

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that results in the deletion of 312 amino acids from each CTD, and to the presence of alternative acceptor splice sites on mGlu1b (11), mGlu1c (12), mGlu1d (13), and mGlu1f (14) messenger RNA (mRNA) transcripts that add either 20, 11, 26, or 20 amino acid residues to the carboxy terminus of each splice variant, respectively. The rat mGlu5 splice variant, mGlu5b (15), results from an in-frame insertion that leads to an additional 32 amino acid residues in the carboxy terminus. Homology screening of a human brain cDNA library also reveals a novel splice variant, mGlu5d, which is 267 amino acids shorter than mGlu5a (16). Finally, subsequent cloning led to the isolation of group I mGlu receptor genes in Mus musculus (mouse), Homo sapiens (human), Pan troglodytes (chimpanzee), Macaca fascicularis (crab-eating macaque), Drosophila melanogaster (fruit fly), Canis familiaris (dog), Gallus gallus (chicken), Oncorhynchus masou (cherry salmon), Tetraodon nigroviridis (spotted green pufferfish), Danio rerio (zebrafish), Bos taurus (cow), and Taeniopygia guttata (zebra finch) (9,17) (http://www.ncbi.nlm.nih.gov/). 1.2. Topology The mGlu receptors are members of the family 3/C GPCRs that include the calcium-sensing receptors (CaRs), the -aminobutyric acid B (GABAB ) receptors, and a broad multigene family of olfactory, taste, and pheromone receptors. This family of receptors is distinct from other GPCRs in that there is no significant sequence homology to the classic rhodopsin-type seventransmembrane domain (7TMD) GPCRs. Whereas the mGlu receptor proteins contain high proportions of hydrophobic residues grouped into seven domains, several TMD prediction algorithms indicate that the mGlu receptors might have a topology that is distinct from the classic 7TMD GPCRs. Molecular studies do provide evidence that the topology of mGlu receptors is consistent with that of GPCRs, but reveal that the mGlu receptors share a distinctive 7TMD signature in common only with other family 3/C GPCR members (18). The family 3/C receptors are also distinct in that they contain a large extracellular ATD where the ligand binds, in contrast to classical GPCRs, where the ligand binds within the 7TMD region. The mGlu receptor ATD has a high degree of secondary structure conservation with the leucine-isoleucine-valine– binding proteins, leucine-binding proteins, and acetamide-binding proteins, which are all periplasmic amino acid–binding proteins (19). Thus, the mGlu receptors share an evolutionary link with the periplasmic-binding protein family and share a common mechanism of ligand binding and processing that involves an equilibrium between closed and open forms (19,20). The ATD is linked to the first transmembrane domain by a cysteine-rich region whose functional significance is unclear; however, deletion mutagenesis studies of this region result in improper folding or cellular targeting of the ATD of group III mGlu

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receptors (21). Queries of the mGlu1 and mGlu5 protein sequences at the NCBI Conserved Domain Database (22) show three conserved domains within the group I mGlu receptors: (1) an atrial natriuretic factor (ANF) receptor family contains an extracellular ligand-binding domain (LBD) found in a wide range of receptors, including the bacterial amino acid–binding proteins, (2) a LivK domain is found in ABC-type branched-chain amino acid transport systems and is involved in amino acid transport and metabolism, and (3) a seven-TM3 domain that is a signature of the family 3/C 7TMD receptors. 1.3. Dimers GPCRs were presumed to function as monomeric proteins; however, studies reveal that several receptors can dimerize. The group I mGlu receptors form homodimers by specific, intermolecular disulfide-dependent interactions in the extracellular ATD (23), and mutagenesis studies have shown that cysteine residues in the ATD are involved in the homodimerization of mGlu1 (24) and mGlu5 (25). X-ray crystallography studies show that the mGlu1 LBD comprises ∼520 amino acids and forms a clamshell-like bilobate domain through intersubunit disulfide bonds and hydrophobic interactions (20). One protomer, or bilobate domain, of a dimeric LBD can adopt an open conformation and a closed conformation. In the absence of glutamate, both protomers are in the open conformation. In the presence of glutamate, one protomer is in the open conformation and the other protomer is in the closed conformation. It is interesting that the open-closed conformation has been observed in the absence of glutamate, which implies that the open–closed protomer conformations are in equilibrium in aqueous solution without ligand. Although glutamate binding stabilizes the closed conformation of one protomer, the glutamate-bound open conformation observed in the crystal structure suggests that there is an allosteric interaction within the dimeric LBD in that the closed conformation in one protomer negatively affects the binding mode of the other protomer. Indeed, subsequent studies reveal a strong negative cooperativity of glutamate binding between each subunit in the dimeric LBD and provide the first direct evidence that the dimeric LBD of mGlu receptors exhibits intersubunit cooperativity of ligand binding (26). Recent work shows that ligand binding to mGlu1a changes the dimeric allocation of the cytoplasmic regions (27). In addition, mGlu1a functionally couples to Gq and Gs when one protomer is bound to glutamate, but the presence of gadolinium (Gd3+ ), a ligand whose binding site is distinct from the glutamate-binding site (28), induces a confirmation state in which coupling to Gq is preferred over Gs (29) (Fig. 3), suggesting that mGlu1a can regulate multiple signaling pathways. The group I mGlu receptors also form heterodimers (30) with the family 3/C CaRs that bind polycationic CaR agonists (31,32). Coimmunoprecipitation studies show that mGlu1a and the CaR interact in bovine brain extracts, and in vitro transfection studies in HEK-293 cells show that compared to CaR

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Fig. 3. The group I metabotropic glutamate (mGlu) receptors can differentially activate G protein signaling pathways dependent on the conformational form of the extracellular ligand-binding domain. For instance, glutamate binding to the mGlu1a receptor can activate both the Gq and Gs pathways; however, gadolinium (Gd3+ ) binding to the extracellular domain induces a conformation of the receptor that preferentially activates the Gq pathway. Adapted from Tateyama M, Kubo Y. Dual signaling is differentially activated by different active states of the metabotropic glutamate receptor 1alpha. Proc Natl Acad Sci USA 2006;103(4):1124–1128.

homodimers, CaR–mGlu1a/5 heterodimers exhibit altered trafficking via interactions with Homer 1c, and the CaR becomes sensitive to glutamate-mediated internalization (30). Thus, covalent CaR–group I mGlu receptor heterodimers likely increase the potential for CaRs to modulate neuronal function. 1.4. Expression In situ hybridization reveals expression of mGlu1a (33) and mGlu5a (6) mRNA transcripts throughout the adult rat brain (Fig. 4, top). Immunocytochemistry studies (Fig. 4, center left) corroborate these data and show that mGlu1a protein is enriched within the olfactory bulb, globus pallidus, thalamus, substantia nigra, superior colliculus, and cerebellum, with lower levels present in neocortex, striatum, amygdala, hypothalamus, medulla, and stratum oriens of CA1 and the polymorph layer of dentate gyrus in hippocampus (Fig. 4, bottom left) (34). The mGlu5 receptor is widely expressed throughout the rat brain (Fig. 4, center right) with the highest density in olfactory bulb, caudate/putamen, lateral septum, cortex, and hippocampus (Fig. 4, bottom right), as well as in the striatum, nucleus accumbens, inferior colliculus, and spinal trigeminal nuclei (35,36). mRNA for both group I mGlu receptors is detected in rat retina (37), and protein for mGlu1 and mGlu5 is detected in chick retina (38) and rat retina (39), whereas only mGlu5 is detected in mammalian optic nerve (40) and spinal cord (41). Electron microscopy studies show that mGlu1a is detected in dendrites and spines but not in presynaptic terminals (34,42), although mGlu1a-receptor

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Fig. 4. The group I metabotropic glutamate (mGlu) receptors are differentially distributed throughout the brain. Top The messenger RNA distribution for mGlu1 (left) and mGlu5 (right). Middle: The protein distribution for mGlu1 (left) and mGlu5 (right). Bottom: The protein distribution of mGlu1 (left) and mGlu5 (right) in the hippocampus. Courtesy of Dr. Ryuichi Shigemoto.

labeling has been observed in presynaptic sites in monkey striatum (43). The mGlu5 receptors are also found in dendritic spines and shafts in the hippocampus and cortex, as well as in presynaptic axon terminals, and thus may function as a presynaptic receptor (35). Functional studies corroborate that mGlu5 is located in presynaptic membranes (44,45), as well as in hippocampal interneurons (46), glial cells (47), and spinal cord (48) . The expression of mGlu5 is regulated during development in hippocampal astrocytes: Whereas mGlu5a and mGlu5b are expressed equally in young animals (postnatal day [P] 11–20 rats), the mGlu5a splice variant declines with increasing age and the mGlu5b splice variant is dominant in adult animals (49). Perisynaptic localization of both mGlu1a and mGlu5 has been observed next to postsynaptic densities of asymmetric, type 1 (presumably glutamatergic) synapses (50,51) in both the cerebellum and the hippocampus (52,53). More recently mGlu5 has been detected in nuclear membranes of midbrain and cortical neurons, indicating a potential role in modulating intranuclear signaling pathways (54). RNA for mGlu5a and mGlu5b is also detected in cultured astrocytes, whereas only mGlu5a mRNA is detected in cultured microglia (55).

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In summary, there are six mGlu1 splice variants and two mGlu5 splice variants in rat that encode glutamate-activated GPCRs with large extracellular ligand-binding domains. The mGlu receptors can form homodimers, and each monomer can couple to distinct G proteins or they can form heterodimers with the CaR. Both mRNA and protein for the group I mGlu receptors are abundantly expressed throughout the brain, and in some regions they show very distinct, nonoverlapping expression patterns. Finally, mGlu1 and mGlu5 are located on the postsynaptic density, perisynaptic to iGluRs, whereas mGlu5 is also localized to presynaptic and nuclear membranes in neurons and is the predominant subtype in glial cells.

2. Signaling The group I mGlu receptors predominantly couple to Gq/11, which activates phospholipase C (PLC) and leads to increases in intracellular calcium (Ca2+ ). These signaling molecules activate downstream signaling pathways such as protein kinase C (PKC) and the p44/p42 mitogen-activated protein kinase/extracellular signal–regulated kinase (MAPK/ERK) pathway that in turn activate the cAMP response element–binding protein (CREB) and other transcriptional factors that modulate gene transcription. The group I mGlu receptors modulate neuronal excitability by inhibition or potentiation of several ion channels, depending on the cell type or synapse. Modulation by group I mGlu receptors can occur in multiple ways, including regulation of gene transcription and protein translation that leads to altered excitability of neurons. 2.1. Second Messenger Pathways The mGlu receptors bind to heterotrimeric GTP-binding proteins (G proteins) located on the inner face of the plasma membrane and modify effectors through intracellular signaling molecules (Fig. 5). The group I mGlu receptors primarily couple to the Gq proteins q and 11 , which can directly stimulate Bruton’s tyrosine kinase (56) and activate phospholipase C (PLC) (for G protein review, see ref. 57 ). Thus, when ligands bind to the group I mGlu receptors, they activate the G heterotrimer, causing Gq/11 to dissociate from the G subunits and the receptor. Gq directly activates PLC, which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2 ) into inositol 1,4,5-trisphosphate (IP3 ) and diacylglycerol (DAG). IP3 is a universal calciummobilizing second messenger that binds to receptors on the endoplasmic reticulum to cause release of Ca2+ into the cytoplasm, whereas DAG is membrane bound and activates PKC. Thus, many of the modulatory effects of group I mGlu receptors are mediated by intracellular calcium and PKC. However, studies show coupling of mGlu1a to the Gai/o signaling pathway in baby hamster kidney cells (58) and in Xenopus oocytes, where mGlu1a

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Fig. 5. Schematic of the group I metabotropic glutamate (mGlu) receptor G protein signaling pathways. The group I mGlu receptors predominantly couple to activation of Gq, which activates phospholipase C (PLC), that hydrolyzes phosphoinositide bisphosphate (PIP2 ) into inositol trisphosphate (IP3 ) and diacylglycerol (DAG). These second messengers lead to the release of intracellular calcium (Ca2+ ) from the endoplasmic reticulum and the activation of protein kinase C (PKC). Alternatively, in some instances the group I mGlu receptors regulate cAMP levels via Gi/o inhibition of adenylyl cyclase (AC) activity, although Gs-mediated activation of AC has been demonstrated in heterologous cells. Finally, group I mGlu receptors can couple to an unidentified G protein to stimulate phospholipase D (PLD), which cleaves phosphatidylcholine (PC) into choline (Ch) and phosphatidic acid (PA) and subsequent PKC activation.

activates the G protein–coupled inwardly rectifying potassium channel (GIRK) via a pertussis toxin (PTX)–sensitive G protein pathway (59,60). In addition, mGlu1 and mGlu5 can couple to Gas and cyclic AMP (cAMP) production in LLC-PK1 porcine kidney cells (29,61), and mGlu1a can increase basal cAMP levels by direct coupling with Gs in transfected Chinese hamster ovary (CHO) cells (62). Finally, many GPCRs stimulate the hydrolysis of phosphatidylcholine (PC) by phospholipase D (PLD) into phosphatidic acid (PA) and choline (Ch). PA can act as a signaling molecule itself, or it can be hydrolyzed to DAG. Early studies observed group I mGlu receptor activation of PLD in hippocampus (63–66) and cortex (67–69). PLD activation is likely mediated by mGlu5, because it is expressed in rat cortical astrocytes but not cortical neurons and

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is expressed in hippocampal astrocytes but not in cerebellar astrocytes (68), where, it is interesting to note, mGlu1 is the predominant group I mGlu receptor (33,34,36). The lack of PLD activation by the cloned mGlu receptors expressed in heterologous cells implied that an uncloned PLD-coupled mGlu subtype existed; however, the recent finding that mGlu1a receptors in CHO cells activate PLD via a mechanism that is dependent on extracellular Ca2+ , PKC, tyrosine kinase, and RhoA (70) suggests that PLD-coupled mGlu effects in native cells may involve additional factors that are missing in heterologous cells. The group I mGlu receptor agonist dihydroxyphenylglycine (DHPG) also stimulates casein kinase 1 and cyclin-dependent kinase 5 in neostriatal neurons to increase phosphorylation of DARPP-32 (dopamine and cAMPregulated phosphoprotein, 32 kDa) (71,72). DHPG-mediated phosphorylation of DARPP-32 is blocked by inhibitors of PLC, the calcium chelator BAPTA/AM [1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetra(acetoxymethyl) ester], and the calcineurin inhibitor cyclosporin A, suggesting that DHPG activates casein kinase I via Ca2+ -dependent stimulation of calcineurin and subsequent dephosphorylation of inhibitory autophosphorylation sites on DARPP-32. 2.2. MAPK/ERK Pathway The MAPK/ERK pathway is an evolutionarily conserved signaling cascade involved in several physiologic responses, including cell proliferation, survival, differentiation, and, in neuronal cells, synaptic plasticity (for review, see ref. 73). The MAPK proteins are a family of serine/threonine protein kinases, and the MAPK signaling cascades are organized into three-tiered modules (74,75). MAPKs are phosphorylated and activated by MAPK kinases (MAPKKs), which in turn are phosphorylated and activated by MAPKK kinases (MAPKKKs). The MAPKKKs are activated by interaction with the family of small GTPases and/or other protein kinases connecting the MAPK module to cell surface receptors or external stimuli. Because proteins encoded by the genes ERK1 and ERK2 (p44/p42) are members of the MAPK family, ERK phosphorylation leads to increased activation of gene transcription molecules such as CREB that stimulate protein synthesis. GPCRs activate the small G protein/MAPK cascade through at least three classes of tyrosine kinases. Src-family kinases are recruited following activation of phosphoinositide 3-kinase (PI3K) by G subunits, which is not shown to be necessary for group I mGlu receptor activation of MAPK. They are also recruited by receptor internalization, cross-activation of receptor tyrosine kinases, or by signaling through an integrin scaffold involving Pyk2, a cytoplasmic pralinerich tyrosine kinase implicated in several intracellular signaling pathways, and/or the closely related focal adhesion kinase (FAK) (for review, see

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ref. 76). GPCRs can also use PLC to activate PKC and calcium/calmodulindependent protein kinase II (CaMKII), which can either stimulate or inhibit the downstream MAPK pathway. The group I mGlu receptors also signal via the MAPK/ERK pathway (Fig. 6). It is interesting that activation of mGlu1 or mGlu5 expressed in CHO cells leads to a rapid increase in tyrosine phosphorylation of FAK. In mGlu1-expressing cells, glutamate-induced tyrosine phosphorylation of FAK is dependent on PLC, intracellular Ca2+ , and calmodulin but independent of PKC and CaMKII, and FAK activation leads to a marked increase in actin stress fiber formation (77). Studies show that mGlu5 activation in cultured rat cortical glial cells leads to PKC-independent tyrosine phosphorylation of several proteins, including ERK1/2, and immunoblot analysis revealed that only mGlu5, and no other mGlu receptor, is expressed in these cultures (78). The mGlu5 receptor increases ERK2 phosphorylation in astrocytes by a mechanism that involves the activation of Gq and both receptor and nonreceptor tyrosine kinases and transactivation of the epidermal growth factor receptor but is independent of the activation of PLC (79). mGlu1a and mGlu5a expressed in CHO cells also stimulate the activation of ERK; however, each receptor uses different G proteins to activate ERK, which likely recruit different tyrosine kinases (80). The mGlu1a-mediated ERK response is attenuated by PTX, indicating mGlu1 coupling to Gi/o, whereas the mGlu5a-mediated ERK

Fig. 6. The group I metabotropic glutamate (mGlu) receptors regulate gene expression via activation of the mitogen-activated protein (MAP) kinase pathway. mGlu1 and mGlu5 use multiple second messengers to stimulate activation of MAP kinase (MAPK/extracellular signal-regulated kinase [ERK]-1/2), which in turn can activate cyclic AMP response element binding protein (CREB)– and Elk-mediated gene expression. CaM, calmodulin; CaMK, calmodulin-dependent kinase; FAK, focal adhesion kinase; PKC, protein kinase C; PLC, phospholipase C; MAPKK, MAPK kinase; MAPKKK; MAPKK kinase; MEK1/2, MAPK/ERK kinase 1 and 2.

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response (and phosphoinositide hydrolysis by activation of either receptor) is PTX insensitive and is probably mediated by coupling to Gq. However, group I mGlu receptor coupling to ERK is independent of PI3K, intra- or extracellular Ca2+ concentration, or PKC, suggesting that the normal second messenger systems activated by group I mGlu receptors do not mediate ERK signaling. Recent studies show that a wide variety of neurotransmitter receptors, including mGlu receptors, muscarinic acetylcholine receptors, dopamine receptors, and -adrenergic receptors, couple to MAPK cascades and play an important role in synaptic plasticity in area CA1 of rat hippocampus (81), and implicate the MAPK cascade as important regulator of gene expression in long-term forms of hippocampal synaptic plasticity (82,83). These data underscore the complexity and specificity of signaling pathways used by group I mGlu receptors in cells. 2.3. CREB Substrates that are regulated by MAPK phosphorylation include the cyclic AMP response element–binding protein (CREB), a major transcriptional activator at the calcium and cAMP response-element (CaCRE), and phosphorylated (p) CREB facilitates gene expression (84). MAPK also phosphorylates the transcription factor Elk-1 that is a transcriptional regulator of the serum response element in the upstream promoter region of the CaCRE (85). Group I, but not group II or III, mGlu receptors phosphorylate CREB, Elk-1, and ERK in striatal neurons in a PKC- and CaMKII-dependent manner (86–88). Data suggest that protein phosphatase 1/2A in striatal neurons tonically dephosphorylates CREB and Elk-1 and suppresses constitutive c-fos mRNA and protein expression. Thus, inhibition of phosphatase 1/2A may contribute to the group I mGlu–regulated phosphorylation of these transcription factors and c-fos expression (89). In addition, studies on group I mGlu–mediated stimulation of CREB phosphorylation in CHO cells show that stimulation depends on both extracellular Ca2+ levels and PKC activity but is not on activation of ERK1/2, calmodulin, or CaMKII (90). Finally, CREB phosphorylation appears to play a role in the induction of cortical long-term depression (LTD), which may involve receptor cross-talk between group I and group II mGlu receptors and a convergence of signaling from the mGlu receptors to CREB-mediated transcription. Recent work shows that mGlu2-mediated inhibition of cAMP levels leads to decreased desensitization of mGlu5, and that both group I and II receptors can directly increase CREB phosphorylation via a cAMP- and protein kinase A (PKA)–independent signaling mechanism (91). 2.4. Receptor Interactions Group I mGlu receptors stimulate release of Ca2+ from intracellular stores, which then modulates many signaling pathways, including those

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coupled to multiple receptor systems. 1-Adrenergic receptor activation leads to modulation of group I mGlu receptor–induced calcium oscillations and glutamate release in astrocytes (92). Another type of interaction occurs at the second messenger level, where synergistic signaling is stimulated with simultaneous activation of receptors. For example, activation of D1 dopamine receptors and mGlu5 receptors synergistically increases phosphorylation of ERK2 and CREB in cultured striatal neurons (93), and activation of mGlu5 receptors potentiates the effects of adenosine A2A receptor signaling through the ERK pathway (94,95). In addition, amphetamine is an indirect dopamine receptor agonist and increases glutamate release in the striatum, and acute amphetamine facilitates phosphorylation of CREB, Elk-1, and ERK1/2 signaling proteins and Fos gene expression via a group I mGlu–mediated pathway in the dorsal striatum (96). Studies also indicate that concurrent activation of mGlu5 receptors potentiate N-methyl-d-aspartate (NMDA)– mediated CREB phosphorylation via the PKC signaling pathway (97). Taken together, these studies suggest that coincident activation of multiple signaling pathways may be a mechanism for a cell to determine and encode particularly salient stimuli. Studies also show that estradiol can influence various brain functions by acting on receptors localized to the neuronal membrane surface and activating multiple intracellular signaling pathways and modulatory proteins, including phosphorylation of CREB. Steroids can potentially modulate the cell in several ways, including changing membrane fluidity without binding to any known protein or receptor, acting as nonspecific allosteric modulators for steroid hormone receptors or other proteins present in the cell membrane, or as classic steroid receptors can be localized to either the plasma or nuclear membranes (for review, see ref. 98). In cultured rat hippocampal neurons, estradiol triggers MAPK-dependent CREB phosphorylation in unstimulated neurons and attenuates L-type calcium channel–mediated CREB phosphorylation in depolarized neurons. These positive and negative effects on CREB activity are sex specific and are mediated by membrane-localized estrogen receptors that directly stimulate group I and II mGlu receptors (99). Activation of estrogen receptor leads to glutamate-independent mGlu1a signaling and stimulates CREB phosphorylation through PLC regulation of MAPK, whereas similar glutamateindependent activation of mGlu2/3 by estrogen receptor or inhibits L-type calcium channel–mediated CREB phosphorylation. Thus, signaling by group I and/or group II mGlu receptors may account for many unexplained observations regarding the influence of estradiol on nervous system function. 2.5. Modulation of Voltage-Gated Ion Channels The group I mGlu receptors inhibit and potentiate several voltage- and ligand-gated ion channels through a variety of second messenger systems.

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The reasons that group I mGlu receptors serve as bidirectional modulators of multiple effectors may include (1) that mGlu receptor pharmacology is not yet selective enough for unequivocal identification of receptors involved, (2) that additional uncloned mGlu subtypes still exist in native cells, or (3) the most likely explanation, that effector modulation by different mGlu receptors depends on the proteins that comprise mGlu receptor signaling complexes in distinct cell types. Although these possibilities are addressed throughout the chapter, we provide a brief summary of all the ion channels that are shown to be targets for modulation by group I mGlu receptors (Fig. 7). Group I mGlu receptors modulate calcium channels. Activation of these receptors decreases N-type Ca2+ channel (100) and NMDA receptor (101) activation in a fast, membrane–delimited manner, suggesting that modulation occurs through a direct G protein interaction. Group I mGlu receptor activation also inhibits L-type Ca2+ channels, but this modulation develops much more slowly and is likely mediated by increased intracellular Ca2+ (102,103). Inhibition of P/Q-type channels has also been described (104,105). Facilitation of N-type Ca2+ channels by mGlu receptors occurs in retinal ganglion

Fig. 7. Group I metabotropic glutamate (mGlu) receptors modulate cell excitability through inhibition or potentiation of several voltage-gated and ligand-gated ion channels. These effects are likely determined by interactions of the group I mGlu receptors with other proteins that comprise the synaptic complex in distinct cell types. AHP, afterhyperpolarization; AMPA, -amino-3-hydroxy-5-methyl-4isoxazolepropionate; GIRK, G protein–coupled inwardly rectifying potassium channel; NMDA, N-methyl-d-aspartate.

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cells via a fast, membrane-delimited mechanism (106), and there is evidence for facilitation of L-type Ca2+ channels in cerebellar granule cells (107) and in nucleus tractus solitarius neurons through IP3 -mediated activation of PKC (108). Group I mGlu receptors also modulate potassium channels. Activation of mGlu1 and mGlu5 inhibits IAHP potassium channels that underlie the afterhyperpolarization and spike accommodation in hippocampal neurons (109–111). The group I mGlu receptors also inhibit the IM (110,112,113) and IKleak (114,115), both of which are implicated in modulation of spike firing patterns, suggesting that group I mGlu receptors are important in shaping neuronal response patterns. In addition, group I mGlu receptors have been shown to inhibit GIRK channels (60). Activation of Ca2+ -dependent potassium channels by group I mGlu receptors occurs via the IP3 pathway and release of intracellular Ca2+ mediated by ryanodine receptors (111,116). Several subtypes of Ca2+ -dependent potassium channels are activated by group I mGlu receptors, including the largeconductance BK channels (117), the intermediate-conductance channels (118), and the small-conductance (SK) channels involved in the afterhyperpolarization and spike accommodation (119). Inhibition of these Ca2+ -dependent potassium channels mediates a slow depolarization in some neurons (120–122), whereas activation of mGlu1 leads to activation of GIRK channels in a heterologous expression system (59). Group I mGlu receptors also modulate nonselective cation channels. Ca2+ dependent nonselective cation channels are activated by group I mGlu receptors in hippocampal CA1 pyramidal cells (123–125), CA3 pyramidal cells (126), and cerebellar Purkinje cells (127). However, in some cases the group I mGlu receptors modulate Ca2+ -dependent nonselective cation currents via PLCmediated modulation of the Na+ /Ca2+ exchanger (111,128–130). There is also evidence for activation of Ca2+ -independent nonselective cation channels in hippocampal slice cultures by group I mGlu receptors (131). More recently, group I mGlu receptors have been shown to activate transient receptor potential (TRP) channels through the PLC pathway in the lateral amygdala (132). TRP channel activation by group I mGlu receptors mediates excitatory postsynaptic currents in cerebellar Purkinje neurons (133) and substantia nigra dopamine neurons (134). The TRP channels are permeable to Ca2+ and have been proposed to play a role replenishing intracellular Ca2+ stores (135,136) in addition to their role in synaptic plasticity (132) (see later discussion). Similarly, the slow excitatory postsynaptic potential (sEPSP) or slow excitatory postsynaptic current (sEPSC) at parallel fiber-to-Purkinje neuron synapses is attributable to group I mGlu receptor activation of a nonselective cation channel, and tyrosine phosphorylation inhibits mGlu1 activation of the channel at a point upstream of the channel (137).

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2.6. Modulation of Ligand-Gated Ion Channels Group I mGlu receptors facilitate and inhibit excitatory neurotransmission by modulation of ligand-gated ion channel (-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid [AMPA] and NMDA) activity. AMPA currents are potentiated by group I agonists (138) in a PKC-dependent manner (139). NMDA potentiation by group I mGlu receptors has been described in rat hippocampal slices (140) and is also dependent on PKC (139,141). Facilitation of NMDA receptor currents by mGlu5 modulates bursting patterns of neurons (142,143). However, activation of mGlu receptors can also depress excitatory transmission (144) and NMDA-mediated currents (101,145) in a membrane-delimited (PKC-independent) manner (101). Modulation of AMPA and NMDA channels by mGlu receptors contributes to the cellular mechanisms associated with long-term potentiation (LTP) and long-term depression (LTD) in many brain areas; this mGlu-mediated modulation of synaptic transmission is discussed in subsequent sections. Synaptic release of glutamate activates the ionotropic ligand-gated glutamate channels on a faster time scale than the extrasynaptic mGlu receptors. However, mGlu receptor–mediated EPSPs and inhibitory postsynaptic potentials (IPSPs) have been described in many brain areas (146). Group I mGlu receptor– mediated EPSPs are slow and heterogeneous in that both Ca2+ -dependent and independent pathways activate either excitatory cation conductances (123,130, 147,148) or inhibitory potassium conductances (110). Synaptic activation of group I mGlu receptors elicits IPSPs in dopamine (DA) neurons in the ventral tegmental area by activating release of intracellular Ca2+ and a Ca2+ -dependent potassium conductance (149). These results demonstrate that mGlu receptors play an important but complex role in the regulation of excitability. In summary, activation of group I mGlu receptors leads to the activation of PKC and an increase in intracellular Ca2+ . These important signaling molecules can activate intracellular signaling pathways such as MAPK/ERK that in turn activate CREB and other transcriptional factors and modulate gene transcription. The group I mGlu receptors regulate neuronal excitability by inhibition or potentiation of several voltage- and ligand-gated ion channels. Thus, the group I mGlu receptors regulate neuronal signaling in multiple ways, including modulation of gene transcription and protein translation, as well as modulation of excitability of neurons. In addition, group I mGlu receptor stimulation of intracellular Ca2+ indicates that they regulate a universal signaling molecule and solidifies their importance as mediators of synaptic plasticity.

3. Pharmacology l-Glutamate, also known as l-glutamic acid or (S)-1-aminopropane-1, 3-dicarboxylic acid, is the endogenous excitatory transmitter in the mammalian central nervous system that acts at both iGlu and mGlu receptors (150,151).

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Although the quest for compounds that are selective for individual mGlu subtypes is ongoing, several group I–selective and –nonselective agonists and antagonists have been developed over the years (152), including the most recent compounds, which are allosteric modulators of group I mGlu receptors. Although several mGlu compounds act on other mGlu and iGlu receptor subtypes and hamper the use of these compounds as selective tools, nonetheless they have provided an enormous amount of information regarding mGlu receptor function over the years. Thus, although it is likely that subtypeselective compounds will be developed in the near future, if one were to study group I mGlu receptors in native cells today, the most useful pharmacologic tools would be the group I–selective agonist DHPG, the mGlu1-selective antagonists CPCCOEt and YM-298198, and the mGlu5-selective antagonists MPEP, MTEP, and fenobam, as discussed in what follows. The most widely used group I–selective agonist is DHPG [(S)3, 5-dihydroxyphenylglycine] (66,153–157) (for review, see ref. 158). DHPG activates both mGlu1 (half-maximal effective concentration [EC50 ] 6 μM) and mGlu5 (EC50 2 μM) but has no effect on group II (mGlu2 EC50 >1000 μM, mGlu3 EC50 >106 μM) or group III (mGlu4, 7, 8 EC50 >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 (<20 min) is consistent with a mechanism involving mGlu stimulation of translation in the dendrites, a mechanism that is further supported by the fact that mGlu receptor agonists stimulate protein synthesis in synaptoneurosomes (285). Recent work shows that the activity of tissue plasminogen activator (tPA), a secreted protease required for some forms of long-term synaptic plasticity, is rapidly increased in hippocampal neurons after glutamate stimulation due to group I mGlu receptor–dependent increases in tPA protein synthesis (286). The importance of group I mGlu receptors in hippocampal LTP is corroborated by studies showing that mGlu5 knockout mice do not exhibit NMDA-dependent LTP in hippocampal CA1 and are deficient in memory tasks (287).

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Although there is strong evidence for mGlu receptor–dependent LTD in several brain areas (reviewed in refs. 146 and 262), the hippocampus and cerebellum will be presented as general models of synaptic plasticity. In the hippocampal CA1 region, two forms of LTD can be dissociated. One form is NMDA receptor dependent but mGlu receptor independent, and the other form is mGlu receptor dependent but does not involve activation of NMDA receptors (288,289). The mGlu receptor–dependent form of LTD is induced with lowfrequency stimulation (290,291), increased intracellular Ca2+ (292–294), and activation of PKC (295,296). Studies show that the mGlu receptor–dependent form of LTD is likely due to activation of mGlu5 (155,297) in presynaptic terminals (298). Group I mGlu receptors expressed on presynaptic axonal terminals of GABAergic interneurons have also been shown to inhibit the release of GABA (45,252,260) and elicit LTD of GABA release (299). Bidirectional modulation of NMDA-mediated currents is dependent on activation of mGlu5 receptors and increased levels of intracellular Ca2+ (300). However, Ca2+ -independent pathways have also been shown to mediate LTD in the hippocampus (301,302) via activation of the MAPK/ERK pathway (303), similar to mGlu receptor–mediated LTD in the cerebellum (304). These data suggest that not all group I mGlu receptor effects are due to increases in intracellular Ca2+ and that additional signaling pathways may lead to activation of transcription factors that modulate cellular activity. In the cerebellum, LTD is mediated by activation of postsynaptic mGlu1 receptors (305–308), increases in intracellular Ca2+ levels (292), activation of PKC (296), and/or MAPK/ERK activation (304), which lead to the expression of proteins involved in translation (309) and a decrease in AMPA receptor expression on the plasma membrane (310). Mice lacking mGlu1 receptors show both severe motor and spatial learning deficits (305,311), suggesting that mGlu1 receptor activation in the cerebellum is significant for motor learning. Although the group I mGlu receptors modulate presynaptic neurotransmitter release, the majority of the group I mGlu receptors are localized extrasynaptically in postsynaptic membranes (53,312), suggesting that postsynaptic induction of LTD is expressed as a change in presynaptic neurotransmitter release (313). mGlu5-mediated LTD in the nucleus accumbens is initiated by postsynaptic activation of mGlu5 and increased intracellular Ca2+ levels, which leads to a subsequent decrease in presynaptic glutamate release. This postsynaptic-to-presynaptic signaling is mediated by mGlu receptor– dependent activation of endogenous retrograde messengers that act on presynaptic receptors, such as endogenous cannabinoids acting on CB1 receptors in the striatum (314–316), arachidonic acid (317,318), or nitric oxide (319–321). In summary, the fact that activation of the group I mGlu receptors modulates a host of effectors suggests that regulation of synaptic transmission is complex and dependent on the spatial and temporal expression of receptors and

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channels in the cell. The mechanisms of LTP and LTD vary depending on the synapse, making it difficult to define a general role for mGlu receptors in regulating synaptic transmission. Even so, several recurring themes have emerged concerning the physiologic functions of mGlu receptors in various fields: (1) The modulation of excitatory neurotransmission by mGlu receptors may increase the signal-to-noise ratio to prioritize a particular signal as important for consolidation into a memory, (2) the ability of mGlu receptors to finely tune excitatory transmission and integrate multiple receptor-mediated signals is critical for encoding convergent signals necessary for associative learning, and (3) the fact that mGlu receptors subtly modulate excitatory transmission is important for their potential as therapeutic targets.

7. Functional Roles It is clear that group I mGlu receptors are important for modulating and fine tuning excitatory neurotransmission, as well as for integrating responses from multiple receptor systems in the cell. The functional roles of mGlu1 and mGlu5 in synaptic transmission are presented in the following sections as they pertain to normal brain function in addition to their role in neurologic and neuropsychiatric disorders. Finally, we present evidence for emerging roles of group I mGlu receptors in neurons and nonneuronal cells. These studies again highlight the fact that group I mGlu receptors contribute to the modulation of excitatory transmission in virtually every brain region and are potential therapeutic targets for the treatment or prevention of several neurologic and neuropsychiatric disorders. 7.1. Cognition Group I mGlu receptors play a prominent role in the regulation of synaptic plasticity in many areas of the brain (for reviews, see refs. 265 and 322– 324). Both mGlu1 and mGlu5 receptors are abundantly expressed throughout the hippocampus (324), where they are important in LTP, LTD, and memory formation (265,322,324). Coapplication of inactive doses of inhibitors of mGlu5 and NMDA receptors synergistically impairs working memory and instrumental learning (234), spatial learning (325), and aversive learning paradigms (326). In addition, mGlu1-knockout mice display reduced LTP and deficits in associative and spatial learning (305,311,327), whereas mGlu5knockout mice have impaired NMDA-dependent LTP in hippocampal CA1 and are deficient in memory tasks (287). In the hippocampal dentate gyrus, mGlu1 and mGlu5 have different but complementary roles in synaptic plasticity. Inhibition of mGlu1 receptor activation impairs the induction and expression of LTP and reference

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memory (327,328), whereas inhibition of mGlu5 receptor activation preferentially blocks the maintenance and protein synthesis–dependent phase of LTP and working memory (329). In hippocampal CA1, induction of LTD is mediated by activation of both group I mGlu receptors (289,313) and is dependent on protein synthesis (330–332) and internalization of iGlu receptors (333). 7.2. Fear Conditioning Regulation of synaptic plasticity and fear conditioning by mGlu5 has been studied in detail in the lateral amygdala. Intraamygdala administration of the specific mGlu5 antagonist MPEP impairs the acquisition, without affecting the expression or consolidation, of contextual fear conditioning (334,335). These data suggest that mGlu5 and mGlu5-dependent facilitation of NMDA currents (334,336) are important for mediating the morphologic changes involved in the memory (337) but not for the maintenance (338) of fear conditioning. Activation of several second messengers is involved, including PKC (339), PKA (340), and CaMKII (341,342). Consolidation of fear conditioning into long-term memory in the amygdala likely involves activation of CREB by PKA and MAPK (338,343,344) and subsequent increases in gene transcription and translation. Taken together, these studies provide a link between mGlu5 and the signaling events that are crucial for the initiation phase of learning and memory and the structural changes in synapses that underlie maintenance of memory in fear conditioning (345). 7.3. Pain Glutamate neurotransmission is important in the transduction of pain and development of hyperalgesia (for review, see ref. 346). Both mGlu1and mGlu5 receptors are expressed in nociceptive primary afferents and contribute to formalin-induced inflammatory pain (347,348) through activation of the MAPK/ERK signaling pathway (349,350). Studies show long-term increases in expression of mGlu1 and mGlu5 in neurons and astrocytes after spinal cord injury (351), suggesting that permanent alterations in dorsal horn receptor expression may play important roles in transmission of nociceptive responses in the spinal cord following injury. Group I receptors promote hyperexcitability of dorsal horn neurons during peripheral inflammation (352–355). Pain processing in the brain also involves activation of mGlu receptors in several areas associated with perception and the emotional aspects of pain. In arthritic pain models, mGlu5 receptor function in the amygdala is increased in postsynaptic neurons with a simultaneous upregulation of functional presynaptic mGlu1 receptors (356), suggesting that in the amygdala, mGlu5 is involved in processing acute nociception, whereas mGlu1 receptors encode

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plasticity during prolonged pain states (357). Because the amygdala is an important structure in pain and emotional processing, understanding the cellular mechanisms that underlie synaptic plasticity in this area may have important therapeutic effects for emotional disorders, as well as for chronic pain states (358). Endogenous opioids can be recruited within the brain to inhibit pain by activating a descending antinociceptive pain pathway from the ventrolateral periaqueductal gray (PAG) to the rostral ventral medulla (RVM) and subsequent modulation of incoming afferent activity in the spinal cord (359,360). Activation of group I mGlu receptors in the PAG elicits antinociception (361,362) via presynaptic inhibition of GABA release in the ventrolateral PAG (363,364) and increases excitability of GABA and glutamate-containing neurons via a postsynaptic mechanism (364). Because group I mGlu receptors are primarily postsynaptic in the PAG (52,363,365), it has been proposed that retrograde signaling, possibly by mGlu5 receptor–mediated production and release of endocannabinoids, may occur in this region (364,366). Thus, activation of group I mGlu receptors is pronociceptive in the periphery but antinociceptive in the PAG, indicating again that these receptors play a complex role in modulating excitatory circuits in the brain. 7.4. Reward Dopamine (DA) neurons in the ventral tegmental area (VTA) project to the ventral striatum or nucleus accumbens (NAcc) and the prefrontal cortex, critical relays in the neural reward/addiction brain circuit (367,368). VTA DA neurons respond to rewarding cues and are thought to be critical for integrating information necessary for predicting reward (369). Brief application of glutamate onto DA neurons induces both depolarization mediated by iGlu receptors and facilitation of burst firing via activation of group I mGlu receptors (148,370,371). The facilitation of burst firing is likely to be mediated by transient receptor potential (TRP)–like channel activation (134,372) and/or inhibition of Ca2+ -dependent potassium (SK-subtype) channels (373). Electrical stimulation of the VTA produces a slow hyperpolarizing postsynaptic potential (IPSP) in DA neurons that is dependent on mGlu1 activation of IP3 and cADPR (cyclic ADP-ribose)/ryanodine–mediated intracellular Ca2+ release (374) and activation of K+ channels (149,375). This mGlu1-mediated hyperpolarization induces a pause in DA neuron firing that inhibits NMDA receptor–induced burst firing (374). However, the question remains as to what responses are evoked by physiologic stimulation of these receptors. In addition, mGlu1 receptors depolarize GABA neurons in the VTA and increase inhibitory currents onto DA neurons, but mGlu1 receptors on DA neurons reduce the amplitude of these events (260). Although this dual modulation of midbrain DA neurons appears incongruous, these mechanisms may work in concert with one another to provide information about the strength of a stimulus. It is clear

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that the group I mGlu receptors are important for regulating excitability of DA neurons in the VTA and thus play a prominent role in the reward circuitry of the brain. 7.5. Motor Control The cerebellum and the basal ganglia are two brain structures involved in the control of movement, and synaptic transmission in both areas is modulated by group I mGlu receptors. There is dense expression of mGlu1 receptors throughout the cerebellum (376,377), where motor learning is dependent on coincident activation of mGlu1 and an increase in intracellular Ca2+ (311, 378). Cerebellar LTD is mediated through activation of postsynaptic mGlu1 receptors (305,308) and a decrease in AMPA receptor expression on the plasma membrane (310). Mice deficient in mGlu1 receptors have both severe motor and spatial learning deficiencies (311), underscoring the important role of these receptors in motor control. In general, both mGlu1 and mGlu5 receptors are expressed throughout the basal ganglia, especially in the striatum. These receptors potentiate NMDA receptor currents and increase excitability of these neurons, opposing the modulatory effects of DA at multiple levels of the basal ganglia circuitry (264,379,380). Although group I agonists do not directly activate ion channels in medium spiny neurons of the striatum, experiments in mGlu1- and mGlu5-knockout mice indicate that mGlu5 receptors in wild-type animals enhance NMDA receptor currents and NMDA-dependent LTP in these cells (238,381,382). Group I mGlu receptors are also critical for synaptic plasticity in corticostriatal pathways (264,314), where high-frequency stimulation can result in both LTP and LTD, depending on concentrations of Mg2+ used during the electrophysiologic recordings (381). Corticostriatal LTP is dependent on NMDA receptor activation (381,382), as well as on activation of voltagedependent Ca2+ channels (383), whereas LTD in the striatum is dependent on an increase in intracellular Ca2+ and activation of Ca2+ -dependent protein kinases (384). In addition, induction of LTD in the striatum also appears to require coincident activation of D1- and D2-dopamine receptors (385). It is interesting that induction of corticostriatal LTP involves activation of both mGlu1 and mGlu5 receptors (386), suggesting that activation of distinct mGlu receptor subtypes mediates bidirectional output in medium spiny neurons (311,387). The second messenger pathways implicated in striatal synaptic plasticity are similar to other brain areas. Group I mGlu receptors, particularly mGlu5, activate CREB phosphorylation through IP3 -dependent intracellular Ca2+ release (97). In addition, ERK signaling pathways stimulated in a Ca2+ independent manner via coactivation of NMDA receptors and mGlu5, likely via cross-talk involving Homer and postsynaptic density (PSD)-95, result in

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strong activation of signaling pathways (388). Signaling via both the IP3 /Ca2+ dependent and Homer pathways is necessary to activate ERK1/2 sufficiently to induce long-term changes in transcriptional regulation (389). There are still many questions regarding the mechanisms by which mGlu1 and mGlu5 signaling is coordinated, regulated, and in turn regulates synaptic plasticity in the basal ganglia. However, it is clear that these receptors oppose the inhibitory actions of DA throughout the basal ganglia, suggesting that group I mGlu receptors may be therapeutic targets for drugs that alleviate motor control dysfunction (390). 7.6. Role in Glial Cells Cultured astrocytes express kainate/AMPA iGlu receptors and mGlu receptors, and glutamate elicits rapid and transient elevation of mRNA levels for the transcription factor genes c-fos, fosB, c-jun, junB, and zif/268 (391). Group I mGlu5 and the group II mGlu3 receptors are the predominant mGlu receptors expressed in glial precursor cells, suggesting that these neurotransmitter receptors may be involved in the proliferation and differentiation of human glial cells (392). Furthermore, reverse transcriptase-polymerase chain reaction (RT-PCR) experiments show that only mGlu3 and mGlu5 mRNAs are significantly expressed in freshly isolated hippocampal astrocytes (393). Activation of the mGlu receptors in astrocytes regulates glutamine synthetase, a glial-specific enzyme that plays a key role in controlling glutamate concentrations in the CNS (394). The mGlu5a receptor acts as a glial sensor of extracellular glutamate, and activated astrocytes enhance uptake through the glutamate/aspartate transporter (GLAST) and the glutamate transporter (GLT-1) (395). However, studies have demonstrated that the group I mGlu receptors also significantly downregulate GLAST and GLT-1 expression and enhance astrocyte proliferation, whereas the group II mGlu receptors increase expression of GLAST and GLT-1 and reduce proliferation (396,397). These results indicate that mGlu receptors mediate intricate control of glutamate in astrocytes, as well as in neurons. Astrocytes also respond to glutamate with dynamic spatio-temporal changes in intracellular calcium generated by mGlu and iGlu receptors (398). The iGlu receptors evoke a sustained elevation in intracellular Ca2+ associated with depolarization and voltage-dependent Ca2+ influx. In contrast, mGlu receptors evoke an initial IP3 -dependent spatial Ca2+ spike that propagates rapidly from cell to cell and produces intracellular oscillatory waves of various amplitudes and frequencies that propagate within cells and are sustained only in the presence of external Ca2+ . It is interesting that the regulation of mGlu5 by phosphorylation/dephosphorylation is critical for the mGlu5-induced intracellular Ca2+ oscillations in cultured astrocytes (399).

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Coactivation of iGlu and mGlu receptors in astrocytes also leads to glutamate release through a Ca2+ -dependent process mediated by prostaglandins (PG) (400). PGE2 -evoked Ca2+ elevations in astrocytes stimulate glutamate release and a subsequent increase in neuronal Ca2+ levels via activation of neuronal glutamate receptors (401). Together these data suggest a pathway of regulated transmitter release from astrocytes and the existence of integrated glutamatergic cross-talk between neurons and astrocytes. Finally, brain macroglia also express a diverse array of neurotransmitter receptors, and there is evidence for heteroreceptor cross-talk between receptor families. For example, coactivation of adenosine A1 receptors enhances group I mGlu–evoked Ca2+ responses in astrocytes via a Gi/o protein pathway (402). Application of specific growth factors to primary astrocyte cultures dramatically increases expression of mGlu3 and mGlu5, suggesting that mGlu receptor upregulation is involved in astrocytic responses. Evidence to support this idea includes increased expression of mGlu5 and mGlu3 in glial cells, likely activated astrocytes, within the area of neuronal loss in the hippocampus after intracerebroventricular injections of kainate (403) and increased expression of mGlu5 and mGlu3 in activated astrocytes proximal to the area of mechanical injury induced by needle insertion in the cerebral cortex (403). Activation of mGlu3, but not mGlu5, in the presence of the inflammatory mediator interleukin-1 (IL-1) enhances the release of interleukin-6 in astrocytes (404). Cultures differentiated by epidermal growth factor have reduced mGlu5 protein expression in the presence of IL-1. Thus, IL-1 may represent an additional pathway through which mGlu5 expression and function can be modulated in astrocytes under different pathologic conditions associated with an inflammatory response (405).

8. Role in Neurologic and Neuropsychiatric Disorders 8.1. Neurodegeneration Activation of glutamate receptors, particularly NMDA and group I mGlu receptors, initiates excitotoxic effects on central neurons (406). Group I mGlu receptors potentiate NMDA-induced neuronal death, whereas inhibition of group I mGlu receptors reduces both NMDA- and kainate-induced neuronal death in cultured mouse cortical cells (407–410). Potentiation of cell death by group I mGlu receptors may involve enhancement of glutamate release because DHPG is known to increase high potassium-stimulated glutamate release (406). The role of mGlu1 and mGlu5 in neurodegeneration is controversial. Antagonists of the group I receptors are consistently neuroprotective; however agonists can either amplify or attenuate excitotoxic neuronal death. It is reported that at least three variables influence these differential responses to agonists: (1) the presence of the NR2C subunit in the NMDA receptor complex,

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(2) the existence of an activity-dependent functional switch of group I mGlu receptors, similar to that described for the regulation of glutamate release, and (3) the presence of astrocytes expressing mGlu5 receptors (410). The excitotoxic effect of kainate on oligodendrocyte progenitor cells is inhibited by the selective group I mGlu receptor agonist DHPG, suggesting that group I mGlu receptors limit oligodendrocyte progenitor cell degeneration during acute brain insults (411). The group I mGlu receptors also inhibit programmed cell death in neurons by blocking induction of caspase 3 activity by nitric oxide (412). 8.2. Neuroprotection Studies show that 1-amino-cyclopentane-1,3-dicarboxylate (ACPD), an mGlu receptor–nonselective agonist, and the group I–selective agonist DHPG effectively attenuate oxygen-glucose deprivation-induced cell death in cultured cerebellar granule cells (413). Activation of mGlu1 receptors reduces NMDA receptor–dependent cell death in organotypic hippocampal cultures via activation of PLC. This neuroprotection is associated with suppression of NMDA currents and prolongation of GABAA receptor–mediated currents. Antisense inhibition of Rab5b, a gene coding for a small GTPase associated with endocytosis, significantly reduces mGlu1-mediated neuroprotection, indicating that mGlu1 can regulate nerve cell susceptibility to injury (414). Both mGlu-specific agonists and antagonists have been used to evaluate the role of mGlu receptors in excitotoxic cell death due to neurodegeneration, NMDA excitotoxicity, cerebral ischemia, and traumatic brain injury. Several studies show that antagonists of group I mGlu receptors are neuroprotective, presumably by blocking group I mGlu–mediated increases in intracellular calcium (415). Conversely, agonists of group II and group III mGlu receptors are neuroprotective, presumably by inhibiting glutamate release from presynaptic terminals. However, whereas mGlu ligands confer neuroprotection in vitro, they have limited applicability in animal models due to low penetration of the blood–brain barrier. The recent development of more potent and selective mGlu antagonists such as MPEP for mGlu5 and BAY36-7620 (3aS,6aS)-6a-naphtalen-2-ylmethyl-5-methylidenhexahydro-cyclopental[c]furan-1-on) for mGlu1 (416) have allowed for stringent assessment of the role of individual mGluR subtypes in nervous system disorders. Studies show that the mGlu5-selective antagonist MPEP is neuroprotective against NMDA toxicity in cortical and hippocampal cells (417), -amyloid peptide toxicity (417), and transient ischemia (418,419), whereas selective blockade of group I mGlu receptors in the striatum induces neuroprotection by enhancing GABAergic transmission (420).

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8.3. Epilepsy Group I mGlu receptor activation elicits persistent ictaform discharges in guinea pig hippocampal slices, providing an in vitro model of epileptogenesis. The induction of persistent ictaform bursts is prevented by cysteine sulfinic acid (CSA), an agonist at presynaptic group I mGlu receptors, particularly the mGlu5 subtype (44). Intracellular recordings performed from CA3 stratum pyramidale show that CSA-mediated suppression of group I mGlu– induced epileptogenesis is PKC dependent and suggest that CSA mediates its effect by driving PLD-mediated activation of PKC, which then desensitizes the PLC-coupled group I mGlu receptors and prevents group I mGlu–induced epileptogenesis (421). Increased hippocampal excitability is a hallmark of human temporal lobe epilepsy (TLE), and changes in expression of group I and III mGlu receptors have been described in human TLE and in several mouse models of epilepsy, suggesting that differential expression of the excitatory group I mGlu receptors and the inhibitory group III mGlu receptors may increase susceptibility to seizures and hippocampal damage (422). 8.4. Traumatic Brain Injury Early studies showed that the mGlu receptor antagonist -methyl-4carboxyphenylglycine (MCPG), administered into the left lateral ventricle prior to fluid percussion traumatic brain injury (TBI) in the rat, significantly reduced motor deficits measured on days 1–5 after injury and learning/memory deficits measured on days 11–15 after injury. Thus, mGlu receptors may contribute to deficits occurring after TBI, and blockade of mGlu1, mGlu5, and/or mGlu2 may reduce these pathophysiologic responses (423). Subsequent studies support a role for group I mGlu receptors in astrocytes in TBI through aberrant PI production and uncoupling of the PLC signaling pathway (424) and depletion of calcium stores (425) that may be related to induction of reactive gliosis. 8.5. Amyotrophic Lateral Sclerosis Amyotrophic lateral sclerosis (ALS) is a terminal neurodegenerative disease characterized by a selective loss of motor neurons and intense gliosis in lesioned areas of the brain and spinal cord. One current theory of ALS progression is that glutamate-mediated excitotoxicity results from impaired astroglial uptake. A recent study examined the regulation of glutamate transporters by mGlu5 in activated astrocytes derived from transgenic rats carrying an ALS-related mutated human superoxide dismutase 1 (hSOD) transgene (426). Cells carrying the hSOD1 mutation showed a threefold higher expression of functional mGlu5 in the spinal cord of advanced ALS animals. Furthermore, cells from wild-type

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animals treated with the group I agonist DHPG displayed immediate PKCdependent uptake of aspartate by GLT-1 that was absent in transgenic cells. Thus, the failure of mGlu5 to increase glutamate/aspartate uptake in astrocytes in an animal model of ALS supports the theory that glutamate excitotoxicity may underlie the pathogenesis of ALS. 8.6. Fragile X Syndrome Fragile X syndrome is the most common form of inherited mental retardation and includes deficits such as cognitive impairment, developmental delay, attention deficit disorder, anxiety, seizures, and obsessive-compulsive behaviors (for reviews, see refs. 427 and 428). Fragile X syndrome occurs as a result of decreased expression of the fragile X mental retardation protein (FMRP), a repressor protein involved in regulating localization and translation of mRNAs in dendrites (427,428). mGlu5-mediated increases in intracellular Ca2+ and PKC activation are necessary for localization of FMRP and Fmr1 mRNA into dendrites (427), suggesting that local protein synthesis regulated by FMRP may be involved in synaptic plasticity. Fmr1-knockout mice display a phenotype that is consistent with deficits found in humans with fragile X syndrome and are generally consistent with overactive group I mGlu receptor signaling (428). In particular, Fmr1-knockout mice exhibit potentiated LTD in the cerebellum. Normally, activation of mGlu5 would increase expression of FMRP and stabilize spines by inhibiting translation of proteins in dendrites that are important for degeneration of the spine. In the Fmr1-knockout mice, this regulation is lost; mGlu5 is overactive, and AMPA and NMDA are lost from the spines (333). A further consequence of this loss of regulation appears to be a morphologic change in the spines so that they become elongated, possibly as a first step to elimination or pruning (429). The possibility that overactive group I mGlu receptor signaling might explain the majority of the deficits in fragile X syndrome patients has received much attention recently, and mGlu5 antagonists are being pursued as potential treatments for these deficits (430). As mentioned earlier, people suffering from fragile X syndrome have many deficits that can be attributed to changes in group I mGlu receptor signaling throughout the brain. It is interesting that behavioral deficits in Drosophila expressing a mutation in the fly ortholog of fmr1 can be rescued with the mGlu5-selective antagonist MPEP, especially when given early in development (431). These results suggest that although fragile X syndrome is caused by a deficit in a regulatory protein, downstream regulation of the affected signaling pathway may be sufficient to restore normal brain functioning (428). Therefore, the deficits of fragile X syndrome may be thought of as deficits in group I mGlu receptor signaling. Examples include prolonged epileptiform discharges induced by altered group I mGlu receptor–mediated synaptic responses in fragile X mice (432) and impairment

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of synaptic plasticity in cerebellum and striatum of Fmr1-knockout mice (430), again suggesting that overactivation of group I mGlu signaling is detrimental to normal synaptic function. The fragile X model provides insights into other disorders that may involve mGlu receptor signaling, including anxiety, movement disorders, pain, and drug addiction. 8.7. Parkinson’s Disease Parkinson’s disease (PD) is a neurodegenerative disorder characterized by motor deficiencies such as tremor, rigidity, and bradykinesia. PD is caused by a progressive loss of dopamine neurons in the substantia nigra pars compacta (SNpc) that project to the striatum. DA neurons in the SNpc produce a net inhibitory control over the output of the striatum that is diminished as PD progresses (for review, see ref. 379). As a result, glutamate-mediated excitotoxicity is thought to mediate neurodegeneration of DA neurons (433). The abundant expression of group I mGlu receptors in the basal ganglia and their functional role in modulating locomotor activity make these receptors prime targets for treatment of PD (324), in which akinetic dyskinesias may result from a disruption in the balance between inhibitory dopaminergic and excitatory glutamatergic signaling in the basal ganglia (379). Evidence supporting this idea comes from reversal of these symptoms with mGlu5 receptor antagonists (434) such as MTEP and MPEP that reduce Parkinsonian-like symptoms (171,435–438) and MPEP that is neuroprotective (439) in animal models of PD. Another promising target for PD is the adenosine receptor because simultaneous activation of mGlu5 and adenosine receptors in the striatum causes a synergistic increase in GABA release (440) and locomotor behaviors (441). These studies suggest that the simultaneous use of antagonists of mGlu5 and adenosine A2A receptors may serve as complementary anti-Parkinsonian therapies (441,442). 8.8. Alzheimer Disease Cerebral deposition of plaques containing amyloid -peptide (A ) has traditionally been considered the central feature of Alzheimer disease (AD). Proteolytic processing of the -amyloid precursor protein (APP) is regulated by neurotransmitters, including group I mGlu receptors that increase the release of soluble APP via a PKC-dependent mechanism (443). Antagonists of group I mGlu receptors block glutamate-mediated release of APPs from hippocampal neurons and astrocytes (444–446). In addition, impaired mGlu receptor/PLC signaling in the cerebral cortex in AD and dementia associated with Lewy bodies correlates with AD-related changes (447). Finally, mGlu5 modulation of the PI3K/Akt/Gsk3 pathway reverses -amyloid-induced neuronal toxicity in the hippocampus (448). Thus, amyloid formation enhanced by a deficiency

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in glutamate signaling suggests that selective mGlu agonists may facilitate synaptic efficacy and serve as a treatment for AD. 8.9. Huntington Disease Huntington disease is caused by a polyglutamine expansion in the huntingtin protein (Htt) and is associated with excitotoxic death of striatal neurons. The Htt-binding protein optineurin (which is also linked to normal-pressure open-angled glaucoma) antagonizes agonist-stimulated mGlu1a signaling by promoting desensitization of mGlu1a (449). Because GRK2 protein is relatively low in striatal tissue, optineurin may substitute for GRK2 in the striatum to mediate group I mGlu desensitization. These studies identify a novel mechanism for mGlu desensitization and a link between altered glutamate receptor signaling and Huntington disease. 8.10. Pick Disease Recent work examined the role of group I mGlu receptors in Pick disease (PiD), which is a neurodegenerative disease with abnormal accumulation of phospho-tau protein (450). There is reduced expression and activity of PLC1 in PiD cortex, as well as reduced expression of PLC, a substrate of tyrosine kinase that is also reduced in PiD. There is also a marked decrease in expression of PKC and increased expression of PKC. In contrast, [3 H]glutamate binding to mGlu receptors is augmented in PiD, mainly due to increased expression of mGlu1. Together, these results represent the first data to show abnormal mGlu receptor signaling in the cerebral cortex of PiD patients and demonstrate a selective vulnerability of group I mGlu receptor signaling molecules such as PLC and PKC in PiD. 8.11. Anxiety Anxiety disorders such as panic attacks, posttraumatic stress disorder, obsessive-compulsive syndrome, and phobias are the most common forms of mental illness (451). These conditions are attributed to excessive excitability within specific brain circuits and are generally treated with benzodiazepines that modulate inhibitory GABAA receptors in the brain. However, this class of drug produces significant side effects such as sedation, memory impairment, and potential for abuse (452). Recent studies provide increasing evidence that drugs that target mGlu receptors are beneficial anxiolytics (for reviews, see ref. 451 and 453). In particular, group I antagonists and group II agonists have proved useful in animal models of anxiety (451). The selective mGlu5 antagonist fenobam was originally developed by McNeil Laboratories as a potential nonbenzodiazepine anxiolytic drug with an unknown target (454,455). Fenobam used in a double-blind, placebo-controlled clinical trial was found

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to be as efficacious as benzodiazepines for anxiety (456). However, fenobam has not been further developed for use in humans due to psychostimulant-like side effects (457) and metabolism issues (446). Further work with fenobam indicates that it is a selective and noncompetitive inhibitor of mGlu5 receptors with potent antianxiety effects in rodent models of anxiety (172). The mGlu5 receptor antagonist MPEP administered intraperitoneally or orally is also an effective anxiolytic in rodent models of anxiety (335,458,459) and stressinduced hyperthermia (460). Although high doses of MPEP interfere with some measures of memory and LTP (329), systemic administration of MPEP produces anxiolytic effects at doses that do not impair working memory or spatial learning (461), suggesting that mGlu5 antagonists may have significant therapeutic advantages over benzodiazepines. These anxiolytic effects appear to be mediated by blockade of mGlu5 receptors in hippocampus and amygdala (334,462) and may be related to mGlu5 participation in LTP/LTD synaptic plasticity in these areas (262). 8.12. Drug Addiction Modulation of synaptic plasticity in the striatum, and particularly the ventral striatum or nucleus accumbens (NAcc), has been the focus of many studies on drug addiction because these areas have an important role in the acquisition of conditioned learning, as well as sensitization to drugs of abuse (for reviews, see refs. 463 and 464). In addition, they receive the bulk of the projections from VTA DA neurons. The NAcc also receives glutamatergic projections from the prefrontal cortex known to play a critical role in the development of addictive behaviors (368). Changes in dopaminergic neuron firing can be produced by administration of many drugs of abuse, and, in many cases, sensitization to drugs of abuse is attenuated with glutamate receptor blockers, suggesting that VTA-glutamate neurotransmission is important in regulating activity of DA neurons (367). Not surprisingly, mGlu receptors have been shown to play several important roles in drug addiction. As mentioned previously, activation of mGlu1 receptors modulates burst firing in DA neurons (148,370,371,374). Amphetamine promotes phasic firing of DA neurons by inhibiting mGlu1mediated hyperpolarizations (465) and can increase ERK/CREB signaling through activation of mGlu5 (96). Chronic cocaine use inhibits presynaptic mGlu1 receptor–mediated increases in glutamate release and elevation of locomotion in the NAcc, probably via downregulation of the scaffolding protein Homer-1b/c (466,467). Long-lasting motor stimulation and stereotypical behaviors are caused by administration of the group I mGlu agonist DHPG into the striatum (468, 469), and intracranial self-stimulation thresholds are increased with the mGlu5 antagonist MPEP, suggesting that group I receptors positively regulate brain reward (470,471). An mGlu5 receptor–dependent LTD is observed in the bed

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nucleus of the stria terminalis and is mediated by activation of ERK1. Repeated administration of cocaine blocks this LTD, suggesting that mGlu5 receptors are important for synaptic plasticity induced by psychostimulants (472). This idea is supported in mice lacking mGlu5, which do not self-administer cocaine and do not display sensitized locomotor activity with cocaine (387). Therefore, mGlu5 appears to play an important role in regulating self-administration of drugs of abuse and drug consumption (471,473,474), whereas the mGlu1 receptors selectively regulate drug-induced increases in brain reward, possibly due to modulation of compensatory changes or plasticity that occur with drug administration (475). 8.13. Additional Roles for mGlu Receptors in Neurons and Nonneuronal Cells It is clear that group I mGlu receptors play important roles in the regulation of synaptic transmission; however, there are additional distinct cellular functions mediated by mGlu receptors in both neurons and nonneuronal cells. For instance, group I and II mGlu receptors regulate cellular metabolism. Activation of group I mGlu receptors increase net metabolic flux through the Krebs cycle, as well as glutamate/glutamine cycle activity, in guinea pig cortex, whereas inhibition of these receptors significantly decreases metabolism, consistent with a neuroprotective role (476). The mGlu receptors are expressed in and regulate gastric rhythm generation in the stomach (477,478), and group I (mGlu1/5] and group II (mGlu2/3] receptors are expressed in pancreas, where they facilitate hormone release (479). Recent studies also show a role for glutamate receptors at multiple levels of the cardiovascular system. Immunocytochemistry experiments reveal expression of mGlu1a and mGlu5, as well as mGlu2/3, in rat heart (480) and monkey heart (481), where they are preferentially localized to nerve terminals, ganglion cells, and elements of the conducting system. Thus, preferential localization of these receptors to different components of the conducting system and cardiac neural structures suggests that they play a role in cardiac physiology. In the midbrain, stimulation of group I mGlu receptors is involved in the maintenance of arterial pressure (482,483) and enhanced cardiovascular responses in hypertensive rats (484). Studies also show that activation of group I and II mGlu receptors in the spinal cord evokes prolonged cardiovascular effects (485) and increases mean blood pressure (486). In addition, group I mGlu receptors mediate decreases in blood flow induced by electrical stimulation of the lower incisor in the rat (487). Electrophysiologic studies in bone cells have demonstrated that mGlu1b receptors modulate NMDA receptor–mediated increases in intracellular Ca2+ . This is the first description of mGlu effects in bone, suggesting that complex glutamatergic signaling occurs in this tissue (488). NMDA receptors are

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involved in osteoclast formation and bone resorption, leading to a hypothesis that manipulation of glutamate actions in bone may provide new therapeutic targets for pathologies associated with modifications of bone remodeling (489–491). Subsequent immunocytochemistry of osteoblasts and osteoclasts revealed expression of iGlu receptors (NMDA, AMPA, and kainate) and mGlu receptors, as well as glutamate transporters, in human osteoblast-like osteosarcoma cells (492). Recent evidence has also emerged that immune cells (thymocytes and lymphocytes) express several types of glutamate receptors that induce functional changes in these cells (for review, see ref. 493). RNA and protein expression studies show that mGlu1 and mGlu5, as well as mGlu2 and mGlu3, are expressed in whole mouse thymus, isolated thymocytes, and the TC-1S thymic stromal cell line (494), as well as in resting and activated lymphocytes from human peripheral blood, where they perform distinct functions (495). In blood lymphocytes, mGlu5 is constitutively expressed and couples to adenylate cyclase, whereas mGlu1 is expressed only on activation of the T cell receptor–CD3 complex and preferentially activates the MAPK cascade. Thus, the differential expression of group I mGlu receptors in resting and activated lymphocytes suggests that these group I receptors have distinct roles in the regulation of T cell physiologic function and in human lymphocytes. Expression of mGlu1 and mGlu5 is detected in vascular endothelial cells, where they increase endothelial permeability (496). Many compounds derived from the large neutral amino acid phenylglycine are highly selective for specific isoforms of mGlu receptors but only become useful therapeutics for CNS diseases such as ischemic disorders, stroke, and epilepsy if they cross the blood–brain barrier efficiently. Thus, one goal is to find potential strategies of synergistic optimization of phenylglycine-derived therapeutics with respect to desired activity at the CNS target combined with carrier-mediated delivery to overcome the blood–brain barrier (497). Finally, studies in a transgenic mouse melanoma model reveal that ectopic expression of mGlu1 in melanocytes is sufficient to induce melanoma development in vivo (498). Stimulation of mGlu1 receptors in vitro cell lines from independent mouse melanoma tumors (499) leads to IP3 accumulation and activation of ERK. This stimulation is inhibited by the mGlu1-specific antagonist LY367385 [(+)-2-methyl-4-carboxyphenylglycine] or by dominantnegative mutants of mGlu1. In addition, ERK activation by mGlu1 is PKC dependent but cAMP and PKA independent. Thus, signaling cascades mediated by mGlu1 in melanoma cells may be potential therapeutic targets for melanoma (500). In summary, the group I mGlu receptors are key players in the normal functioning of the nervous system and are implicated in the cause of, or treatment for, several neurologic and neuropsychiatric disorders. In addition,

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roles for mGlu receptors as signaling mediators in nonneuronal cells outside of the brain are beginning to emerge and highlight the importance of these receptors in the immune system and cardiovascular responses. The group I mGlu receptors have proven to be exciting targets for new therapeutic drugs due to their role as modulators of excitatory neurotransmission rather than mediators of signaling. However, because these receptors are practically ubiquitous throughout the brain, further research into the functions of mGlu receptors under various physiologically relevant conditions is needed to design effective drugs for specific disorders. In addition, whereas roles for the group II and III mGlu receptors are discussed in the following chapters, it is obvious that the physiologic output of a brain circuit responding to glutamate will be dependent on the integration of glutamate signals mediated by all of the glutamate receptors present in a given synapse, mGlu and iGlu receptors, as well as the molecular components of signaling complexes.

9. Future Directions 9.1. Glutamate Receptor Regulation of/by MicroRNAs One of the most exciting scientific findings in the last few years has been the discovery of a large number of previously overlooked non–protein-coding regulatory RNAs, termed microRNAs (miRNAs) and small interfering RNAs (siRNAs) (for review, see ref. 501). These small, ˜21- to 25-nucleotide RNAs regulate posttranscriptional gene expression in plants, animal, and viruses. Most miRNA genes are transcribed by RNA polymerase II (502), leading to primary miRNA transcripts that are transported to the cytoplasm and cleaved by an endonuclease, such as Dicer, to produce a mature miRNA. One strand of the miRNA then associates with an RNA-induced silencing complex that, in mammalian cells, binds to mRNA to inhibit translation or binds to existing miRNAs to potentiate translation of proteins. Our recent studies show that ischemic preconditioning in mice induced by transient middle cerebral artery occlusion that likely activates glutamate pathways leads to altered expression of miRNAs (503). Given the role of group I mGlu receptors in regulating transcription factors, including CREB, it is conceivable that activation of group I mGlu receptor pathways will lead to altered expression of miRNAs, which in turn lead to long-term changes in protein expression, such as those necessary for memory consolidation (Fig. 9). Altered miRNA expression mediated by activation of other signaling pathways may in turn regulate group I mGlu receptor expression. Studies show increased repression of translation of mRNAs that are targets of multiple miRNAs, suggesting that concerted actions of miRNAs are more powerful repressors of protein expression (504). Furthermore, the miRNA target prediction program developed by the Memorial Sloan-Kettering Cancer Center Computational

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Fig. 9. Future roles for group I metabotropic glutamate (mGlu) receptors include the regulation of microRNA (miRNA) expression, which in turn may lead to the subsequent regulation of protein expression, independent of changes in messenger RNA. CREB, cyclic AMP response element–binding protein; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; RISC, RNA-induced silencing complex.

Biology Group (http://www.microrna.org/) shows that both mGlu1 and mGlu5, but not other mGlu receptor subtypes, serve as potential targets for regulation by miR-181a and miR-181b. Thus, protein expression levels of mGlu1 and mGlu5 may be regulated by a concerted action of miR-181a and miR-181b on group I mRNAs in response to the altered expression of these miRNAs via activation of group I mGlu receptors (homologous regulation) or via activation of other signaling pathways (heterologous regulation). These new and exciting mechanisms of cellular regulation will without doubt play a significant role in the regulation of neuronal signaling and will likely not be limited to glutamate but will also involve other signaling molecules.

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11 Group II Metabotropic Glutamate Receptors (mGlu2 and mGlu3) Michael P. Johnson and Darryle D. Schoepp

Summary The group II metabotropic glutamate (mGlu2 and mGlu3) receptors are among the minority of glutamate receptors that primarily inhibit synaptic transmission by coupling to Gi/o, inhibiting adenylate cyclase and potassium channels. Our understanding of their physiologic role has advanced considerably with the discovery of systemically active and selective agonists, antagonists, and, most recently, subtype-selective allosteric modulators. The mGlu2/3 agonists, such as LY354740 (and its prodrug LY544344), show activity in a number of preclinical models of anxiety, psychosis, and drug abuse and as neuroprotectants. The anxiolytic actions of group II agents have subsequently been conﬁrmed in humans, and represent an exciting breakthrough in the glutamate ﬁeld. Emerging preclinical results with mGlu2/3 antagonists and potentiators of the mGlu2 receptor indicate that, despite a presynaptic localization distal to the synaptic active zone, signiﬁcant glutamatergic tone can occur under some physiologic conditions. This chapter describes some mGlu2/3-selective pharmacologic tools, brieﬂy discusses the different synaptic and glial localizations, and notes how these unique receptors may regulate synaptic transmission. Key Words: Group II metabotropic glutamate; LY354740; anxiolytic; antipsychotic; presynaptic; glial; inhibitory; heteroautoreceptor

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

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1. Structure (Genes, Diversity) Unlike many of the other ionotropic and metabotropic glutamate (mGlu) receptors, the group II receptors (including mGlu2 and mGlu3) show surprisingly little diversity in genomic structure, expression of variants, and coding sequences within the animalia phylum (Table 1). Indeed, there are no reports confirming any splice variants for either the human or rodent mGlu2 or mGlu3 receptors. The gene encoding the human mGlu2 receptor (GRM2) is localized on chromosome 3q21.2 and has five exons. The resulting mRNA encodes a typical family 3 G protein–coupled receptor (GPCR) with 872 amino acids, including a seven-transmembrane region and a glutamate-site containing an amino terminus (for more details on protein structure see Chapter 14). Similarly, the GRM3 gene encodes the human mGlu3 receptor, is localized to 7q21.1–q21.2, has six exons, and encodes an 879-amino acid family 3 GPCR. The human mGlu2 and mGlu3 receptors are relatively homologous, with an amino acid identity of 66% overall and an even higher 70% and 75% identity within the critical glutamate-binding region and the seven-transmembrane region, respectively. Homology across mammalian species is quite high; for instance, amino acid identity between rat and human is 97% and 96% for mGlu2 and mGlu3, respectively. Indeed, the human group II mGlu receptors have an unusual degree of amino acid identity (46%–48%) for even Drosophila melanogaster (the fruit fly) mGluRA (P-91685) as well. Taken together, this suggests that evolutionary pressure has maintained the group II mGlu receptors, implying their fundamental importance. Table 1 Homology and Genomic Structure of Group II Metabotropic Glutamate (mGlu) Receptors

mGlu2 Human (1,2) Rat (3,4) Mouse mGlu3 Human (5,6) Rat (3,4) Mouse (7) mGluA Drosophila (8)

Amino acids

Accession number

Homology to human (%)

Chromosomal localization

872 872 872

Q14416 P31421

100 97 92

3q21.2 8q32 9 F1

879 879 879

Q14832 P31422 Q9QYS2

100 96 93

7q21.1–q21.2 4q32 5 A1-h

976

P91685

46 vs. mGlu2 48 vs. mGlu3

102F4

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2. Signaling Both the mGlu2 and mGlu3 receptors are generally inhibitory in their actions. When expressed in recombinant cell systems or examined in native tissue, they typically couple to Gi/o, thus decreasing intracellular cAMP, stimulating G protein inwardly rectifying potassium channels (GIRKs), and/or inhibiting the voltage-gated calcium channels (VGCCs) (1,9–12). Thus, when localized presynaptically, group II mGlu receptors decrease evoked neurotransmitter release (e.g., via VGCC inhibition), and on postsynaptic membranes, they limit depolarization (e.g., via stimulation of GIRKs) or postsynaptic excitability (via regulation of cAMP elements).

3. Pharmacology The group II mGlu receptors are typical of family 3 GPCRs in that the ligand-binding domain (contained within the N-terminus) is segmented from the transmembrane (TM) and intracellular domains. The TM and intracellular domains mediate signal transduction and/or localization on cellular membrane. Thus, orthosteric/glutamate-site ligands bind completely within the N-terminus. However, it is clear now that a second domain of interaction with smallmolecule pharmacologic agents is possible within the TM region, analogous to family 1 GPCR’s orthosteric domain. This allosteric site(s) of the group II receptors could result in a continuum of activity, including inverse agonism, noncompetitive antagonism, or full/partial agonist activity. In addition, allosterically interacting small molecules can also give rise to a unique pharmacologic activity, for example, acting as “potentiators” of a receptor. Here “potentiator” is defined as an allosteric modulator that, despite possibly increasing the affinity or the maximum amount bound by an agonist, still require an orthosteric agonist for the receptor activation to occur. Within group II mGlu receptor pharmacology there are some excellent examples of both orthosteric and allosteric ligands. 3.1. NAAG: A Second Neurotransmitter for mGlu3? Given the three-dimensional structure of the group II receptors and sequence similarity, it is perhaps not surprising that very few subtype-selective orthosteric agonists or antagonists have been discovered. An exception is the neuropeptide N-acetylaspartylglutamate (NAAG), which appears to preferentially activate mGlu3 versus mGlu2 receptors (for a review see ref. 13). Indeed, since it was first discovered and described as a neurotransmitter (14,15) it has been well accepted that one of the likely receptors for NAAG is the mGlu3 receptor. That said, one must remember that mGlu3 receptors have a much higher affinity for glutamate (nanomolar range) than NAAG (micromolar

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range). In addition, glutamate carboxypeptidases (GCPII and III) are widely distributed on glial and postsynaptic membranes, rapidly converting NAAG to N-acetyl-aspartate and glutamate. So, the relative roles of NAAG itself and glutamate derived from NAAG degradation are often difficult to determine. Nevertheless, the effects of glutamate carboxypeptidase inhibitors in animal models of excitotoxicity, pain, and even psychosis might suggest that NAAG, presumably through activation of mGlu3 receptors, can play a significant role in certain pathologic states. 3.2. Orthosteric Agonists for mGlu2 and mGlu3 2R,4R-4-Aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC) (Fig. 1) is typical of group II selective agonists in that it is a conformationally constrained rigid glutamate analog. It is similar in structure to 1-aminocyclopentane-1, 3-dicarboxylic acid (1S,2R-ACPD), in which the cyclopentane has been replaced with a pyrrolidine ring (16). However, whereas 1S,2R-ACPD is an agonists at the group I and II mGlu receptors, 2R,4R-APDC is a selective group II agonist with no appreciable effects on group I or group III mGlu receptors (17,18). In rat brain slices, 2R,4R-APDC selectively suppresses forskolinstimulated cAMP formation at concentrations (1–100 mM) having no effect on phosphoinositide hydrolysis per se (18). 2R,4R-APDC inhibits the development of kindled seizures in rats and does not induce spontaneous nocioceptive behaviors, unlike the group I agonist (R,S)-3,5-dihydroxyphenylglycine (3,5DHPG) (19–21). The potency (millimolar) and selectivity of 2R,4R-APDC

Fig. 1. Orthosteric agonists of the group II metabotropic glutamate receptors. See text for abbreviations.

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make it a useful in vitro tool. However, the discovery of more potent and systemically acting mGlu2/3 agonists essentially limits the use of this compound to in vitro studies or direct injection into the tissue. 2´,3´-Dicarboxycyclopropylglycine (DCG-IV) and 2-(carboxycyclopropyl)glycine (L-CCG-I) are two other commonly used group II–preferring orthosteric agonists found in the literature. DCG-IV is a relatively potent (nanomolar) and selective agonist for group II mGlu receptors (22,23). However, at higher millimolar concentrations DCG-IV also has antagonist activity at group I and III mGlu receptor subtypes (22) and has been reported to activate N-methyld-aspartate (NMDA) receptors in native tissues (24–26). In rat mGlu2 recombinant cell lines, 3 H-DCG-IV binds with relatively high affinity (KD = 160 nM) (27), and in rat brain homogenates to an apparent single site with KD = 180 nM and Bmax = 780 fmol/mg protein (28). This binding was potently displaced by the group II antagonist LY341495 (see later discussion) and group II agonist LY354740. However, it was not until the discovery of (1S,2S,5R,6S)-2aminobicyclo[3.1.0.]hexane-2,6-dicarboxylate monohydrate (LY354740) (29, 30) that a systemically active group II–selective agonist was described (for a review see ref. 31). LY354740 potently activates mGlu2 and mGlu3 receptors in vitro, with no appreciable effects on other mGlu or ionotropic glutamate receptor subtypes (30,32). In rat hippocampus, LY354740 suppresses forskolinstimulated cAMP formation with about the same potency as was observed in human mGlu3-expressing cell (33). In rat brain tissue, 3 H-LY354740 binds with high affinity (KD = 8 nM), and specific 3 H-LY354740–labeled sites exhibit a pharmacology and distribution consistent with the selective labeling of group II mGlu receptors (34). Consistent with the role of group II mGlu receptors in modulating glutamate neurotransmission, systemic LY354740 prevents veratridine-evoked glutamate and aspartate release in free-moving rats undergoing microdialysis of the striatum (35). Two related conformationally constrained heterocyclic compounds with either oxygen (LY379268) or sulfur (LY389795) in the bicyclohexane ring are also highly potent and systemically active mGlu2/3 agonists (36). In functional assays with human mGlu2- or mGlu3-expressing cells, LY379268 and LY389795 are three to eight times more potent than LY354740. As with LY354740, LY379268 and LY389795 are active with systemic dosing in several animal models of epilepsy, psychosis, ischemia, and pain. 3.3. Group II-Selective Orthosteric Antagonists One of the earliest and most widely utilized group II selective antagonists was a simple analog of an earlier group II agonist. (2S,1´S,2´S)-2-methyl-2(2´-carboxycyclopropyl)glycine (MCCG) (Fig. 2) is the -methyl derivative of

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Fig. 2. Orthosteric antagonists of the group II metabotropic glutamate receptors. See text for abbreviations.

the potent group II mGlu receptor agonist L-CCG-I. In neonatal spinal cord, MCCG blocks the presynaptic depressant effects of L-CCG-I while having minimal effects on the depressant effects of the group III–selective agonist L-AP4 and no effect on the depolarizing response to 1S,3R-ACPD, indicating no appreciable group I or III mGlu receptor activity (37). This group II– selective antagonist profile has subsequently been confirmed in human mGlu1, mGlu2, and mGlu4 cloned receptors (38,39). However, in adult rat cerebral cortex slices, MCCG acted as an agonist/partial agonist, suppressing forskolinstimulated cAMP formation to 80% of that observed with L-CCG-I (40). This may suggest a partial agonist activity for MCCG on rat mGlu3 receptors that most likely contribute, along with mGlu2 receptors, to cAMP inhibition. One of the most useful pharmacologic tools for group II mGlu receptors is the group II-preferring antagonist -9´-xantheylmethyl-2(carboxycyclopropyl)-glycine (LY341495) (41,42). Full characterization of LY341495 across human mGlu receptor subtypes indicates that LY341495 is a competitive antagonist with low nanomolar potency at mGlu2 and mGlu3 receptors but will antagonize group I and group III receptors at higher concentrations (high nanomolar to millimolar) (39,43). Thus, careful attention to the exposure level/concentration allows LY341495 to be used as a selective group II antagonist or at higher levels (micromolar) as a group I, II, and III antagonist. For instance, 3 H-LY341495 selectively binds to recombinant human and native rat mGlu2 and mGlu3 receptors (KD = 1–2 nM), with a pharmacologic profile indicative of binding to the mGlu2/3 receptor subtypes (44,45). This makes 3 H-LY341495 a highly useful ligand for quantifying group II receptor expression, determining the affinity of other orthosteric compounds for mGlu2or mGlu3-binding sites, and investigating any changes in group II receptor localization with different physiologic/pathologic states. LY341495 is also systemically active and, when relatively low parenteral doses (0.3–1 mg/kg) are used, it has been shown to block the pharmacologic actions of several selective group II mGlu receptor agonists. For instance, in mice, a 0.3- mg/kg intraperitoneal dose of LY341495 reversed the anxiolytic actions of LY354740

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while having no anxiolytic or anxiogenic effects when administered alone (41). Similarly, the inhibitory actions of LY379268 against phencyclidine (PCP)evoked motor activation were completely reversed in rats with LY341495 (1 mg/kg subcutaneously) (46). The potent and selective actions of LY341495 make it a useful pharmacologic agent with which to study group II mGlu receptor functions. (1R,2R,3R,5R,6R)-2-Amino-3-(3,4-dichlorobenzyloxy)6-fluorobicyclo[3.1.0] hexane-2,6-dicarboxylic acid (MGS0039) represents another rigid glutamate analog with antagonist activity at mGlu2 and mGlu3 receptors. Like LY341495, MGS0039 has low nanomolar potency at both mGlu2 and mGlu3 with high nanomolar potency for the mGlu7 receptor and micromolar potency for mGlu4 and mGlu6 receptors. However, MGS0039 has no appreciable affinity for mGlu1, whereas LY341495 will block mGlu1 and mGlu5 at micromolar concentration (47,48). MGS0039 has been shown to be active when administered systemically to mice and rats (49–53). The potency of MGS0039 for the mGlu8 receptor has not been reported. Given that many of the mGlu2/3 agonists retain some potency for the mGlu8 receptor and the antagonist LY341495 blocks mGlu8 receptors more potently than either mGlu4 or 7, one cannot assume that MGS0039 lacks significant activity at mGlu8. Furthermore, in many pathways there are overlapping or complementary distributions and functions for the mGlu2 and mGlu8 receptors—for instance, the perforant pathways (54,55). Thus, when interpreting the actions of either LY341495 or MGS0039, careful consideration must be given to the possibility that mGlu8 plays a significant role. 3.4. mGlu2 Allosteric Potentiators The first reported series of mGlu2 receptor potentiators are typified by 2,2,2trifluoro-N-[4-(2-methoxyphenoxy)phenyl]-N-(3-pyridinylmethyl) ethanesulfonamide (4-MPPTS or LY487379), shown in Fig. 3 (56,57). As with the other family 3 GPCR potentiators, 4-MPPTS and its close analog 2,2,2-trifluoro-N[3-(2-methoxyphenoxy)phenyl]-N-(3-pyridinylmethyl)-ethanesulfonamide) (3MPPTS) increase the affinity of orthosteric agonists such as glutamate and 3 H-DCG-IV (56–58). Chimeric and point mutation of mGlu2 receptors indicate that the binding domain of the potentiators are within the transmembrane domain, with a critical role for an asparagine in TM V, which is unique for mGlu2 versus mGlu3 (56,58). Indeed, these allosteric potentiators of the mGlu2 receptor are remarkably selective, with no appreciable activity at any of the other mGlu’s including the mGlu3 receptor. Further exploration led to other potent potentiators, including 2,2,2-trifluoro-N-[3-(cyclopentyloxy)phenyl]N-(3-pyrindinylmethyl)-ethanesulfonamide (cyPPTS) (24 nM) (57,59,60). Indeed, cyPPTS typifies one important aspect of potentiators, namely an ability to act in a state-dependent manner (see Fig. 4).

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Fig. 3. Allosteric modulators: metabotropic glutamate (mGlu) 2 receptor potentiators. See text for abbreviations.

Several of the mGlu2 potentiators from this series, including 4MPPTS, 2,2,2-trifluoro-N-[1-methyl-butoxy)phenyl]-N-(3-pyrindinylmethyl) ethanesulfonamide (222-TEMPS), N-[4-(4-carboxyamidophenoxy)phenyl]N-(3-pyridinylmethyl)-ethanesulfonamide (4-APPES), and N-[4´-cyanobiphenyl-3-yl]-N-(3-pyridinylmethyl)-ethanesulfonamide (CBiPES), have been active in model(s) of anxiety and/or psychosis (57,61). For instance, high doses of either 4-MPPTS or APPES were anxiolytic-like in a rat potentiated startle paradigm, as was CBiPES in reducing stress-induced hyperthermia in mice (57,58). CBiPES also reduced the PCP-induced locomotor activity model commonly used to test for antipsychotics (57), and pretreatment with 222TEMPS limits ketamine-induced hippocampal norepinephrine release in rats (61). Although they are excellent tools for in vitro work, these first mGlu2 potentiators were limited in their pharmacokinetic characteristics and often required unusually high doses to reach efficacious levels in vivo. A second series of mGlu2 potentiators has been described and is typified by 1-(2-hydroxy-4-{4-[4-(2H-tetrazol-5-yl)-phenoxy]-butoxy}-3propyl-phenyl)-ethanone (compound 1, Fig. 3) (62,63). Compound 1 is reported to be a moderately potent but highly selective mGlu2 potentiator. However, despite reasonable plasma levels with systemic dosing, the brain to plasma ratio was very low, indicative of poor brain penetration. Thus,

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Fig. 4. The state-dependent activity of potentiators. A. Representative (average of 10 individual neurons) corticostriatal excitatory postsynaptic potentials (EPSPs) in rat brain slice preparation using a whole-cell patch-clamp technique. At lowerfrequency (0.06 or 1 Hz) stimulation of the corticostriatal projections, 2,2,2-trifluoro-N[3-(cyclopentyloxy)phenyl]-N-(3-pyrindinylmethyl)-ethanesulfonamide (cyPPTS) (0.3 or 1 μM) did not significantly suppress the EPSPs. However, when the frequency of stimulation, and presumably the amount of glutamate released, was increased, cyPPTS significantly suppressed the resulting EPSPs. B. The orthosteric agonist LY354740 was effective in suppressing the EPSPs even when stimulated at a lower frequency (0.6 Hz). Reprinted with permission from Johnson MP, Barda D, Britton TC, et al. Metabotropic glutamate 2 receptor potentiators: receptor modulation, frequency-dependent synaptic activity, and efficacy in preclinical anxiety and psychosis model(s). Psychopharmacology (Berl) 2005;179(1):271–283.

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this series may be limited as a tool for exploring central activity with mGlu2 potentiation after dosing peripherally. In addition, two other mGlu2 potentiators have been described: (±)-6,7-dichloro-2,3-dihydro-2-propyl-5[{3-[(4-pyridinylthio)methyl]phenyl}methoxy]-1H-Inden-1-one (compound 2, Fig. 3) and (±)-5-[(4´-carboxy-[1,1´-biphenyl]-3-yl)methoxy]-2,3-dihydro-6,7dimethyl-2-pentyl-1H-Inden-1-one (compound 3, Fig. 3) (64,65). The brain penetration for compounds 2 and 3 was greatly improved, and systemic doses were effective in blocking ketamine-induced locomotor activity. More experiments are needed, but these initial results suggest that CBiPES and compound 3 may be useful pharmacologic tools with which to explore the utility of mGlu2 potentiators.

4. Interaction Partners Few trafficking or modulatory proteins have been shown to directly interact with either the mGlu2 or mGlu3 receptors (for review see ref. 66). Like the other mGlu receptors, it is believed the intracellular carboxy-terminus is the primary site of trafficking information and regulation of receptor internalization/deactivation. The carboxy terminus of mGlu3 has been shown to interact with the calcium-sensing protein calmodulin (67), and phosphorylation of the mGlu2 receptor carboxy terminus by protein kinase C (at sites shared with mGlu3) is reported to inhibit G protein interaction (68). In addition, a segment of the mGlu3 carboxy terminus, but not the mGlu2, has been shown to interact with protein phosphatase 2C, acting as a substrate at the phosphorylated serine 845 (a cAMP-dependent protein kinase A site) (69). There are also reports of astrocyte mGlu3 receptors interacting with other GPCR’s such as the adrenergic 2 or the adenosine A2a receptor (70), although it is somewhat unclear whether this represents a direct interaction or the result of complex cross-talk among various activated second message systems.

5. Modulation of Synaptic Transmission As previously described, the majority of data indicate presynaptic mGlu2/3 receptors can negatively modulate both excitatory and inhibitory synaptic transmission via inhibition of select calcium channels and/or activation of potassium channels (for a review see ref. 71). An illustration of this modulation can be seen in the corticostriatal synapse and how the excitatory postsynaptic potentials are differentially modulated by the mGlu2/3 agonist LY354740 and the mGlu2 potentiator cyPPTS (57,59). Specifically, the orthosteric agonist blocks excitatory postsynaptic potentials (EPSPs) resulting from presynaptic stimulation irrespective of the frequency of stimulation (Fig . 4). In contrast, cyPPTS showed a clear dependence on the frequency of presynaptic stimulation. A

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likely explanation for this differential response is the potentiator’s dependence on greater synaptic glutamate released with higher-frequency stimulation. In essence, cyPPTS acts to sensitize the mGlu2-mediated presynaptic inhibition of glutamate release, altering only higher-frequency synaptic transmission. In contrast, mGlu2 agonists might be expected to continuously inhibit mGlu2sensitive glutamatergic pathways and/or result in GPCR desensitization and possible drug tolerance responses in vivo. Thus potentiators would have a unique ability to modulate overexcitation without inducing an inhibition of normal or basal responses. When considering the in vivo actions of potentiators, the degree of tone on the target receptor becomes a critical factor. In the case of the group II mGlu receptors the behavioral and biochemical activity seen with the orthosteric antagonists LY341495 and MGS0039 suggests that mGlu2 and/or mGlu3 receptor are activated under certain conditions. For instance, bilateral intra-accumben injection of LY341495 significantly increased the locomotor activity in rats following habituation to a novel environment, an effect partially reversed with the mGlu2/3 agonist APDC (72,73). Similarly, both MGS0039 and LY341495 block normal “compulsive” behavior in mice, as indicated in a “marble-burying” paradigm (51). MGS0039 and LY341495 decreased immobility in the rat forced-swim and mouse tail-suspension tests (52), and subchronic (14 day) repeated MGS0039 showed an intriguing increase in cell division within the dentate gyrus of the mouse hippocampus (53), activities attributed to the efficacy with antidepressants. Furthermore, systemic MGS0039 increased the firing rate of the dorsal raphe nucleus and increased extracellular serotonin in the medial prefrontal cortex (50). It is interesting that many of the observations may be attributable to alterations in arousal. In freely moving rats, LY341495 increased waking time and decreased both the non-rapid eye movement (NREM) and rapid eye movement (REM) sleep times. Electroencephalographic (EEG) monitoring showed a decrease in lowfrequency and an increase in high-frequency power with LY341495 and a unique state-dependent increase in awake but not NREM theta power (74). The opposing ability of the mGlu2/3 agonist to suppress REM sleep and decrease high-frequency EEG power (75), however, would argue that the mGlu2/3 receptors are not saturated. Taken together, the observations with the group II antagonists suggest that under certain conditions there is significant tone on these receptors and are suggestive of the arousal state of the animal being critically regulated by the relative degree of mGlu2/3 activity. The ability of mGlu2 potentiators to decrease spontaneous locomotor activity in mice (57), as well as show activity in various animal models of psychiatric disorders, further implies that there are conditions in which tone on the mGlu2 receptor plays some compensatory role in the animal.

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Direct alterations in synaptic transmission, however, are not limited to spinal cord or brain nuclei synapses. There are multiple reports of mGlu2/3 receptors in peripheral tissues often associated with peripheral nerve endings including the leptomeninges (76), heart, gastrointestinal tract, testes, kidney, uterus, ovary, and thymus (for a review see refs. 77 and 78). Although the relative roles of the mGlu2/3 receptors at these peripheral nerve endings have largely been unexplored, there is some evidence to suggest that the mGlu2/3 receptors act to modulate sensory perception. For instance, local injection of the mGlu2/3 agonist APDC blocks prostaglandin E2–induced thermal hyperalgesia and mechanical sensitization and reverses carrageenan-induced mechanical allodynia (79,80). Similarly, direct injection of APDC or DCG-IV decreased the orofacial mechanical allodynia induced with interleukin-1, an effect blocked by pretreatment with LY341495 (81). Whether this response is mediated by direct inhibition of sensory fiber afferents or represents an indirect action via activation of nonneuronal cell group II mGlu receptors (see later discussion below) is unclear.

6. Modulation of Signaling Systems Much of the focus on group II receptors has been on the direct modulation of central or peripheral nervous system synaptic transmission presumably through these pre- and postsynaptic neuronal mGlu receptors. However, as noted earlier, extensive evidence exists for mGlu2/3 receptors on nonneuronal cells, especially for the mGlu3 receptor. Group II receptors are localized on and show functional activity in microglia, astrocytes, and neuronal progenitor cells. Within these cells mGlu3-mediated inhibition of intracellular signaling pathways, such as mitogen-activated protein kinase and phosphatidylinositol3-kinase (82–84), most likely regulates cell survival and growth, as well as altering cellular phenotype. For instance, isolated adult human glial precursor cells have been found to express mGlu3 (and mGlu5) (85), and U373 cells, of human glioma origin, alter expression of the glutamate transporters with mGlu2/3 stimulation (84). In the human glioma cell line U87MG, an mGlu2/3 antagonist inhibited cellular proliferation, and when transplanted into a mouse, decreased the size of resulting brain tumor (82). Cultured mouse neuronal progenitor cells similarly express mGlu3 (and mGlu5), and blockade of these receptors reduced the number of dividing progenitor cells in the dentate gyrus of the hippocampus (86). There is also evidence for mGlu3 expression and/or functional activity on activated microglia or reactive astrocytes (83,87–90). Thus, there is a growing body of evidence linking mGlu3 (and in some cases mGlu2) to a role in controlling cell differentiation and/or nonneuronal cell function. Determination of the relative significance of these findings will most likely have to await more potent and selective mGlu3 pharmacologic tools.

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7. Function (in Vitro and in Vivo) Many studies have depended on the available group II–selective (but lacking subtype selectivity for mGlu2 or mGlu3) pharmacologic agents such as LY35740 (see Section 3). A distinct role of each subtype is not always clear. However, the propensity of data suggests that mGlu2 receptors have a predominant presynaptic localization somewhat distal from the synaptic cleft, that is, parasynaptic. Here, mGlu2 receptors most likely function to monitor glutamate that has diffused from the synaptic space following highfrequency stimulation and/or with reductions in glutamate uptake by glial or neuronal transporters. Thus, mGlu2 receptors provide negative feedback to prevent excessive glutamate release. For instance, the presynaptic localization of mGlu2 to glutamate terminals and functional consequences of mGlu2 receptor activation have been well defined within the hippocampal formation. Here, mGlu2 immunoreactivity localizes to the medial perforant and mossy fiber terminal subfields in the hippocampus (90a). Unilateral entorhinal cortex lesions containing perforant path neurons lead to ipsilateral loss of mGlu2/3 immunoreactivity (90a) and 3 H-LY354740 binding (90b) within perforant path terminal fields of the hippocampus. Furthermore, mGlu2 receptor–knockout mice lacked mGlu2/3 immunostaining within stratum lucidum of CA3 (mossy fiber terminal field) or stratum lacunosum moleculare of CA1 (medial perforant path terminal field) of hippocampus (90c). It is worth noting that long-term depression induced by mossy fiber low-frequency stimulation, a form of synaptic plasticity, was abolished in CA3 synapses from mGlu2-knockout mice. Thus, in some synaptic fields, presynaptic mGlu2 receptors may be essential as a homeostatic mechanism to prevent excessive glutamate release that might lead to pathologic conditions of overexcitation. In contrast, the functions of mGlu3 receptors are less well understood. However, using an mGlu3-preferring antibody and mGlu2 receptor–knockout mice, Tamaru and colleagues (91) successfully localized mGlu3 to pre- and postsynaptic membranes, with a widespread localization on glial cells as well. More specifically, the postsynaptic mGlu3 receptors are in or close to the synaptic specialization of neurons. What specific role they play here is unclear. Like mGlu2 receptors, presynaptic mGlu3 receptors are localized distal from the synaptic cleft (92), often identified as GABAergic terminals. Indeed, it is now recognized that mGlu3 receptors can negatively modulate GABA release and thus also have hetereosynaptic functions (93). When group II pharmacologic agents are used in vivo, numerous functions are revealed, including regulation of many neurotransmitters such as glutamate, -aminobutyric acid (GABA), dopamine, norepinephrine, and acetylcholine (for review see ref. 94; also see ref. 95). Group II agents have shown activity in a number of animal models of psychiatric, neurologic, and neurodegenerative

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diseases. One of the most advanced areas of study is that of anxiety disorders, in which the extensive preclinical work has now been validated in early clinical trials (for a review see ref. 96). Moreover, the preclinical evidence also indicates mGlu2/3 could be a therapeutic target for psychosis (97), pain (79,98), neurodegeneration (99,100), and epilepsy (101), among several other diseases associated with glutamate overexcitation.

8. Summary and Future Perspectives Among the different classes of mGlu receptors, the pharmacology of group II receptors has advanced considerably in the last decade. Many highly potent, selective, structurally and mechanistically distinct pharmacologic tools have been described, allowing for preclinical and early clinical explorations of their functions and therapeutic applications. Furthest advanced is the mGlu2/3 receptor agonist LY35470 (LY544344 as prodrug), which has been shown to be anxiolytic in generalized anxiety disorder in humans, and was rationally designed for this application based on preclinical activity in animal models. This compound and more potent analogs such as LY379268 and LY404039 have also been shown to have activity in certain models of psychosis and drug abuse and as novel neuroprotectants, and clinical information on certain of these indications may be on the horizon. Issues that need to be further addressed for this class include their activity across different psychiatric and neurologic conditions in humans and their long-term safety in animals and humans. Many earlier studies suggested that there is very little endogenous tone at the group II receptors under physiologic conditions, and the dogma in the field was that these receptors functioned as a negative-feedback mechanism to monitor glutamate primarily under conditions of excessive or pathologically induced release (hyperglutamatergic states). This implied that there might be few if any therapeutic applications for group II receptor antagonists because blockade of mGlu2/3 receptors should be relatively silent normally and in fact might worsen pathologic conditions of excessive glutamate release. In support of this, mGlu2/3 (and maybe mGlu8) receptor blockade with LY341495 has been reported to be anxiogenic in mice (102) and worsen drug withdrawal to morphine in rats (103). A relatively minor role for mGlu2 and mGlu3 receptors independently in controlling physiologic glutamate release was also supported by data in mGlu2- and mGlu3-knockout animals, in which where the phenotype was not much different than in wild-type animals (other than removing mGlu2/3 receptor agonist pharmacology) (104). However, more recent data with systemically active mGlu2/3 antagonists (LY341495 and MSG0039) suggest that there may be considerable tone at these receptors

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Fig. 5. Enhanced glutamate neurotransmission by blockade of metabotropic glutamate (mGlu) 2/3 receptors with LY341495. A. On depolarization, glutamate (Glu) is released from nerve terminals into the synaptic space, where it acts on postsynaptic ionotropic receptors (N-methyl-d-aspartate [NMDA], -amino-3-hydroxy-5-methyl-4isoxazolepropionic acid [AMPA], kainate subtypes) to gate ions (Na+ , Ca2+ ) that

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even under physiologic conditions, and this represents a novel mechanism for increasing glutamatergic tone (Fig. 5). With the advent of high-throughput screening methods, newer positive modulators for mGlu2 receptors have also been described, and these agents share some of the pharmacologic properties of orthosteric mGlu2/3 receptor agonists. The applications of these agents needs further study and may depend on newer agents with better druglike properties than those described so far. The discovery of mGlu3-selective agents has seemingly lagged behind, but such compounds would be useful because it is not usually clear what the roles of these two receptors are in isolation from modulation of the other. The glial localization of mGlu3 receptor is particularly intriguing and may have quite novel functions relative to what is known for mGlu2 receptors. Looking ahead, there is considerable knowledge from which to build on to accelerate our understanding of the functions of mGlu2 and mGlu3 receptors. Much of this information has been based on the use of less-than-optimal tools, and thus the discovery of better tools is still a key factor for future advances in this area. Fig. 5. (Continued) function to enhance excitation and promote plasticity in neurons. The majority of synaptic glutamate is likely removed from the synapse by excitatory amino acid transporters (EAATs), which are expressed in glial cells proximal to the synaptic space. Synaptic glutamate not taken up by transport into glia can diffuse from the synaptic space, where presynaptic mGlu2 and/or mGlu8 receptors function to monitor this space and to limit further release of glutamate via presynaptic negative feedback (–). mGlu2/3 and possibly mGlu8 receptors may also be present on heterosynapses, where they function to negatively (–) modulate the release of other neurotransmitters such as -aminobutyric acid (GABA). Glial cells also express mGlu3 receptors, where their function is less understood but might also involve some sort of feedback mechanism for modulation of excitability. DA, dopamine. B. Administration of the potent, systemically active mGlu2/3 receptor antagonist LY341495 blocks presynaptic mGlu2 receptors (and at higher doses possibly mGlu8 receptors as well) and prevents negative feedback in populations of synapses in many brain regions. This results in enhanced release of glutamate (possibly in a use-dependent manner) that acts on ionotropic glutamate receptor to enhance postsynaptic excitation (as can be measured by increased cFos expression in vivo in rats). Subsequent to this enhanced depolarization of postsynaptic cells (direct, or indirectly mediated, depending on the phenotype of the postsynaptic cell), there is also increased release of other neurotransmitters (NT). including glutamate, GABA, serotonin (5HT), DA, norepinephrine (NE), and histamine. The release of neurotransmitters by LY341495 may also be due to actions at heterosynapses containing mGlu2/3 or 8 receptors. In any case, this may represent a novel approach to enhancing ionotropic glutamate neurotransmission, possibly by a presynaptic mechanism.

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12 Group III Metabotropic Glutamate Receptors (mGlu4, mGlu6, mGlu7, and mGlu8) Volker Neugebauer

Summary G protein-coupled group III metabotropic glutamate receptors (mGluRs) include four subtypes that are negatively coupled to adenylyl cyclase (mGluRs 4, 7, and 8) or positively to a cGMP phosphodiesterase (mGluR6). Typically, they exert inhibitory inﬂuences on neuronal activity. This chapter reviews the characteristics of group III mGluRs and their normal functions and role in nervous system disorders. The therapeutic potential of group III mGluRs resides in their antiepileptic (mGluRs 4 and 7), neuroprotective (mGluRs 4, 7, and 8), anti-parkinsonian (mGluRs 4 and 7), anxiolytic (mGluR4), and analgesic effects. However, possible side effects may need to be considered because group III mGluRs can inhibit normal synaptic transmission and memory processes, and mGluR6 is important for visual transmission. This chapter emphasizes that the analysis of the role of individual group III mGluR subtypes is required to determine their potential therapeutic value. Key Words: Glutamate; G protein; signaling; synaptic transmission; affective disorders; neurotoxicity; neurodegeneration; Parkinson; pain; epilepsy.

1. Structure, Localization, and Distribution Group III metabotropic glutamate receptors (mGluRs) include four subtypes (mGluRs 4, 6, 7, and 8), which share about 70% sequence homology, similar principal signaling mechanism (inhibition of stimulated cyclic AMP formation From: The Receptors: The Glutamate Receptors Edited by: R. W. Gereau and G. T. Swanson © Humana Press, Totowa, NJ

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via Gi), and basic pharmacologic properties (l-2-amino-4-phosphonobutanoate [L-AP4] as the prototypical agonist) (1–7). Several splice variants have been identified (mGlu4a and 4b; mGlu7a and 7b; and mGlu8a and 8b), which differ with regard to the intracellular C-terminal (CT) but not the extracellular N-terminal domains (see Section 1.1). Therefore, group III mGlu splice variants likely retain the same G protein coupling selectivity and pharmacologic properties, which do not reside in the CT domain (see later discussion) (8). The functional significance of group III mGlu splice variants remains to be determined. 1.1. Structure Like other mGluRs of the class 3 G protein–coupled receptors (GPCRs), group III mGluRs are characterized by a seven-transmembrane domain topology and a large N-terminal extracellular domain (ECD). The ECD forms a bilobed structure called a “Venus fly-trap” module and contains the ligandbinding site for (orthosteric) agonists (2–4,9–13). Agonist affinity is determined by the binding-site residues at position lysine 74 in mGlu4, glutamine 58 in mGlu6, and asparagine 74 in mGlu7, but residues outside of the binding pocket also contribute to agonist affinity and pharmacologic profile (14). All mGluRs form monomers or homodimers stabilized by disulfide bonds both in native tissues and heterologous expression systems (6,12,13). It has been suggested that the closure of one ECD on agonist binding is sufficient for receptor activation, but the closure of both ECDs is required for full activity (15). The second (and possibly third) intracellular loop determines G protein specificity and the transduction mechanism. The intracellular CT domain interacts directly with intracellular proteins, which are involved in receptor trafficking, synaptic anchoring, cell signaling, and constitutive (basal) receptor activity (4,6,9,16). The CT domain within the group III mGluRs is largely conserved, although splice variants show differences, but it is significantly different (no homology) from the CT domains of group I and group II mGluRs (8,17). Therefore, splice variants could be subjected to different subcellular targeting, trafficking, and modulation by signaling molecules (see Section 5). 1.2. Subsynaptic Localization Group III mGlu4, 7, and 8 are predominantly presynaptic receptors that are situated in (mGlu7) or near (mGlu4 and 8) the active zone of the synapse (1,18–22). There is evidence to suggest that each of these subtypes (mGlu4, 7, and 8) can function as an autoreceptor on glutamatergic and as a heteroreceptor on GABAergic terminals (1,6,20,21). Group III mGluRs, including mGlu4 and possibly mGlu7, have also been found on postsynaptic sites in some neurons (1,19,23). mGlu6 is exclusively expressed postsynaptically on ON-bipolar cells

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in both rod and cone systems of the retina (3). Expression of group III mGluRs in glial cells is somewhat controversial. Cultured rat microglia express mRNA and receptor protein for group III mGlu4, mGlu6, and mGlu8 but not mGlu7 (24). In human postmortem brain tissue, no mGlu4 or mGlu8 immunoreactivity was detected in control tissue, but mGlu8 was found in microglial cells in active multiple sclerosis (MS) lesions, and mGlu4 and mGlu8 were detected in reactive astrocytes at the borders of some chronic active MS lesions (25). 1.3. Distribution in the Nervous System mGlu7 is more widely distributed than the other group III mGlu subtypes, whereas mGlu6, being confined to the retina, shows the most restrictive expression (3,22,23,26,27). High levels of mGlu7 mRNA were found in the olfactory system (particularly in the tufted and mitral cells of the olfactory bulb), medial septal nucleus, locus coeruleus, and trigeminal and dorsal root ganglia. Moderate levels were detected in all neocortical layers, cerebellar Purkinje cells, throughout the limbic system, including septal and amygdalar nuclei, and in other subcortical regions such as thalamus, hypothalamus, and basal ganglia. Several brainstem areas also showed moderate expression of mGlu7 mRNA; they include sensory regions, parabrachial nucleus, solitary tract nucleus, periaqueductal gray, reticular formation, raphe nuclei, ventral tegmental area, and pontine nuclei. Moderate expression of mGlu7 mRNA was also detected in the dorsal horn of the spinal cord (26). Similarly, mGlu7a-like immunoreactivity is widely distributed in the brain of rat and mouse, with particularly high expression levels in relay nuclei of sensory pathways. Intense mGlu7a expression was seen in the olfactory bulb, anterior olfactory nucleus, olfactory tubercle, piriform cortex and entorhinal cortex, periamygdaloid cortex, amygdalo-hippocampal area, hippocampus, neocortex (layer I), thalamic nuclei, globus pallidus, cerebellum, superior colliculus, locus coeruleus, dorsal cochlear nucleus, and superficial spinal dorsal horn. The distribution of mGlu7b was more restricted and included substantia innominata, hippocampus, ventral pallidum, and globus pallidus (22,23). High or moderate expression of mGlu4 but not mGlu7 was found in the granule cells of the olfactory bulb and cerebellum. Both mGlu4 and mGlu7 mRNA were expressed moderately or intensely in the olfactory tubercle, superficial layers of the entorhinal cortex, dentate gyrus, septofimbrial nucleus, intercalated nuclei of the amygdala, medial mammillary nucleus, many thalamic nuclei, including the relay, intralaminar, and midline nuclei, in the pontine nuclei, and in the trigeminal and dorsal root ganglia. Regions with little or no significant mRNA expression of mGlu4 but intense or moderate expression of mGlu7 include mitral and tufted cells of the olfactory bulb, neocortical regions, cerebellar Purkinje cells, cingulate cortex, piriform cortex, perirhinal cortex, hippocampal areas CA1–3, some septal nuclei, bed nucleus of the

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stria terminalis, ventral pallidum, most of the amygdalar nuclei, and nearly all hypothalamic nuclei, sensory brainstem regions, motor nuclei of cranial nerves, reticular formation, raphe nuclei, and spinal dorsal horn. No significant expression of mGlu4 and mGlu7 was found in the subthalamic nucleus, substantia nigra pars compacta, red nucleus, pterygopalatine ganglion, and superior cervical ganglion (19,26). Compared to mGlu4 and mGlu7, the distribution of mGlu8 is more restricted (20,27). Most prominent expression was detected in the olfactory bulb, piriform cortex, pontine gray, and reticular nucleus of the thalamus. Lower expression levels were found in the neocortex (layers V/VI), septum, hippocampus (particularly lateral perforant path to dentate gyrus), amygdala, mammillary body, and cerebellum (20,27). Although anatomic data are lacking that show a localization of mGlu8 in the spinal cord and on primary afferents, there is pharmacologic evidence for a presynaptic action of mGlu8 on the central terminals of primary afferents (28). mGlu6 is restrictedly expressed at the postsynaptic site of ON-bipolar cells in both rod and cone systems (3).

2. Signaling Group III mGluRs (like group II mGluRs) are negatively coupled to adenylyl cyclase through Gi proteins, thereby inhibiting (stimulated) cyclic AMP (cAMP) formation and cAMP-dependent protein kinase A (PKA) activation (2,4–6,29). However, there is evidence that not all effects of group III mGluR activation are mediated by a reduction of cAMP formation. An alternative signaling mechanism is the coupling of native group III mGluRs to the mitogenactivated protein kinase (MAP kinase) and phosphatidylinositol-3-kinase (PI3 kinase) pathways through a pertussis toxin (PTX)–sensitive Gi protein (30). Activation of group III mGluRs increased the phosphorylation and activity of the MAP kinases ERK1 and ERK2 and increased the phosphorylation of the PI3 kinase target protein kinase B (PKB/AKT) (30). Native mGlu6 is positively coupled via a G protein to a cGMP phosphodiesterase (PDE) in the retina (3). Activation of the PDE produces hydrolysis of cGMP, which leads to the closure of a cGMP–dependent cation channel and hyperpolarizes the cell. Activation of group III mGluRs can inhibit N-, L- and P/Q-type calcium channels, with the inhibition of N-type channels being most commonly observed (29,31). These effects may not involve a diffusible intracellular messenger. Group III mGluRs can activate potassium channels, including G protein–coupled inwardly rectifying potassium (GIRK) channels through mGlu4, 6, 7, and 8 (27,29). GIRK activation by group III mGluR agonists hyperpolarized zebra finch neurons and produced an inward potassium current in oocytes (in the presence of high extracellular potassium); hyperpolarization

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and potassium current were G protein dependent and not blocked by 1, 2-bis (2-aminophenoxy)ethane-N,N,N , N -tetraacetic acid (BAPTA) (27,29,32,33).

3. Pharmacology The prototypic agonist and key diagnostic molecule for group III mGluRs is L-AP4, which activates with low micromolar potency mGlu4, 6, and 8, whereas concentrations two to three orders of magnitude higher are required for mGlu7 (1–4,6,29). Other widely used agonists with actions on all or several group III mGlu subtypes are O-phospho-l-serine (L-SOP) and (R,S)-4phosphonophenylglycine [(RS)-PPG]. (RS)-PPG shows about a 25-fold selectivity for mGlu8 over mGlu4 or mGlu6 (34). In recent years, subtype-selective agonists have become available. (S)-3, 4-Dicarboxyphenylglycine [(S)-3,4-DCPG] activates mGlu8 with nanomolar potency (EC50 = 31 ± 2 nM) and has >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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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