NMDA receptors are well known to play an important role in synaptic development and plasticity. Functional NMDA receptors are
synapses on pyramidal neurons in the lateral amygdala, whereas NMDA EPSCs at immature synapses are slow and blocked by NR2B-
to NR2A subunits is greater in the central amygdala than in the lateral amygdala. These results show that the subunit composition
Glutamate, the major excitatory transmitter in the mammalian
CNS, activates three classes of ionotropic receptors: AMPA, kai-
nate, and NMDA receptors that can be separated on biophysical
and pharmacological grounds (Dingledine et al., 1999). AMPA
and kainate receptors are functional at negative membrane po-
tentials and mediate synaptic transmission at resting membrane
potentials. In contrast, NMDA receptors require glutamate, gly-
cine, and membrane depolarization for their activation (McBain
and Mayer, 1994). These receptors are largely inactive during
involved in learning and memory (Cull-Candy et al., 2001).
NMDA receptors are heteromeric complexes assembled from
alternatively spliced gene (Nakanishi et al., 1992; Laurie and See-
tains the glycine binding site (Kuryatov et al., 1994). The NR2
subunit contains the glutamate binding site and is found as four
different isoforms encoded by different genes NR2A–NR2D
pharmacological and kinetic properties (Kutsuwada et al., 1992;
Dingledine et al., 1999; Yamakura and Shimoji, 1999). When
activated by glutamate, receptors containing NR2A subunits
have rapid offset kinetics, whereas receptors containing NR2B,
NR2C, and NR2D subunits have slower kinetics with time con-
stants of NR2B ? NR2C ? NR2D (Vicini et al., 1998). In central
neurons, NR2A and NR2B subunits are the most abundant, and
and NR2B subunits with NR1/NR2A and NR1/NR2B combina-
tions are also present (Sheng et al., 1994; Luo et al., 1997). NR2C
subunit expression is largely restricted to cerebellar granule cells,
and NR2D subunits have not been detected at synapses (Cull-
Candy et al., 2001).
NR2 subunit expression is developmentally regulated such
that at birth NR2B is the predominant subunit. NR2A subunit
expression begins around day 3, after which there is a gradual
Sheng et al., 1994; Zhong et al., 1995). With maturation, the
change in subunit composition results in EPSCs that have faster
kinetics and are markedly less sensitive to NR2B-selective antag-
onists (Carmignoto and Vicini, 1992; Hestrin, 1992; Flint et al.,
Correspondence should be addressed to Dr. Pankaj Sah, Queensland Brain Institute, School of Biomedical Sciences,
6876 • TheJournalofNeuroscience,July30,2003 • 23(17):6876–6883
receptor-mediated plasticity at glutamatergic synapses (Cull-
Candy et al., 2001; Philpot et al., 2001).
The amygdala, a complex of nuclei in the temporal lobe, has
consistently been shown to have a key role in the acquisition and
expression of fear conditioning (LeDoux, 2000; Maren, 2001).
Sensory information reaches the amygdala via the lateral amyg-
dala (LA), is processed locally, and is then transmitted to the
amus and brainstem are responsible for the behavioral responses
Sensory inputs to LA pyramidal neurons form dual component
glutamatergic synapses (Mahanty and Sah, 1999; Weisskopf and
LeDoux, 1999) that undergo long-term potentiation after fear
conditioning (Quirk et al., 1995; McKernan and Shinnick-
Gallagher, 1997). Infusion of the NMDA receptor antagonist
APV into the basolateral amygdala blocks the acquisition of fear
conditioning (Miserendino et al., 1990). It has been proposed
derlies the cellular changes that mediate associative fear condi-
tioning (LeDoux, 2000; Maren, 2001). A recent study has shown
that administration of the NR2B-specific receptor antagonist if-
sion in the amygdala are not known.
Here, we examine the composition of synaptic NMDA recep-
tors in the LA and CeA. We show that synaptic NMDA receptors
in the LA are largely NR1/NR2B multimers at birth, but with
maturation the NR2B subunits are largely replaced by receptors
containing NR2A subunits. In contrast, in the central nucleus,
NR2B multimers into adulthood. Thus, whereas all synapses ini-
tially begin with NMDA receptors that contain NR2B subunits,
they undergo different developmental changes, and the subunit
combinations present at synapses in CeA and LA are different in
tone (50 mg/kg), and coronal slices (400 ?m) were prepared using stan-
dard methods (Mahanty and Sah, 1999). For electrophysiological exper-
iments, slices were transferred to the recording chamber after a 1-hr
recovery and superfused with oxygenated (95% O2-5% CO2) physiolog-
ical solution containing (in mM): 118 NaCl, 2.5 KCl, 25 NaHCO3, 1.2
NaH2PO4, 1.3 MgCl2, 2.5 CaCl2, and 10 glucose at 33–35°C. Picrotoxin
(100 ?M) was included in the perfusing ringer to block inhibitory trans-
mission. For Western blots and real-time PCR experiments, tissue from
the lateral and central nucleus were dissected from the slices under a
the central amygdala (CeL) and LA using the blind approach. Electrodes
were filled with intracellular solution containing (mM): CsGluconate,
had a junction potential of ?13 mV, and all records have been corrected
for this. Access resistance was 7–20 M? and was monitored throughout
the experiment. Data were discarded if access resistance changed by
?10% during an experiment. Signals were recorded using an Axopatch
at 10 kHz (ITC 16; Instrutech, Port Washington, NY). Data were ac-
quired and analyzed using Axograph 4.6 (Axon Instruments). All traces
shown are averages of 10–15 sweeps.
For recordings in the central nucleus, bipolar stainless steel electrodes
(FHC Inc., Bowdoinham, ME) were placed in the basolateral complex,
and a separate stimulating electrode was placed on afferents entering the
inputs, the data between them have been pooled. For recordings in the
LA, stimulating electrodes were placed in the external capsule to activate
cortical afferents and dorsomedially to the central nucleus to activate
thalamic afferents (Mahanty and Sah, 1999; Weisskopf and LeDoux,
1999). For hippocampal recordings, pyramidal neurons were recorded
from area CA1, and a stimulating electrode was placed in the stratum
radiatum to activate Schaffer collaterals. Stimuli (50 ?s; 10–30 V) were
applied by constant–voltage-isolated stimulators (DS2A; Digitimer,
Welwyn Garden City, UK). For comparison of deactivation kinetics of
averaged, and the current decays were fitted to a double exponential
equation of the form:
decay time constants. The weighted time constant was calculated as:
?W? ?If/?If? Is???f? ?Is/?If? Is???s
For experiments testing MK-801 block, cells were held at ?30 mV in the
presence of 15 ?M CNQX, and synaptic inputs were activated every 10
sec. After a baseline of 5 min, 5 ?M MK-801 was added to the bath, and
synaptic stimulation was stopped to ensure that the drug concentration
equilibrated. After 3 min, synaptic inputs were activated every 10 sec
until MK-801 completely blocked the synaptic current. The progressive
block by MK-801 was fit to a single exponential.
Semiquantitative real time PCR. Total RNA was prepared using Trizol
(Invitrogen, San Diego, CA) and reverse transcribed using a RT-PCR kit
system (Corbett Research, Mortlake, Australia) and Quantitec
SYBRGreen PCR kit (Qiagen, Hilden, Germany). Primers used were
as follows: NR2A (CAATCTGACTGGATCACAGAGC), (CTGTCCT-
(CCGGGGTTGTTGTGGTGGTGTC). No other products were ampli-
fied because melting curves showed only one peak in each sample. Fluo-
rescence signals were measured over 50 PCR cycles. The cycle number
(Ct) at which the signals crossed a threshold set within the logarithmic
in cycle threshold between NR2A and NR2B subunits. A ?Ctof ?1
of both primers was tested by serial dilutions of the RNA for both sub-
units and plotting ?Ctagainst log(total RNA). The slope of this line was
linear (slope ? 0.0371; r2? 0.956), showing that the efficiencies of the
two sets of primers were similar.
Western blot. Tissue from the LA and CeA was homogenized in a lysis
buffer containing 5% SDS, 0.01% Bromophenol Blue, 8 M Urea, 0.1 mM
EDTA, and 40 mM Tris, pH 6.8. Proteins were separated by SDS-PAGE
(4–12% gradient gel; Invitrogen) and electrophoretically transferred to
an Immobilon-P membrane (Millipore, Bedford, MA). Blots were incu-
bated with primary antibody (1:1000 rabbit polyclonal antibodies anti-
radiograms were analyzed and quantified using National Institutes of
Health image 1.6. Western blots were done independently from four
animals using CeA and LA tissue from three slices in each animal.
Drugs used were CNQX, D-APV (Tocris Cookson, Bristol, UK), ifen-
prodil, MK-801, and picrotoxin (Sigma-Aldrich). All values are ex-
pressed as mean ? SEM, and statistical comparisons were done using
two-tailed t tests or ANOVA, with significance set at p ? 0.05.
Whole-cell recordings were made from neurons in the LA and in
LopezdeArmentiaandSah•NMDAReceptorsinAmygdalaNeuronsJ.Neurosci.,July30,2003 • 23(17):6876–6883 • 6877
in the central nucleus were made from the
lateral subdivision (CeL) (Cassell et al.,
tial of ?70 mV, stimulation of afferents
evoked a rapidly rising and decaying syn-
aptic current. Depolarization of the
postsynaptic cell revealed a slowly decay-
1A). The current–voltage relationship
(I–V) of the fast component was linear
for AMPA receptor-mediated currents,
whereas the I–V of the slower component
showed a region of negative slope resis-
tance (Fig. 1B), typical for currents medi-
ated by NMDA receptors (Hestrin et al.,
1990). The fast component was selectively
onist CNQX, whereas the slower compo-
nent was blocked by the NMDA receptor
antagonist D-APV (Fig. 1C). Thus, excita-
tory inputs to neurons in the CeL activate
typical dual component glutamatergic
In recordings from animals aged P21–
P30, the AMPA receptor-mediated EPSC
in CeL neurons had a 10–90% rise time of
4.4 ? 0.3 msec (n ? 20). The NMDA receptor component of the
EPSC had a 10–90% rise time of 5.0 ? 0.1 msec (n ? 27). The
decay of the EPSC (measured at ?30 mV) was best fit by two
exponentials with time constants, ?fast? 95 ? 14 msec and ?slow
? 253 ? 29 msec, with the amplitude of the fast component
the LA also activate dual component glutamatergic synapses
(Mahanty and Sah, 1999; Weisskopf and LeDoux, 1999). The
10–90% rise time of the NMDA receptor current in LA neurons
was 5.1 ? 0.2 msec (n ? 39), and the decay was best fit with two
exponentials with a ?fast? 35 ? 3 msec and ?slow? 190 ? 11
msec, the amplitude of the fast component being 65 ? 2% (n ?
rons was similar for both cortical (84 ? 4 msec; n ? 34) and
thalamic (86 ? 6 msec; n ? 19) inputs. These values are signifi-
cantly faster ( p ? 0.001) than those obtained in CeL neurons
(Fig. 1D), suggesting a different synaptic NMDA receptor sub-
unit composition between the two nuclei of the amygdala.
current in adult CeL neurons suggests that these receptors pref-
erentially contain NR2B subunits (Flint et al., 1997; Vicini et al.,
1998; Tovar and Westbrook, 1999). We next tested the effects of
the selective NR2B antagonists (1S, 2S)-1-(4-hydroxyphenyl)-
and ifenprodil (Williams, 1993; Chenard and Menniti, 1999) on
the amplitude of the evoked EPSC by 68 ? 5% (n ? 9); similar
results were seen with ifenprodil (10 ?M; 64 ? 7%; n ? 6). This
cini et al., 1998; Tovar and Westbrook, 1999), indicating that
units. The kinetics of the EPSC in the presence of the NR2B
blockers was not different from that recorded in control condi-
tions (Fig. 2A2), consistent with the presence of a single popula-
tion of NR1/NR2B-containing receptors at these synapses. In
CP-101,606 or ifenprodil reduced the evoked EPSC by 34 ? 4%
(n ? 9; Fig. 2B) and 36 ? 19% (n ? 3), respectively. Unlike CeL
EPSC in the presence of CP-101,606 (Fig. 2B) or ifenprodil. The
contribution of the fast component of the EPSC increased from
were not because of a reduction in transmitter release, because
neither CP-101,606 (Fig. 2B, inset) nor ifenprodil (data not
shown) had any effects on AMPA receptor-mediated EPSCs.
Together, these results indicate that NMDA receptors at syn-
apses on CeL neurons contain largely NR2B subunits, whereas
the contribution of NR2B subunits to NMDA receptor synapses
in the LA is much lower (Flint et al., 1997; Vicini et al., 1998;
Tovar and Westbrook, 1999). To confirm this finding, we tested
for differences in the expression levels of NR2A and NR2B sub-
units between the two nuclei. We first tested for differences in
mRNA levels for the two subunits using semiquantitative real-
time PCR. The difference in cycle threshold between NR2A and
NR2B subunits (?Ct; see “Materials and Methods”) for the cen-
tral nucleus was 1.1 ? 1.7 as compared with ?3.4 ? 0.8 (n ? 3)
more abundant than NR2A mRNA in the central nucleus,
whereas NR2A mRNA is more abundant in the lateral nucleus.
Consistent with the PCR results, Western blots also showed that
CeA as compared with the LA (Fig. 3C).
rons, NMDA receptors have a subunit composition similar to
that seen at immature synapses, whereas at synapses in the LA,
mental change in subunit composition, as has been described in
decay is much faster in LA neurons. The histogram shows the average weighted decay time constants (see “Materials and
6878 • J.Neurosci.,July30,2003 • 23(17):6876–6883LopezdeArmentiaandSah•NMDAReceptorsinAmygdalaNeurons
other forebrain regions (Carmignoto and Vicini, 1992; Hestrin,
1992; Monyer et al., 1994; Flint et al., 1997). In recordings from
LA neurons in P5 animals, the NMDA receptor-mediated EPSC
was similar to that recorded from adult CeL neurons and had a
weighted decay time constant of 151 ? 16 msec (n ? 8; Fig.
4A,B). Application of CP-101,606 (5 ?M) blocked the EPSC by
77 ? 7% (n ? 3), showing that synaptic NMDA receptors in the
LA in P5 animals contain NR2B subunits. In contrast, NMDA
neurons from 3- to 4-week-old animals (compare Fig. 1), had a
weighted decay constant of 90 ? 10 msec (n ? 4), and CP-
101,606 blocked them by 39 ? 15% (n ?
3) (Fig. 4B). To check whether the slow
decay of EPSCs in CeL neurons in P21 an-
imals could be because of a delayed matu-
ration of these synapses, NMDA receptor-
mediated EPSCs in the central nucleus
were also recorded from P80 animals.
These EPSCs had a weighted decay time
to that in P21 animals (Fig. 4C). Thus,
NMDA receptors in CeL neurons do not
change with maturation, whereas those in
the LA pyramidal neurons do.
It has recently been shown that at de-
velopmentally immature synapses, where
units, transmitter release probability is
high (Chavis and Westbrook, 2001). With
development, as NR2B subunits are re-
placed by NR2A subunits, there is a con-
comitant reduction in release probability
(Chavis and Westbrook, 2001). Because
their immature phenotype with regard to
the subunit composition of synaptic
NMDA receptors, we tested whether these
synapses would also display a higher re-
lease probability as compared with inputs
to LA neurons. One measure of the probability of glutamate re-
lease is the progressive block of synaptic NMDA currents by the
irreversible NMDA open-channel blocker MK-801 (Huettner
ulation in the presence of MK-801, synapses with high-release
ability (Rosenmund et al., 1993; Chavis and Westbrook, 2001).
We tested the rate of progressive block of synaptic currents by
MK-801 in LA and CeL neurons. In both the CeL and LA, the
block of EPSCs by MK-801 was well fit with a single exponential,
with similar release probabilities. The time constant of MK-801
block at synapses on CeL neurons was 23 ? 3 stimuli (n ? 7).
This is significantly faster ( p ? 0.05) than at inputs to LA neu-
rons, in which the time constant was 48 ? 7 stimuli (n ? 8; Fig.
4D). This result is consistent with the release probability being
significantly higher at inputs to the central nucleus as compared
with inputs to neurons in the LA.
As described previously, the presence of synaptic NMDA re-
ceptors that predominantly have NR2B subunits results in
NMDA receptor EPSCs with much slower kinetics. To test
whether the slower decay kinetics impact on temporal summa-
compared the effects of repetitive stimulation at synapses in the
LA and CeL neurons. As shown in Figure 5, repetitive activation
of synapses in CeL neurons led to much larger summation of the
NMDA EPSC compared with inputs to the LA.
These results show that, as in other parts of the forebrain,
NMDA receptors at synapses on pyramidal neurons in the LA
undergo a developmental change from those containing NR2B
subunits to those containing largely NR2A subunits. Expressed
NMDA receptors that contain NR1/NR2A subunits are not af-
fected by NR2B antagonists (Williams et al., 1993; Vicini et al.,
1998; Tovar and Westbrook, 1999). However, at synapses in the
picrotoxin. Application of 5 ?M CP-101,606 partially blocks the EPSC, and the remaining EPSC is blocked by D-APV (30 ?M),
NMDA receptors at synapses in CeA neurons contain NR2B subunits. A1, Normalized amplitude of NMDA EPSCs
LopezdeArmentiaandSah•NMDAReceptorsinAmygdalaNeuronsJ.Neurosci.,July30,2003 • 23(17):6876–6883 • 6879
LA, the NMDA receptor component is partially blocked by CP-
101,606 (Fig. 2). This result suggests that receptors containing
NR2B subunits are present at mature synapses in the LA. In hip-
are thought to contain largely NR2A subunits (Tovar and West-
at hippocampal pyramidal neurons with those in the LA. The
kinetics of the NMDA receptor-mediated EPSC at Schaffer col-
lateral synapses in area CA1 was similar to the kinetics in LA
neurons (Fig. 6A). EPSCs recorded in CA1 pyramidal neurons
that in LA neurons (Fig. 6A). However, unlike in LA neurons,
neither CP-101,606 (5 ?M; n ? 6) nor ifenprodil (10 ?M; n ? 7)
had any effect on the NMDA EPSC in CA1 pyramidal neurons
because of problems of access of these compounds in hippocam-
pal slices, because application of these compounds to EPSCs in
slices from P5 animals (n ? 2; Fig. 6B) and the response to
exogenously applied NMDA in adult slices (n ? 2; data not
shown) showed a robust blockade by CP-101,606 of 80%. Thus,
as in visual cortical neurons (Stocca and Vicini, 1998) and cul-
tured hippocampal neurons (Tovar and Westbrook, 1999),
NMDA receptors at Schaffer collateral synapses in the adult hip-
pocampus express NMDA receptors that are insensitive to
NR2B-selective antagonists. Receptors containing NR1/NR2B
subunits are present on mature neurons but are extrasynaptic.
At most glutamatergic synapses, the abundant NR2B subunits
present at birth are gradually replaced by NR2A subunits during
development (Carmignoto and Vicini, 1992; Hestrin, 1992; Flint
et al., 1997; Stocca and Vicini, 1998). Thus, at newly formed
synapses, NMDA receptors largely consist of NR1/NR2B sub-
from neurons in the CeL at P14 and P80. D, Release probability is higher at NR2B-containing
rons as compared with LA neurons. A, NMDA receptor-mediated EPSCs recorded at ?30 mV
netically similar to those in LA neurons, but are pharmacologically different. A, Normalized
NMDA EPSCs recorded in slices from P21–P30 rats from CA1 pyramidal neurons and an EPSC
subunits. Block of the NMDA receptor-mediated EPSC by the NR2B-selective antagonist CP-
(top traces) and P5 animals (lower traces). CP-101,606 has no effect on EPSCs at mature
6880 • J.Neurosci.,July30,2003 • 23(17):6876–6883LopezdeArmentiaandSah•NMDAReceptorsinAmygdalaNeurons
units. At mature synapses, NR2A-containing receptors are tar-
geted to synapses, whereas receptors containing only NR2B sub-
units are largely excluded from the postsynaptic membrane
tal profile, we have shown that in pyramidal neurons of the LA,
developmentally immature synapses express NMDA receptors
composed of NR1/NR2B subunits. With development, there is a
both NR2A and NR2B subunits. In contrast, at synapses on CeL
neurons, there is no developmental change, and NR2B-
containing receptors present at immature synapses are main-
tained into adulthood. This lack of change in the physiology of
synaptic currents is consistent with the higher expression level of
NR2B subunits in the central nucleus. Furthermore, the higher-
release probability seen at synapses in the CeL suggests that these
synapses maintain their immature phenotype into adulthood
(Chavis and Westbrook, 2001).
In agreement with the relative abundance of NR2B subunits
early in development, coexpression of NR1 and NR2B subunits
results in NMDA receptors that have kinetics and pharmacolog-
ical properties similar with those of NMDA receptor EPSCs at
immature synapses. As for the EPSCs, when activated by gluta-
mate, NR1/NR2B-containing receptors have slow offset kinetics
and are significantly blocked by NR2B-selective antagonists (Vi-
cini et al., 1998; Tovar and Westbrook, 1999). The presence of
NR2A subunits accelerates the offset kinetics and reduces their
sensitivity to NR2B-selective antagonists (Vicini et al., 1998; To-
var and Westbrook, 1999). At inputs onto CeL neurons, because
onists changes with development, it is clear that NR2A subunits
contrast, at synapses on pyramidal neurons in the LA, NMDA
EPSCs at immature synapses have properties consistent with
those of receptors containing NR1/NR2B subunits. However,
with development, these EPSCs have faster decay kinetics and a
cating that at mature synapses in LA neurons NMDA receptors
contain NR2A subunits (Flint et al., 1997; Stocca and Vicini,
1998; Tovar and Westbrook, 1999).
For hippocampal synapses in culture, the developmental
change in NMDA subunit composition is accompanied with a
reduction in release probability (Chavis and Westbrook, 2001).
Consistent with the presence of abundant NR2B subunits at syn-
apses on CeL neurons, the release probability at these synapses
seems to be higher than that at synapses in the LA. Release prob-
ability was assessed by progressive block by MK-801 of the
NMDA EPSC (Huettner and Bean, 1988; Rosenmund et al.,
of the NMDA receptor (Rosenmund et al., 1993; Chen et al.,
1999). As compared with receptors containing NR1/NR2B sub-
significantly higher peak open probability (Chen et al., 1999; for
review, see Prybylowski et al., 2002). This result indicates that at
terminals with similar release probabilities, the rate of MK-801
block at synapses containing NR2B subunits should be slower
than at synapses that contain predominantly NR2A subunits.
Thus, our finding of a faster block of the EPSC in CeL neurons
(that contain NR2B subunits) is likely to have been underesti-
mated and is consistent with these synapses maintaining a more
noted, however, that comparisons of channel open probability
have only been done on expressed receptors containing either
taining both subunits has not been determined. If the receptors
present at synapses in the LA consist of heterotrimers (NR1/
NR2A/NR2B; see below), it remains possible that the slower
block by MK-801 results from a difference in open probability.
At mature synapses in the LA, NMDA receptor EPSCs have
sistent with the presence of NR2A subunits (Flint et al., 1997;
Stocca and Vicini, 1998; Tovar and Westbrook, 1999). However,
unlike in the hippocampus in which the NMDA EPSC is insensi-
tive to NR2B-selective antagonists (Fig. 6), EPSCs in LA pyrami-
dal neurons are partially blocked by NR2B-selective antagonists
ically similar to EPSCs in hippocampal neurons, are sensitive to
NR2B-selective antagonists whereas those in the hippocampus
At other central synapses, the developmental change from
NR2B to NR2A subunits results in NMDA receptor EPSCs that
have faster kinetics and are insensitive to NR2B antagonists
(Stocca and Vicini, 1998; Rumbaugh and Vicini, 1999), consis-
tent with the presence of NMDA receptors that contain, exclu-
we have shown that at synapses in the adult hippocampus,
NMDA receptor-mediated EPSCs have rapid kinetics and are
insensitive to NR2B-selective antagonists. However, expression
et al., 1998; Tovar et al., 2000). Experiments with recombinant
NMDA receptors expressed in isolated cell systems have shown
that coexpression of NR1 subunits with both NR2A and NR2B
subunits results in functional receptors that share kinetic and
tors. Whereas some of these properties result from the presence
of both NR1/NR2A and NR1/NR2B subunits, it has been sug-
NR1/NR2A/NR2B subunits (Stocca and Vicini, 1998; Tovar and
Westbrook, 1999; Prybylowski et al., 2002). In agreement with
this suggestion, biochemical studies have also shown that recep-
tors containing both NR2A and NR2B subunits are present in
central neurons (Sheng et al., 1994; Chazot and Stephenson,
1997; Luo et al., 1997). Although one study suggested that these
The exact subunit composition of functional NMDA recep-
tors has been much debated. At least two molecules of glutamate
and two of glycine are required for NMDA receptor activation
(Benveniste and Mayer, 1991; Clements and Westbrook, 1991),
so at least four subunits must co-assemble in the functional re-
receptors could be either pentamers or tetramers containing ei-
ther two or three NR1 subunits and two or three NR2 subunits
(Behe et al., 1995; Premkumar and Auerbach, 1997; Laube et al.,
1998; Hawkins et al., 1999; Schorge and Colquhoun, 2003). If
two NR2 subunits (Behe et al., 1995; Laube et al., 1998; Schorge
and Colquhoun, 2003), then our data are consistent with the
properties of expressed NR1/NR2A receptors seem to be too
rapid to explain the kinetics of the EPSC recorded at synapses in
LopezdeArmentiaandSah•NMDAReceptorsinAmygdalaNeurons J.Neurosci.,July30,2003 • 23(17):6876–6883 • 6881
either the hippocampus or LA. Thus, receptors at synapses on
pyramidal neurons in the LA and those in hippocampal CA1
sensitivity of the EPSC to NR2B-selective antagonists at synapses
in the LA suggests the additional presence of NR1/NR2B-
containing receptors at these synapses. However, this possibility
is difficult to reconcile with the kinetic similarities between EP-
SCs in the LA and those recorded from hippocampal CA1 neu-
rons and the fact that the kinetics of the slow component of the
subunits expressed alone (Vicini et al., 1998). Thus, the exact
composition of the synaptic NMDA receptors in LA neurons
remains to be elucidated. It is notable that although expressed
NMDA receptors that lack NR2B subunits are insensitive to low
small block by ifenprodil (Tovar et al., 2000). It is, therefore,
conceivable that other factors besides the presence of NR2B sub-
pounds, explaining the difference in sensitivity of the EPSC to
ifenprodil and CP-101,606 between hippocampal neurons and
those in the LA.
the fear conditioning (LeDoux, 2000; Davis and Whalen, 2001).
A converging body of literature over the last 15 years has sug-
gested that activation of NMDA receptors within the basolateral
amygdala is essential in the acquisition, and perhaps the mainte-
nance, of fear conditioning (Blair et al., 2001; Walker and Davis,
2002). Recent data have shown that infusion of the selective
sition of fear conditioning, and it has been suggested that the site
of action of ifenprodil is in the LA (Rodrigues et al., 2001). Our
subunits in the central nucleus suggest that another likely site of
action of ifenprodil for this effect is in the CeA. Interestingly,
in the lateral nucleus (Swanson and Petrovich, 1998; Puelles,
2001). Thus, it is seems possible that glutamatergic synapses on
neurons with different embryonic origins may have different de-
Behe P, Stern P, Wyllie DJ, Nassar M, Schoepfer R, Colquhoun D (1995)
Determination of NMDA NR1 subunit copy number in recombinant
NMDA receptors. Proc R Soc Lond B Biol Sci 262:205–213.
Benveniste M, Mayer ML (1991) Kinetic analysis of antagonist action at
and glycine. Biophys J 59:560–573.
Blair HT, Schafe GE, Bauer EP, Rodrigues SM, LeDoux JE (2001) Synaptic
plasticity in the lateral amygdala: a cellular hypothesis of fear condition-
ing. Learn Mem 8:229–242.
Brimecombe JC, Boeckman FA, Aizenman E (1997) Functional conse-
aspartate receptors. Proc Natl Acad Sci USA 94:11019–11024.
Carmignoto G, Vicini S (1992) Activity-dependent decrease in NMDA re-
ceptor responses during development of the visual cortex. Science
CassellMD,GrayTS,KissJZ (1986) Neuronalarchitectureintheratcentral
nucleus of the amygdala: a cytological, hodological, and immunocyto-
chemical study. J Comp Neurol 246:478–499.
Chavis P, Westbrook G (2001) Integrins mediate functional pre- and
postsynaptic maturation at a hippocampal synapse. Nature 411:317–321.
Chazot PL, Stephenson FA (1997) Molecular dissection of native mamma-
onstration of NMDA receptors comprising NR1, NR2A, and NR2B sub-
units within the same complex. J Neurochem 69:2138–2144.
ChenN,LuoT,RaymondLA (1999) Subtype-dependenceofNMDArecep-
tor channel open probability. J Neurosci 19:6844–6854.
Chenard BL, Menniti FS (1999) Antagonists selective for NMDA receptors
containing the NR2B subunit. Curr Pharm Des 5:381–404.
Clements JD, Westbrook GL (1991) Activation kinetics reveal the number
of glutamate and glycine binding sites on the N-methyl-D-aspartate re-
ceptor. Neuron 7:605–613.
Cull-Candy S, Brickley S, Farrant M (2001) NMDA receptor subunits: di-
versity, development and disease. Curr Opin Neurobiol 11:327–335.
DavisM,WhalenPJ (2001) Theamygdala:vigilanceandemotion.MolPsy-
DingledineR,BorgesK,BowieD,TraynelisSF (1999) Theglutamaterecep-
tor ion channels. Pharmacol Rev 51:7–61.
Flint AC, Maisch US, Weishaupt JH, Kriegstein AR, Monyer H (1997)
NR2A subunit expression shortens NMDA receptor synaptic currents in
developing neocortex. J Neurosci 17:2469–2476.
Hawkins LM, Chazot PL, Stephenson FA (1999) Biochemical evidence for
the co-association of three N-methyl-D-aspartate (NMDA) R2 subunits
in recombinant NMDA receptors. J Biol Chem 274:27211–27218.
Hestrin S (1992) Developmental regulation of NMDA receptor-mediated
synaptic currents at a central synapse. Nature 357:686–689.
HestrinS,NicollRA,PerkelDJ,SahP (1990) Analysisofexcitatorysynaptic
J Physiol (Lond) 422:203–225.
Huettner JE, Bean BP (1988) Block of N-methyl-D-aspartate-activated cur-
rent by the anticonvulsant MK-801: selective binding to open channels.
Proc Natl Acad Sci USA 85:1307–1311.
Kew JN, Richards JG, Mutel V, Kemp JA (1998) Developmental changes in
NMDA receptor glycine affinity and ifenprodil sensitivity reveal three
distinct populations of NMDA receptors in individual rat cortical neu-
rons. J Neurosci 18:1935–1943.
Kuryatov A, Laube B, Betz H, Kuhse J (1994) Mutational analysis of the
glycine-binding site of the NMDA receptor: structural similarity with
bacterial amino acid-binding proteins. Neuron 12:1291–1300.
Kutsuwada T, Kashiwabuchi N, Mori H, Sakimura K, Kushiya E, Araki K,
Meguro H, Masaki H, Kumanishi T, Arakawa M, Mishina M (1992)
Molecular diversity of the NMDA receptor channel. Nature 358:36–41.
Laube B, Kuhse J, Betz H (1998) Evidence for a tetrameric structure of re-
combinant NMDA receptors. J Neurosci 18:2954–2961.
LaurieDJ,SeeburgPH (1994) Regionalanddevelopmentalheterogeneityin
splicing of the rat brain NMDAR1 mRNA. J Neurosci 14:3180–3194.
LeDoux JE (2000) Emotion circuits in the brain. Annu Rev Neurosci
Luo J, Wang Y, Yasuda RP, Dunah AW, Wolfe BB (1997) The majority of
N-methyl-D-aspartate receptor complexes in adult rat cerebral cortex
contain at least three different subunits (NR1/NR2A/NR2B). Mol Phar-
MahantyNK,SahP (1999) Excitatorysynapticinputstopyramidalneurons
of the lateral amygdala. Eur J Neurosci 11:1217–1222.
Maren S (2001) Neurobiology of Pavlovian fear conditioning. Annu Rev
McBain CJ, Mayer ML (1994) NMDA receptor structure and fucntion.
Physiol Rev 74:723–760.
McKernan MG, Shinnick-Gallagher P (1997) Fear conditioning induces a
lasting potentiation of synaptic currents in vitro. Nature 390:607–611.
Miserendino MJD, Sananes CB, Melia KR, Davis M (1990) Blocking of ac-
quisition but not expression of conditioned fear-potentiated startle by
NMDA antagonists in the amygdala. Nature 345:716–718.
MonyerH,BurnashevN,LaurieDJ,SakmannB,SeeburgPH (1994) Devel-
ties of four NMDA receptors. Neuron 12:529–540.
Nakanishi N, Axel R, Shneider NA (1992) Alternative splicing generates
functionally distinct N-methyl-D-aspartate receptors. Proc Natl Acad Sci
PhilpotBD,SekharAK,ShouvalHZ,BearMF (2001) Visualexperienceand
deprivation bidirectionally modify the composition and function of
NMDA receptors in visual cortex. Neuron 29:157–169.
Premkumar LS, Auerbach A (1997) Stoichiometry of recombinant
N-methyl-D-aspartate receptor channels inferred from single-channel
current patterns. J Gen Physiol 110:485–502.
6882 • J.Neurosci.,July30,2003 • 23(17):6876–6883LopezdeArmentiaandSah•NMDAReceptorsinAmygdalaNeurons
Prybylowski K, Fu Z, Losi G, Hawkins LM, Luo J, Chang K, Wenthold RJ,
Vicini S (2002) Relationship between availability of NMDA receptor
subunits and their expression at the synapse. J Neurosci 22:8902–8910.
Puelles L (2001) Thoughts on the development, structure and evolution of
B Biol Sci 356:1583–1598.
Quirk GJ, Repa C, LeDoux JE (1995) Fear conditioning enhances short-
ings in the freely behaving rat. Neuron 15:1029–1039.
Rodrigues SM, Schafe GE, LeDoux JE (2001) Intra-amygdala blockade of
the expression of fear conditioning. J Neurosci 21:6889–6896.
LR (2003) The measurement of adenosine and estrogen receptor ex-
ysis. Brain Res Brain Res Protoc 11:9–18.
Rosenmund C, Clements JD, Westbrook GL (1993) Nonuniform probabil-
ity of glutamate release at a hippocampal synapse. Science 262:754–757.
Rumbaugh G, Vicini S (1999) Distinct synaptic and extrasynaptic NMDA
receptors in developing cerebellar granule neurons. J Neurosci
Schorge S, Colquhoun D (2003) Studies of NMDA receptor function and
stoichiometry with truncated and tandem subunits. J Neurosci
Sheng M, Cummings J, Roldan LA, Jan YN, Jan LY (1994) Changing sub-
unit composition of heteromeric NMDA receptors during development
of rat cortex. Nature 368:144–147.
Stocca G, Vicini S (1998) Increased contribution of NR2A subunit to syn-
aptic NMDA receptors in developing rat cortical neurons. J Physiol
SwansonLW,PetrovichGD (1998) Whatistheamygdala?TrendsNeurosci
Tovar KR, Westbrook GL (1999) The incorporation of NMDA receptors
with a distinct subunit composition at nascent hippocampal synapses in
vitro. J Neurosci 19:4180–4188.
Tovar KR, Sprouffske K, Westbrook GL (2000) Fast NMDA receptor-
mediated synaptic currents in neurons from mice lacking the epsilon2
(NR2B) subunit. J Neurophysiol 83:616–620.
(1998) Functional and pharmacological differences between recombi-
nant N-methyl-D-aspartate receptors. J Neurophysiol 79:555–566.
Walker DL, Davis M (2002) The role of amygdala glutamate receptors in
fear learning, fear-potentiated startle, and extinction. Pharmacol Bio-
chem Behav 71:379–392.
WeisskopfMG,LeDouxJE (1999) DistinctpopulationsofNMDAreceptors
J Neurophysiol 81:930–934.
Williams K (1993) Ifenprodil discriminates subtypes of the N-methyl-D-
aspartate receptor: selectivity and mechanisms at recombinant hetero-
meric receptors. Mol Pharmacol 44:851–859.
Williams K, Russell SL, Shen YM, Molinoff PB (1993) Developmental
switch in the expression of NMDA receptors occurs in vivo and in vitro.
Yamakura T, Shimoji K (1999) Subunit- and site-specific pharmacology of
the NMDA receptor channel. Prog Neurobiol 59:279–298.
Zhong J, Carrozza DP, Williams K, Pritchett DB, Molinoff PB (1995) Ex-
pression of mRNAs encoding subunits of the NMDA receptor in devel-
oping rat brain. J Neurochem 64:531–539.
LopezdeArmentiaandSah•NMDAReceptorsinAmygdalaNeuronsJ.Neurosci.,July30,2003 • 23(17):6876–6883 • 6883