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Progress in Neurobiology Vol. 54, pp.

581 to 618, 1998


# 1998 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
0301-0082/98/$19.00

PII: S0301-0082(97)00085-3

GLUTAMATE RECEPTORS IN THE MAMMALIAN


CENTRAL NERVOUS SYSTEM

SEIJI OZAWA*, HARUYUKI KAMIYA and KEISUKE TSUZUKI


Department of Physiology, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi,
Gunma, Japan 371

(Received 9 October 1997)

AbstractÐGlutamate receptors (GluRs) mediate most of the excitatory neurotransmission in the mamma-
lian central nervous system (CNS). In addition, they are involved in plastic changes in synaptic trans-
mission as well as excitotoxic neuronal cell death that occurs in a variety of acute and chronic
neurological disorders. The GluRs are divided into two distinct groups, ionotropic and metabotropic
receptors. The ionotropic receptors (iGluRs) are further subdivided into three groups: a-amino-3-
hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) receptor
channels. The metabotropic receptors (mGluRs) are coupled to GTP-binding proteins (G-proteins), and
regulate the production of intracellular messengers. The application of molecular cloning technology has
greatly advanced our understanding of the GluR system. To date, at least 14 cDNAs of subunit proteins
constituting iGluRs and 8 cDNAs of proteins coznstituting mGluRs have been cloned in the mammalian
CNS, and the molecular structure, distribution and developmental change in the CNS, functional and
pharmacological properties of each receptor subunit have been elucidated. Furthermore, the obtained
clones have provided valuable tools for conducting studies to clarify the physiological and pathophysiolo-
gical signi®cances of each subunit. For example, the generation of gene knockout mice has disclosed criti-
cal roles of some GluR subunits in brain functions. In this article, we review recent progress in the
research for GluRs with special emphasis on the molecular diversity of the GluR system and its impli-
cations for physiology and pathology of the CNS. # 1998 Elsevier Science Ltd

CONTENTS
1. Introduction 582
2. Ionotropic receptors 583
2.1. Molecular diversity 583
2.1.1. Classi®cation 583
2.1.2. Multiplicity of genes, and splicing and editing variants 583
2.1.3. Structure (membrane topology, etc.) 583
2.2. AMPA receptors 584
2.2.1. Distribution 585
2.2.2. Channel properties 586
2.2.2.1. Ion selectivity and recti®cation properties 586
2.2.2.2. Kinetics 587
2.2.2.3. Single-channel properties 587
2.2.2.4. Relation between functional and molecular properties of native receptors 588
2.2.3. Pharmacology 588
2.2.3.1. Agonist binding site 588
2.2.3.2. Competitive antagonists 590
2.2.3.3. Drugs that a€ect desensitization 590
2.2.3.4. Channel blockers 590
2.2.4. Physiology 591
2.2.4.1. Determinants of EPSC kinetics 591
2.2.4.2. Functional signi®cance of Ca2+ permeability 591
2.2.5. Pathophysiology 592
2.3. Kainate receptors 592
2.3.1. Distribution 593
2.3.2. Channel properties 593
2.3.2.1. Ion selectivity and recti®cation properties 593
2.3.2.2. Kinetics 594
2.3.2.3. Single-channel properties 594
2.3.3. Pharmacology 594
2.3.4. Physiology 594
2.3.5. Pathophysiology 595

* E-mail: ozawas@sb.gunma-u.ac.jp.

581
582 S. Ozawa et al.

2.4. NMDA receptors 596


2.4.1. Distribution 596
2.4.2. Channel properties 597
2.4.2.1. Ca2+ permeability 597
2.4.2.2. Voltage-dependent block by Mg2+ 597
2.4.2.3. Molecular determinants of ion permeation 598
2.4.2.4. Kinetics 599
2.4.2.5. Single-channel properties 599
2.4.2.6. Relation between functional and molecular properties of native receptors 600
2.4.3. Pharmacology 600
2.4.3.1. Glycine as a co-agonist 600
2.4.3.2. Binding sites for agonists and co-agonists 600
2.4.3.3. Drugs that a€ect NMDA receptor function 601
2.4.4. Physiology 601
2.4.4.1. Kinetics of EPSC 601
2.4.4.2. Targeted disruption of subunit gene 602
2.4.5. Pathophysiology 602
2.4.5.1. Neuronal cell death 602
2.4.5.2. Psychiatric disturbance 603
2.4.5.3. Neuropathic pain 603
3. Metabotropic receptors 603
3.1. Molecular diversity 604
3.1.1. Multiplicity of genes, and splicing variants 604
3.1.2. Structure (membrane topology, etc.) 604
3.1.3. Classi®cation 605
3.1.4. Pharmacology 605
3.1.5. Transduction mechanisms 605
3.2. Physiology 606
3.2.1. Regulation of neuronal excitability 606
3.2.2. Presynaptic inhibition 606
3.2.3. Synaptic plasticity 607
3.3. Pathophysiology 608
4. Concluding remarks 608
Acknowledgements 608
References 609

1. INTRODUCTION (NMDA) receptor channels. On the other hand, the


metabotropic receptors (mGluRs) are coupled to
Glutamate receptors (GluRs) mediate most of the
GTP-binding proteins (G-proteins) and modulate
excitatory neurotransmission in the mammalian cen-
tral nervous system (CNS). They also participate in the production of intracellular messengers.
plastic changes in the ecacy of synaptic trans- The application of molecular cloning technology
mission underlying memory and learning, and the has caused dramatic changes in the study of the
formation of neural networks during development GluR system. The ®rst iGluR was cloned in 1989
(see Mayer and Westbrook, 1987b; Dingledine et with the expression-cloning approach (Hollmann et
al., 1988; Monaghan et al., 1989 for reviews). al., 1989). The cloning of the ®rst mGluR was also
Ironically, glutamate and related excitatory amino accomplished using the same technique in 1991
acids are toxic to central neurons. Excessive acti- (Houamed et al., 1991; Masu et al., 1991). To date,
vation of GluRs during stress to the brain, such as at least 14 cDNAs of iGluRs and 8 cDNAs of
ischemia, head trauma and epileptic seizures leads mGluRs have been identi®ed in the mammalian
to the death of central neurons. The glutamate neu- CNS. In recent years, both physiology and pathol-
rotoxicity may also be involved in the geneses of ogy of GluR systems have been investigated exten-
various neurodegenerative diseases (see Rothman sively by using various techniques that manipulate
and Olney, 1987; Choi, 1988; Choi and Rothman,
expressions of GluR genes. The aim of this review is
1990; Meldrum and Garthwaite, 1990 for reviews).
to summarize recent ®ndings on GluRs, putting
Thus, the GluRs are intimately involved in both the
emphasis on describing the physiological and patho-
physiology and pathology of brain functions.
The GluRs are categorized into two distinct logical signi®cances of the molecular diversity of
classes, ionotropic and metabotropic receptors (see GluRs. We will ®rst describe recent ®ndings on
Nakanishi, 1992; Seeburg, 1993; Hollmann and iGluRs in terms of their molecular diversity, distri-
Heinemann, 1994 for reviews). The ionotropic recep- bution in the CNS, ion channel properties, pharma-
tors (iGluRs) contain cation-speci®c ion channels, cology, and physiological as well as
and are further subdivided into three groups: pathophysiological signi®cances. Then, we will
a-amino-3-hydroxy-5-methyl -4 - isoxazolepropionate describe the molecular diversity, physiology and
(AMPA), kainate and N-methyl-D-aspartate pathophysiology of mGluRs.
Glutamate Receptors in the CNS 583

2. IONOTROPIC RECEPTORS
2.1. Molecular Diversity
2.1.1. Classi®cation
Traditionally, iGluRs have been divided into
three major subtypes, AMPA, kainate and NMDA
receptors, on the basis of agonist speci®cities.
However, since neither agonist nor antagonist
clearly distinguished between AMPA and kainate
receptors, they were often collectively referred to as
non-NMDA receptors. Cloning studies have demon-
strated that they are distinct receptor complexes
although they can be activated by the same agonists,
notably AMPA receptors are activated by kainate
and kainate receptors with certain subunit compo-
sitions by AMPA. In recent years, several antagon-
ists that di€erentially block either AMPA or kainate
receptors have been developed.

2.1.2. Multiplicity of Genes, and Splicing and Editing


Variants
Molecular cloning and expression studies have
revealed that the diversity of iGluRs is much larger Fig. 1. Dendrogram of the members of the ionotropic glu-
than expected from electrophysiological and phar- tamate receptor family. Unrooted neighbor-joint tree of 14
macological studies. Expression cloning in Xenopus iGluR subunit proteins of the rat constructed by a clustal
oocyte has lead to the identi®cation of two iGluR w computer program (Thompson et al., 1994). The value,
subunits, an AMPA receptor subunit (GluR1) and 100% minus the sum of the length of the horizontal solid
the principal NMDA receptor subunit (NR1) lines between the two subunits, indicates % identity in the
amino-acid sequence between them. For example, the %
(Hollmann et al., 1989; Moriyoshi et al., 1991).
identity in the amino-acid sequence between GluR1 and
Additional cDNAs encoding iGluR subunits were GluR2 is obtained as follows. The distance of GluR1 and
cloned by homology cloning and by polymerase GluR2 to each nearest node is 15% and 14%, respectively
chain reaction (PCR)-based strategies. So far, 14 on the dendrogram, and the distance between these two
cDNAs, 4 for AMPA receptor subunits (GluR1, nodes is 2%, resulting in a total of
GluR2, GluR3 and GluR4), 5 for kainate receptor 15% + 14% + 2% = 31% distance between GluR1 and
subunits (GluR5, GluR6, GluR7, KA1 and KA2), GluR2. Therefore, the identity in the amino-acid sequence
and 5 for NMDA receptor subunits (NR1, NR2A, between them is (100% ÿ 31%) = 69%. The amino-acid
NR2B, NR2C and NR2D), have been isolated. identities among group 1 (GluR1±GluR7, KA1, KA2),
group 2 (NR1) and group 3 (NR2A±NR2D) are low, ap-
Phylogenetic trees of these 14 iGluR subunits are il-
proximately 20%, and they are combined with dashed lines
lustrated in Fig. 1. In addition, two cDNAs for d which are not used for estimating the distance. Data used
subunits (d1 and d2), of which the functions are pre- for constructing this dendrogram were obtained from
sently unknown, have been cloned (see Nakanishi, DNA Data Bank of Japan (DDBJ) with accession num-
1992; Seeburg, 1993; Hollmann and Heinemann, bers, X17184(GluR1), M85035(GluR2), M85036(GluR3),
1994 for reviews). In addition to multiplicity of M85037(GluR4), Z11713(GluR5), Z11548(GluR6),
genes, the molecular diversity of iGluRs is further M83552(GluR7), X59996(KA1), Z11581(KA2),
increased by variants due to alternative splicing and X63255(NR1), M91561(NR2A), M911562(NR2B),
RNA editing. M91563(NR2C), and D13213(NR2D).
In most cases, the iGluR subunits were cloned
independently and almost simultaneously in several indicated the intracellular location of the C-terminus
laboratories, and therefore given di€erent names to (Petralia and Wenthold, 1992; Tingley et al., 1993).
identical subunits. In this review, we use gene names In an attempt to determine the transmembrane top-
consistent with those introduced for the ®rst cloned ology model of the iGluR subunit, Hollmann et al.
representative of each subfamily. (1994) constructed a series of mutants of the AMPA
receptor subunit, GluR1, by introducing N-glycosy-
2.1.3. Structure (Membrane Topology, etc.) lation consensus sequences at di€erent sites along
iGluR subunits have in common a large extra- the entire length of the protein and analyzed these
cellular N-terminus domain and four hydrophobic mutant receptors for glycosylation, since glycosyla-
membrane segments (M1±M4). From analogy to tion at any given site can be taken as proof of the
other ligand-gated ion channels, such as the nic- extracellular localization of the respected site. Based
otinic acetylcholine (ACh) receptor and GABAA on these analyses, they have concluded that the
receptor, it was initially proposed that M1±M4 are receptor has only three transmembrane domains,
transmembrane segments (TMI±TMIV) and the C- which correspond to previously proposed TMI,
terminus is extracellular. However, this conventional TMIII and TMIV. According to this three-trans-
model has been revised in order to accomodate a membrane domain model, M2 previously assigned
variety of later ®ndings. For example, both im- for TMII does not span the membrane, but is con-
munocytochemical and biochemical studies have sidered to either lie in close proximity to the intra-
584 S. Ozawa et al.

cellular surface of the plasma membrane or make a A transmembrane topology of the AMPA recep-
hairpin turn within the membrane. Furthermore, the tor subunit, GluR2, constructed according to the
entire region between M3 and M4, previously new model proposed by Hollmann et al. (1994) is
believed to be intracellular, is extracellular, and the illustated in Fig. 2.
C-terminus is intracellular. Several lines of evidence
obtained using chimeric proteins and site-directed
2.2. AMPA Receptors
mutations support the notion that this new model is
applicable not only for AMPA receptor subunits, AMPA receptors mediate fast excitatory neuro-
but also for kainate and NMDA receptor subunits transmission in most of the synapses in the CNS.
(Kuryatov et al., 1994; Stern-Bach et al., 1994; These receptors were initially named quisqualate
Bennett and Dingledine, 1995; Wo and Oswald, receptors. However, they were renamed AMPA
1995; Hirai et al., 1996; Laube et al., 1997). receptors, since quisqualate was found to act on

Fig. 2. Structure of AMPA receptor subunit GluR2. The 862 amino acids of the mature GluR2 protein
shown in single-letter code are placed according to the three transmembrane domain topology model
proposed by Hollmann et al. (1994). The two RNA editing sites, glutamine(Q)-to-arginine(R) at position
586 and arginine(R)-to-glycine(G) at position 743, are indicated by ®lled squares. The box around
amino acids 744±781 indicates the region where alternative splicing variants, ¯ip and ¯op, occur. The
nine amino acids in the ¯ip version indicated by double arrows inside the box are changed to those out-
side of the box in the ¯op version.
Glutamate Receptors in the CNS 585

mGluR and AMPA acted more speci®cally on ined in more detail using in situ hybridization
AMPA receptors. histochemistry. Systematic surveys have revealed re-
Using the expression cloning technique, gional expression patterns of GluR1±GluR4 in
Hollmann et al. (1989) isolated the ®rst iGluR adult rat brains (KeinaÈnen et al., 1990; Sommer et
clone, GluR1, from a rat brain cDNA library. With al., 1990; Monyer et al., 1991). In the hippocampus,
sequence information of GluR1, the closely related the GluR1, GluR2 and GluR3 mRNAs are abun-
receptor genes, GluR2, GluR3 and GluR4, were dantly expressed in the pyramidal cell layer and den-
cloned by several groups. The four AMPA receptor tate gyrus with no apparent gradient of expression
subunits, GluR1±GluR4, are of similar size (0900 between CA1 and CA3. Expression of GluR4
amino acids), and share 68±73% amino acid mRNA is much less abundant than that of GluR1±
sequence identity (Boulter et al., 1990; KeinaÈnen et GluR3 mRNAs, and is relatively higher in CA1 and
al., 1990; see Seeburg, 1993; Hollmann and dentate gyrus than in CA3±CA4. In the cerebral
Heinemann, 1994; Bettler and Mulle, 1995; for cortex, the expression patterns of GluR1, GluR3
reviews). Each of the GluR1±GluR4 subunits exists and GluR4 mRNAs di€er among layers, while
in two di€erent forms, ``¯ip'' and ``¯op'', created by GluR2 mRNA is uniformly found in all layers. Only
alternative splicing of a 115-base pair (bp) region low levels of GluR1 and GluR3 occur in layers III
immediately preceding M4 (see Fig. 2, column and IV, whereas GluR4 expression appears to be
drawn with thick lines) (Sommer et al., 1990). RNA strong in this region. In the cerebellum, GluR1
editings further increase the diversity of receptor mRNA is expressed in Purkinje cells, but not in
subunits. A glutamine residue (Q; CGA) in M2 is granule cells. GluR2 mRNA is abundant in
encoded in the genes for GluR1±GluR4. However, Purkinje cells and granule cells. GluR3 mRNA is
GluR2 cDNA clones from adult animals contain an expressed in Purkinje cells, stellate-basket cells, with
arginine (R; CGG) at this position termed the Q/R no detectable expression in granule cells. GluR4
site (Fig. 2, a ®lled square in the M2 segment of mRNA is expressed at high level in granule cells.
GluR2; also see Fig. 4) (Sommer et al., 1991). This Both GluR1 and GluR4, but not GluR2 and
codon change due to the adenosine (A)-to-guanosine GluR3, are expressed in Bergmann glial cells
(G) alteration is generated by site-directed nuclear (KeinaÈnen et al., 1990).
RNA editing, and only low levels of unedited RNA Developmental and regional di€erences in ex-
are present in fetal brain (Burnashev et al., 1992b; pressions of two alternative splice variants, ¯ip and
Higuchi et al., 1993). RNA editing at the second site ¯op, have been studied using in situ hybridization
occurs in positions termed R(arginine; AGA)/ histochemistry (Sommer et al., 1990; Monyer et al.,
G(glycine; GGA) sites immediately preceding the 1991). AMPA receptor subunits are expressed pre-
alternative spliced modules, ``¯ip'' and ``¯op'' of dominantly in the ¯ip form in embryonic brains.
GluR2, GluR3 and GluR4 (Fig. 2, the other ®lled The ¯op forms are expressed at low levels prior to
square) (Lomeli et al., 1994). postnatal day 8, and gradually increase throughout
AMPA receptors are either homomeric or hetero- the brain, reaching adult levels by postnatal day 14.
meric oligomers composed of these multiple subu- Thus, excitatory neurotransmission in the adult
nits. Remarkable di€erences in functional properties brain appears to be mediated mainly by AMPA
of native AMPA receptors are consequences of receptors carrying the ¯op module. However, it has
di€erent assemblies of these subunits. been noted that certain neuronal populations, exem-
pli®ed by hippocampal CA3 pyramidal cells, express
only ¯ip modules even in the adult stage (Monyer et
2.2.1. Distribution al., 1991).
AMPA receptors are distributed ubiquitously The GluR2 RNA editing to replace the gene-
throughout the CNS, although regional di€erences encoded Q to R is developmentally regulated
in the distribution are conspicuous. The regional (Sommer et al., 1991). At the early developmental
di€erence in densities of AMPA-binding receptors stage (embryonic day 14), a small percentage of the
was previously examined with [3H]AMPA binding GluR2 does not undergo editing, and therefore the
studies (Monaghan et al., 1984; Olsen et al., 1987; unedited form coexists with the edited form. In post-
Insel et al., 1990). Brain regions that are rich in high natal stages, however, virtually all GluR2 exists in
anity [3H]AMPA binding include the hippo- the edited form (Sommer et al., 1991; Burnashev et
campus, which has slightly higher densities in CA1 al., 1992b). The editing at the R/G site in GluR2±
than CA3, and higher densities in the pyramidal cell GluR4 is also developmentally regulated. The extent
layer than stratum radiatum and stratum oriens. of R/G editing in the embryonic brain is generally
Levels of binding in the molecular layer of the den- small, but increases markedly during development
tate gyrus and the super®cial layer of the cerebral up to 55% to 0100% in the adult brain (Lomeli et
cortex are also very high. Intermediate levels of al., 1994).
binding are found in deeper layer cortex and in the A comprehensive study of AMPA receptor immu-
caudate-putamen. Lower levels are found in the nohistochemistry was performed on sections of rat
diencephalon, midbrain and brainstem. In the cer- brain, which were immunolabeled with antibodies
ebellum, binding levels are low as a whole, but more made against peptides corresponding to the C-term-
binding is found in the molecular layer than in the inal portions of GluR1, GluR2/3 and GluR4
granule layer (Monaghan et al., 1984; Olsen et al., (Petralia and Wenthold, 1992). Regional distribution
1987). patterns of AMPA receptor subunits in the brain
After the molecular identi®cation of receptor sub- were generally consistent with those revealed by in
units, distributions of AMPA receptors were exam- situ hybridization studies. The subcellular distri-
586 S. Ozawa et al.

butions of AMPA receptors were examined using with observations by Iino et al. (1990). They have
anti-GluR1, anti-GluR2/3 and anti-GluR4 in the indicated that the functional properties of recombi-
cerebral cortex and hippocampus of rat brain nant AMPA receptors depend on their subunit com-
(Petralia and Wenthold, 1992). Immunolabeling positions. Homomeric receptors assembled from
with these antibodies is detected in the cytoplasm GluR2 subunits display little permeability to Ca2+
and on the plasma membrane of the cell body and (PCa/Palkali-metal10.1) and an outward recti®cation.
dendrites. The labeling in the cytoplasm has a spotty In contrast, homomeric receptors assembled from
distribution with accumulation on the outer mem- either GluR1, GluR3 or GluR4 subunits are highly
brane of mitochondria and nucleus and on microtu- permeable to Ca2+ (PCa/Palkali-metal12) and display
bules. It is possible that at least a part of labelings a strong inward recti®cation. In heteromeric recep-
represents receptors being transported to and from tors, GluR2 is dominant in determining both Ca2+
the plasma membrane. The receptors on the plasma permeability and recti®cation properties.
membrane are localized predominantly at the post- Coexpression of GluR2 subunits with GluR1,
synaptic densities. These postsynaptic labelings seem GluR3 and/or GluR4 results in the formation of
to be con®ned to the intracellular side of the mem- recombinant receptors with little Ca2+ permeability
brane (Petralia and Wenthold, 1992). This obser- and an outward recti®cation (see Seeburg, 1993;
vation suggests an intracellular location for the Hollmann and Heinemann, 1994; Jonas and
antigenic site (C-terminal portion), being consistent Burnashev, 1995 for reviews).
with the three transmembrane topology model for The unique properties of GluR2 can be traced to
iGluR subunit proteins (Hollmann et al., 1994). a single amino acid residue in the M2 segment
Developmental changes in the subcellular localiz- (Hume et al., 1991; Mishina et al., 1991; Burnashev
ation of GluR1 and GluR2/3 were examined in cul- et al., 1992b). This residue is a positively charged
tured rat hippocampal neurons by arginine (R) in GluR2, whereas it is a glutamine (Q)
immunohistochemistry (Craig et al., 1993). GluR1 with a neutral charge in the other AMPA receptor
and GluR2/3 are relatively uniformly distributed in subunits. This site in M2 has been referred to as the
somata, axons and minor processes at the early Q/R site (see Seeburg, 1993 for review). When the
stage in cultured neurons obtained from embryonic arginine in this site in GluR2 is replaced with gluta-
day 18 rat. When the dendrites begin to develop, the mine using site-directed mutagenesis, the mutant
receptor subunits become polarized to the dendrites. GluR2(Q) shows properties similar to those of the
In matured pyramidal cells (obtained from embryo- wild-type GluR1, GluR3 and GluR4 subunits both
nic day 18 and 2±4 weeks in culture), clusters of in the Ca2+ permeability and recti®cation proper-
GluR1 and GluR2/3 are restricted to a subset of ties. The three-transmembrane domain model
postsynaptic sites in the dendritic spines. The target- suggests that the Q/R site is located near the intra-
ing of these receptor subunits appears to occur in cellular entrance of the AMPA receptor channel,
two stages that develop sequentially: ®rst, exclusion rather than near the extracellular entrance as
from the axon; second, enrichment at postsynaptic expected from the four-transmembrane domain
sites (Craig et al., 1993). model (Fig. 2). The positive charge of arginine in
this position hinders the permeation of divalent cat-
2.2.2. Channel Properties ions in AMPA receptors.
With regard to recti®cation properties, physiologi-
2.2.2.1. Ion selectivity and recti®cation properties cal concentrations of intracellular polyamines, such
AMPA receptor channels had been considered to as spermine and spermidine, mediate inward recti®-
be permeable only to Na+ and K+, and almost cation of Ca2+-permeable AMPA receptors with
impermeable to Ca2+ in central neurons. However, glutamine in the Q/R site (Bowie and Mayer, 1995;
it was found that AMPA receptors displayed a sub- Donevan and Rogawski, 1995; Isa et al., 1995;
stantial permeability to Ca2+ (PCa/Palkali-metal12.3 Kamboj et al., 1995; Koh et al., 1995). The dissipa-
according to the Goldman±Hodgkin±Katz (GHK) tion of these naturally occurring polyamines from
equation) and a strong inward recti®cation in a inside of the test cell or in the excised patch causes a
small population of cultured rat hippocampal neur- loss of the inward recti®cation without a€ecting
ons (type II neurons) (Iino et al., 1990; Ozawa and Ca2+ permeability in Ca2+-permeable AMPA
Iino, 1993). In contrast, AMPA receptors displayed receptors. It is likely that polyamines enter the chan-
a slight outward recti®cation and little permeability nel pores from the intracellular side at positive po-
to Ca2+ (PCa/Palkali-metal < 0.18) in most neurons tentials and block ion ¯ows when the Q/R site is
(type I neurons). The permeability sequence for the occupied by glutamine. Since they are polyvalent
AMPA receptor in type II neurons to ®ve divalent cations, the presence of a positively charged arginine
cations (Ca2+, Sr2+, Ba2+, Mg2+, and Mn2+) was in this site prevents them from approaching the
Ba2+ (1.3) > Ca2+ (1.0) > Sr2+ (0.9) > Mg2+ binding site.
(0.8) > Mn2+ (0.7). The corresponding sequence for As described in the initial part of Section 2.2, the
the NMDA receptor was Ba2+ (1.2) > Ca2+ arginine in the Q/R site is not encoded on the
(1.0) > Sr2+ (0.8) > Mn2+ (0.3)>>Mg2+ (<0.002). GluR2 genomic DNA, but is introduced by RNA
Thus, the type II AMPA receptor displays a much editing to replace the gene-encoded glutamine. In
weaker selectivity among divalent cations than the adult brains, almost all GluR2 are expressed in the
NMDA receptor (Iino et al., 1990). edited form. Thus, Ca2+ entry into central neurons
The results obtained from expression studies of through AMPA receptors is prevented by the RNA
GluR1±GluR4 using Xenopus oocytes or human editing. In embryonic brains, a small percentage of
embryonic kidney (HEK 293) cells are in accordance GluR2 mRNA does not undergo editing, and there-
Glutamate Receptors in the CNS 587

fore unedited Ca2+-permeable AMPA receptors is determined almost exclusively by desensitization


could exist during brain development. kinetics in this case, not depending on the duration
of agonist application (Mosbacher et al., 1994).
2.2.2.2. Kinetics
AMPA, glutamate and kainate are representative 2.2.2.3. Single-Channel properties
agonists of AMPA receptors. Except kainate, these Single-channel properties of the non-NMDA
agonists evoke rapidly and profoundly desensitizing receptors, both AMPA and kainate receptors, are
responses in AMPA receptors (Kiskin et al., 1986; less well characterized than those of the NMDA
Trussell et al., 1988; Tang et al., 1989). Kinetics of receptor due to a combination of three diculties.
desensitization of AMPA receptors were investi- (1) single-channel conductances of non-NMDA
gated in outside±out membrane patches including receptors are of smaller amplitude than those of
either native or recombinant receptors by rapid ap- NMDA receptors; (2) the open time of non-NMDA
plication of agonists via a piezo-driven solution channels is of briefer duration than that of NMDA
exchange device (Colquhoun et al., 1992; channels and (3) non-NMDA receptors adopt more
Mosbacher et al., 1994; Geiger et al., 1995). Time multiple conducting states than NMDA receptors.
constants of desensitization of current responses to The issue is further complicated by the fact that
1±10 mM glutamate ranged from 1 to 16 ms in out- both AMPA and kainate lack speci®city for activat-
side±out patches derived from a variety of central ing AMPA and kainate receptors, respectively.
neurons (Tang et al., 1989; Trussell and Fischbach, Previous experiments on a variety of central neur-
1989; Colquhoun et al., 1992; Hestrin, 1992; Trussell ons have indicated that non-NMDA receptors are
et al., 1993; Barbour et al., 1994; Geiger et al., 1995; linked to channels with a wide range of conduc-
HaÈusser and Roth, 1997). This large di€erence in tances; some channel events are too small to be
the desensitization kinetics seems to be due to the resolved as discrete channel openings (low conduc-
subunit composition of AMPA receptors contained tance responses), whereas others have clearly resol-
in each membrane patch. Mosbacher et al. (1994) vable multiple conductance levels (high conductance
measured desensitization of di€erent recombinant responses) (Cull-Candy and Usowicz, 1987, 1989a,b;
homo- and heteromeric AMPA receptors. Among Jahr and Stevens, 1987; Ascher and Nowak, 1988a;
homomeric GluR1, GluR3 and GluR4 receptors, Cull-Candy et al., 1988; Ozawa et al., 1991b; Wyllie
the GluR4 ¯op channel showed the fastest desensiti- et al., 1993). Low conductance responses are
zation time constant, 0.9 ms, and GluR3 ¯ip the detected as noise increases, and the single channel
slowest one, 4.8 ms when 1 mM glutamate was conductance values are estimated to be as low as
applied rapidly. In heteromeric receptors assembled 1 pS (much lower in some cases, 0140 fS) with the
with either the ¯ip or ¯op forms of GluR2 and use of noise analysis (Cull-Candy et al., 1988;
GluR4, GluR2 ¯op/GluR4 ¯op channel showed the Ozawa et al., 1991b; Wyllie et al., 1993).
fastest time constant, 0.8 ms, whereas GluR2 ¯ip/ The low conductance responses are activated
GluR4 ¯ip channel the slowest one, 6.1 ms. Except mainly by kainate. Wyllie et al. (1993) have shown
for GluR1 channel, the channels assembled with that AMPA produces discrete openings with two
¯op variants showed a faster desensitization time conductance levels of 6 and 10 pS in the outside±out
constant than those with ¯ip variants. It is thus patch where kainate produces only low conductance
likely that alternative splicing of AMPA receptor responses in rat cerebellar granule cells. A simple ex-
subunits regulates channel kinetics which a€ect the planation for this result is that the low conductance
shape of synaptic currents (Mosbacher et al., 1994). responses are due to activation of kainate receptors.
Desensitization kinetics of AMPA receptors are However, the possibility that a single GluR channel
also regulated by RNA editing at the R/G site. may open to di€erent conductance levels, depending
Receptors assembled with the edited forms showed on the agonist, cannot be ruled out. It is also poss-
a slower desensitization rate than those with the ible that although kainate activates 6/10 pS events,
unedited forms (Lomeli et al., 1994). their open times are too short to be detected as full
The rate of deactivation for both native and openings. In patches where kainate produces high
recombinant AMPA receptors was estimated by conductance responses with 010, 020 and 030 pS
measuring decay time constants of responses pro- openings, AMPA and glutamte also activate re-
duced by 1 ms pulses of 1 mM glutamate. These sponses with the same conductances (Wyllie et al.,
values ranged from 0.9 to 3.3 ms in native AMPA 1993). Since the relative proportion of these conduc-
receptors (Geiger et al., 1995). In general, there was tance levels is constant among di€erent patches for
a positive correlation between desensitization and all three agonists, it seems likely that three multiple
deactivation time constants in native AMPA recep- conductances originate from a single AMPA recep-
tors. For example, both desensitization and deacti- tor channel activated by AMPA, kainate and gluta-
vation time constants in hippocampal CA3 mate (Wyllie et al., 1993). Kinetic analysis of single
pyramidal cells (15.2 ms and 3.0 ms, respectively) channel events in high conductance responses has
are much slower than the corresponding values in indicated that the mean open time is as brief as
relay neurons of the medial nucleus of the trapezoid 0.5 ms for all agonists, and that few bursts of open-
body (MNTB, 1.7 ms and 0.9 ms, respectively) ings occur, in other words, most channel activations
(Geiger et al., 1995). However, in recombinant are composed of single openings (Wyllie et al.,
GluR4 ¯op receptors, the desensitization time con- 1993).
stant is as fast as the deactivation time constant It is reasonable to assume that the single-channel
(0.6±0.9 ms). Thus, the time course of current decay properties of AMPA receptors depend on the subu-
588 S. Ozawa et al.

nit composition. Swanson et al. (1997) have investi- cipal neurons and interneurons of the hippocampus
gated the e€ects of RNA editing at the Q/R site, and neocortex, auditory relay neurons, and
and splice variation of ¯ip and ¯op cassette on the Bergmann glial cells, in acute brain slices of rats
single-channel properties of recombinant AMPA (Jonas et al., 1994; Geiger et al., 1995; Lambolez et
receptors formed from GluR2 and GluR4 subunits. al., 1996). These studies have led to two conclusions.
Native AMPA receptors expressed in rat cerebellar (1) The relative abundance of GluR2 mRNA is the
granule cells are known to be composed of both ¯ip main determinant of the Ca2+ permeability of
and ¯op forms of GluR2 and GluR4, with the ¯op native AMPA receptors. The range of Ca2+ per-
isoform increasing with age (Monyer et al., 1991; meability (PCa/PNa) in native AMPA receptors var-
Mosbacher et al., 1994). In these recombinant ied from 0.07 to 2.8, and this value is inversely
AMPA receptors, the single-channel conductance is correlated with the relative abundance of GluR2
highest for the Ca2+-permeable heteromeric chan- (Geiger et al., 1995; Jonas and Burnashev, 1995). (2)
nels [GluR2(Q) (unedited form of GluR2) ¯op/ Deactivation and desensitization time constants
GluR4 ¯ip], lowest for the Ca2+-impermeable show a signi®cant positive correlation with the rela-
homomeric channels formed entirely of edited tive abundance of GluR2 and GluR2 ¯ip mRNA
GluR2 ¯ip or ¯op, and intermediate for the Ca2+- and a negative correlation with the relative abun-
impermeable heteromeric channels formed of both dance of GluR4 and GluR4 ¯op mRNA (Geiger et
edited and unedited subunits (GluR2 ¯ip/GluR4 ¯ip al., 1995).
and GluR2 ¯op/GluR4 ¯ip). For the ®rst group the
single-channel conductance levels activated by gluta- 2.2.3. Pharmacology
mate are 8, 15 and 24 pS, whereas they are <400 fS
The AMPA receptor has at least three separate
for the second group. For the third group, they are
binding sites at which agonists or antagonists can
4 and 10 pS, resembling the 6 and 10 pS levels of the
act: glutamate binding, desensitization and intra-ion
native low-conductance channels in rat cerebellar
channel binding sites. The glutamate binding site is
granule cells (Wyllie et al., 1993; Swanson et al.,
the site for competitive antagonists such as 2,3-dihy-
1997). These results indicate that the single-channel
droxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline
conductance is determined at least partly by the ex-
(NBQX) and 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,
pression of edited GluR2.
4H)-quinoxalinedione (YM90K). AMPA receptors
show a rapid desensitization. A number of sub-
2.2.2.4. Relation between functional and molecular stances such as cyclothiazide (CTZ) or aniracetam
properties of native receptors a€ect this desensitization mechanism. Another
group of substances such as joro spider toxin
To correlate the subunit expression of AMPA
(JSTX) and its analoges bind to the third site within
receptors with their functional properties in central
the ion channel and block ion ¯ows.
neurons, a method combining the whole-cell patch-
clamp recording with reverse-transcription (RT) fol-
lowed by PCR ampli®cation was adopted 2.2.3.1. Agonist binding site
(Lambolez et al., 1992; Bochet et al., 1994; Ozawa The protein domains in both AMPA and kainate
and Rossier, 1996). Experiments using this method receptors that determine the agonist binding speci-
(abbreviated as patch-clamp RT±PCR hereafter), ®city have been investigated by exchanging portions
the general procedure of which is outlined in of the AMPA receptor subunit GluR3 and the kai-
Fig. 3(A), revealed that GluR1 and GluR4 subunits nate receptor subunit GluR6 (Stern-Bach et al.,
were expressed, but no GluR2 was detected in type 1994). A total of 26 chimeric subunits were con-
II cultured hippocampal neurons expressing AMPA structed with reciprocal exchanges between GluR3
receptors with high Ca2+ permeability and strong and GluR6. They were analyzed by measuring ago-
inward recti®cation. On the other hand, the GluR2 nist-induced currents in Xenopus oocytes injected
was expressed at a high level together with GluR1 in with the speci®c cRNAs and the ability of
type I neurons [Fig. 3(B) and (C)]. Thus, a high [3H]kainate and [3H]AMPA to bind to membrane
Ca2+ permeability and a strong inward recti®cation fractions of transfected HeLa cells. These analyses
of native AMPA receptors expressed in type II neur- have led to a conclusion that two discontinuous seg-
ons can be explained by either a lack of or very little ments of 0150 amino acid residues each, the ®rst
expression of the GluR2 subunit. Furthermore, ¯ip± segment (S1) adjacent and N-terminal to M1 and
¯op analysis of GluR1 and GluR4 subunits in type the second segment (S2) located between M3 and
II neurons revealed that these subunits were M4, determine the agonist-binding properties of
expressed predominantly in the ¯op forms. In type AMPA and kainate receptors (Stern-Bach et al.,
II hippocampal neurons, rapid application of a high 1994). It has been suggested that S1 and S2 consti-
concentration (1 mM) of AMPA elicited current re- tute two lobes that together form an amino acid-
sponses with extremely fast desensitization kinetics binding pocket analogous to the two lobes of the
(Bochet et al., 1994). These results are in accordance bacterial lysine-arginine-ornithine-binding protein
with those obtained in recombinant receptorsÐthat (LAOBP) (Oh et al., 1993; O'Hara et al., 1993;
AMPA receptors assembled with the ¯op form show Stern-Bach et al., 1994). This model is supported by
a faster desensitization rate than those with the ¯ip the result of Uchino et al. (1992) that mutagenesis
form (Mosbacher et al., 1994). of a number of residues in S1 of GluR1 alters the re-
With the use of patch-clamp RT±PCR, the re- sponses to agonists. Furthermore, the model is com-
lation between functional and molecular properties patible with the three-transmembrane topology
of native AMPA receptors was investigated in prin- model of iGluR described in Section 2.1.3 in that
Glutamate Receptors in the CNS 589

Fig. 3. Molecular analysis of native AMPA receptors at the single cell level using patch-clamp reverse
transcription (RT)-polymerase chain reaction (PCR) method. (A) Experimental procedures of patch-
clamp RT±PCR method. Electrophysiological properties of AMPA receptor were ®rst analyzed in
whole-cell patch-clamp con®gurations. Then, the cell contents which include mRNAs of AMPA receptor
subunits were aspirated into the patch pipette. The harvested cell contents were expelled to a test tube
for reverse transcription (RT) reaction. The cDNA products were then subjected to PCR ampli®cation.
The cDNAs were ampli®ed 107ÿ1011 at the ®rst 40 PCR cycles. The second PCR was performed to
obtain enough ampli®ed product (>100 ng/ml) for restriction analysis. For the PCR, a common primer
pair for GluR1±GluR4, which ampli®es GluR1±GluR4 without changing the initial proportion, was
used. The sizes of the ®nal PCR products were approximately 750 bps. Four restriction endonucleases,
Bgl 1, Bsp 1286I, Eco 47III, and Eco RI that selectively digest GluR1, GluR2, GluR3, and GluR4 PCR
products, respectively, were chosen, and the restriction reaction was analyzed by agarose gel electrophor-
esis. Vertical bars in the left, lower column indicate the positions of restriction sites on the GluR1±
GluR4 fragments. For more details see Lambolez et al. (1992) and Bochet et al. (1994). (B), (C)
Recti®cation properties and subunit composition of AMPA receptors in cultured rat hippocampal neur-
ons. (B) Current-voltage (I±V) relations of responses of AMPA receptors in type I and type II neurons.
The I±V relation in the type I neuron displays a slight outward recti®cation, whereas that in the type II
neuron shows a strong inward recti®cation. Inset: Whole-cell currents evoked by ionophoretic appli-
cations of kainate at holding potentials of ÿ60, ÿ40, ÿ20, 0, +20, +40 and +60 mV in type I and type
II neurons. Kainate almost exclusively activated AMPA receptors in these experimental conditions. (C)
Restriction analysis of AMPA receptor subunits in four type I (left) and four type II (right) neurons.
Note the absence of digestion product with the enzyme speci®c for GluR2 fragment in the four type II
neurons and presence of these fragments in the four type I neurons. The Hae III digests of FX174 were
used as molecular weight markers (from Bochet et al., 1994).
590 S. Ozawa et al.

the agonist-binding sites, S1 and S2, should be aniracetam were further investigated by Partin et al.
located extracellularly. (1996). The kinetic analyses of responses of GluR1
AMPA receptors with various mutations at position
2.2.3.2. Competitive antagonists 750 have suggested that both modulators bind at or
near this critical site within the ¯ip/¯op domain, but
Quinoxalinediones such as 6-cyano-7-nitroqui-
that they act by distinct mechanisms: aniracetam
noxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxa-
slows desensitization by decreasing the closing rate
line-2,3-dione (DNQX) are potent competitive
constant for ion channel gating, whereas CTZ either
antagonists at non-NMDA receptors (Honore et al.,
abolishes or reduces desensitization by stabilizing a
1988). Both CNQX and DNQX potently displace
nondesensitized agonist-bound closed state (Partin
[3H]AMPA binding (IC50=00.3±0.5 mM), but they
et al., 1996).
also signi®cantly displace [3H]kainate (IC50=0 1.5±
Atypical 2,3-benzodiazepines such as 1-(4-amino-
2.0 mM) (Sheardown et al., 1990). NBQX is a more
phenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzo-
selective competitive antagonist for AMPA recep-
diazepine (GYKI 52466) and 1-(4-aminophenyl)-4-
tors, which has approximately 30-fold selectivity for
methyl-7,8-methylenedioxy-5H-(3N-methylcarba-
AMPA over kainate receptors (IC50= 0 0.15 vs
mate)-2,3-benzodiazepine (GYKI 53655) are highly
04.8 mM) (Sheardown et al., 1990). Recently,
selective noncompetitive antagonists of AMPA
another selective competitive antagonist of AMPA
receptors (Tarnawa et al., 1989; Donevan and
receptors, YM90K has been synthesized by displace-
Rogawski, 1993; Palmer and Lodge, 1993; Zorumski
ment of the cyano group of CNQX with a 1-imida-
et al., 1993). It has been suggested that they counter-
zolyl group (Ohmori et al., 1994).
act the e€ect of CTZ by increasing the rate of desen-
sitization of AMPA receptors by binding to the
2.2.3.3. Drugs that a€ect desensitization CTZ binding site or a neighbouring site (Palmer and
Two categories of drugs potentiate responses of Lodge, 1993; Zorumski et al., 1993; Desai et al.,
AMPA receptors by slowing the rate of desensitiza- 1995). However, a more recent study indicates that
tion. The ®rst are the pyrrolidinones aniracetam, their action sites are not identical to the CTZ site,
piracetam, and the related compound 1-(1,3-benzo- since the serine (S) to glutamine (Q) mutated GluR1
dioxol-5-ylcarbonyl)-piperidine (1-BCP) (Ito et al., ¯ip, which is not a€ected by CTZ, is similarly antag-
1990; Isaacson and Nicoll, 1991; Ozawa et al., onized by these drugs (Partin and Mayer, 1996).
1991a; Tang et al., 1991; Vyklicky et al., 1991;
Gouliaev and Senning, 1994; Staubli et al., 1994;
Desai et al., 1995). The second are the benzothiadia- 2.2.3.4. Channel blockers
zines CTZ, diazoxide, and 7-chloro-3-methyl-3-4- A variety of spider and wasp toxins such as
dihydro-2H-1,2,4 benzothiadiazine (IDRA21) JSTX, argiotoxin and philanthotoxin are known to
(Yamada and Rothman, 1992; Patneau et al., 1993; block glutamatergic synaptic transmission (Kawai et
Zivkovic et al., 1995). Very recently, a novel sulfo- al., 1982; Abe et al., 1983; Jackson and Usherwood,
nylamino compound, 4-[2-(phenyl-sulfonylami- 1988; Washburn and Dingledine, 1996). JSTX, the
no)ethylthio]-2,6-di¯uoro-phenoxyacetamide toxin from Nephila clavata, has a polyamine moiety
(PEPA), which is structurally distinct from pyrroli- connected to a phenyl ring, and carries 2±3 positive
dinone or benzothiadiazine compounds, has been charges at physiological pH (Aramaki et al., 1987).
found to potentiate responses of AMPA receptors It has been shown that JSTX speci®cally blocks
by either abolishing or markedly slowing the rate of inwardly rectifying and Ca2+-permeable AMPA
desensitization (Sekiguchi et al., 1997). Since most receptors lacking the edited GluR2 subunit
of these drugs positively modulate glutamatergic (Blaschke et al., 1993; Iino et al., 1996). At low con-
synaptic transmission, it is expected that they have centrations (<3mM), the toxin abolished almost
the potential for therapeutic use as memory- and completely responses of the Ca2+-permeable AMPA
cognition-enhancing drugs (Isaacson and Nicoll, receptors without a€ecting outwardly rectifying
1991; Ozawa et al., 1991a; Tang et al., 1991; Ca2+-impermeable AMPA receptors assembled with
Yamada and Tang, 1993). GluR2. This e€ect of toxin is use-dependent and
With regard to the action site of CTZ, Partin et highly voltage-dependent. At positive potentials, the
al. (1994) have found that the ¯ip and ¯op splice toxin exerted no blocking action. An analysis of vol-
variants of homomeric GluR1 AMPA receptors tage-dependency of the blocking e€ect has indicated
show strikingly di€erent sensitivity to CTZ; recep- that the site for the toxin binding is located near the
tors composed of ¯op forms are much less a€ected intracellular entrance of the channel pore. It is likely
by CTZ than those of ¯ip forms. They have further that the positive charge of the toxin is an important
shown that although the ¯ip and ¯op forms of factor for the blockade, and positively-charged argi-
GluR1 subunits have di€erences in amino acid nine at the Q/R site of the edited GluR2 shields the
sequence at three separate areas, the large di€erence binding site from the positively charged toxin (Iino
in the sensitivity to CTZ is fully explained by a et al., 1996). These results are consistent with the
single amino acid at position 750, namely, the di€er- three transmembrane topology model for AMPA
ence is reversed completely by exchange of serine- receptor channels which predicts that the Q/R site is
750 (¯ip) and glutamine-750 (¯op) (Partin et al., located near the intracellular entrance of the channel
1995). These results suggest that a part of the bind- pore rather than the extracellular entrance
ing site for CTZ includes residue 750 in GluR1. (Hollmann et al., 1994). Argiotoxin, philanthotoxin,
The roles of a single residue at position 750 of and 1-naphthyl acetyl spermine, a synthetic ana-
GluR1 in controlling sensitivity to CTZ, and also logue of JSTX, also selectively block the Ca2+-per-
Glutamate Receptors in the CNS 591

meable AMPA receptors in a highly voltage-depen- tivation and desensitization between patches taken
dent manner (Herlitze et al., 1993; Washburn and from these two types of neurons, it has been
Dingledine, 1996; Koike et al., 1997). suggested that the complex geometry of synapses
between parallel ®bers and spines of Purkinje cells
2.2.4. Physiology disturbs the di€usion of transmitter, thereby
prolonging EPSCs (Barbour et al., 1994). In such
2.2.4.1. Determinants of EPSC kinetics cases, the time course of the EPSCs are in¯uenced
At most central synapses, both AMPA and by desensitization kinetics.
NMDA receptors are activated during synaptic As described in Section 2.2.2.2, kinetics for desen-
transmission. AMPA receptors mediate fast neuro- sitization and deactivation of AMPA receptors
transmission. Their rapid kinetics are suitable for depend on which subunit gene is expressed. Thus,
this purpose. In contrast, neurotransmission the time course of fast EPSCs is ®nely regulated in
mediated by NMDA receptors occurs more slowly di€erent synapses by assembling di€erent subunits
and lasts for a much longer period, since their kin- into the receptors.
etics are much slower than those of AMPA recep-
tors.
The time course of EPSCs mediated by AMPA 2.2.4.2. Functional signi®cance of Ca2+ permeability
receptors depends on two factors, that is, the gluta- Native AMPA receptors are likely to be hetero-
mate concentration transient at the synapse and the mers composed of GluR1±GluR4 subunits. Since
properties of the postsynaptic receptors. The GluR2, which determines the Ca2+ permeability in
amount of glutamate released from the presynaptic recombinant AMPA receptors, is ubiquitously
terminal and the rate at which it is removed by dif- expressed in the CNS, native AMPA receptors in
fusion and/or uptake, determine the transmitter con- most central neurons exhibit low permeability to
centration in the synaptic cleft. The anity of the Ca2+. However, native AMPA receptors with high
receptors for glutamate and their deactivation and Ca2+ permeability have been reported in a variety
desensitization kinetics control the time course of of cells in the CNS. Among these cells, the Ca2+
synaptic currents produced by the transmitter avail- permeability is highest in type II cultured rat hippo-
able in the synaptic cleft. It seems likely that campal neurons and cerebellar Bergmann glia, fol-
removal of transmitter by di€usion occurs very lowed by hippocampal interneurons, MNTB relay
rapidly at central synapses, as is the case in the frog neurons and neocortical interneurons in rat brain
neuromuscular junction. An experimental estimation slices (Iino et al., 1990; Burnashev et al., 1992a;
has indicated that the peak glutamate concentration Jonas et al., 1994; Geiger et al., 1995; Isa et al.,
at central glutamtergic synapses reaches 1.1 mM and 1996; Itazawa et al., 1997). It has also been noted
decays with a time constant of 1.2 ms (Clements et that even in similar types of neuron, such as hippo-
al., 1992). This estimation is supported by the obser- campal and neocortical interneurons, the Ca2+ per-
vations that in outside±out patches of both hippo- meability varies from cell to cell. The patch-clamp
campal and visual cortical neurons a brief (1 ms) RT±PCR experiments indicate that the relative
application of 1 mM glutamate produces a response abundance of GluR2 expressed in each neuron
that mimics the time course of EPSCs (Colquhoun determines the degree of Ca2+ permeability (Bochet
et al., 1992; Hestrin, 1992). It seems likely that the et al., 1994; Jonas et al., 1994; Geiger et al., 1995).
sojourn of the transmitter in the synaptic cleft is Most brain neurons with the Ca2+-permeable
very brief in most synapses, and that deactivation AMPA receptors are GABAergic and express a
rather than desensitization plays a major role in Ca2+ binding protein, parvalbumin (Bochet et al.,
determining the decay rate of EPSCs since the time 1994; Jonas et al., 1994; Leranth et al., 1996; Kondo
constant for the decay of EPSCs is faster than that et al., 1997).
for desensitization in most cases tested. For The Ca2+-permeable AMPA receptors are
example, Hestrin (1992, 1993) has shown that the involved in the excitatory synaptic transmission in a
decay time constants are 02 ms for both deactiva- population of hippocampal and neocortical nonpyr-
tion and EPSCs, but 8 ms for desensitization in amidal, and spinal dorsal horn neurons (McBain
visual cortical neurons. At some synapses, however, and Dingledine, 1993; Gu et al., 1996; Isa et al.,
there is evidence that desensitization plays a role in 1996; Itazawa et al., 1997). It is expected that these
determining the time course of EPSCs, as described receptors provide a synaptically activated route for
below. First, CTZ, which markedly reduces desensi- Ca2+ entry, and play a role in modulating a long-
tization but has little e€ect on deactivation, pro- term synaptic function. This notion is supported by
longs some EPSCs (Yamada and Rothman, 1992; the observation that the amplitude of miniature
Yamada and Tang, 1993). A striking e€ect of this EPSCs is enhanced for a prolonged period after
drug has been reported at the chick calyceal Ca2+ entry through Ca2+-permeable AMPA recep-
synapses of the magnocellullaris (Trussell et al., tors in spinal dorsal horn neurons (Gu et al., 1996).
1993). It has also been reported that EPSCs evoked Very recently, mice lacking the GluR2 subunit
in two types of cerebellar neurons, Purkinje cells have been produced by gene targeting (Jia et al.,
and interneurons (stellate and basket cells), by 1996). Surprisingly, most of the GluR2-lacking mice
stimulation of parallel ®bers have markedly di€erent survived and the adult mutants appeared healthy
kinetics: the Purkinje cell EPSCs decay slowly and fully capable of caring for themselves. In these
(t =0 7.3 ms), whereas the interneuron EPSCs mice, the Ca2+ permeability of AMPA receptors in
have much faster kinetics (t =01.5 ms). Since hippocampal CA1 pyramidal cells increased 9-fold.
there are no di€erences in the kinetics of both deac- The long-term potentiation (LTP) in the CA1
592 S. Ozawa et al.

synapse was markedly enhanced and became non- Mechanisms for reduced ischemic damage by
saturable. Furthermore, the LTP could be induced AMPA receptor antagonists would be as follows.
in the presence of blockers of both NMDA recep- Firstly, blockade of the AMPA receptor prevents
tors and high-voltage activated Ca2+ channels (Jia membrane depolarization, which in turn attenuates
et al., 1996). These results suggest that Ca2+ entry the activations of the NMDA receptor and voltage-
through Ca2+-permeable AMPA receptors can par- activated Ca2+ channel, thereby reducing Ca2+
ticipate in the regulation of synaptic plasticity in cer- entry. Secondly, blockade of the AMPA receptor
tain conditions. directly reduces Ca2+ entry through the AMPA
receptor lacking GluR2. It should also be taken into
account that the AMPA receptor assembled with
2.2.5. Pathophysiology GluR2 is slightly permeable to Ca2+ (PCa/Palkali-
There is overwhelming evidence that the gluta- metal (0.1) (Iino et al., 1990; Jonas and Burnashev,
mate-induced excessive Ca2+ entry under pathologi- 1995). Prolonged periods of activation of generally
cal conditions leads to neuronal cell death (see Choi, poorly Ca2+-permeable AMPA receptors could pro-
1988; Choi and Rothman, 1990 for reviews). vide neurons with sustained load of Ca2+, which
Neurological disorders in which this mechanism would eventually cause severe cell injury. This mech-
may be involved include global and focal ischemia, anism may underly the vulnerability of cerebellar
hypoglycemia, physical trauma, drug abuse, meta- Purkinje cells (Brorson et al., 1995).
bolic poisoning, certain food toxicities, epilepsy, To elucidate functional signi®cances of the Q/R
AIDS-related dementia, Parkinson disease, motor site editing in the GluR2 subunit, Brusa et al. (1995)
neuron diseases (including amyotrophic lateral scler- generated heterozygous mice to harbor an editing-
osis), Huntington disease, and Alzheimer disease incompetent GluR2 allele by targeting the editing
(see Lee, 1996 for review). The glutamate-induced site complementary sequence (ECS) in intron 11. A
Ca2+ entry may occur through any of three routes: double-stranded RNA structure found in the pre-
NMDA receptor channels, Ca2+-permeable AMPA mRNA between the editing site in exon 11 and the
receptor channels and voltage-dependent Ca2+ ECS is indispensable for the nuclear process of the
channels. Initially, the NMDA receptor, which is Q/R site editing (Higuchi et al., 1993). In these
highly permeable to Ca2+, was thought to play mutant mice, the abundance of GluR2 mRNA was
major roles in causing neuronal cell death in various 70% of that in their wild-type littermates and 025%
pathological conditions. The excessive activation of of the transcripts were unedited at the Q/R site. The
the NMDA receptor can cause severe neuronal Ca2+ permeability of AMPA receptors in principal
injury, and speci®c antagonists of this receptor neurons such as hippocampal CA1 pyramidal cells,
powerfully protect against NMDA neurotoxicity neocortical pyramidal cells and cerebellar Purkinje
(Rothman and Olney, 1987). However, since potent cells in these mice was 05±7 times higher than in
AMPA receptor antagonists such as NBQX, that in the wild-type mice. These mutant mice devel-
YM90K and GYKI 52466 have become available, oped severe seizures and died by postnatal day 20
evidence has been accumulating that AMPA recep- (Brusa et al., 1995). This result contrasts with the
tor antagonists are e€ective in preventing neuronal report by Jia et al. (1996) that the GluR2-ablated
cell death caused by brain ischemia (Sheardown et mice showed no signs of seizure and adult mutants
al., 1990; Le-Peillet et al., 1992; Yatsugi et al., appeared healthy. Thus, the overexpression of une-
1996). This suggests that AMPA receptors play a dited GluR2 di€ers from the disruption of GluR2 in
role in the pathogenesis of ischemic neuronal cell its pathophysiological signi®cances, although both
death. manipulations increase the Ca2+ permeability of
Transient but severe global or forebrain ischemia AMPA receptors in principal neurons in the brain.
causes injury in speci®c populations of neurons, es-
pecially in the hippocampal CA1 pyramidal cells 2.3. Kainate Receptors
(Kirino, 1982). Speci®c blockers of the NMDA
receptor are protective in moderate but not severe Although kainate is a potent agonist of the
ischemia (Buchan and Pulsinelli, 1991), whereas AMPA receptor, it also activates a distinct class of
NBQX is e€ective in preventing delayed CA1 cell iGluRs, i.e. kainate-preferring receptors (kainate
death following severe ischemia (Sheardown et al., receptors). A family of kainate receptors has been
1990; Buchan et al., 1991). It has been reported that cloned by using low-stringency hybridization screen-
following severe transient forebrain ischemia, ing with AMPA receptor subunit probes, and ®ve
GluR2 gene expression is preferentially reduced in subunits, termed GluR5, GluR6, GluR7, KA1, and
CA1 hippocampal neurons at a time point that pre- KA2, have been identi®ed (see Seeburg, 1993;
ceded their degeneration (Pellegrini-Giampietro et Hollmann and Heinemann, 1994; Bettler and Mulle,
al., 1992, 1994). Since the Ca2+ permeability of the 1995 for reviews). GluR5±GluR7 may represent the
AMPA receptor is increased by a reduced expression low anity kainate-binding site with KD of
of GluR2, the result suggests that the increased 050 nM, whereas KA1±KA2 correspond to the
Ca2+ entry caused by a switch in AMPA receptor high anity kainate-binding site (KD=0 5 nM) in
subunit gene expression is a mechanism underlying the neuronal membranes revealed by earlier radioli-
delayed death of CA1 neurons. This notion is sup- gand binding studies. GluR5±GluR7 are of similar
ported by the ®nding that after ischemia the EPSCs size (0900 amino acids) and share 75±80% amino
in CA1 neurons in the hippocampus of gerbil are acid sequence identity with each other, and 040%
mediated by Ca2+-permeable AMPA receptors with AMPA receptor subunits GluR1±GluR4.
(Tsubokawa et al., 1995). KA1±KA2 are somewhat larger than GluR5±GluR7
Glutamate Receptors in the CNS 593

(0970 amino acids), and share 70% amino acid suggested their presence in dendrites (Siegel et al.,
sequence identity with each other, and 040% with 1995). Using somewhat more speci®c antibodies
either GluR1±GluR4 or GluR5±GluR7. As in the against GluR6±GluR7 and KA2, immunolabeling
case of GluR2, RNA editing occurs at the Q/R site was also detected in some postsynaptic densities
in the M2 segment of GluR5±GluR6, but not in (Petralia et al., 1994a). Presynaptic localization of
GluR7. In contrast to GluR2, the Q/R site editing is the receptors has been suggested by the staining
incomplete during development, and signi®cant with the antibody against GluR6±GluR7 in pre-
amounts of both edited and unedited versions coex- sumptive unmyelinated axons in the CA3 region,
ist in adult brain. Editing at two additional sites in possibly mossy ®bers. This is in agreement with the
M1, i.e. I(isoleucine)/V(valine) and Y(tyrosine)/ ®nding obtained by autoradiographic studies that
C(cysteine) sites, has also been found in the GluR6 high-anity kainate binding in stratum lucidum of
subunit. Alternative splicing of GluR5 further adds the CA3 region is markedly reduced following the
to receptor diversity (see Seeburg, 1993 for review). selective lesion of the dentate granule cells (Represa
Despite their widespread distribution throughout et al., 1987).
the CNS, physiological signi®cances of kainate
receptors largely remain unknown, since the nonde- 2.3.2. Channel Properties
sensitizing kainate-induced response at AMPA
Since kainate elicits large nondesensitizing current
receptors precludes detection of the smaller and
responses at AMPA receptors, the native kainate
rapidly desensitizing response of kainate receptors.
receptor-mediated response was dicult to detect in
Recently, however, the kainate receptor-mediated
isolation until very recently. Therefore, the channel
responses have been studied using novel pharmaco-
properties have mostly been examined in recombi-
logical tools which selectively interact with either
nant kainate receptors. In expression studies, kai-
AMPA or kainate receptors.
nate-evoked currents are observed in homomeric
receptors of GluR5 or GluR6 subunit (Egebjerg et
2.3.1. Distribution al., 1991; Sommer et al., 1992), while functional
channels are not formed by GluR7, KA1 or KA2
Radioligand binding studies using [3H]kainate
expression alone (Werner et al., 1991; Bettler et al.,
have revealed speci®c kainate binding sites with
1992; Herb et al., 1992; Lomeli et al., 1992).
both low (KD= 050 nM) and high (KD= 05 nM)
Homomeric GluR5 or GluR6 channels show rapid
anities. These binding sites are abundant through-
desensitization to the continuous application of kai-
out the entire CNS, although some brain areas such
nate. When KA2 subunit is combined with either
as the hippocampal CA3 region and the granular
GluR5 or GluR6, the time course of desensitization
layer of the cerebellum show intense labeling (Foster
and current-voltage (I±V) relation changes from
et al., 1981; Monaghan and Cotman, 1982; Represa
those of homomeric GluR5 or GluR6 channels
et al., 1987; Miller et al., 1990).
(Herb et al., 1992). Speci®cally, heteromeric chan-
More recently, kainate receptor subunits have
nels of KA2 and the unedited version of GluR5(Q)
been localized at the mRNA level using in situ hy-
at the Q/R site show more rapid desensitization and
bridization histochemistry (Wisden and Seeburg,
a less inwardly rectifying I±V relation than the
1993; Bahn et al., 1994), and at the protein level
homomeric GluR5(Q) channels. Furthermore,
using immunohistochemistry (Huntley et al., 1993;
GluR6/KA2 channels can be activated by AMPA,
Vickers et al., 1993; Roche and Huganir, 1995;
which does not activate homomeric GluR6 recep-
Siegel et al., 1995). Each subunit, which forms kai-
tors. Since in situ hybridization studies (Wisden and
nate receptors, is di€erentially distributed in the
Seeburg, 1993; Bahn et al., 1994) suggest that non-
CNS. The KA1 mRNA occurs mainly in the CA3
functioning subunits (GluR7 or KA1±KA2) co-loca-
region and dentate gyrus of the hippocampus. The
lize with functioning subunits (GluR5±GluR6) in
KA2 transcript is almost universally expressed. The
the same populations of neurons, the native kainate
GluR5 mRNA expression is found in the cingulate
receptors may be heteromers composed of di€erent
and piriform cortex, the subiculum, various septal
combinations and ratios of these subunits.
nuclei, and cerebellar Purkinje cells. The GluR6
mRNA is most abundant in cerebellar granule cells,
with lower levels in caudate-putamen and hippo- 2.3.2.1. Ion selectivity and recti®cation properties
campus. The GluR7 transcripts are present in the In contrast to AMPA receptors, homomeric
deep cerebral cortex, cingulate cortex, subiculum, GluR6 receptors show substantial Ca2+ per-
caudate-putamen, reticular thalamus, and stellate/ meability. RNA editing at the Q/R site in the M2
basket cells in the cerebellum. The combined ex- region plays a role in determining the Ca2+ per-
pression pattern of these ®ve subunits approximates meablity as well as the recti®cation properties
the autoradiographic pattern of [3H]kainate, (KoÈhler et al., 1993). Unlike AMPA receptors, both
suggesting that kainate binding sites represent the edited and unedited versions of GluR6 at the Q/R
distribution of kainate-preferring receptors. This sites expressed channels with relatively high Ca2+
idea is also supported by the ®ndings that kainate permeability. Although one group reported that the
and AMPA receptor subunits can coexist in the PCa/Palkali-metal value was greater in the edited form
same neurons (Mackler and Eberwine, 1993), but than in the unedited form (KoÈhler et al., 1993),
they do not seem to coassemble with each other another group reported the opposite (Egebjerg and
(Wenthold et al., 1994). Heinemann, 1993). As for the recti®cation proper-
Immunohistochemical studies using nonselective ties, the edited GluR5±GluR6 homomers (Egebjerg
monoclonal antibodies against GluR5±GluR7 et al., 1991) or heteromers coexpressed with KA2
594 S. Ozawa et al.

(Herb et al., 1992) form channels with either a linear subunits have a KD of 50±100 nM for kainate
or slightly outwardly rectifying I±V relation, (Bettler et al., 1992; Sommer et al., 1992), similar to
whereas the unedited GluR5±GluR6 homomers or the binding anity of the low anity kainate-bind-
the heteromers with KA2 showed a strong inward ing site with KD of 050 nM. The homomeric KA1
recti®cation (Herb et al., 1992; Sommer et al., 1992). shows a KD of 3.9 nM (Werner et al., 1991) and the
KA2 has a KD of 015 nM (Herb et al., 1992), simi-
2.3.2.2. Kinetics lar to those of the high anity kainate-binding site
One striking di€erence in channel properties in the brain (KD=0 5 nM). Therefore, it seems
between AMPA and kainate receptors exists in the likely that GluR5±GluR7 represent the low anity
desensitization pro®les of responses to continuous kainate binding site, whereas KA1±KA2 correspond
applications of kainate. Until recently, it has been to the high anity kainate-binding site in the neur-
believed that kainate produces nondesensitizing cur- onal membranes.
rents at AMPA receptors and rapid and complete Useful pharmacological tools to di€erentiate
desensitizing currents at kainate receptors, allowing between kainate and AMPA receptors are the drugs
a clear-cut functional distinction between these which a€ect desensitization pro®les of these recep-
receptors. However, studies using the fast drug ap- tors. Concanavalin A (ConA) is a lectin that selec-
plication device have revealed the presence of a tively removes desensitization of kainate receptor-
small, and rapidly desensitizing component in kai- mediated responses, but does not a€ect AMPA
nate-activated current at AMPA receptors (Patneau receptor-mediated responses (Partin et al., 1993;
et al., 1993). Furthermore, kainate receptor- Wong and Mayer, 1993). In contrast, CTZ has been
mediated currents pharmacologically isolated by shown to reduce rapid desensitization of AMPA
using a noncompetitive AMPA receptor antagonist receptors by kainate and consequently potentiates
GYKI 53655 contain substantial steady-state re- the responses of AMPA receptors to kainate with-
sponses (030% of peak) in cultured hippocampal out a€ecting kainate receptors (Patneau et al.,
neurons (Wilding and Huettner, 1997). Thus, the 1993).
desensitization pro®le does not necessarily give an Selective antagonists of kainate receptors which
absolute distinction between the AMPA and kainate do not block AMPA receptors may help to reveal
receptors. As a whole, however, it is still true that their functions in physiological and pathological
kainate-activated currents at the kainate receptor conditions. Unfortunately, the classical non-NMDA
are desensitized more markedly than those at the receptor antagonist CNQX (Honore et al., 1988)
AMPA receptor. a€ects both AMPA and kainate receptor-mediated
responses. A newly developed non-NMDA antagon-
2.3.2.3. Single-Channel properties ist, 5-nitro-6,7,8,9-tetrahydrobenzo[g]indole-2,3-
dione-3-oxime (NS-102), which selectively displaces
Single-channel properties of kainate receptors low anity [3H]kainate-binding (Johansen et al.,
were studied in the expression system using HEK 1993), reversibly suppresses kainate receptor-
293 cells transfected with kainate receptor subunits mediated responses with almost no e€ect on AMPA
GluR5, GluR6, and KA2 (Howe, 1996; Swanson et receptors in cultured hippocampal neurons (Lerma
al., 1996). It was concluded that the single-channel et al., 1993). NS-102 also blocks recombinant
conductance is signi®cantly reduced by Q/R site GluR6 responses in HEK 293 cells with minimal
editing. Homomeric unedited GluR6(Q) receptors e€ects on GluR2/4 receptors (Verdoorn et al., 1994).
exhibited directly resolvable single-channel conduc- Another compound 2S,4R-4-methylglutamate (SYM
tance of 8, 15, 25 pS and the noise analysis gave a 2081), a newly developed glutamate analogue, has
mean conductance of 5.4 pS, whereas the edited ver- recently been shown to desensitize selectively and
sion GluR6(R) showed an extremely low conduc- potently kainate receptor-mediated currents
tance of 225 fS (Swanson et al., 1996). A similar low (Wilding and Huettner, 1997; Zhou et al., 1997).
conductance value for the GluR6(R) receptor The AMPA-receptor selective noncompetitive an-
(0230±260 fS) was reported by another author tagonist GYKI 52466 (Donevan and Rogawski,
(Howe, 1996). GluR5(Q) exhibited resolvable con- 1993) and its more potent analogue GYKI 53655
ductance of 5, 9, 14 pS, while GluR5(R) had an (Paternain et al., 1995) have been used to isolate
extremely low (<200 fS) conductance (Swanson et native kainate receptor-mediated responses. The
al., 1996). It was also shown that the co-assembly noncompetitive manner of antagonism these drugs
with non-functioning subunit KA2 could a€ect the display suggests that the blocking action would not
single-channel conductance value as well as kinetic be a€ected by phasic changes in glutamate concen-
properties. A several fold increase in the single-chan- trations in the synaptic cleft during neurotrans-
nel conductance was shown for the heteromeric mission. Therefore, these compounds may be useful
GluR5(R)/KA2 and GluR6(R)/KA2 relative to to reveal functional roles of the kainate receptor in
homomeric GluR5(R) and GluR6(R), respectively synaptic transmission (see Lerma et al., 1997 for
(Howe, 1996; Swanson et al., 1996). The coexpres- review).
sion of KA2 with GluR5(Q) resulted in a signi®cant
shortening of burst length as compared to that of
homomeric GluR5(Q) (Swanson et al., 1996). 2.3.4. Physiology
Until recently, our knowledge on the functional
2.3.3. Pharmacology properties of the kainate receptor was severely lim-
Binding assays using recombinant kainate recep- ited, mainly because of the lack of speci®c pharma-
tor subunits have indicated that the GluR5±GluR7 cological tools discriminating kainate from AMPA
Glutamate Receptors in the CNS 595

receptors. The functional native kainate receptors transmission. It has been reported that autaptic re-
were ®rst characterized in rat dorsal root ganglion sponses in primary cultures as well as the population
(DRG) neurons (Huettner, 1990), where high levels synaptic responses at CA1 and dentate gyrus
of GluR5 as well as KA2 mRNA have been synapses in slice preparations are completely
observed (Bettler et al., 1990; Herb et al., 1992). blocked by the AMPA receptor-selective antagonist
Since there is a close correspondence in pharmaco- GYKI 53655 at its saturating concentration
logical and electrophysiological characteristics (100 mM), suggesting that fast synaptic transmission
between recombinant GluR5 receptors and native does not involve the kainate receptor activation (see
kainate receptors found in DRG neurons, GluR5 Lerma et al., 1997 for review). However, recent stu-
may be a major component of the native receptors dies have demonstrated that high-frequency stimu-
of these neurons. Except for the case of DRG neur- lation of the hippocampal mossy ®bres generates
ons, the characterization of kainate receptor- slow excitatory postsynaptic currents mediated by
mediated currents, despite their widespread distri- kainate receptors in hippocampal CA3 neurons
bution, has been hampered by the larger AMPA which express many of the kainate receptor subunits
receptor-mediated currents. The diculty in record- (Castillo et al., 1997; Vignes and Collingridge,
ing kainate receptor-mediated currents could be also 1997).
due to their desensitizing properties as well as their In addition to postsynaptic contributions, several
possible localization on distal dendrites or axon lines of evidence suggest presynaptic roles for kai-
terminals (Represa et al., 1987; Huntley et al., 1993; nate receptors. Earlier studies by Represa et al.
Petralia et al., 1994a). Although apical dendrites of (1987) suggested that kainate binding sites are
hippocampal pyramidal cells represent strong immu- highly localized on hippocampal mossy ®ber term-
nolabeling of kainate receptor subunits (Good et al., inals in the CA3 region. Since selective destruction
1993; Petralia et al., 1994a), only AMPA receptor- of the mossy ®bers reduces the convulsive e€ect of
mediated responses were detected in outside±out kainate on CA3 neurons (Debonnel et al., 1989;
patches derived from the dendrites of these neurons Gaiarsa et al., 1994), it has been proposed that kai-
in slice preparations (Jonas and Sakmann, 1992; nate acts at presynaptic kainate receptors on mossy
Spruston et al., 1995). ®ber terminals and generates epileptiform activity.
In the presence of GYKI 52466, that selectively This idea is supported by the ®nding that unmyeli-
blocks AMPA receptor-mediated responses, Lerma nated presumptive mossy ®bers are labeled by the
and colleagues have detected the functional kainate GluR6±GluR7 antibody (Petralia et al., 1994a).
receptor-mediated responses in cultured embryonic With regard to functional roles of presynaptic kai-
hippocampal neurons (Lerma et al., 1993; Paternain nate receptors, Chittajallu et al. (1996) have recently
et al., 1995). In the majority of cells, the electro- shown that kainate suppresses KCl-stimulated
physiological properties were similar to those of [3H]glutamate release from hippocampal synapto-
homomeric GluR6(Q) receptors. The kainate recep- somes and also suppresses the NMDA receptor-
tor-mediated current rapidly and almost completely mediated synaptic responses measured in the CA1
desensitized in response to continuous application of region of the slice preparations in the presence of
kainate, and the I±V relation displayed a strong AMPA receptor antagonist GYKI 52466. Based on
inward recti®cation. A more recent study using the these observations, it was suggested that kainate
patch-clamp RT±PCR has shown that most of the receptors on the presynaptic terminals at hippocam-
cultured neurons express GluR6 mRNA and a few pal CA1 synapses negatively regulate the synaptic
cells express GluR5, but GluR7 and KA1±KA2 sub- release of glutamate. On the other hand, several
units are not detected (Ruano et al., 1995). di€erent groups have reported that kainate enhances
Furthermore, analysis of the editing state of the Q/ transmitter release from synaptosomes prepared
R site of the GluR6 subunit has revealed that the from the hippocampal CA3 region (Gannon and
unedited variant is predominantly expressed, Terrian, 1991; Malva et al., 1995, 1996). Therefore,
although in some cases both unedited and edited modulating actions of presynaptic kainate receptors
variants coexist in the same cell. In addition, this may vary from synapse to synapse (i.e. suppression
study has revealed that the recti®cation properties of vs potentiation on CA1 and CA3 synapses, respect-
kainate responses are directly correlated with the ively). Presynaptic localization of the functional kai-
relative abundance of edited vs unedited versions of nate receptors is also described in C-®bers of the
GluR6 in a single neuron (Ruano et al., 1995). primary a€erents (Agrawal and Evans, 1986).
In contrast to the above results, native kainate
receptor-mediated currents isolated by using GYKI
53655 in cultured hippocampal neurons from 2- to
5-day-old postnatal rats show incomplete desensiti- 2.3.5. Pathophysiology
zation to kainate (leaving 030% steady-state cur- Kainate has a potent convulsive action when
rents of the peak currents). Furthermore, the I±V applied in vivo. Intracerebral or parenteral adminis-
relation of kainate receptors from neonatal neurons tration of kainate in rats results in limbic seizures
has been shown to be almost linear (Wilding and and a pattern of brain damage which resembles
Huettner, 1997). The reason for these di€erences those of human temporal lobe epilepsy patients (see
between embryonic and neonatal origins of the cul- Nadler, 1981; Ben-Ari, 1985 for reviews). Domoate
tured neurons are currently unknown. intoxication following ingestion of contaminated
The presence of the functional kainate receptors mussels has been reported to cause a clinical syn-
in hippocampal neurons suggests the involvement of drome which includes seizures as well as anterograde
the native kainate receptor in the fast glutamatergic memory de®cits (Teitelbaum et al., 1990). Since
596 S. Ozawa et al.

some neuronal populations, i.e. CA3 hippocampal form functional NMDA receptor channels by them-
pyramidal cells or thalamic reticular neurons, are selves, when one of them is coexpressed with NR1,
highly sensitive to kainate-induced damage (Nadler current responses of the heteromeric receptors
et al., 1978) and their distribution approximates increase by several orders. Since recombinant het-
those of kainate binding sites, involvement of kai- eromeric NMDA receptors display di€erent proper-
nate receptors in the epileptic seizure has been pro- ties depending on which of the four NR2 subunits
posed. Actually, several non-NMDA antagonists are assembled with NR1, the NR2 subunits can be
protect against certain forms of epileptic seizures regarded as modulatory subunits, whereas NR1
(Chapman et al., 1991). Since selective lesion of hip- serves as a fundamental subunit to form heteromeric
pocampal mossy ®bers reduces not only the convul- NMDA receptors (see Nakanishi, 1992; Seeburg,
sive e€ect of kainate on CA3 neurons (Debonnel et 1993; Mori and Mishina, 1995 for reviews).
al., 1989; Gaiarsa et al., 1994), but also the high a- NR1 and NR2A±NR2D subunits are composed
nity kainate binding sites localized presumably at of 938 (main isoform among splice variants, see
presynaptic mossy ®ber terminals (Represa et al., below), 1464, 1482, 1250 and 1323 amino acids, re-
1987), it has been proposed that the kainate-induced spectively. NR1 shows low but signi®cant homology
convulsion is due to the presynaptic action on (25±28% amino acid sequence identity) with other
mossy ®ber terminals which leads to a massive glu- iGluR subunits and shares a similar hydrophobicity
tamate release (Gaiarsa et al., 1994). The obser- pro®le to them. Amino acid sequence identities
vation that kainate enhances glutamate release from between the NR1 and NR2 subfamilies are as low as
synaptosomes prepared from the hippocampal CA3 018%, and those among the NR2 families are as
region (Gannon and Terrian, 1991; Malva et al., high as 040% to 050% (see Nakanishi, 1992;
1995, 1996) is consistent with this notion. In ad- Hollmann and Heinemann, 1994; Mori and
dition, a loss of CA3 and hilar neurons was induced Mishina, 1995 for reviews). In spite of a substantial
by over-expression of GluR6 using a viral vector in divergence in the amino acid sequences of NR2 sub-
organotypic slice cultures (Bergold et al., 1993). units from NR1, they possess structural character-
Interestingly, kindling, an experimental model of istics similar to those of NR1 as will be elaborated
epilepsy, causes sprouting of aberrant mossy ®bers in Sections 2.4.2 and 2.4.3.
in hippocampal CA3 region (see Ben-Ari and For the NR1 subunit, the existence of eight
Represa, 1990 for review). The involvement of kai- alternative splice variants, that is, the main isoform,
nate receptors in such a morphological plasticity has four variants with N-terminal insertion and three
to be tested in future studies using more speci®c variants with C-terminal deletion, have been
pharmacological tools as well as gene targeting tech- reported. The relative abundance of these variants
nology. are 067, 015 and 018%, respectively (Durand et
al., 1993; Sugihara et al., 1992; Yamazaki et al.,
2.4. NMDA Receptors 1992; Hollmann et al., 1993; see Mori and Mishina,
1995 for review).
NMDA receptors mediate excitatory neurotrans-
mission in the CNS in di€erent ways from AMPA
receptors. They are characterized by voltage-depen- 2.4.1. Distribution
dent block by Mg2+ (Mayer et al., 1984; Nowak et To determine NMDA receptor distribution,
al., 1984), a high permeability to Ca2+ ligand binding studies were conducted by using a
(MacDermott et al., 1986; Mayer and Westbrook, number of di€erent radioligands, which speci®cally
1987a), and slow gating kinetics (Lester et al., 1990). bind to NMDA receptors such as [3H]glutamate,
These unique properties have attracted the interest [3H]1-(1-(2-thienyl)-cyclohexyl)piperidine ([3H]TCP),
of neuroscientists, since they provide the NMDA [3H]3 -((2)- 2 -carboxypiperazin -4-yl)-propyl-1-phos
receptor with a molecular basis for synaptic plas- phonic acid ([3H]CPP) and [3H]5-methyl-10,11-dihy-
ticity. dro-5H-dibenzo[a,d]cyclohepten-5,10-imine
Using the expression cloning technique, ([3H]MK-801) (Monaghan et al., 1983; Maragos et
Moriyoshi et al. (1991) have cloned the fundamental al., 1988; Subramaniam and McGonigle, 1991; see
NMDA receptor subunit, NMDA1R (NR1), from a Monaghan et al., 1989 for review). Results of these
rat brain cDNA library. Upon expression in studies have shown that NMDA receptors are found
Xenopus oocytes, this subunit was capable of form- throughout the brain but predominantly within the
ing homomeric receptor channels that displayed forebrain. The highest levels in the entire brain are
characteristic NMDA properties. However, the found in the CA1 region of the hippocampus.
amplitude of current responses obtained with the Furthermore, quantitative comparisons of the distri-
homomeric NR1 receptors in oocytes was much bution of NMDA receptors determined by binding
smaller than that obtained with brain mRNA, sites of the di€erent ligands have suggested the pre-
suggesting the existence of additional subunits to sence of multiple pharmacologically distinct types of
form heteromeric receptors. Additional four recep- NMDA receptors (see Monaghan et al., 1989 for
tor subunits, NMDAR2A (NR2A)-NMDAR2D review).
(NR2D) (also termed e1±e4 by Mishina and col- Following the molecular cloning of NMDA recep-
leagues), were cloned in the laboratories of Seeburg, tor subunits, the distribution of each subunit was
Mishina and Nakanishi by PCR and cross hybridiz- examined using in situ hybridization histochemistry
ation techniques (Ikeda et al., 1992; Kutsuwada et (Moriyoshi et al., 1991; Kutsuwada et al., 1992;
al., 1992; Meguro et al., 1992; Monyer et al., 1992; Monyer et al., 1992, 1994; Watanabe et al., 1992;
Ishii et al., 1993). Although NR2 subunits do not see review Mori and Mishina, 1995). In the adult
Glutamate Receptors in the CNS 597

rodent, the NR1 mRNA is distributed ubiquitously According to this equation, the ratio of the per-
throughout the brain. In contrast, the four NR2 meability coecients of Ca2+ and alkali-metal (Cs+
transcripts display distinct regional patterns. The or Na+), PCa/Palkali-metal was estimated to be 4.0
NR2A mRNA is distributed widely in the brain, but (Mayer and Westbrook, 1987a) or 6.2 (Iino et al.,
more so in the cerebral cortex, hippocampus, and 1990) in cultured central neurons when ionic con-
cerebellum. The NR2B transcript is selectively pre- centrations were used for the calculation. If ionic ac-
sent in the forebrain with high level expression in tivities were used instead of concentrations, the
the cerebral cortex, hippocampus, septum, caudate- value became 10.6 or 14.3. To predict the actual
putamen and olfactory bulb. The NR2C mRNA is Ca2+ in¯ow through the NMDA channel in physio-
expressed predominantly in the granule cell layer of logical conditions using this information, however,
the cerebellum, with weak expression in the olfac- the following two points should be kept in mind.
tory bulb and thalamus. Low levels of the NR2D Firstly, the GHK equation is based on the assump-
transcript are detected in the thalamus, brain stem tion that there is no interaction among ions per-
and olfactory bulb. The NR2C and NR2D tran- meating through the channel. This is obviously not
scripts are found in a subset of hippocampal neur- the case for the NMDA channel. The single-channel
ons, most likely interneurons (Monyer et al., 1994). conductance of the NMDA channel decreases as the
Expression patterns of the NR2 subunits are also Ca2+ concentration in external saline is increased,
regulated developmentally in rodent brains despite that the Ca2+ permeability of the NMDA
(Watanabe et al., 1992; Monyer et al., 1994). NR2B channel is much higher than that to the alkali-metal
and NR2D mRNAs occur prenatally, whereas cations (Ascher and Nowak, 1988b; Gibb and
NR2A and NR2C mRNAs are ®rst detected around Colquhoun, 1992; Jahr and Stevens, 1993; Tsuzuki
birth. The most prominent change is the switch et al., 1994; Premkumar and Auerbach, 1996; Iino
from NR2B to NR2C expression which occurs in et al., 1997). It is likely that permeation through the
the cerebellar granule cells. NR2B mRNAs, which NMDA channel involves binding of permeant cat-
are abundantly expressed in the cerebellar granule ions, and Ca2+ binds to the site within the channel
cells on embryonic day 7, almost disappear, and are more tightly and stays there longer than the alkali-
replaced by NR2C mRNAs. Since the functional metal cation, therefore reducing the amplitude of
properties of NMDA receptor channels such as the the single-channel current. This inhibitory e€ect of
degree of voltage-dependent Mg2+ block and deacti- Ca2+ on the single-channel conductance can be well
vation kinetics depend on which of the four NR2 is explained by assuming a model for one-ion channel
assembled, it is conceivable that di€erent spatial and based on the Eyring±LaÈuger theory (LaÈuger, 1973;
temporal patterns of expressions of the NR2 genes Iino et al., 1997). Secondly, the relative Ca2+ per-
are designed for ®ne tuning of NMDA receptor meability of the NMDA channel is usually measured
functions in both embryonic and adult brains. in external solutions containing unphysiologically
An immunohistochemical study of the distri- high concentrations of Ca2+ (>10 mM). To esti-
bution of the NR1 subunit was performed on sec- mate the proportion of whole-cell current carried by
tions of adult rat brain, which were immunolabeled Ca2+ (fractional Ca2+ current) through nonspeci®c
with a selective antibody recognizing 30 C-terminal cationic channels, Neher and colleagues have devel-
amino acid residues (Petralia et al., 1994b). The re- oped a technique combining whole-cell patch-clamp
gional pattern of the expression obtained by this recording with ¯uorescence measurement using fura-
study agrees with the results of in situ hybridization 2 (for review see Neher, 1995). With the use of this
studies in that most neurons throughout the brain technique, Burnashev et al. (1995) measured frac-
express NR1 transcripts. In this study, the ultra- tional Ca2+ current through recombinant NMDA
structural localization of immunostaining was also channels expressed in HEK 293 cells. When the
examined in the hippocampus, cerebral cortex, and HEK cells were bathed in physiological external sol-
cerebellar cortex. Major stainings were localized in ution containing 135 mM Na+, 5.4 mM K+ and
postsynaptic densities apposed by unstained presyn- 1.8 mM Ca2+, fractional Ca2+ currents through
aptic terminals with mainly round vesicles, and in NR1/NR2A and NR1/NR2C NMDARs were 11
associated dendrites. and 8.2%, respectively. The corresponding values
predicted by the relative Ca2+ permeabilities
2.4.2. Channel Properties obtained according to the GHK equation were 14
2+ and 10%.
2.4.2.1. Ca permeability
The NMDA receptor channel has characteristic
ion permeation properties. The alkali-metal cations, 2.4.2.2. Voltage-Dependent block by Mg2+
Na+, K+ and Cs+ ions, permeate through the In external medium containing physiological con-
channel with a low selectivity, but major di€erences centrations of Mg2+ (01 mM), the NMDA recep-
from non-NMDA receptor channels exist in the per- tor-mediated current is maximal between ÿ20 and
meation properties for Ca2+ and Mg2+. Ca2+ is ÿ30 mV, and is reduced at more hyperpolarized po-
highly permeant, whereas Mg2+ is a potent blocker tentials despite the increased electrical driving force.
of the NMDA channel (Mayer et al., 1984; Nowak The inward current is negligible at ÿ80 mV, and the
et al., 1984; MacDermott et al., 1986; Mayer and I±V relation of the NMDA response thus exhibits a
Westbrook, 1987a; Ascher and Nowak, 1988b). clear negative slope conductance between ÿ30 and
The relative Ca2+ to the alkali-metal cation per- ÿ80 mV. The negative slope conductance is elimi-
meability in the NMDA receptor has been most nated by removing Mg2+ from the external solution
commonly estimated by using the GHK equation. (Mayer et al., 1984; Nowak et al., 1984).
598 S. Ozawa et al.

Single-channel studies have shown that the


NMDA receptor has a conductance of 40±50 pS in
salines containing no Mg2+ (Nowak et al., 1984;
Ascher and Nowak, 1988b). In the presence of
Mg2+, the single-channel current occurs in bursts of
short-lasting openings separated by brief closures,
which re¯ect a fast transition between blocking and
unblocking of the channel by Mg2+ (Nowak et al.,
1984; Ascher and Nowak, 1988b). The block by
Mg2+ may be explained by assuming that the pore
of the channel has a wide mouth located near the
extracellular space in which hydrated cations enter
easily, and a narrow constriction located deep in the
membrane through which only dehydrated Mg2+
ions can pass. The apparent electrical distance of
the site of Mg2+ block from the outside of the mem-
brane was calculated to be between 0.64 and 1.0
(Ascher and Nowak, 1988b; Jahr and Stevens, 1990; Fig. 4. Amino acid sequences of the M2 segments of
Premkumar and Auerbach, 1996). Since the speed of AMPA and NMDA receptor subunits. Amino-acid
the replacement of water molecules immediately sur- sequences of the M2 segments of AMPA receptor subunits
rounding the ion is much slower for Mg2+ than for (GluR1±GluR4) and NMDA receptor subunits (NR1 and
NR2A±NR2D). Boxes indicate the Q(glutamine)/
other physiological ions (Na+, K+ and Ca2+), the
R(arginine) site of the AMPA receptor subunits and the
permeation of Mg2+ through the channel would be N(asparagine) site of the NMDA receptor subunits. The
hindered more markedly than that of the permeant amino-acid residues in these sites determine permeations of
ions. This notion is supported by the fact that Ni2+ divalent cations both in AMPA and NMDA receptor
and Co2+, around which water molecules are channels. N + 1 site, one position downstream to the C-
replaced as slowly as Mg2+, mimic the e€ect of terminus from the N-site, is occupied by asparagine in
Mg2+, but not Cd2+, Sr2+ and Ba2+ around which NR2A±NR2D, and also contributes to the formation of
the rate of water exchange is 1000 times faster than ion selectivity ®lter of the NMDA channel. For more
Mg2+ (Diebler et al., 1969; Mayer and Westbrook, detail, see Wollmuth et al. (1996).
1985; Ascher and Nowak, 1988b; see Mayer and
Westbrook, 1987b for review). More membrane units guarantees the hallmark of the NMDA recep-
hyperpolarization would increase the probability tor that the channel is highly permeable to Ca2+
that Mg2+ occupies the entrance of the constriction with no permeability to Mg2+. As described in
region, thereby increasing the degree of the Mg2+ Section 2.2.2.1, the selectivity of the Ca2+-per-
block. meable AMPA receptors for divalent cations is very
low, and the receptors display a substantial per-
meability to Mg2+. In the mutants of AMPA recep-
2.4.2.3. Molecular determinants of ion permeation tors containing an asparagine in the Q/R site, the
Inspection of the primary structures of the relative permeability ratio between Ca2+ and Mg2+,
NMDA receptor subunits, NR1 and NR2A±NR2D, PCa/PMg, becomes higher than in those containing
has revealed that the positions corresponding to the glutamine (Burnashev et al., 1992b).
Q/R site in the M2 segment of the AMPA receptor Although the amino-acid residues in the N site
subunits are occupied by asparagine (N) in the play crucial roles in determining the Mg2+ block in
NMDA receptor subunits (Fig. 4) (see Nakanishi, the NMDA channel, they are not the sole factor
1992; Seeburg, 1993; Hollmann and Heinemann, determining the degree of the Mg2+ block. This
1994; Mori and Mishina, 1995 for reviews). This N property di€ers depending on the particular NR2
site governs both Ca2+ permeability and Mg2+ subunit coexpressed with NR1, that is, the NR1±
block in the NMDA channel (Burnashev et al., NR2A and NR1±NR2B channels are more sensitive
1992c; Mori et al., 1992; Sakurada et al., 1993). to Mg2+ than the NR1±NR2C and NR1±NR2D
Replacing the asparagine in this site of the NR1 channels (Kutsuwada et al., 1992; Monyer et al.,
subunit with glutamine by site-directed mutagenesis 1992, 1994). In external solution containing 1 mM
leads to a reduction of Ca2+ permeability and a les- Mg2+, the current response was largest at ÿ25 mV
ser degree of Mg2+ block. The importance of the in the former two receptor channels, whereas it was
asparagine is more convincingly supported by the around ÿ45 mV in the latter two. Furthermore, the
result that replacing this amino acid with arginine block by Mg2+ on the inward current was much
almost completely abolishes both Ca2+ permeability stronger in the range between ÿ25 and ÿ80 mV for
and Mg2+ block (Burnashev et al., 1992c; Sakurada the former than the latter (Monyer et al., 1994).
et al., 1993). Asparagine in the corresponding site of The profound e€ects of the amino-acid residues in
the NR2 subunits (NR2A and NR2B) also plays an the N site on the permeation of divalent cations
important role in the permeability of the divalent strongly suggest that structural elements including
cations. Replacing the asparagine in this site of this site constrict the permeation pathway of the
NR2 with glutamine markedly reduces the Mg2+ NMDA channel, serving as an ion selectivity ®lter.
block and causes a permeability to Mg2+ The cross-sectional diameter of the constriction in
(Burnashev et al., 1992c; Mori et al., 1992). Thus, the wild-type NR1±NR2A receptor was estimated
asparagine in the N site of the NMDA receptor sub- to be 0.55 nm by measuring the relative permeability
Glutamate Receptors in the CNS 599

of di€erently sized organic cations (Villarroel et al., subunit of NR2 was assembled with NR1. The
1995; Wollmuth et al., 1996). The e€ects of mutating values were 0120 ms, 0400 ms, 0380 ms and
the amino-acid residues at the N sites and the adja- 04800 ms, for NR2A±NR2D, respectively (Monyer
cent two sites of both NR1 and NR2A subunits on et al., 1994).
the pore size were further investigated (Fig. 4).
When the NR1 N-site arginine was replaced by the
smaller glycine residue (G), the pore size of the 2.4.2.5. Single-Channel properties
mutated NR1(N598G)±NR2A channel was The native NMDA receptor has 050 pS openings
increased to 0.75 nm. In the NR2 subunit, both N and 040 pS sublevels in various central neurons
site and N + 1 site, one position downstream to the (Nowak et al., 1984; Jahr and Stevens, 1987; Ascher
C-terminus from the N site, are occupied by aspara- et al., 1988). As described in Section 2.4.2.1, how-
gine (Fig. 4). When the asparagine was replaced ever, the single-channel conductance decreases as
with glycine at these two sites, the e€ect of the the extracellular Ca2+ concentration is increased.
N + 1 site mutation (NR2A(N596G)) was much For example, in rat CA1 pyramidal cells, the con-
more prominent than that of the N site mutation ductance of the main state is 51 pS in 1 mM Ca2+
(NR2A(N595G)), increasing the pore size from and 148 mM Na+ solution, and is reduced to 42 pS
0.55 nm to 0.67 nm. The double mutation, when the Ca2+ concentration is raised to 2.5 mM
NR1(N598G)±NR2A(N596G), increased the pore (Gibb and Colquhoun, 1992). In addition to this 50/
size to 0.87 nm, the sum of the increase produced by 40 pS state, a lower conductance state (038/18 pS)
the individual mutations (Wollmuth et al., 1996). has been reported in cultured cerebellar neurons
The pore size of the wild NR1±NR2A channel is (Cull-Candy and Usowicz, 1987). Farrant et al.
similar to that of the native NMDA receptor esti- (1994) have shown that the single-channel properties
mated in cultured rat hippocampal neurons of the NMDA receptor in cerebellar granule cells
(00.45  0.57 nm) (Zarei and Dani, 1995). This size markedly changes during early development. At an
is smaller than that of the nicotinic acetylcholine early stage (before postnatal day 13), most openings
receptor (00.70±0.74 nm, Dwyer et al., 1980). were to the 50/40 pS state. In contrast, the majority
of channel openings (65%) were to the lower con-
ductance state (033/20 pS) at postnatal days 19±23.
2.4.2.4. Kinetics Furthermore, the mean open time of the 50 pS state,
It was reported that desensitization of the NMDA 3.4 ms, was reduced to 0.85 ms for the 33 pS con-
channel in whole-cell recordings of cultured central ductance state (Farrant et al., 1994).
neurons was reduced by high concentrations of gly- Expression studies have shown that both NR1±
cine and low levels of Ca2+. In the external Mg2+- NR2A and NR1±NR2B NMDA receptors have
free solution containing 10 mM glycine and 0.2 mM 050/40 pS openings (Stern et al., 1992; Tsuzuki et
Ca2+, very little desensitization was observed in re- al., 1994), whereas NR1±NR2C and NR1±NR2D
sponse to continuous application of either NMDA receptor openings have lower conductances (035/
or glutamate (Vyklicky et al., 1990). In outside±out 20 pS) (Stern et al., 1992; Wyllie et al., 1996). These
patches, however, NMDA responses exhibited clear results strongly suggest that the changes in the single
desensitization irrespective of concentrations of gly- channel properties of the NMDA receptor in cer-
cine and/or Ca2+. Even in the solution containig ebellar granule cells during the early development is
10 mM glycine and 0.01 mM Ca2+, the steady-state due to developmental changes of expressions of the
response declined to about 20% of the initial peak NR2 subunits. This notion is further supported by
amplitude with a time constant of 0210 ms (Sather an elegant experiment by Takahashi et al. (1996a)
et al., 1990). Although the reason for this di€erence using mutant mice in which the NR2A gene is dis-
between the two conditions is unknown, it should rupted by gene targeting. They have shown that at
be noted that even in the outside±out con®guration, postnatal day 7 the single-channel conductance of
the rate of desensitization in the native NMDA the NMDA receptor in cerebellar granule cells is
receptor is much slower than in the AMPA recep- predominantly 050/40 pS. At postnatal day 30, the
tor. low-conductance (034/18 pS) channels become
Both activation and deactivation kinetics are also dominant, and very few high-conductance channels
much slower in NMDA receptors than AMPA are detected. The virtual absence in expression of
receptors. When NMDA currents were evoked by 5- the high-conductance NMDA channels at this stage
ms glutamate pulses in outside±out patches of cul- in the NR2A-ablated mice contrasts with their con-
tured hippocampal neurons of neonatal rats, the tinuous presence in wild-type mice. It is most likely
10±90% rise-time was 010 ms, and the decay could that the high-conductance channels are produced
be ®tted by a sum of two exponentials (tfast190 ms predominantly by NR1±NR2B and NR1±NR2A
and tslow1260±600 ms) (Lester et al., 1990). During combinations in immature and mature animals, re-
the long decaying period, bursts of channel openings spectively, since in situ hybridization studies have
persisted which indicates that the agonist remains shown that NR2A is expressed relatively late post-
bound to the receptor for this period (Lester et al., natally whereas NR2B expression is transient during
1990). the earlier stage in cerebellar granule cells
Di€erences in the decay time constants among (Watanabe et al., 1992; Monyer et al., 1994). In the
recombinant NR1±NR2 NMDA channels were esti- mutant mice lacking NR2A expression which should
mated following 300-ms glutamate pulses in Mg2+- occur during the development, no high-conductance
free solution containing 10 mM glycine. The time channel is produced after the NR2B-to-NR2C subu-
constants di€ered markedly depending on which nit switch.
600 S. Ozawa et al.

2.4.2.6. Relation between functional and molecular unless the glassware being used to make the sol-
properties of native receptors utions is baked at high temperatures for several
Using the patch-clamp RT±PCR technique, hours to destroy glycine. After such a treatment,
Audinat et al. (1994) have investigated a correlation NMDA responses were not detected without the ad-
between the functional properties of NMDA recep- dition of glycine to the external solution in Xenopus
tors and expression of the NR2A±NR2B mRNAs in oocytes injected with mRNA from the rat brain.
single granule cells of rat cerebellar slice cultures. NMDA responses are detectable in culture and
They have found that the cells mainly express brain slice preparation because of the presence of
NR2A mRNA after 15±40 days under normal cul- endogenous glycine. However, these NMDA re-
ture conditions, whereas they maintain a predomi- sponses are almost completely abolished by 7-chlor-
nant expression of NR2B in cultures chronically okynurenic acid (7Cl Kyn) that competitively
exposed to tetrodotoxin (TTX). Accordingly, the displaces glycine from its binding site (Kemp et al.,
NMDA responses in the normal culture were only 1988; Vyklicky et al., 1990).
weakly inhibited by ifenprodil, which selectively sup- D-serine and D-alanine are active at the strych-
presses NR1/NR2B receptor but not NR1/NR2A nine-insensitive glycine-binding site, and act as co-
receptor (Williams, 1993), whereas those exposed to agonists at the NMDA receptor (see Kemp and
TTX were strongly inhibited. This result suggests Leeson, 1993 for review). The e€ect of D-serine has
that the switch of expression of NR2 subunits recently been compared with that of glycine using a
depends on neural activities (Audinat et al., 1994). Xenopus oocyte expression system (Matsui et al.,
Plant et al. (1997) identi®ed GABAergic neurons 1995). The ED50 value of D-serine is 3±4 times
in the medial septum of rat brain slices by detecting lower than that of glycine in any combination of
the presence of mRNA for the GABA-synthesizing NR2A to NR2D with NR1. Furthermore, an in vivo
enzyme, glutamic acid decarboxylase (GAD), after microdialysis study has revealed that the extracellu-
patch-clamp recordings. Among the GABAergic lar concentration of free D-serine in the rodent fron-
neurons, 96% expressed NR2B, either alone (20%), tal cortex is 6.5 mM, being high enough to saturate
or together with NR2A (40%), or with NR2C the glycine-binding site of the NMDA receptor
(28%). In a separate batch of cells, 75% expressed (Matsui et al., 1995). These results suggest that D-
NR2D. These results suggest that the majority of serine also acts as an endogenous co-agonist of the
neurons express NR2B and NR2D, usually together NMDA receptor in the rodent brain.
with either NR2A or NR2C. Despite the expression
of NR2D in most neurons, main functional proper- 2.4.3.2. Binding sites for agonists and co-agonists
ties, such as the sensitivity to Mg2+ and ifenprodil,
and the time course of synaptic current suggest that The binding sites for glutamate and glycine on the
most receptors are composed of NR1 and NR2B recombinant NR1±NR2B receptor have been ident-
subunits. It is possible that triplet receptors com- i®ed by site-directed mutagenesis (Kuryatov et al.,
posed of NR1 + NR2B + NR2D have functional 1994; Hirai et al., 1996; Laube et al., 1997).
properties similar to those of NR1 + NR2B alone Substitution of NR2B residues in the region preced-
(Gibb and Wyllie, 1997). Alternatively, NR2B ing M1 and the loop domain separating M3 and M4
mRNA could be expressed much more abundantly causes marked decreases in the anity of glutamate
than NR2D, and therefore most receptors would be without signi®cantly changing the glycine response
formed by a NR1±NR2B combination. To examine (Laube et al., 1997). In contrast, mutation of the
this possibility, it is necessary to establish a method corresponding regions of the NR1 subunit strongly
that allows a quantitative estimate of mRNA ex- reduces the potency of glycine without signi®cantly
pression at the single cell level. a€ecting the glutamate binding (Kuryatov et al.,
1994; Hirai et al., 1996). In both subunits, the
2.4.3. Pharmacology mutated regions have signi®cant and structural
homology to the bacterial amino-acid protein
2.4.3.1. Glycine as a co-agonist LAOBP, as is the case for the proposed agonist-
Johnson and Ascher (1987) have demonstrated binding sites for AMPA/kainate receptors. Using
that the NMDA response is markedly potentiated the sequence homology and the known three-dimen-
by glycine in cultured central neurons. The e€ect of sional structure of LAOBP, Laube et al. (1997) have
glycine is detectable with low concentrations constructed a high-resolution model of the gluta-
(ED50=0.1±0.7 mM), and does not involve the mate and glycine binding sites of the NMDA recep-
strychnine-sensitive receptor which mediates the in- tor.
hibitory glycine action (Johnson and Ascher, 1987; The amino-acid residues determining the anity
Kleckner and Dingledine, 1988; Vyklicky et al., of glutamate are strictly conserved in all NR2 subu-
1990). It later turned out that glycine is not simply a nits (Laube et al., 1997). The location of the gluta-
strong potentiator of the NMDA response, but is mate binding site on the NR2 subunits appears to
absolutely required for the NMDA receptor channel be incompatible with the fact that the NR1 cDNA
to enter the open state, thus playing a role as a co- has been cloned by expression cloning in Xenopus
agonist. Although NMDA responses are detectable oocytes of NMDA channels that are activated by
in nominally glycine-free solution in various prep- co-application of glutamate and glycine (Moriyoshi
arations, this is due to background contamination et al., 1991). It could be that Xenopus oocytes con-
of the experimental solutions by glycine. Kleckner tain NR2-like proteins which are capable of forming
and Dingledine (1988) have shown that nominally the functional NMDA channel with NR1 (Laube et
glycine-free solutions contain 20±50 nM glycine al., 1997). In mammalian cells, coexpression of NR1
Glutamate Receptors in the CNS 601

and NR2 subunits is essential for forming functional EPSC) and the results obtained by gene targeting
receptors (Grimwood et al., 1995). technology.

2.4.3.3. Drugs that a€ect NMDA receptor function 2.4.4.1. Kinetics of EPSC
According to their site of action on the NMDA At many of the excitatory synapses in the CNS,
receptor, drugs are divided into four groups, i.e. the EPSCs have both AMPA and NMDA receptor-
those acting at: (1) the glutamate/NMDA recog- mediated components. The NMDA±EPSC has a
nition site; (2) the strychnine-insensitive glycine- much slower rise and decay times relative to the
binding site; (3) the intra-ion channel binding site; AMPA±EPSC (Hestrin et al., 1990a; Lester et al.,
and (4) modulatory sites such as the redox modula- 1990; Keller et al., 1991; Ozawa et al., 1991a). For
tory site, the proton sensitive site, the Zn2+ site, and example, Lester et al. (1990) have reported that at
polyamine site (see Sucher et al., 1996 for review). glutamatergic synapses in cultured rat hippocampal
Recent experiments using heterologous expression neurons the 10±90% rise-time of the NMDA±EPSC
of recombinant NMDA receptor subunits have is 07 ms, and that the decay phase is ®tted by
revealed that the pharmacological diversity of double exponentials, the time constants of the fast
NMDA receptors is determined by their subunit and slow components being 063 ms and 250±
composition. For example, Mishina and colleagues 545 ms, respectively. The decay times beween the
have shown that the sensitivity to the competitive NMDA± and AMPA±EPSCs di€er more than 100-
antagonist of the glutamate/NMDA recognition fold (see Section 2.2.4.1). Several lines of evidence
site, D-2-amino-5-phosphonovalerate (D±APV), is suggest that AMPA and NMDA receptors are co-
in the order of the NR1/NR2A > NR1/ localized and commonly activated by a nearly in-
NR2B > NR1/NR2C > NR1/NR2D receptors, stantaneous rise and a fast decay of the transmitter,
whereas the sensitivity to the competitive antagonist glutamate, liberated into the synaptic cleft (Jones
of the glycine-binding site, 7Cl Kyn, is in the and Baughman, 1991; Clements et al., 1992). It is
order of the NR1/NR2C > NR1/NR2B > NR1/ thus likely that the AMPA receptor has a relatively
NR2A 1NR1/NR2D receptors. They have further low anity and becomes unbound very quickly after
shown that the NR1/NR2A and NR1/NR2B recep- the clearance of the transmitter, whereas the
tors are more sensitive to the intra-channel blockers, NMDA receptor has much higher anity, resulting
Mg2+ and MK-801, than NR1/NR2C and NR1/ in prolonged binding during which the channel can
NR2D receptors, whereas the sensitivities to phency- open repeatedly (Hestrin et al., 1990b; Lester et al.,
clidine (PCP) and ketamine are similar among them 1990; Lester and Jahr, 1992). This notion is sup-
(Ikeda et al., 1992; Kutsuwada et al., 1992, see Mori ported by the two ®ndings obtained in glutamatergic
and Mishina, 1995 for review). synapses in cultured hippocampal neurons (Lester et
al., 1990). First, the rapid application of the com-
petitive antagonist D-APV at saturating concen-
2.4.4. Physiology trations did not interfere with the slow time course
The NMDA receptor contributes to excitatory of the NMDA±EPSC once it had been initiated.
neurotransmission in many of central synapses. As This suggests that no rebinding of the transmitter
described above, the NMDA receptor is equipped occur during the long-lasting decay phase of the
with three characteristic features. (1) at resting po- EPSC. Second, a brief pulse of high concentrations
tentials it remains blocked by Mg2+. Ionic currents of glutamate (4 ms, 0.1 mM) to an outside±out
through the receptor occur only when the neuronal patch membrane caused openings of NMDA chan-
membrane is depolarized. (2) signi®cant amounts of nels that lasted for several hundreds of milliseconds
extracellular Ca2+ enter the cell interior during the and had a very similar decay time course to the
activation of the receptor and (3) the NMDA recep- synaptically evoked NMDA±EPSC. This suggests
tor-mediated neurotransmission occurs slowly and that a brief pulse of glutamate in the synaptic cleft
lasts for a prolonged period. Because of these prop- is sucient to produce the long-lasting NMDA±
erties, the NMDA receptor serves as a molecular ap- EPSC.
paratus that can detect the coincidence of the In addition to slow unbinding of glutamate, the
presynaptic activity and postsynaptic depolarization desensitization of the NMDA receptor also appears
at the synapse, and inject the postsynaptic cell with to contribute to the slow decay kinetics of the
a sucient amount of the second messenger ion, NMDA±EPSC. If the time course of the decay of
Ca2+, that will initiate plastic changes in the the NMDA±EPSC were determined simply by
strength of synaptic connection. Possible mechan- unbinding of the agonist, the EPSC would have a
isms and physiological implications of the synaptic single exponential decay. In most experiments, how-
plasticities in which the NMDA receptor is inti- ever, the decay phase has been shown to be ®tted by
mately involved, such as LTP and long-term de- double exponentials (Lester et al., 1990; Keller et
pression (LTD) in the hippocampus and the al., 1991; Lester and Jahr, 1992; Takahashi et al.,
neocortex, have been well documented by other 1996a). This two-component decay has been pro-
authors (Bliss and Collingridge, 1993; Bear and posed to be due to a signi®cant contribution of the
Malenka, 1994; Nicoll and Malenka, 1995; Chen desensitization of the NMDA receptor (Lester and
and Tonegawa, 1997; Kaczmarek et al., 1997). In Jahr, 1992). It is conceivable that following repeated
this section, we describe only the two aspects of channel openings by a brief application of agonist a
NMDA receptor functions, that is, the kinetics of substantial number of receptors enter a desensitized
the NMDA receptor-mediated EPSC (NMDA± state rather than becoming unbinding, and from this
602 S. Ozawa et al.

desensitized state the channel can reenter the open campal CA1 synapse (Sakimura et al., 1995). The
state before the agonist ®nally dissociates from the disruption of the NR2C gene results in the disap-
receptor. This transition from desensitized to open pearance of low-conductance NMDA receptor chan-
states would account for the presence of the late nels (<37 pS) normally expressed in matured
component of the EPSC (Lester and Jahr, 1992). cerebellar granule cells (Ebralidze et al., 1996). To
As described in Section 2.4.2.4, the time constant generate mice lacking both NR2A and NR2C,
for deactivation of the NMDA channel di€ers, NR2A and NR2C mutant mice were mated with
depending on the subunit composition (Monyer et each other (Kadotani et al., 1996). The NMDA
al., 1994). This may partly account for di€erences in receptor-mediated components in cerebellar granule
the decay kinetics of the NMDA±EPSC in di€erent cells were virtually abolished in mice lacking both
regions and di€erent developmental stages. NR2A and NR2C. The mice could manage simple
Recently, Takahashi et al. (1996a) have shown that coordinated tasks but failed in more sophisticated
targeted disruption of the NR2A subunit slows the tasks, such as staying on a quickly rotating rod.
decay time of the NMDA±EPSC at cerebellar Tsien et al. (1996a, 1996b) produced CA1-
mossy ®ber-granule cell synapses. It is known that restricted NR1-ablated mice. They crossed Cre
the recombinant NR1/NR2A receptor has the low- transgenic mice in which Cre expression was
est anity for agonist (Kutsuwada et al., 1992; Ishii restricted to CA1 pyramidal cells with fNR1 mice in
et al., 1993) and the fastest deactivation kinetics which multiple exons of the NR1 gene were ¯anked
among the NR1/NR2A±NR2D receptors (Monyer by a pair of loxP sequences inserted into the adja-
et al., 1994). This result suggests that expression of cent intron regions. In the resulting litter, the NR1
the NR2A subunit that occurs postnatally in the gene was disrupted only in the CA1 pyramidal cells
CNS may underlie developmental acceleration in the by the Cre/loxP recombination. The loxP insertions
kinetics of the NMDA±EPSC (Takahashi et al., did not interfere with normal expression of the NR1
1996a). gene in the other part of the brain. These mice grew
normally in contrast to the conventional NR1
mutant mice that died neonatally. However, NMDA
2.4.4.2. Targeted disruption of subunit gene receptor-mediated currents and the LTP were abol-
To examine functional roles of NMDA receptor ished in the CA1 synapses. These mice exhibited
subunits, the ®ve subunit genes, NR1 and NR2A± impaired spatial memory, indicating that the
NR2D, have been disrupted by gene targeting. The NMDA receptor-mediated synaptic plasticity in the
disruption of subunit genes causes a variety of mor- CA1 region is necessary for the proper represen-
phological and functional defects. tation of space (McHugh et al., 1996).
Targeted disruption of NR1 or NR2B results in
the early death of the mutant mice (Forrest et al.,
1994; Li et al., 1994; Kutsuwada et al., 1996). Thus, 2.4.5. Pathophysiology
NR1 and NR2B are essential for the survival of
Abnormal functioning of the NMDA receptor
newborn mice, although they showed no gross ana-
may lead to a variety of neurological disorders.
tomical abnormalities in the brain. Respiratory fail-
Overactivation of the NMDA receptor such as in
ure and the impairment of suckling response
ischemic insults, head trauma, or epileptic seizures
appeared to be the causes of death in NR1 and
triggers a cascade of cellular events culminating
NR2B mutant mice, respectively. Both subunits
neuronal cell death (see Rothman and Olney, 1987;
seem to be involved in the development of neural
Choi, 1988; Choi and Rothman, 1990 for reviews).
networks in the CNS. Tactile hairs (whiskers) on the
On the other hand, hypofunction of NMDA recep-
snout of rodents are arranged in an array and col-
tors elicits a psychotomimetic state that closely re-
lectively form a unique sense organ. This sense
sembles schizophrenia (see Javitt and Zukin, 1991
organ is connected to the brain via the trigeminal
for review). Furthermore, the NMDA receptor plays
nerve, and in the brainstem trigeminal nuclei the
a particular role in pathogenesis of neuropathic pain
whisker-speci®c neural patterns, termed barrelettes,
(see Dickenson, 1990; Urban et al., 1994 for
are formed. The barrelettes are absent in the NR1
reviews). Here, we brie¯y describe these topics.
or NR2B mutant mice, indicating that NMDA
receptors are involved in the detailed patterning of
target neurons (Li et al., 1994; Kutsuwada et al.,
1996). 2.4.5.1. Neuronal cell death
NR1, NR2B and NR2D subunit mRNAs are Glutamate neurotoxicity may be mainly mediated
expressed in the embryonic brains. However, the by an excessive entry of extracellular Ca2+. Since
NR2D-ablated mice grow normally (Ikeda et al., the NMDA receptor channel is highly permeable to
1995a). It could be possible that the role of the Ca2+, overactivation of this subtype of iGluR has
NR2D subunit in the development is compensated been considered to be most responsible for the exci-
by the NR2B subunit. totoxic neuronal cell death. In vitro hypoxia, selec-
NR2A and NR2C subunit mRNAs are not tive antagonists of the NMDA receptor, either
expressed in the embryonic brains. Both NR2A- and competitive or noncompetitive antagonists, attenu-
NR2C-ablated mice developed normally and dis- ate the neuronal injury in cortical or hippocampal
played normal fertility (Sakimura et al., 1995; cultures (see Choi, 1988; Choi and Rothman, 1990
Ebralidze et al., 1996). The disruption of the NR2A for reviews). Recently, it has been shown that non-
gene results in a signi®cant reduction of the NMDA neuronal cells transfected with recombinant NR1/
receptor channel current and the LTP at the hippo- NR2A or NR1/NR2B undergo to cell death when
Glutamate Receptors in the CNS 603

exposed to glutamate, and that they are protected MK-801-induced stereotyped behavior and ataxia in
by NMDA receptor antagonists (Anegawa et al., the rat (Contreras, 1990; Tanii et al., 1994).
1995; Raymond et al., 1996). Furthermore, Furthermore, glycine has been reported to improve
decreased cytotoxity was observed when a mutated negative symptoms signi®cantly in patients with
NR1 subunit (N598R) with no Ca2+ permeability chronic schizophrenia (Javitt et al., 1994).
was used in coexpressions with NR2A or NR2B
(Boeckman and Aizenman, 1996). In vivo ischemic 2.4.5.3. Neuropathic pain
brain damage, Simon et al. (1984) have ®rst demon- Activation of a€erent C ®bers with nociceptive
strated that direct intrahippocampal injection of the stimuli produces pain sensations that are enhanced
competitive NMDA antagonist, 2-amino-7-phos- during pathological conditions. The pronounced
phonoheptanoate (APH), reduces the loss of CA1 pain symptoms such as reduced pain threshold (allo-
pyramidal neurons produced by transient forebrain dynia) and increased responses to pain (hyperalge-
ischemia in the rat. A number of di€erent labora- sia) are considered to be due to plastic changes that
tories have con®rmed and further extended this occur in the CNS. Activity-dependent increases in
result by using various types of NMDA antagonists, excitability are induced in the spinal dorsal horn
although some negative results have been reported neurons by repetitive stimulation of C ®bers. This
with global ischemia models (Buchan and Pulsinelli, phenomenon was ®rst described and termed
1990, 1991). ``windup'' by Mendell and Wall (1965), and is now
On the basis of the above studies, the NMDA an- considered as a neural basis underlying the develop-
tagonists have been considered for use as neuropro- ment and maintenance of the pronounced pain
tective therapeutic agents. The antagonists that symptoms. NMDA antagonists, ketamine and D-
readily penetrate blood±brain barrier (BBB) seemed APV, have been shown to consistently reduce the
to be suitable for this purpose, and therefore the windup in the rat dorsal horn nociceptive neurons,
neuroprotective e€ects of PCP and related agents suggesting that the NMDA receptor contributes to
such as MK-801 were examined extensively. this phenomenon (Davies and Lodge, 1987;
However, the severe psychotomimetic and memory- Dickenson and Sullivan, 1987). It may be speculated
impairing e€ects of these agents manifested in ani- that a plastic change in the excitatory synaptic
mal experiments have hindered the clinical use of transmission between the C ®ber and the dorsal
these noncompetitive NMDA antagonists. horn nociceptive neurons occurs in a manner analo-
Furthermore, it was found that these agents cause a gous to the LTP in the CA1 region of the hippo-
neurotoxic reaction consisting of pathomorphologi- campus, and ``pain memory'' is produced in the
cal changes in speci®c pyramidal neurons. sensory pathway. Both competitive and noncompeti-
Competitive NMDA antagonists were reported to tive NMDA antagonists are e€ective in reducing
also produce similar psychotomimetic and cytotoxic various types of neuropathic pains in experimental
e€ects (see Olney, 1994 for review). animals, such as allodynia evoked by tight ligation
of the lumbar spinal nerves (a model of chronic
nerve injury pain) and the formalin paw test (a
2.4.5.2. Psychiatric disturbance
model of hyperalgesia caused by in¯ammation of
The noncompetitive NMDA antagonist PCP has the peripheral tissue) in the rat (Qian et al., 1996;
been known as a psychotomimetic agent. The use of Chaplan et al., 1997). These results strongly suggest
PCP in humans has been observed to produce both that NMDA antagonists would be useful thera-
a hallucinatory-paranoid state and de®cit sympto- peutic agents for preventing the development of neu-
matologies which are indistinguishable from those ropathic pains. Consistent with this notion,
of schizophrenic patients (see Javitt and Zukin, 1991 ketamime attenuates the development of hyper-
for review). Ketamine that inhibits NMDA recep- algesia in human subjects (Park et al., 1995;
tors noncompetitively and is used in human anesthe- Warncke et al., 1997).
sia also causes PCP-like psychotic e€ects (Krystal et
al., 1994). MK-801, a more selective and potent
ligand of the PCP site, displays strong neurobeha- 3. METABOTROPIC RECEPTORS
vioral e€ects such as stereotypy, ataxia and cata-
lepsy in animal experiments designed to examine its Glutamate activates not only iGluRs, but also
potential as a neuroprotective therapeutic agent. mGluRs coupled to G-proteins (see Schoepp and
Competitive NMDA antagonists such as CPP and Conn, 1993; Nakanishi, 1994; Pin and Duvoisin,
selfotel have been reported to cause similar psycho- 1995 for reviews). It was ®rst reported that gluta-
tic symptoms in humans (see Olney, 1994 for mate activates inositol phosphate metabolism
review). These results suggest that NMDA receptor directly in striatal (Sladezek et al., 1985) and cer-
hypofunction might occur in schizophrenia, and ebellar granule cell cultures (Nicoletti et al., 1986b,
that the transient psychosis induced by NMDA an- 1988) as well as in hippocampal slices (Nicoletti et
tagonists is not only a useful experimental model for al., 1986a). Thereafter, Sugiyama et al. (1987) have
clarifying the etiology of this disease (see Javitt and demonstrated that Xenopus oocytes injected with rat
Zukin, 1991; Moghaddam, 1994; Thornberg and cerebral mRNA express an oscillatory Clÿ current
Saklad, 1996 for reviews), but also provides a cue upon application of glutamate that is due to inosi-
for the development of a novel class of antipsychotic tol-1,4,5-trisphosphate (IP3)-mediated intracellular
agents. It has been shown that D-serine, an en- Ca2+ release (Murphy and Miller, 1988).
dogenous agonist for the strychnine-insensitive gly- Monitoring the glutamate-induced Clÿ currents in
cine site of the NMDA receptor, reduces PCP- and Xenopus oocytes, two groups independently cloned a
604 S. Ozawa et al.

structurally unique receptor protein mGluR1 extracellular domain. Recently, an extracellular


(Houamed et al., 1991; Masu et al., 1991). Later, a Ca2+-sensing receptor of the parathyroid gland was
whole family of related mGluR subtypes, i.e. found to share a signi®cant sequence homology with
mGluR2 through mGluR8, was isolated (see Section mGluRs (Brown et al., 1993). Therefore, it has been
3.1.1). Even though structurally related, these proposed that the mGluRs form a unique superfam-
mGluR subtypes are highly heterogenous in their ily of the G-protein-coupled receptors which
agonist selectivity, signal transduction mechanisms includes the parathyroid Ca2+-sensing receptor. A
and distribution in the brain. newly isolated mGluR from salmon brain has been
shown to be activated by both glutamate and extra-
cellular Ca2+ (Kubokawa et al., 1996).
3.1. Molecular Diversity
3.1.1. Multiplicity of Genes, and Splicing Variants
Using PCR-mediated ampli®cation and cross-hy- 3.1.2. Structure (Membrane Topology, etc.)
bridization screening with the mGluR1 sequence,
All mGluRs are considerably large proteins (854±
additional seven related genes, termed mGluR2±
1179 amino acids) with a large hydrophilic N-term-
mGluR8 have been identi®ed (Abe et al., 1992;
inal region of 0550 amino acids, a core region of
Tanabe et al., 1992, 1993; Nakajima et al., 1993;
0250 amino acids including seven transmembrane
Okamoto et al., 1994; Duvoisin et al., 1995;
domains, and a C-terminal region, the length of
Saugstad et al., 1997). Several of them (mGluR1,
which varies with subtype (see Nakanishi, 1992;
mGluR4 and mGluR5) have been reported to gener-
Hollmann and Heinemann, 1994; Pin and Duvoisin,
ate di€erent mRNAs by alternative splicing (Pin et
1995 for reviews). It has been proposed that the N-
al., 1992; Tanabe et al., 1992; Pickering et al., 1993;
terminal domain is extracellular and the C-terminal
Simoncini et al., 1993; Joly et al., 1995; Minakami
intracellular, respectively, since the N-terminal
et al., 1995).
domain of mGluR1 contains several consensus sites
The eight subtypes mGluR1±mGluR8 are closely
for N-glycosylation and the C-terminal domain has
related in primary structure (Fig. 5). The amino acid
the potential phosphorylation sites (Masu et al.,
sequences of the mGluRs show more than 40%
1991). In all mGluR subtypes, 21 cysteine residues
homology (see Pin and Duvoisin, 1995 for review),
are conserved, 19 of which are located in the puta-
and all have seven putative transmembrane regions
tive extracellular portions, suggesting that the rigid
like other G-protein-coupled receptors such as
three dimensional conformation of the extracellular
muscarinic and adrenergic receptors. Even though
portions is important for mGluR functions.
the membrane topology is similar, the mGluR
Several lines of evidence suggest that the N-term-
family seems to be quite di€erent from the other G-
inal large extracellular domain contains glutamate-
protein-coupled receptors, since it shows no
binding sites. It was proposed that the N-terminal
sequence homology with the other G-protein-
half of this domain is involved in the agonist selec-
coupled receptors and possesses an unusually large
tivity and serves as a glutamate-binding site by a
study using a series of chimeric receptors at the
extracellular domains of mGluR1 and mGluR2
(Takahashi et al., 1993). Structural homology with
bacterial periplasmic binding proteins was used to
predict the tertiary structure of the N-terminal
domains and the glutamate-binding sites. The model
was tested by mutation analysis, and some mutants
were a€ected in their functional anities to gluta-
mate and quisqualate, suggesting that these sites are
critical for glutamate-binding of mGluRs (O'Hara
et al., 1993).
It has been proposed that the C-terminal domain
plays a role in determining the potency of agonists,
since natural deletions of the C-terminal domain in
mGluR1 splicing variants (mGluR1b and
mGluR1c), which have shorter C-terminal tails than
mGluR1a, result in a decrease in agonist potency in
Fig. 5. Dendrogram of the members of the metabotropic the expression system (Flor et al., 1996). It is likely
glutamate receptor family. Unrooted neighbor-joint tree of that the C-terminal domain regulates the transduc-
8 mGluR subunit proteins of the rat constructed by a clus- tion mechanisms of the mGluR.
tal w computer program (Thompson et al., 1994). The The sequences of the ®rst and the third intracellu-
value, 100% minus the sum of the length of the horizontal lar loop (i1 and i3) are highly conserved among all
solid lines between the two subunits, indicates % identity the mGluRs, suggesting that these domains are
in the amino-acid sequence between them, as explained in
involved in G-protein activation. The less conserved
Fig. 1. Data used for constructing this dendrogram were
obtained from DNA Data Bank of Japan (DDBJ) with second intracellular loop (i2) and the intracellular
accession numbers, X57569(mGluR1), M92075(mGluR2), portion adjacent to the seventh transmembrane
M92076(mGluR3), M92077(mGluR4), D10891(mGluR5), domain (i4) were suggested to determine the speci®c
D13963(mGluR6), D16817(mGluR7), and coupling of mGluR1 to phospholipase C (PLC) (Pin
U63288(mGluR8). et al., 1994).
Glutamate Receptors in the CNS 605

3.1.3. Classi®cation agonist potency for group II is: DCG-IV rL-


The eight mGluR subtypes are classi®ed into CCG-I > Glu > ACPD > Ibo > Quis. Recently,
three subgroups on the basis of their amino acids (2S,3S,4S)-2-methyl-2- (carboxycyclopropyl) - glycine
sequence homology. These are termed group I (MCCG) has been proposed as a speci®c antagonist
(mGluR1 and mGluR5), group II (mGluR2 and of group II mGluRs (KnoÈpfel et al., 1995; Salt and
mGluR3), and group III (mGluR4, mGluR6, Eaton, 1995).
mGluR7 and mGluR8) (see Pin and Duvoisin, 1995 Group III receptors (mGluR4, mGluR6, mGluR7
for review). The members of the same subgroup and mGluR8) are characterized by their sensitivity
share 070% amino acid sequence identity, while the to L-AP4 (Nakajima et al., 1993; Tanabe et al.,
homology between di€erent subgroups is 045%. 1993; Okamoto et al., 1994; Duvoisin et al., 1995;
This classi®cation also holds in their pharmacology Saugstad et al., 1997) as well as insensitivity to
as well as in their signal transduction mechanisms. ACPD. The sensitivity of L-AP4 for mGluR7 is
In the expression systems, group I mGluRs somewhat lower than for other members of group
(mGluR1 and mGluR 5) are activated potently with III. While the signal transduction systems for cloned
quisqualate and are coupled to the activation of the mGluRs of both group II and III are similar (inhi-
PLC pathway (Abe et al., 1992; Aramori and bition of adenylyl cyclase), L-AP4 does not activate
Nakanishi, 1992). Group II (mGluR2 and mGluR3) group II receptors. It has also been proposed that a-
and group III (mGluR4, mGluR6, mGluR7 and methyl-L-AP4 (MAP4) is a selective antagonist of
mGluR8) are both negatively coupled with the ade- group III receptors (Johansen and Robinson, 1995;
nylyl cyclase system (Tanabe et al., 1992, 1993; KnoÈpfel et al., 1995; Salt and Eaton, 1995).
Nakajima et al., 1993; Okamoto et al., 1994; 3.1.5. Transduction Mechanisms
Duvoisin et al., 1995). Group II mGluRs are
potently activated by (2S,1'R,2'R,3'R)-2-(2,3-dicar- Signal transduction mechanisms of the mGluR
boxycyclopropyl)glycine (DCG-IV; Ishida et al., subtypes were examined in the expression systems.
1993; Hayashi et al., 1993), whereas L-2-amino-4- Consistent with the pharmacological classi®cation,
phosphonobutyrate (L-AP4) is a selective agonist the mGluRs of the same subgroups show similarity
for group III mGluRs (Nakajima et al., 1993; in transduction mechanisms.
Tanabe et al., 1993; Okamoto et al., 1994; Duvoisin Activation of group I receptors (mGluR1 and
et al., 1995; Saugstad et al., 1997). mGluR5) stimulates PLC and the subsequent IP3
formation leads to the release of Ca2+ from intra-
cellular stores (Masu et al., 1991; Abe et al., 1992;
3.1.4. Pharmacology Aramori and Nakanishi, 1992). The mGluR1 trans-
The pharmacological pro®les of mGluR subtypes fected into CHO cells also stimulates cAMP for-
were characterized using Xenopus oocytes, chinese mation as well as arachidonic acid release (Aramori
hamster ovary (CHO) cells, baby hamster kidney and Nakanishi, 1992), whereas mGluR5 does not
(BHK) cells, and HEK 293 cells transfected with the couple with the stimulatory cAMP pathway (Abe et
cloned mGluRs. In such heterologous expression al., 1992). Another di€erence between mGluR1 and
systems, it was possible to express the cloned mGluR5 is the sensitivity to pertusis toxin (PTX).
mGluR subtypes exclusively in pure forms. The blocking action of PTX is weak for mGluR5
Glutamate, quisqualate and ibotenate have been (015% maximal inhibition) compared with mGluR1
shown to activate mGluRs as well as iGluRs. Some (040%). Another group reported that the PTX sen-
glutamate analogues, such as 1-aminocyclopentane- sitivity could di€er between the splicing variants
1,3-dicarboxylic acid (ACPD) and L-AP4, are (Pickering et al., 1993). The stimulation of PI hy-
speci®c for mGluRs, while their potencies di€er for drolysis by mGluR1a showed PTX-sensitive and -
each mGluR subtype. insensitive components, whereas the mGluR1b dis-
Group I mGluRs (mGluR1 and mGluR5) show played only the toxin-insensitive component.
the following rank order of agonist potency: All other mGluRs of group II and group III are
Quis > Glu rIbo > ACPD. 3,5-dihydrophenylgly- coupled to the inhibition of adenylyl cyclase. Group
cine (DHPG; Ito et al., 1992) has been shown to be II mGluRs show strong inhibition of forskolin-
a highly selective agonist for group I receptors induced cAMP formation (Tanabe et al., 1992,
(Schoepp et al., 1994; Gereau and Conn, 1995a,b). 1993). Maximal inhibition of cAMP formation was
No group I-speci®c antagonist has been identi®ed, somewhat smaller for the group III receptors. All
while some phenylglycine derivatives such as (+)-a- these e€ects of group II and group III are strongly
methyl-4-carboxyphenylglycine ((+)-MCPG) are inhibited by PTX, suggesting that the G-proteins
antagonists of mGluR1 as well as of mGluR2 which involved in the coupling are of the Gi family
belongs to group II receptors (Eaton et al., 1993; (Tanabe et al., 1992, 1993; Nakajima et al., 1993;
Hayashi et al., 1994). (S)-4-Carboxyphenyl-glycine Okamoto et al., 1994; Duvoisin et al., 1995).
((S)-4CPG) has been proposed as a relatively speci®c The signal transduction mechanisms studied in the
antagonist for group I (mGluR1a), although it acts heterologous expression systems may di€er from
on group II (mGluR2) at higher concentrations those of the native receptors expressed in situ, since
(Hayashi et al., 1994). the G-proteins and the subsequent e€ector proteins
DCG-IV as well as (2S,1'S,2'S)-2-(carboxycyclo- could vary among cell types (see Nakanishi, 1994 for
propyl)-glycine (L-CCG-I) were shown to be selective review). Therefore, the identi®cation of the native
agonists of group II mGluRs (mGluR2 and mGluR subtypes involved in variable physiological
mGluR3) (Nakagawa et al., 1990; Hayashi et al., and pathological functions using signal transduction
1992, 1993; Ishida et al., 1993). The rank order of pharmacology must be evaluated with caution.
606 S. Ozawa et al.

3.2. Physiology ductance (Nawy and Jahr, 1990, 1991), the role of
mGluR6 in ON response was suggested as follows:
Physiological functions of mGluRs have been stu- Photoreceptors hyperpolarize in response to light
died extensively using agonists (i.e. ACPD) and an- and reduce glutamate release from their terminals.
tagonists (i.e. 2-amino-3-phosphonopropionic acid Consequently, the activity of the mGluR6-phospho-
(AP3) and MCPG) of the mGluRs in various prep- diesterase system in ON-bipolar cells is depressed
arations in vivo and in vitro. The speci®c mGluR during the light stimulus, and the resultant increase
subtypes involved in certain physiological functions in cyclic GMP activates cationinc channels, thereby
have been elucidated by pharmacological studies causing the membrane depolarization in ON-bipolar
using subgroup-speci®c agonists (i.e. DHPG, DCG- cells. Direct evidence for the role of mGluR6 has
IV and L-AP4) or antagonists (i.e. MCCG and been demonstrated by targeted disruption of the
MAP4), as well as anatomical studies using subtype- mGluR6 gene (Masu et al., 1995). In the mGluR6-
speci®c mRNA probes or antibodies raised against ablated mice, ON responses were completely abol-
the cloned mGluR subtypes. In addition, targeted ished without any obvious change in OFF responses
disruptions of the mGluR subtype genes have been which is mediated by AMPA receptors. Detailed
utilized to elucidate synaptic and behavioral func- behavioral analysis revealed that the mGluR6 dis-
tions of the mGluRs (Aiba et al., 1994a,b; Conquet ruption reduced the sensitivity of pupillary responses
et al., 1994; Hsia et al., 1995; Masu et al., 1995; to light stimulus and impaired the ability to drive
Pekhletski et al., 1996; Yokoi et al., 1996). optokinetic nystagmus in response to visual con-
trasts (Nakanishi, 1996).
3.2.1. Regulation of Neuronal Excitability In hippocampal CA3 neurons of slice cultures,
The mGluR agonists induce a slow depolarizaion mossy ®ber stimulations induced possible mGluR-
accompanied by an increase in ®ring rate in many mediated EPSCs with a slow time course [a rise time
neurons including those of the hippocampus, neo- of around 5 s and lasting up to 60 s] (Charpak and
cortex, and cerebellum. These excitatory e€ects GaÈhwiler, 1991). These EPSCs were associated with
could be attributed to direct actions on several K+ the inhibition of K+ conductance. Miller et al.
channels. The depolarizing e€ects may be due to the (1995) reported relatively fast EPSCs mediated by
reduction of a voltage-independent leak K+ current mGluRs (but still slower than those mediated by
(IK,leak: GueÂrineau et al., 1994) and a voltage-depen- ionotropic AMPA and NMDA receptors; a 10±90%
dent K+ current IM (Charpak et al., 1990), both of rise time of 15±30 ms) from CA3 neurons in hippo-
which contribute to the resting membrane conduc- campal slice cultures as well as in acute slices. These
tances. The increase in ®ring rate may be due to an EPSCs were associated with the activation of a non-
inhibition of a Ca2+-dependent K+ current IAHP selective cationic conductance. mGluR-mediated
(Charpak et al., 1990) that generates the afterhyper- EPSPs in response to burst activities of presynaptic
polarization following an action potential. In ad- neurons were demonstrated also in CA1 cells of slice
dition, mGluR activation has been shown to preparations (Bianchi and Wong, 1995). In cerebel-
broaden the action potentials in hippocampal neur- lar slices, repetitive stimulation of the parallel ®bers
ons by slowing the repolarization (Hu and Storm, induced slow EPSPs in Purkinje cells (Batchelor and
1991). Activation of mGluRs in hippocampal neur- Garthwaite, 1993), mediated by group I mGluRs
ons also results in an increase in Ca2+-activated (possibly by mGluR1) (Batchelor et al., 1997). It has
nonspeci®c cationic conductance (CreÂpel et al., been shown that a single climbing ®ber activation
1994: GueÂrineau et al., 1995). Several studies strongly potentiates these mGluR-mediated exci-
attempted to pharmacologically characterize roles of tations at the parallel ®ber synapses for 02 min
the speci®c mGluR subtypes in the regulation of the (Batchelor and Garthwaite, 1997). This heterosynap-
neuronal excitability. It was shown that activation tic modulatory mechanism was suggested to be a
of group I mGluR (possibly mGluR5) elicits the novel form of temporal integration of the synaptic
membrane depolarization with increased input re- activities in Purkinje cells.
sistance and blockade of spike frequency adaptation
in hippocampal CA1 neurons (Davies et al., 1995;
Gereau and Conn, 1995a).
Roles of the mGluRs in glutamatergic synaptic 3.2.2. Presynaptic Inhibition
transmission have been demonstrated in several In addition to the direct excitatory postsynaptic
synapses. In particular, the functional role of e€ects, mGluR activation was demonstrated to sup-
mGluR6 in the glutamatergic transmission from press both excitatory and inhibitory transmission at
photoreceptors to ON-bipolar cells in the retina has synapses by presynaptic mechanisms (see Nakanishi,
been established by Nakanishi and colleagues (see 1994 for review). Electron-microscopic analysis
Nakanishi, 1995 for review). mGluR6, cloned from using speci®c antibodies raised against several
a rat retinal cDNA library, is expressed selectively mGluR subtypes revealed their localization on the
at the bipolar-cell layer of the retina. presynaptic terminals (Bradley et al., 1996;
Immunohistochemical and immunoelectron micro- Shigemoto et al., 1996; Yokoi et al., 1996). These
scopic analyses have disclosed that mGluR6 is presynaptic mGluRs may play important roles in ac-
restrictedly localized at the postsynaptic site of ON- tivity-dependent regulation of the synaptic func-
bipolar cells (Nomura et al., 1994). Since electro- tions, since they have been reported to not only
physiological studies indicated that both glutamate serve as ``autoreceptors'' to suppress excitatory
and L-AP4 (group III mGluR agonist) hyperpol- transmission (Forsythe and Clements, 1990; Baskys
arize ON-bipolar cells by decreasing membrane con- and Malenka, 1991; Herrero et al., 1992; Scanziani
Glutamate Receptors in the CNS 607

et al., 1997), but also to induce plasticity due to the 3.2.3. Synaptic Plasticity
presynaptic mechanism (Kobayashi et al., 1996). Since both LTP and LTD in the hippocampus
The mGluR subtypes involved in the presynaptic and LTD in the cerebellum occur at the glutamater-
inhibitory actions seem to di€er among various gic synapses, the possible interaction of mGluRs
synapses. In the early 1980s, L-AP4 was shown to with these synaptic events were studied in many lab-
suppress glutamatergic transmission in several oratories.
regions including the hippocampus, olfactory bulb,
Ca2+ in¯ux through NMDA receptor channels is
retina, and spinal cord. This compound was ®rst
essential for the induction of LTP in the CA1 region
regarded as a speci®c ligand for a new GluR sub-
of the hippocampus. Additional contributions of
type (Foster and Fagg, 1984), and later it turned out
mGluRs in CA1 LTP were examined using their
to act as an agonist of group III mGluRs. In the
selective agonists (McGuiness et al., 1991; Otani and
hippocampus, transmission at the lateral perforant
Ben-Ari, 1991; Bortolotto and Collingridge, 1992,
path-dentate granule cell synapse was potently
1995; Behnisch and Reymann, 1993; O'Connor et
inhibited by L-AP4 (Koerner and Cotman, 1981).
al., 1995) and antagonists (Bashir et al., 1993;
Synaptic transmission at mossy ®ber-CA3 synapses
Chinestra et al., 1993; Bortolotto et al., 1994;
of guinea pig, but not in rat, was also suppressed by
Manzoni et al., 1994; Selig et al., 1995). Targeted
this drug (Yamamoto et al., 1983; Lanthorn et al.,
disruption of mGluR1 was also conducted for this
1984). This e€ect of L-AP4 was suggested to be of
presynaptic origin, because of the absence of e€ects purpose (Aiba et al., 1994a; Conquet et al., 1994).
on the miniature EPSPs and the quantal size of the Unfortunately, these studies produced contradictory
evoked EPSPs (Cotman et al., 1986; Yamamoto et conclusions. At present, it is believed that mGluRs
al., 1991). are not necessary for the induction of CA1 LTP,
Group II-speci®c agonist DCG-IV has been although some modulatory e€ects on the threshold
reported to suppress not only inhibitory trans- for the LTP induction can not be excluded comple-
mission in the accessory olfactory bulb (Hayashi et tely (Selig et al., 1995).
al., 1993) and in the hippocampus (Poncer et al., As for the LTP at the hippocampal mossy ®ber-
1995), but also excitatory transmission at the hippo- CA3 synapse, which is known as a unique form of
campal mossy ®ber-CA3 synapse both in rat and plasticity for its NMDA-independent induction
mouse (Kamiya et al., 1996; Yokoi et al., 1996) and mechanisms, two groups have reported con¯icting
the corticostriatal synapse (Lovinger and McCool, results. Although using the same strategy, one
1995). demonstrated impairment of mossy ®ber LTP in
The presence of multiple mGluR subtypes (of mGluR1-de®cient mice (Conquet et al., 1994),
group II and group III) has been demonstrated in whereas the other reported no e€ect of the disrup-
the lateral perforant path-dentate granule cell tion of mGluR1 gene (Hsia et al., 1995). The reason
(Bushell et al., 1996) and mossy ®ber-CA3 synapses for this discrepancy is presently unknown. In con-
of the guinea pig (Yoshino et al., 1996). trast, the activation of the mGluR2 located on the
Hippocampal CA1 synapses are also suggested to be presynaptic mossy ®ber terminals has been shown to
regulated by multiple presynaptic mGluRs, although be involved in the induction of LTD at the mossy-
the reports of two groups reached con¯icting con- ®ber synapse (Kobayashi et al., 1996; Yokoi et al.,
clusions (Gereau and Conn, 1995b; Vignes et al., 1996).
1995). A direct link between mGluR activation and
The mechanisms underlying the mGluR-mediated synaptic plasticity has been demonstrated for the
presynaptic inhibitory e€ects are not fully under- parallel ®ber synapse in the cerebellar cortex.
stood. It was suggested that inhibition of presyn- Application of quisqualate together with Purkinje
aptic Ca2+ channels possibly underlies the cell depolarization induces LTD at this synapse
suppression of the transmitter release by mGluR (Kano and Kato, 1987). Since quisqualate activates
agonists. Activation of mGluRs has been demon- group I mGluRs potently and mGluR1 is localized
strated to inhibit both L-type (Lester and Jahr, at the distal dendritic spines of the Purkinje cell
1990; Chavis et al., 1994) and N-type (Swartz and (Shigemoto et al., 1994), the involvement of
Bean, 1992; Ikeda et al., 1995b) Ca2+ channels. L- mGluR1 in cerebellar LTD has been suggested. In
AP4 inhibits synaptic transmission as well as fact, LTD was severely impaired in mGluR1-ablated
somatic Ca2+ channels in primary cultures of the mice (Aiba et al., 1994b: Conquet et al., 1994), and
olfactory bulb (Trombley and Westbrook, 1992). mGluR1-speci®c antibodies blocked LTD induction
Moreover, ``whole-terminal'' recordings of the Ca2+ in cultured Purkinje cells (Shigemoto et al., 1994).
currents from the large calyx-type presynaptic term- A contribution of mGluR2 to the formation of
inals in the brainstem slices (Takahashi et al., olfactory memory in mice has been shown by mor-
1996b) and ¯uorometric measurement of presyn- phological, electrophysiological and behavioral stu-
aptic Ca2+ transients in hippocampal slices dies (Hayashi et al., 1993; Kaba et al., 1994; see
(Yoshino and Kamiya, 1995) have directly demon- Nakanishi, 1995 for review). Female mice have an
strated that the inhibition of presynaptic Ca2+ olfactory recognition memory located at the dendro-
in¯ux accounts for the suppression of transmitter dendritic synapse between granule cells (GABAergic
release by mGluR activation. This idea was also neurons) and mitral cells (glutamatergic neurons) in
supported by the ®nding that the activation of pro- the accessory olfactory bulb (AOB). As a result of
tein kinase C (PKC) by phorbol esters suppresses this memory, males made familiar by mating are
mGluR-mediated inhibition of both Ca2+ currents recognized by the female, thereby mitigating preg-
and synaptic transmission (Swartz et al., 1993). nancy block. This olfactory memory is considered to
608 S. Ozawa et al.

be formed as plastic changes of GABAergic trans- ACPD application (Sacaan and Schoepp, 1992;
mission from granule cells to mitral cells in the McDonald et al., 1993). Another group reported
accessory olfactory bulb (Brennan et al., 1990). bidirectional e€ects of ACPD; at low concentration
Hayashi et al. (1993) have disclosed the speci®c of ACPD (5 mM), a facilitatory e€ect and at higher
localization of mGluR2 in granule cell dendrites dose, a suppressive e€ect (Burke and Hablitz, 1995).
that form dendrodendritic synapses with mitral cells. Since L-AP4 has only a suppressive e€ect on the sei-
Using the selective agonist DCG-IV, they have zure discharge (Burke and Hablitz, 1995), these
further demonstrated that the granule cell mGluR2 opposite e€ects of ACPD may be due to the contri-
presynaptically suppresses inhibitory GABAergic bution of multiple types of mGluRs. Consistent
transmission to the mitral cell. Since this modula- with this notion, Tizzano et al. (1995) have reported
tory e€ect of mGluR2 is con®ned to the synapses of that a group I-selective agonist DHPG enhances
excited mitral cells, this modulation would relieve limbic seizures, whereas group II (L-CCGI)- and
the recurrent inhibition of the excited mitral cells group III-selective agonists protect from seizures.
but would maintain lateral inhibition of unexcited
neighboring mitral cells. This mechanism would not
only enhance the signal-to-noise ratio between the 4. CONCLUDING REMARKS
excited and unexcited mitral cells, but also result in
a more profound excitation of the excited mitral During the last decade, our understanding of the
cells that might induce long-lasting changes in GluRs in the mammalian CNS has been advanced
synaptic eciency in the olfactory system (see enormously by the application of molecular cloning
Nakanishi, 1995 for review). In fact, Kaba et al. technology. This technology has revealed that the
(1994) have found that the activation of mGluR2 by molecular diversity of the GluRs is much larger
infusing DCG-IV into the AOB permits the for- than expected from the previous electrophysiological
mation of a speci®c olfactory memory without the and pharmacological studies. To date, at least 14
occurrence of mating in female mice. iGluR subunits and 8 mGluR subtypes have been
identi®ed, and their molecular structure, distribution
3.3. Pathophysiology in the CNS together with developmental changes,
functional and pharmacological properties have
Since mGluRs are involved in a variety of neur- been elucidated. One may speculate that the brain
onal activities, mGluR activation may regulate cer- has developed a large number of receptor subtypes,
tain pathological conditions of the brain. To date, each ®nely tuned to play a particular role in the
regulatory e€ects of mGluRs in glutamate-induced synaptic transmission at di€erent neuron types as
neurotoxicity as well as in epileptiform activity have well as in di€erent developmental stages. In an
been reported. However, these studies are compli- attempt to understand physiological signi®cances of
cated by the fact that the activation of mGluRs each receptor subunit, a variety of mutant mice
could have both excitatory and inhibitory e€ects on have been generated by gene targeting technology.
neuronal activities. Morphological, electrophysiological and behavioral
The neuronal death was attenuated by group II analyses on such gene knockout mice have disclosed
(mGluR2, mGluR3)-selective agonist DCG-IV critical roles of some receptor subunits in the neural
(Bruno et al., 1994, 1995: Buisson et al., 1996), but development, synaptic plasticity, learnig and mem-
not by group III (mGluR4, mGluR6-mGluR8) ago- ory, and motor coordination. Recent improvements
nist L-AP4 (Pizzi et al., 1996), indicating that group of the gene targeting technology that allow a gene
II receptors were responsible for this e€ect. Since deletion in a region-speci®c and/or temporally con-
group II receptors are involved in the presynaptic trolled fashion will further contribute to elucidate
inhibition at many synapses, the inhibition of gluta- functional signi®cances of the molecular diversity
mate release may account for this protective e€ect. (see Chen and Tonegawa, 1997 for review).
In contrast, the e€ect of ACPD is controversial. Moreover, recent developments in gene transfer
Several reports suggest a neuroprotective action of technology using the adenoviral vector have enabled
ACPD (Koh et al., 1991; Chiamulera et al., 1992; one to introduce almost any receptor subunit gene
Birrell et al., 1993; Copani et al., 1995). However, into a given target cell (Le Gal La Salle et al., 1993).
ACPD was also reported to enhance, rather than Sudo et al. (1997) have expressed functional AMPA
attenuate, the glutamate-mediated neuronal injury receptors using this viral vector. The combination of
(McDonald and Schoepp, 1992). This was supported gene knockout and gene transfer technologies will
by the ®nding that the injection or the systemic ad- be most powerful in elucidating both physiological
ministration of ACPD resulted in seizures and brain and pathophysiological roles of each receptor subu-
damage (Sacaan and Schoepp, 1992; McDonald et nit. The combined use of these two technologies will
al., 1993). These apparently opposite results may be also be useful to seek possibilities of gene therapy
due to nonspeci®c actions of ACPD on various for neurological disorders which may be caused by
types of mGluRs. malfunctions of the GluRs.
Another consequence of mGluR activation in vivo
is the epileptiform activities. ACPD-induced syn-
AcknowledgementsÐWe are most grateful to Professor
chronized oscillations of excitatory networks in the John W. Phillis of Wayne State University for his continu-
hippocampal CA3 region (Taylor et al., 1995) as ing encouragement in ®nishing this review. We would also
well as of inhibitory circuits in CA1 (Whittington et like to thank the Human Frontier Science Program and the
al., 1995) have been reported. These synchronized Ministry of Education, Science, Sports and Culture of
bursts may underlie the seizure discharge induced by Japan for support.
Glutamate Receptors in the CNS 609

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