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Cellular/Molecular
A Pathway-Specific Function for Different AMPA Receptor
Subunits in Amygdala Long-Term Potentiation and
Fear Conditioning
Yann Humeau,
1,2
* Daniel Reisel,
3
* Alexander W. Johnson,
4
* Thilo Borchardt,
5
Vidar Jensen,
6
Christine Gebhardt,
1
Verena Bosch,
5
Peter Gass,
7
David M. Bannerman,
3
Mark A. Good,
4
Øivind Hvalby,
6
Rolf Sprengel,
5
and Andreas Lu¨thi
1
1
Friedrich Miescher Institute for Biomedical Research, CH-4058 Basel, Switzerland,
2
Unite´ Mixte de Recherche 7168, Centre National de la Recherche
Scientifique, F-67084 Strasbourg, France,
3
Department of Experimental Psychology, University of Oxford, Oxford OX1 3UD, United Kingdom,
4
School of
Psychology, Cardiff University, Cardiff CF10 3YG, United Kingdom,
5
Department of Molecular Neurobiology, Max Planck Institute for Medical Research,
D-69120 Heidelberg, Germany,
6
Molecular Neurobiology Research Group, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway, and
7
Central Institute of Mental Health Mannheim, J5, D-68159 Mannheim, Germany
The AMPA receptor subunit glutamate receptor 1 (GluR1 or GluR-A) contributes to amygdala-dependent emotional learning. It remains
unclear, however, to what extent different amygdala pathways depend on GluR1, or other AMPA receptor subunits, for proper synaptic
transmission and plasticity, and whether GluR1-dependent long-term potentiation (LTP) is necessary for auditory and contextual fear
conditioning. Here, we dissected the role of GluR1 and GluR3 (GluR-C) subunits in AMPA receptor-dependent amygdala LTP and fear
conditioning using knock-out mice (GluR1
⫺/⫺
and GluR3
⫺/⫺
). We found that, whereas LTP at thalamic inputs to lateral amygdala (LA)
projection neurons andat glutamatergic synapses in the basal amygdala was completely absent in GluR1
⫺/⫺
mice, both GluR1 and GluR3
contributed to LTP in the cortico-LA pathway. Because both auditory and contextual fear conditioning were selectively impaired in
GluR1
⫺/⫺
but not GluR3
⫺/⫺
mice, we conclude that GluR1-dependent synaptic plasticity is the dominant form of LTP underlying the
acquisition of auditory and contextual fear conditioning, and that plasticity in distinct amygdala pathways differentially contributes to
aversive conditioning.
Key words: lateral amygdala; basal amygdala; fear conditioning; synaptic transmission; LTP; GluR1; GluR3
Introduction
One basic mechanism that is thought to be involved in memory
formation is synaptic plasticity mediated by AMPA receptors
containing the glutamate receptor 1 (GluR1) subunit (Zamanillo
et al., 1999; Reisel et al., 2002). Mice lacking the GluR1 subunit
both fail to express hippocampal CA3-to-CA1 long-term poten-
tiation (LTP) and exhibit impaired spatial working memory.
However, both LTP and spatial working memory are restored on
selective reexpression of GluR1 in hippocampal pyramidal neu-
rons (Mack et al., 2001; Schmitt et al., 2005). GluR1 is involved in
many forms of activity-dependent synaptic plasticity in brain
regions other than the hippocampus (Malinow and Malenka,
2002). However, the existence of GluR1-independent forms of
LTP (Hoffman et al., 2002; Jensen et al., 2003) indicates that other
subunit combinations, such as GluR2/3 (GluR-B/C), may also
play an important role (Malinow and Malenka, 2002) (but see
Meng et al., 2003).
Here, we studied the function of GluR1 and GluR3 in projec-
tion neurons in two nuclei of the basolateral amygdaloid complex
(BLA), the lateral (LA) and the basal (BA) amygdala. The BLA
plays an important role during the formation of a Pavlovian as-
sociation between a conditioned stimulus (CS) and an aversive
unconditioned stimulus (US) (LeDoux, 2000; Davis and Whalen,
2001; Maren, 2001). Whereas the LA is thought to represent the
site of convergence between a discrete auditory CS and the US
during auditory fear conditioning (Romanski and LeDoux, 1992;
Nader et al., 2001), the BA, by means of its strong anatomical and
functional interactions with the hippocampal formation (Maren
and Fanselow, 1995; Pitkanen et al., 2000), may constitute an
important substrate integrating BLA and hippocampus-
dependent context–US associations (Kim and Fanselow, 1992;
Phillips and LeDoux, 1992; Calandreau et al., 2005).
In vivo and in vitro electrophysiological studies have identified
cellular correlates of auditory fear conditioning, such as NMDA
receptor-dependent LTP (Maren and Quirk, 2004; Sigurdsson et
al., 2007). The mechanisms underlying amygdala LTP have been
studied most extensively in the thalamo- and cortico-LA path-
ways (Sigurdsson et al., 2007), where both GluR1/2 and GluR2/3
subunit combinations are thought to be expressed (Farb and Le-
Received June 7, 2007; revised Aug. 22, 2007; accepted Aug. 26, 2007.
This work was supported by the Volkswagen Foundation, Centre National de la Recherche Scientifique, and the
Novartis Research Foundation. We thank G. Casassus for helpful discussions and comments on this manuscript, and
Peter H. Seeburg for his generous support.
*Y.H., D.R., and A.W.J. contributed equally to this work.
Correspondence should be addressed to Andreas Lu¨thi, Friedrich Miescher Institute for Biomedical Research,
Maulbeerstrasse 66, CH-4058 Basel, Switzerland. E-mail: andreas.luthi@fmi.ch.
DOI:10.1523/JNEUROSCI.2603-07.2007
Copyright © 2007 Society for Neuroscience 0270-6474/07/2710947-10$15.00/0
The Journal of Neuroscience, October 10, 2007 • 27(41):10947–10956 • 10947
Doux, 1997, 1999; Radley et al., 2007). Whereas in vivo studies
indicate that the thalamo-amygdala pathway is rapidly potenti-
ated during the acquisition of conditioned fear (Quirk et al.,
1995, 1997), the relative contribution of these two pathways to
the acquisition of auditory fear conditioning is still a matter of
debate. Furthermore, a recent study has demonstrated, using vi-
ral transfection techniques, that disrupting GluR1 trafficking
blocks thalamo-amygdala LTP, but only partially reduces the ac-
quisition of auditory fear conditioning (Rumpel et al., 2005). We
therefore examined the contribution of GluR1 and GluR3 to syn-
aptic transmission and plasticity in different amygdala pathways.
Although our study reveals pathway-specific involvements of
GluR1- and GluR3-dependent mechanisms to glutamatergic
synaptic transmission and plasticity, the former appears to dom-
inate during auditory and contextual fear conditioning.
Materials and Methods
Gene-targeted mice
The gene-targeted mouse lines for the GluR1 (GluR-A) and GluR3
(GluR-C) knock-out were generated in the laboratory of R. Sprengel and
were published and described in detail by Zamanillo et al. (1999) and
Sanchis-Segura et al. (2006), respectively. In brief, in both targeting
events mouse R1-embryonic stem cells (Nagy et al., 1993) were used to
manipulate the Gria1 and Gria3 alleles. R1-embryonic stem cell clones
that were identified by Southern blot as being positive for the targeting
events were injected into C57BL/6/N blastocysts to produce chimeric
animals. The chimeric male mice were then backcrossed for more than
six generations with C57BL/6/N animals. Both the GluR1 and the GluR3
knock-out mice are kept as a heterozygous line. For individual experi-
ments, the colonies of wild-type (WT) and knock-out mice were simul-
taneously produced by mating of heterozygous mice.
Whole-cell recordings
Standard procedures were used to prepare 350- to 400-
m-thick coronal
slices from 4- to 5-week-old male wild-type, GluR1
⫺/⫺
, and GluR3
⫺/⫺
mice following a protocol approved by the Veterinary Department of the
Canton of Basel-Stadt (Humeau et al., 2003). Briefly, the brain was dis-
sected in ice-cold artificial CSF (ACSF), mounted on an agar block, and
sliced with a vibratome (Leica VT 1000; Leica, Wetzlar, Germany) at 4°C.
Slices were maintained for 45 min at 35°C in an interface chamber con-
taining ACSF equilibrated with 95% O
2
/5% CO
2
and containing the
following (in m
M): 124 NaCl, 2.7 KCl, 2 CaCl
2
, 1.3 MgCl
2
, 26 NaHCO
3
,
0.4 NaH
2
PO
4
, 18 glucose, 4 ascorbate, and then for at least 45 min at
room temperature before being transferred to a superfusing recording
chamber. Whole-cell recordings from LA projection neurons were per-
formed at 30 –32°C. Neurons were visually identified with infrared
videomicroscopy using an upright microscope equipped with a 40⫻ ob-
jective (Olympus, Tokyo, Japan). Patch electrodes (3–5 M⍀) were pulled
from borosilicate glass tubing and normally filled with a solution con-
taining the following (in m
M): 120 K-gluconate, 20 KCl, 10 HEPES, 10
phosphocreatine, 4 Mg-ATP, and 0.3 Na-GTP (pH adjusted to 7.25 with
KOH or CsOH, respectively, 295 mOsm). For voltage-clamp experi-
ments, K-gluconate was replaced by equimolar Cs-gluconate. All exper-
iments were performed in the presence of the GABA
A
receptor blocker
picrotoxin (100
M). In current-clamp recordings, the membrane poten-
tial was kept manually at ⫺70 mV. Data were recorded with an Axopatch
200B, filtered at 2 kHz, and digitized at 10 kHz. In all experiments, series
resistance was monitored throughout the experiment by applying a hy-
perpolarizing current or voltage pulse. If the series resistance changed by
⬎15%, the data were not included in the analysis. Data were acquired
and analyzed with ClampEx9.2, ClampFit9.2 (Molecular Devices, Palo
Alto, CA), and the Mini Analysis Program (Synaptosoft, Decatur, GA).
Monosynaptic EPSPs exhibiting constant 10 –90% rise times and laten-
cies were elicited by stimulation of afferent fibers with a bipolar twisted
platinum/10% iridium wire (25
m diameter). Although we never ob-
served any antidromic spikes, we cannot exclude the possibility that some
fibers originating from LA projection neurons were stimulated. Minia-
ture EPSCs (mEPSCs) were recorded in the presence of the sodium chan-
nel blocker TTX (1
M)at⫺70 mV and analyzed off-line by a computer
program (Mini Analysis Program). mEPSC amplitudes and decay time
constants were obtained by monoexponentially fitting the decay of each
individual event. To construct cumulative histograms, an equal number
of events was randomly selected from each neuron and pooled. To record
quantal events from defined afferent pathways, stimulation-evoked EP-
SCs were desynchronized by replacing extracellular Ca
2⫹
with Sr
2⫹
(2
m
M). The threshold for Sr
2⫹
-mEPSC detection was set to 5 pA and at
least 120 events were analyzed per cell. To compare the AMPA/NMDA
ratio of evoked synaptic transmission, the AMPA component was mea-
sured as the EPSC peak amplitude at ⫺70 mV; the NMDA component
was determined by measuring the current amplitude at 100 ms after
EPSC onset at ⫹30 mV. LTP was induced by pairing afferent stimulation
(four times 100 Hz; 1 s; 0.1 Hz) with postsynaptic depolarization to ⫺20
mV. LTP was quantified for statistical comparisons by normalizing and
averaging EPSP slopes during the last 5 min of experiments relative to the
5 min of baseline before LTP induction. All values are expressed as
means ⫾ SEM. Statistical comparisons were done with paired or un-
paired Student’s t test as appropriate (two-tailed p ⬍ 0.05 was considered
significant).
Extracellular field recordings
Adult (4 – 6 months of age) wild-type, GluR1
⫺/⫺
, and GluR3
⫺/⫺
mice
were killed with desflurane. The brains were removed and cooled to
0 – 4°C in ACSF of the following composition (in m
M): 124 NaCl, 2 KCl,
1.25 KH
2
PO
4
, 1.3 MgSO
4
, 2.5 CaCl
2
, 26 NaHCO
3
, 12 glucose, bubbled
with 95% O
2
–5% CO
2
, pH 7.4. All experiments were conducted accord
-
ing to the Norwegian Animal Welfare Act and the European Union’s
Directive 86/609/EEC. Coronal slices (400
m) containing the amygdala
were cut with a vibroslicer in 4°C O
2
/CO
2
-bubbled ACSF. Slices were
placed in an interface chamber exposed to humidified gas at 28–32°C
and perfused with ACSF. Orthodromic synaptic stimuli (50
s; ⬍ 300
A; 0.1 Hz) were delivered alternately through two tungsten electrodes:
one placed in the internal capsule, close to the medial border of the lateral
nucleus of the amygdala (thalamic pathway), and the other in the capsula
externa (cortical pathway). Extracellular synaptic responses were moni-
tored by two glass electrodes (filled with ACSF), placed in the LA. After
stable synaptic responses had been obtained in both pathways for at least
15 min, one pathway was tetanized (100 Hz for 1 s repeated four times at
5 min intervals). The second pathway served as a control pathway and
never exhibited any significant changes. In a subset of experiments, one
of the stimulation electrodes was placed at the medial border of the BA
and a recording electrode within the BA. Six consecutive responses (1
min) were averaged and normalized to the mean value recorded 1– 4 min
before the first tetanization. Data from the different pathways were
pooled across animals of the same genotype and are presented as mean ⫾
SEM. Statistical significance was evaluated using a paired two-tailed t
test, whereas LTP levels between genotypes were statistically compared
by linear mixed-model analysis.
Behavior
Subjects. All experiments were conducted in two replications with 10- to
12-month-old male GluR1
⫺/⫺
(n ⫽ 9), GluR3
⫺/⫺
(n ⫽ 9), and wild-type
littermate (n ⫽ 13) mice. The wild-type group consisted of six littermates
of the GluR1
⫺/⫺
mice and seven littermates of the GluR3
⫺/⫺
mice. There
was no significant difference between the two groups of wild-type mice
originating from the two colonies (GluR1 and GluR3) in any of the
behavioral measures. Subjects were housed individually in a holding
room with a 12 h light/dark cycle (lights on from 7:00 A.M. to 7:00 P.M.),
with food and water ad libitum. All experiments were conducted under
the auspices of the United Kingdom Home Office Project and personal
licenses held by the authors.
Apparatus. Behavioral procedures were performed in two identical
standard operant chambers (internal dimensions, 12.5 cm wide by 12.5
cm deep by 30 cm high; Coulbourn Instruments, Allentown, PA), housed
in sound attenuating boxes. Each chamber had two aluminum walls, a
transparent Perspex wall and a Perspex door that served as a fourth wall.
The two aluminum walls were divided into three sections, and the ceiling
was also aluminum and contained an infrared detector measuring the
10948 • J. Neurosci., October 10, 2007 • 27(41):10947–10956 Humeau et al. • AMPA Receptor Subunits in Amygdala Function
animal’s locomotor activity. The chambers received ambient illumina-
tion from a house light operated at 24 V located on the middle section at
the top of the right side wall. A speaker was mounted at the top of the left
side aluminum wall located in the middle section, through which a com-
puter generated tone (80 dB) was emitted. The subject’s behavior was
recorded using a digital camera directed at the transparent wall and
attached to a VCR.
Fear conditioning. Mice were transferred to the operant chamber and,
after an initial acclimatization period of 6 min, were presented with three
pairings of the auditory conditioning stimulus with footshock (0.4 mA;
2 s). The cue was presented for 30 s, and the shock was administered for
the last 2 s, coterminating with the auditory cue. Pairings were separated
by 2 min, and mice were removed from the chamber 30 s after the last
shock presentation. Twenty-four hours after training, mice were tested
for CS-induced conditioned responses. For each chamber, the sections of
the side aluminum walls were replaced with sections containing check-
erboard patterns, which were also attached to each Perspex wall, and
additionally fresh sawdust was scattered on the floor of each chamber.
After an initial acclimatization period of 6 min, the CS was presented for
8 min (CS test). Twenty-four hours later, an additional test was per-
formed for context conditioning. The chambers were altered to the orig-
inal configuration used during conditioning. Subjects were placed in the
chamber for 8 min (context test).
Scoring. During each stage of fear conditioning, the mouse’s tendency
to freeze was scored. Observation was performed using a time-sampling
procedure. Every 5 s, each mouse was judged as either freezing or active.
Freezing was defined as the absence of visible movement, except for
respiration (Blanchard and Blanchard, 1969). Scoring began ⬃10 s after
the mouse was placed in the chamber and continued until the session had
finished. From this observation, a percentage freezing score was calcu-
lated by dividing the number of intervals the
subject was judged to be freezing by the total
number of observations. All scoring was con-
ducted by observers who were “blind” with re-
spect to the critical aspects of the manipulation;
that is, they were unaware of the genotype of the
mice and their conditioning history. The inter-
rater reliability between observers who used
this scoring procedure was ⬃95%. To provide
an independent assessment of changes in loco-
motor activity, we also recorded the activity of
the mice using an infrared movement detector
(Coulbourn Instruments; model H24-61MC;
set to mouse sensitivity) mounted (22 cm above
the floor) in the ceiling of the apparatus. This
device calculated the animal’s movement in
“movement units”; each unit corresponding to
whether movement was detected during a 20
ms period (maximum movement unit pulses
was 50 pulses per second).
Reagents
AP5 was from Tocris-Cookson (Bristol, UK).
TTX was from Latoxan (Valence, France). All
other drugs were from Fluka/Sigma (Buchs,
Switzerland).
Results
Reduction of AMPA receptor-mediated
synaptic transmission at thalamo- and
cortico-LA synapses in GluR1
ⴚ/ⴚ
mice
We first analyzed the GluR1 and GluR3
contribution to AMPA receptor-mediated
synaptic transmission by recording mEP-
SCs in LA projection neurons in the pres-
ence of TTX (Fig. 1). GluR1- and GluR3-
deficient mice (Zamanillo et al., 1999;
Sanchis-Segura et al., 2006) exhibited a
similar decrease in mEPSC amplitude and
mEPSC frequency indicating that GluR1 and GluR3 AMPA re-
ceptor subtypes contribute to excitatory transmission (Fig. 1)
(wild type: ⫺12.4 ⫾ 0.9 pA, n ⫽ 20; GluR1
⫺/⫺
: ⫺8.1 ⫾ 0.4 pA,
n ⫽ 14, p ⬍ 0.01; GluR3
⫺/⫺
: ⫺9.1 ⫾ 0.5 pA, n ⫽ 6, p ⬍ 0.01; wild
type: 5.8 ⫾ 1.5 Hz, n ⫽ 20; GluR1
⫺/⫺
: 2.8 ⫾ 0.9 Hz, n ⫽ 14, p ⬍
0.05; GluR3
⫺/⫺
: 2.1 ⫾ 0.5 Hz, n ⫽ 6, p ⬍ 0.05). In addition,
mEPSCs exhibited a slower decay time constant with no differ-
ence in the 10 –90% rise time in GluR3
⫺/⫺
mice (Fig. 1c) (wild
type: 5.0 ⫾ 0.3 ms, n ⫽ 20; GluR1
⫺/⫺
: 5.4 ⫾ 0.1 ms, n ⫽ 14, NS;
GluR3
⫺/⫺
: 6.1 ⫾ 0.2 ms, n ⫽ 6, p ⬍ 0.05).
The comparable overall reduction in AMPA receptor currents
at synapses on LA projection neurons in GluR1
⫺/⫺
and
GluR3
⫺/⫺
mice appeared, however, to be input-specific, because
AMPA receptor-mediated synaptic transmission at thalamo- and
cortico-LA synapses was affected only in GluR1
⫺/⫺
mice.
Whereas GluR1
⫺/⫺
mice showed a dramatic reduction in the
AMPA/NMDA receptor ratio at both thalamo- and cortico-LA
synapses, in GluR3
⫺/⫺
mice the AMPA/NMDA ratio was not
different from wild-type mice in either pathway (Fig. 2) (tha-
lamic: wild type, 6.5 ⫾ 1.6, n ⫽ 8; GluR1
⫺/⫺
, 2.7 ⫾ 0.3, n ⫽ 9, p ⬍
0.05; GluR3
⫺/⫺
, 8.4 ⫾ 1.2, n ⫽ 13, NS; cortical: wild type, 7.6 ⫾
1.9, n ⫽ 8; GluR1
⫺/⫺
, 2.4 ⫾ 0.3, n ⫽ 9, p ⬍ 0.05; GluR3
⫺/⫺
, 8.0 ⫾
0.9, n ⫽ 13, NS). Because the mEPSC amplitude and frequency
were equally affected in both knock-out mice, the regular AMPA/
NMDA ratio in GluR3
⫺/⫺
mice indicates that either a different
population of synapses (other than thalamo- and cortico-LA syn-
Figure 1. Reduced mEPSC amplitude and frequency in GluR1
⫺/⫺
and GluR3
⫺/⫺
mice. a, Representative sample traces from
WT, GluR1
⫺/⫺
, and GluR3
⫺/⫺
mice. Calibration: 12 pA, 350 ms. b, Histograms illustrating the relative reduction in large
amplitude events in GluR1
⫺/⫺
and GluR3
⫺/⫺
animals relative to wild-type controls. Histograms are normalized to the total
number of events recorded in each genotype. The gray line indicates mEPSC amplitude distribution in wild-type mice. The traces
represent averaged mEPSC waveforms obtained from all events. Calibration: 7 pA, 5 ms. c, Cumulative distributions of mEPSC
amplitude, interevent intervals, and
decay
for all genotypes containing 300 randomly selected events from each cell (wild type,
n ⫽ 20 cells; GluR1
⫺/⫺
, n ⫽ 14; GluR3
⫺/⫺
, n ⫽ 6). The insets show mean ⫾ SEM for mEPSC amplitude, frequency, and
decay
.
Thetracesillustrateincreased decay in GluR3
⫺/⫺
mice(tracesarepeak-scaled averages obtained from all cells).Calibration,5ms.
*p ⬍ 0.05; **p ⬍ 0.01.
Humeau et al. • AMPA Receptor Subunits in Amygdala Function J. Neurosci., October 10, 2007 • 27(41):10947–10956 • 10949
apses) contributed to the loss of mEPSCs in absence of GluR3 or,
alternatively, that there was a concomitant reduction in NMDA
receptor-mediated transmission in GluR3
⫺/⫺
mice.
To exclude a reduction in the NMDA receptor component, we
examined synaptic efficacy in terms of quantal amplitude of
AMPA receptor-mediated transmission at thalamo- and
cortico-LA synapses. We recorded stimulation-induced desyn-
chronized mEPSCs (in the presence of extracellular Sr
2⫹
instead
of Ca
2⫹
). Consistent with the reduced TTX–mEPSC amplitude
and the marked reduction in the AMPA/NMDA ratio,
stimulation-induced mEPSCs at thalamo- and cortico-LA syn-
apses were significantly smaller in GluR1
⫺/⫺
mice compared
with wild-type mice (Fig. 3a– c) (thalamic: wild type, ⫺14.0 ⫾ 0.2
pA, n ⫽ 4; GluR1
⫺/⫺
, ⫺8.2 ⫾ 1.8 pA, n ⫽ 5, p ⬍ 0.05; cortical:
wild type, ⫺16.9 ⫾ 0.4 pA, n ⫽ 4; GluR1
⫺/⫺
, ⫺9.9 ⫾ 1.1 pA, n ⫽
5, p ⬍ 0.01). In GluR3
⫺/⫺
mice, however, quantal amplitudes
were not different from wild-type animals (thalamic: ⫺13.8 ⫾
1.9 pA, n ⫽ 5, NS; cortical: ⫺16.4 ⫾ 1.7 pA, n ⫽ 5, NS) (Fig.
3a– c). There was no difference in the decay kinetics between
groups (Fig. 3d). Thus, at thalamic and cortical inputs to LA
projection neurons, the lack of GluR1 leads to a pronounced
reduction in the efficacy of AMPA receptor-mediated synaptic
transmission, which also might include a reduction in the num-
ber of functional synapses. For GluR3
⫺/⫺
mice, we observed that
the amplitude of Sr
2⫹
mEPSCs was unchanged compared with
control animals, suggesting that pathways other than the
thalamo- and cortico-LA pathways are affected in the absence of
GluR3.
LTP in the thalamo-amygdala pathway depends on GluR1
We analyzed whether deletion of either the GluR1 or the GluR3
subunit affected LTP in the neural pathways associated with au-
ditory cued fear conditioning (Sigurdsson et al., 2007). In slices
from wild-type mice, the average field EPSP (fEPSP) slope in the
thalamic pathway was significantly increased 40 – 45 min after the
last tetanization when compared with the pretetanic control
value (117 ⫾ 5%; n ⫽ 22; p ⬍ 0.01) (Fig. 4a). In GluR1
⫺/⫺
mice,
however, we failed to observe a persistent potentiation (102 ⫾
5%; n ⫽ 27; p ⫽ 0.77), whereas the amount of LTP in GluR3
⫺/⫺
mice was similar to wild-type animals (112 ⫾ 6%; n ⫽ 17; p ⫽
0.64) (Fig. 4a).
In GluR1
⫺/⫺
mice, the failure to observe tetanization-
induced LTP in the thalamo-amygdala pathway could be ex-
plained by an insufficient depolarization during the induction
caused by the reduced AMPA receptor-mediated responses.
We therefore used whole-cell recordings to compensate for a
possible deficit in postsynaptic depolarization and induced
LTP by pairing a similar presynaptic stimulation pattern (1 s;
100 Hz; four times; 0.1 Hz) with postsynaptic depolarization
to ⫺20 mV. In addition, we blocked GABA
A
receptor-
mediated inhibition, thereby facilitating the induction of LTP
(Wigstro¨m and Gustafsson, 1985; Bissie`re et al., 2003). In gen-
eral, thalamo-LA LTP induced by pairing presynaptic stimu-
lation with postsynaptic depolarization in the presence of a
GABA
A
receptor antagonist (Fig. 4b) appeared to be more
stable than LTP induced by tetanic stimulation alone (Fig. 4a).
This may indicate that the extent of postsynaptic depolariza-
tion during LTP induction at thalamo-LA synapses has an
impact on LTP stability or, alternatively, that thalamo-LA fEP-
SPs contain an additional nonsynaptic component that decays
over time. Similar to the extracellular results, however, whole-
cell recordings revealed an almost complete lack of LTP in the
thalamo-amygdala pathway in GluR1
⫺/⫺
mice (GluR1
⫺/⫺
:
114 ⫾ 8% of baseline, n ⫽ 9, NS), whereas the magnitude of
LTP was of similar size in wild-type and GluR3
⫺/⫺
mice (wild
type: 159 ⫾ 21% of baseline, n ⫽ 12, p ⬍ 0.05; GluR3
⫺/⫺
:
149 ⫾ 9% of baseline, n ⫽ 5, p ⬍ 0.05) (Fig. 4b). Thus, LTP
expression at thalamo-amygdala synapses depends on the
presence of the GluR1 subunit.
Figure 2. GluR1
⫺/⫺
mice exhibit a decreased AMPA/NMDA ratio of evoked synaptic transmission at thalamo- and cortico-LA synapses. a, Placement of stimulation and recording electrodes. b,
Sample traces depicting evoked EPSC waveforms at thalamo- and cortico-LA synapses recorded at ⫺70 mV and at ⫹30 mV in wild-type, GluR1
⫺/⫺
, and GluR3
⫺/⫺
mice. The AMPA component
was obtained by measuring the EPSC peak amplitude at ⫺70 mV; the NMDA component was determined by measuring the current amplitude at 100 ms after EPSC onset at ⫹30 mV (arrows). c,
Averaged data illustrating the significant ( p ⬍ 0.05) reduction in the AMPA/NMDA ratio at cortical and thalamic afferents in GluR1
⫺/⫺
mice. Number of experiments is indicated on bars. *p ⬍
0.05. Error bars indicate SEM.
10950 • J. Neurosci., October 10, 2007 • 27(41):10947–10956 Humeau et al. • AMPA Receptor Subunits in Amygdala Function
LTP in the cortico-amygdala pathway depends on GluR1
and GluR3
Next, we examined whether LTP in the cortico-amygdala path-
way was similarly GluR1 dependent. Cortical afferents to the LA
were stimulated by placing a stimulation electrode in the external
capsule above the LA (Humeau et al., 2003). Whereas LTP was
readily induced in wild-type animals (125 ⫾ 3%; n ⫽ 34; p ⬍
0.01), we failed to obtain LTP in both GluR1
⫺/⫺
and GluR3
⫺/⫺
mice (GluR1
⫺/⫺
: 104 ⫾ 3%, n ⫽ 33, NS; GluR3
⫺/⫺
: 105 ⫾ 4%,
n ⫽ 26, NS) (Fig. 5a). To exclude a failure in LTP induction, we
used the same pairing procedure as in the thalamo-amygdala
pathway. Although LTP was not completely abolished, we ob-
served a substantial reduction in this pathway both in GluR1- and
GluR3-deficient mice (wild type: 195 ⫾ 28%, n ⫽ 14, p ⬍ 0.01;
GluR1
⫺/⫺
: 137 ⫾ 13%, n ⫽ 7, p ⬍ 0.05; GluR3
⫺/⫺
: 142 ⫾ 5%,
n ⫽ 5, p ⬍ 0.01) (Fig. 5b). This suggests that both GluR1 and
GluR3 subunits contribute to LTP expression at cortico-LA syn-
apses. Moreover, because quantal amplitude at cortico-LA syn-
apses was reduced in GluR1- but not in GluR3-deficient mice, a
reduced synaptic efficacy and/or density in this pathway can
hardly account for the similar reduction in LTP in the two geno-
types. Thus, in contrast to thalamo-amygdala LTP, LTP in the
cortico-amygdala pathway does not solely depend on the GluR1
subunit.
LTP within the BA is GluR1 dependent
Based on previous studies indicating a role for the BA in both
contextual and cued fear conditioning (Anglada-Figueroa and
Quirk, 2005; Calandreau et al., 2005; Goosens and Maren, 2006),
we next analyzed whether deletion of either the GluR1 or the
GluR3 subunit affected LTP within the BA.
As at thalamo-LA synapses, we found that
LTP in the BA was normal in GluR3
⫺/⫺
mice, but completely absent in GluR1
⫺/⫺
mice (wild type: 125 ⫾ 4%, n ⫽ 12, p ⬍
0.05; GluR1
⫺/⫺
: 103 ⫾ 3%, n ⫽ 16, p ⫽
0.23; GluR3
⫺/⫺
: 124 ⫾ 8%, n ⫽ 13, p ⬍
0.05) (Fig. 6a,b).
GluR1
ⴚ/ⴚ
mice exhibit complete
absence of freezing behavior during
fear conditioning
To study whether GluR1- and/or GluR3-
containing AMPA receptors participate in
emotional learning, we analyzed the ac-
quisition and retention of auditory cued
and contextual fear conditioning. Before
CS–US pairings, all genotypes displayed
similar levels of baseline activity (supple-
mental Fig. 1, available at www.jneurosci.
org as supplemental material). Con-
versely, after the first CS–US pairing,
activity levels decreased as freezing re-
sponses increased for GluR3
⫺/⫺
and wild-
type mice. In contrast, GluR1
⫺/⫺
mice
showed similar activity levels throughout
the conditioning session, indicating major
impairments during the acquisition phase
of fear conditioning in the absence of
GluR1 (Fig. 7a,b). This is substantiated by
the detailed analysis of the activity levels
and freezing responses during the three in-
tertrial intervals (ITIs) preceding CS1,
CS2, and CS3, respectively (Fig. 7c,d). Main effects analysis of
mean activity levels revealed no effect of genotype during the first
ITI period (F ⬍ 1). However, a significant effect of genotype was
revealed during the second and third ITI time bins (smallest
F
(2,32)
⫽ 8.97; p ⫽ 0.001) (Fig. 7c). Multiple post hoc Newman–
Keuls comparisons supported this, showing that during the sec-
ond ITI, wild-type mice differed from both GluR1
⫺/⫺
( p ⬍
0.0001) and GluR3
⫺/⫺
mice ( p ⬍ 0.05). However, during the
third ITI, GluR3
⫺/⫺
and wild-type mice showed similar levels of
activity suppression, suggesting that acquisition of fear condi-
tioning may be delayed in GluR3
⫺/⫺
mice. In contrast, GluR1
⫺/⫺
mice exhibited no change in activity across the ITI period ( p ⬎
0.05) (Fig. 7c). The analysis of the freezing data suggested a sim-
ilar pattern of behavior (Fig. 7d). Thus, whereas GluR3
⫺/⫺
mice
exhibited delayed, but mostly normal levels of activity suppres-
sion and freezing behavior, GluR1
⫺/⫺
animals showed a com
-
plete absence of any CS- or context-induced fear behavior during
the acquisition phase of fear conditioning.
The impaired fear responses of GluR1
⫺/⫺
mice during the
acquisition phase of fear conditioning were not caused by a deficit
in detecting novel auditory stimuli or insensitivity to the foot-
shook as revealed by the unconditioned suppression of activity
elicited by the novel tone and by the initial agitation induced by
the footshock (Bouton and Bolles, 1980) (Fig. 8a). The locomo-
tor activity was scored in 0.5 s time bins for the last4softhefirst
ITI and the first4softhefirst CS tone presentation. An ANOVA
confirmed that there was a main effect of phase (ITI vs CS), which
did not interact with genotype, and a significant interaction be-
tween phase and time bin (F
(3,84)
⫽ 3.54; p ⬍ 0.05). The test of
simple main effects revealed a significant effect of time bin only
Figure 3. GluR1
⫺/⫺
mice exhibit a reduction in quantal amplitude at thalamo- and cortico-LA synapses. a, Sample traces of evoked
EPSCs in the presence of Ca
2⫹
or Sr
2⫹
. In the presence of Sr
2⫹
, asynchronous quantal events are detectable. Calibration: 20 pA, 50 ms;
insets,10 pA, 25 ms. b,Normalized histograms illustrating theselective reduction in mEPSC amplitude in the two pathways in GluR1
⫺/⫺
mice (wild type, n ⫽ 691 events from 5 cells; GluR1
⫺/⫺
, n ⫽ 985 events from 5 cells; GluR3
⫺/⫺
, n ⫽ 713 events from 5 cells). Fits
indicate mEPSC amplitude distribution in wild-type animals. c, Same data plotted as cumulative probability distributions and averaged
means⫾ SEM (insets). d,GluR1
⫺/⫺
andGluR3
⫺/⫺
exhibitedno significant difference inmEPSC kinetics (wildtype, n ⫽ 5;GluR1
⫺/⫺
,
n ⫽ 5; GluR3
⫺/⫺
, n ⫽ 5; p ⬎ 0.05). Superimposed traces represent amplitude-scaled mEPSC waveforms obtained by averaging the
scaled mean mEPSC waveforms from each cell. Calibration, 4 ms. *p ⬍ 0.05; **p ⬍ 0.01.
Humeau et al. • AMPA Receptor Subunits in Amygdala Function J. Neurosci., October 10, 2007 • 27(41):10947–10956 • 10951
during the CS period (F
(3,84)
⫽ 7.83; p ⬍ 0.001) that reflected a
gradual rise in locomotor activity during the CS for all genotypes
suggesting that all genotypes were able to detect the presentation
of the tone. Similarly, the unconditioned reaction to footshock
measured by mean locomotor activity responses, for the first
1.25 s after presentation of the footshock was similar for all geno-
types (Fig. 8b), indicating that all mice were able to detect the US.
Absence of long-term memory of conditioned fear in
GluR1
ⴚ/ⴚ
mice
Fear memory was tested by assessing retention of conditioned
responses 24 h after conditioning by measuring locomotor activ-
ity and freezing responses before and after the CS presentation in
a novel context (Fig. 9a,b). Before the presentation of the audi-
tory CS, both freezing and activity levels were comparable for all
groups. On presentation of the auditory CS, both GluR3
⫺/⫺
and
wild-type mice exhibited freezing responses, indicating a mem-
ory for the CS. GluR1
⫺/⫺
mice, however, showed no change in
activity or freezing responses throughout the duration of the CS
presentation, demonstrating that the GluR1 deletion results in a
complete failure to acquire and/or retain memory for cued audi-
tory fear conditioning (Fig. 9a). This pattern of results was also
evident in the analysis of the locomotor activity data (Fig. 9b).
Before the presentation of the cue, there was an apparent trend
for GluR1
⫺/⫺
mice to show higher baseline levels of activity in the
novel context. However, this difference was not significant. In
contrast, there was a significant difference between the genotypes
in locomotor activity during the tone presentation. Both the WT
and GluR3
⫺/⫺
mice showed lower levels of locomotor activity
than GluR1
⫺/⫺
mice on presentation of the CS.
Twenty-four hours later, retention of contextual fear condi-
tioning was assessed. The experimental chambers were altered
back to the original configuration used during conditioning.
Subjects were placed in the chamber for 8 min. GluR3
⫺/⫺
and
wild-type mice showed a steady suppression of locomotor activ-
ity, as a consequence of the freezing elicited by the context (Fig.
9c,d). In contrast, GluR1
⫺/⫺
mice failed to show enhanced freez
-
Figure 4. Selective absence of thalamo-LA LTP in GluR1
⫺/⫺
but not GluR3
⫺/⫺
mice. a,
TimecourseofthefEPSPslopeatthalamicafferentsin wild-type (n ⫽ 22), GluR1
⫺/⫺
(n⫽ 27),
and GluR3
⫺/⫺
(n ⫽ 17) mice. Whereas wild-type and GluR3
⫺/⫺
animals exhibited signifi
-
cant LTP ( p ⬍ 0.01), LTP could not be induced in GluR1
⫺/⫺
animals ( p ⫽ 0.77). LTP was
induced by tetanic stimulation of thalamic afferents (100 Hz; 1 s; repeated 4 times with 5 min
interval). The depicted traces show averaged fEPSPs before and 45 min after the last tetaniza-
tion. Calibration: 0.5 mV, 5 ms. b, Pairing-induced LTP at thalamic afferents is selectively abol-
ished in GluR1
⫺/⫺
but not GluR3
⫺/⫺
mice. Time course of the EPSP slope at thalamo-LA
synapses in wild-type (n ⫽ 12), GluR1
⫺/⫺
(n ⫽ 9), and GluR3
⫺/⫺
(n ⫽ 5) mice. LTP was
induced by pairing afferent stimulation (4 times; 1 s, 100 Hz; arrow) with postsynaptic depolar-
ization to ⫺20 mV. The depicted traces show averaged EPSPs for 2 min of baseline and 2 min of
LTP (25–30 min after pairing). Calibration: 2 mV, 10 ms.
Figure 5. Equal reduction of LTP at cortico-LA synapses in GluR1
⫺/⫺
and GluR3
⫺/⫺
mice.
a, Time course of the fEPSP slope in the cortico-LA pathway in wild-type (n ⫽ 34), GluR1
⫺/⫺
(n ⫽ 33), and GluR3
⫺/⫺
(n ⫽ 26) mice. LTP in both GluR1
⫺/⫺
and GluR3
⫺/⫺
animals was
absent 40 – 45 min after induction ( p ⬎ 0.05). LTP was induced by tetanic stimulation of
corticalafferents (4 times; 1 s, 100 Hz). The depictedtraces show averaged fEPSPs before and 45
min after the last tetanization. Calibration: 0.5 mV, 5 ms. b, Pairing-induced LTP at cortico-LA
synapsesis equally reduced inGluR1
⫺/⫺
andGluR3
⫺/⫺
mice.Time course of theEPSP slope in
the cortico-LA pathway in wild-type (n ⫽ 14), GluR1
⫺/⫺
(n ⫽ 7), and GluR3
⫺/⫺
(n ⫽ 5)
mice.LTPwasinducedbypairingafferentstimulation (4 times; 1 s; 100 Hz; arrow)withpostsyn-
apticdepolarization to ⫺20 mV. The depicted tracesshow averaged EPSPs for 2 min of baseline
and 2 min of LTP (25–30 min after pairing). Calibration: 2 mV, 10 ms.
Figure 6. Selective absence of LTP in the BA in GluR1
⫺/⫺
but not GluR3
⫺/⫺
mice. a,
Placement of stimulation and recording electrodes. b, Time course of the fEPSP slope in the
basal amygdala of wild-type (n ⫽ 12) and GluR1
⫺/⫺
(n ⫽ 16) mice. Whereas wild-type
animals exhibit robust LTP ( p ⬍ 0.05), LTP is completely abolished in GluR1
⫺/⫺
mice ( p ⬎
0.05). c, GluR3
⫺/⫺
animals (n ⫽ 13) exhibit normal LTP ( p ⬍ 0.05). LTP was induced by
tetanic stimulation of local afferents (100 Hz; 1 s; repeated 4 times with 5 min interval). The
depictedtraces show averaged fEPSPs beforeand 45 min after the last tetanization. Calibration:
0.5 mV, 2.5 ms.
10952 • J. Neurosci., October 10, 2007 • 27(41):10947–10956 Humeau et al. • AMPA Receptor Subunits in Amygdala Function
ing responses and exhibited correspondingly higher levels of lo-
comotor activity relative to GluR3
⫺/⫺
and wild-type control
mice (Fig. 9c,d). In conclusion, our findings demonstrate that
GluR1
⫺/⫺
, but not GluR3
⫺/⫺
mice, are impaired in the forma
-
tion of conditioned fear memories for both contextual and audi-
tory cues.
Discussion
Our electrophysiological analysis revealed a striking difference
between GluR1 and GluR3 function in terms of AMPA receptor-
mediated synaptic transmission and LTP at thalamic and cortical
synapses onto LA projection neurons. In the absence of GluR1,
the postsynaptic efficacy and possibly the number of thalamo-
and cortico-amygdala synapses were strongly reduced. Although
AMPA receptor-mediated synaptic transmission at thalamo- and
cortico-LA synapses appeared normal when GluR3 is missing, we
found a significant reduction in the amplitude and frequency of
mEPSCs recorded in TTX. In principle, this reduction in mEPSC
frequency can be caused by presynaptic and/or postsynaptic
mechanisms (e.g., by a reduction in the release probability or by
removal of postsynaptic AMPA receptors at a subset of synapses).
However, the finding that the postsynaptic quantal amplitude at
thalamo- and cortico-LA inputs was normal, whereas the overall
quantal amplitude of mEPSCs measured in TTX was markedly
reduced, cannot be explained by a presynaptic mechanism. This
indicates that in LA projection neurons postsynaptic GluR3-
containing AMPA receptors play a more prominent role at other
glutamatergic inputs, such as for example at intra-amygdala con-
nections. Thus, different AMPA receptor subunits participate in
a pathway-specific manner to synaptic transmission in LA pro-
jection neurons and the GluR3 content is low or absent at tha-
lamic and cortical inputs under baseline conditions.
Whereas LTP at thalamo-amygdala synapses and fear mem-
ory appeared to be normal in the GluR3
⫺/⫺
mice, LTP and fear
memory were severely impaired in the GluR1
⫺/⫺
mice. These
results are in agreement with previous work indicating that the
thalamo-amygdala pathway is rapidly potentiated during audi-
tory fear conditioning (Quirk et al., 1995, 1997), that local pre-
vention of GluR1 trafficking in the LA interferes with thalamo-
amygdala LTP and auditory fear conditioning (Rumpel et al.,
2005), and that fear conditioning induces rapid expression of
GluR1 (Yeh et al., 2006). Together with previous studies (Hu-
meau et al., 2005; Rumpel et al., 2005), our findings strongly
support a postsynaptic expression mechanism for thalamo-
amygdala LTP (but see Apergis-Schoute et al., 2005).
Although baseline synaptic transmission at cortico-LA syn-
Figure 7. GluR1
⫺/⫺
mice exhibit a complete lack of CS- and context-induced fear behavior
during conditioning. a, Mean infrared activity (IR) levels during the three presentations of the
30 s auditory CS. Whereas WT (n ⫽ 13; p ⬍ 0.05) and GluR3
⫺/⫺
(n ⫽ 9; p ⬍ 0.05) mice
exhibit a significant reduction in activity levels during conditioning, activity levels in GluR1
⫺/⫺
mice(n ⫽ 9) remain unaffected ( p⬎ 0.05).Number of CS is indicatedonthe x-axis. A two-way
ANOVA revealed no main effect on genotype on baseline activity before CS–US pairing time
bin; largest F
(11,308)
⫽ 1.20, p ⬎ 0.20. Analysis of the activity scores during the three CS
presentations revealed a main effect of genotype (F
(2,28)
⫽ 6.68; p ⬍ 0.01), and CS presenta
-
tion (F
(2,28)
⫽ 34.96; p ⬍ 0.0001). There was a significant genotype by CS presentation inter
-
action (F
(4,56)
⫽ 7.43; p ⬍ 0.0001). Analysis of the simple main effects followed by post hoc
Newman–Keuls comparisons revealed a significant effect of genotype during the second and
third CS presentation (smallest F
(2,56)
⫽ 3.18; p ⬍ 0.05) with GluR1
⫺/⫺
mice differing from
GluR3
⫺/⫺
and wild-type groups ( p ⬍ 0.05). b, Percentage of freezing responses during the
three presentations of the 30 s auditory CS. Only WT (n ⫽ 13; p ⬍ 0.05) and GluR3
⫺/⫺
(n ⫽
9;p ⬍ 0.05) animals show a significant increase in freezing levels. Freezing levels of GluR1
⫺/⫺
mice do not increase during conditioning (n ⫽ 9; p ⬎ 0.05). The number of CS is indicated on
the x-axis. There is a main effect of genotype (F
(2,28)
⫽ 61.30; p ⬍ 0.0001) and of CS presen
-
tation (F
(2,28)
⫽ 122.87; p ⬍ 0.0001) and a significant interaction between these two factors
(F
(4,56)
⫽ 30.43; p ⬍ 0.0001). Simple main effects analysis followed by post hoc Newman–
Keuls comparisons revealed a significant effect of genotype during the second and third CS
presentations (smallest F
(2,56)
⫽ 38.57; p ⬍ 0.001), with GluR1
⫺/⫺
mice differing from all
other genotypes ( p ⬍ 0.05). c, Mean IR activity levels during the ITIs. Number of ITIs preceding
CS1,CS2,and CS3 areindicatedon the x-axis.Duringthe ITIs, GluR1
⫺/⫺
micedonot exhibit any
reduction in locomotor activity with conditioning (n ⫽ 9; p ⬎ 0.05), whereas activity levels are
significantly reduced in WT (n ⫽ 13; p ⬍ 0.05) and GluR3
⫺/⫺
(n ⫽ 9; p ⬍ 0.05) mice. A
two-way mixed ANOVA of the activity data revealed a main effect of genotype (F
(2,28)
⫽ 6.47;
p ⬍ 0.01) and of activity time bin (F
(2,56)
⫽ 59.38; p ⬍ 0.0001) and a genotype by ITI time bin
interaction (F
(4,56)
⫽ 22.07; p ⬍ 0.0001). d, Percentage of freezing responses during the ITIs
does not increase in GluR1
⫺/⫺
mice (n ⫽ 9; p ⬎ 0.05), whereas WT (n ⫽ 13; p ⬍ 0.05) and
GluR3
⫺/⫺
(n ⫽ 9; p ⬍ 0.05) mice exhibit a significant increase in freezing levels with a main
effects of genotype (F
(2,28)
⫽ 147.07; p ⬍ 0.0001) and of ITI time bin (F
(2,56)
⫽ 219.52; p ⬍
0.0001)and an interaction between the two factors (F
(4,56)
⫽ 62.47; p ⬍ 0.0001). Simple main
effects analysis followed by multiple post hoc Newman–Keuls comparisons revealed again a
significanteffect of genotype duringthe second andthird ITI bins (smallestF
(2,84)
⫽ 92.93; p ⬍
0.0001), with wild-type mice differing from both knock-out groups during the second ITI bin,
and GluR1
⫺/⫺
differing from GluR3
⫺/⫺
and wild-type mice during the third ITI bin ( p ⬍
0.05). Error bars indicate SD.
Figure8. Comparable CSand US reactivity inwild-type,GluR1
⫺/⫺
,andGluR3
⫺/⫺
mice.a,
Mean infrared activity (IR) levels for the 4 s period before presentation of the first CS during
conditioning,and the 4 s period of CS presentation does notdiffer between WT,GluR1
⫺/⫺
,and
GluR3
⫺/⫺
mice. Activity scores averaged across 0.5 s time bins (WT, n ⫽ 13; GluR1
⫺/⫺
, n ⫽
9; GluR3
⫺/⫺
, n ⫽ 9; p ⬎ 0.05). A two-way ANOVA of the activity data revealed no main effect
of genotype (F
(2,28)
⫽ 2.52; p ⬎ 0.09). There was a main effect of phase (ITI vs CS; F
(1,28)
⫽
13.58; p ⬍ 0.01), main effect of time bin (F
(1,28)
⫽ 14.16; p ⬍ 0.01), and a significant inter
-
actionbetweenthesefactors(F
(3,84)
⫽ 3.54;p ⬍ 0.05), but no interactions involving genotype.
The test of simple main effects revealed a significant effect of time bin only during the CS period
(F
(3,84)
⫽ 7.83;p ⬍ 0.001) that reflected agradualrise in locomotor activity duringtheCS for all
genotypes, suggesting that all genotypes were able to detect the presentation of the tone. b,
Comparable activity levels of wild-type, GluR1
⫺/⫺
, and GluR3
⫺/⫺
animals for the 1.25 s
period after presentation of the first shock. Activity scores averaged across 0.25 s time bins (WT,
n ⫽ 13; GluR1
⫺/⫺
, n ⫽ 9; GluR3
⫺/⫺
, n ⫽ 9; p ⬎ 0.05). A two-way ANOVA with genotype
and time bin as factors revealed no main effect of genotype (F
(2,28)
⫽ 2.36; p ⫽ 0.1121).
However, there was a main effect of time bin, which reflected a reduction in locomotor activity
after offset of the shock (F
(4,112)
⫽ 10.01; p ⬍ 0.0001).
Humeau et al. • AMPA Receptor Subunits in Amygdala Function J. Neurosci., October 10, 2007 • 27(41):10947–10956 • 10953
apses was impaired in GluR1
⫺/⫺
, but not GluR3
⫺/⫺
mice, LTP
expression was equally reduced in the two genotypes. We and
others have shown that cortico-LA LTP can be induced presyn-
aptically and postsynaptically depending on the induction proto-
col (Huang and Kandel, 1998; Tsvetkov et al., 2002, 2004; Hu-
meau et al., 2003, 2005; Shaban et al., 2006; Humeau and Lu¨thi,
2007). Together with our previous studies (Humeau et al., 2003),
the present results indicate that presynaptic and postsynaptic ex-
pression mechanisms may coexist at cortico-LA synapses.
The loss of synaptic plasticity in the BA in GluR1
⫺/⫺
mice is
consistent with the observed impairment in contextual fear con-
ditioning, thereby extending the parallels between the behavioral
effects of GluR1 deletion and lesions of the LA and/or BA, which
also disrupt conditioned freezing to discrete cues and to context
(Phillips and LeDoux, 1992; Nader et al., 2001; Calandreau et al.,
2005; Anglada-Figueroa and Quirk, 2005; Goosens and Maren,
2006). Our results suggest that GluR1-dependent synaptic plas-
ticity may play a general role at different synapses within the
amygdala during the acquisition of both cued and contextual fear
conditioning.
GluR1
⫺/⫺
mice displayed a robust deficit in the acquisition of
conditioned freezing, both to a discrete CS and to the experimen-
tal context. The deficit in fear conditioning was unlikely to be
attributable to differences in baseline activity levels or to differ-
ences in CS or US detection, because unconditioned CS and US
responses were normal. In contrast, GluR3
⫺/⫺
mice revealed a
delayed acquisition of conditioned freezing behavior. However,
after the third CS–US pairing and during CS and context memory
tests, freezing behavior of GluR3
⫺/⫺
mice was equal to wild-type
animals.
The relatively normal conditioned freezing displayed by the
GluR3
⫺/⫺
mice, which exhibit deficient LTP at the cortico-LA
pathway, suggests that AMPA receptor plasticity in this pathway
is not essential for conditioned freezing. It could be argued that
the delayed acquisition of conditioned freezing observed after the
first tone–footshock pairing may reflect a modulatory role for
GluR3-dependent plasticity in the cortico-LA pathway. This dis-
sociation in electrophysiological phenotypes between the two
mouse mutants may therefore explain the different behavior,
suggesting that the deficits in conditioned freezing observed in
GluR1
⫺/⫺
mice are attributable to the reduction of AMPA
receptor-dependent synaptic transmission and/or the absence of
GluR1-dependent synaptic plasticity at thalamo-LA synapses
(Rumpel et al., 2005).
There are a number of important caveats, however. First, rel-
atively normal conditioned freezing in GluR3
⫺/⫺
mice does not
completely preclude a role for plasticity at cortico-LA synapses in
supporting this behavior in normal wild-type mice. The LA re-
ceives auditory inputs from thalamus and cortex (Carlsen and
Heimer, 1988; Farb and LeDoux, 1997, 1999; McDonald, 1998;
Smith et al., 2000), and conditioned freezing to a simple auditory
CS can be mediated by either of these pathways (Romanski and
LeDoux, 1992). It may be the case therefore that plasticity in both
cortical and thalamic pathways can contribute to conditioned
freezing in normal animals. It is also possible that the intact LTP
at thalamo-LA synapses in GluR3
⫺/⫺
mice can compensate and
support the behavior in these mutant mice, and that plasticity in
both thalamic and cortical inputs must be impaired before a be-
havioral deficit becomes evident (as in the GluR1
⫺/⫺
mice). Al
-
ternatively, it has been argued that the cortico-LA pathway is
important for more complex auditory stimuli or for stimulus
discriminations (Thompson, 1962; Jarrell et al., 1987; Shaban et
al., 2006) (but see Armony et al., 1997), and therefore the tha-
lamic pathway is more likely to support conditioning to a simple
tone CS. Indeed, the present data suggest that synaptic plasticity
in the cortico-LA pathway has a more subtle and limited role in
this particular form of conditioned freezing, thus potentially re-
flecting the relatively simple nature of the tone CS used in the
present study.
Second, it is also possible that the deficits in conditioned freez-
ing in the GluR1
⫺/⫺
mice, in particular to the experimental con
-
text, could be explained by a deficit in hippocampal synaptic
plasticity (Zamanillo et al., 1999). Previous studies have shown
that GluR1
⫺/⫺
mice are impaired on hippocampus-dependent
tasks that require the rapid encoding and/or expression of trial-
Figure 9. GluR1
⫺/⫺
but not GluR3
⫺/⫺
mice exhibit severe memory deficits in cued and
contextual fear conditioning. Retention test. a, b, The percentage observations of a freezing
response(a)and the mean IRlocomotor activity responses (b) duringthe baseline period before
the CS and during the CS presentation in WT (n ⫽ 13), GluR1
⫺/⫺
, and GluR3
⫺/⫺
mice (n ⫽
9 each). No differences in baseline freezing (F
(2,28)
⬍ 1; p ⬎ 0.10; two-way ANOVA) or activity
(F
(2, 28)
⫽ 1.52; p ⬎ 0.10; two-way ANOVA) levels was observed. Two-way ANOVAs were
conducted on the freezing response and activity data obtained during presentation of the CS.
Analysis of the freezing data revealed a main effect of genotype (F
(2,28)
⫽ 33.42; p ⬍ 0.0001),
of time bin (F
(15,420)
⫽ 23.79; p ⬍ 0.0001), and an interaction between these two factors
(F
(30,420)
⫽ 6.69; p ⬍ 0.0001). Analysis of the simple main effects followed by post hoc New
-
man–Keulscomparisons revealed a main effect of genotype during freezing bins 13–23 (small-
est F
(2,205)
⫽ 3.56; p ⬍ 0.05) with GluR1
⫺/⫺
differing from GluR3
⫺/⫺
and WT groups ( p
values ⬍ 0.05). Analysis of the infrared activity data, similarly, revealed a main effect of geno-
type (F
(2,28)
⫽ 6.03; p ⬍ 0.01), and of activity time bin (F
(15,420)
⫽ 17.01; p ⬍. 00001), and a
genotype by activity time bin interaction (F
(30,420)
⫽ 1.781; p ⬍ 0.01). Simple main effects
analysis revealed a main effect of genotype during activity bins 13–19 and 21–23 (smallest
F
(2,66)
⫽ 4.37; p ⬍ 0.05). Post hoc Newman–Keuls comparisons revealed differences between
GluR1
⫺/⫺
mice and the remaining groups ( p ⬍ 0.005). c, d, Context extinction test: the
percentage observations of a freezing response (c) and the mean IR locomotor activity re-
sponses conditioning context (d)ofWT(n ⫽ 13), GluR1
⫺/⫺
, and GluR3
⫺/⫺
mice (n ⫽ 9
each). WT and GluR3
⫺/⫺
mice showed a steady suppression of locomotor activity. GluR1
⫺/⫺
mice showed considerably reduced freezing responses and higher levels of locomotor activity
relative to GluR3
⫺/⫺
and WT mice. The analysis of the freezing response data revealed a main
effectof genotype (F
(2,28)
⫽ 17.57; p ⬍ 0.0001) andof time bin (F
(15,420)
⫽ 4.91; p ⬍ 0.0001),
and an interaction between the two factors (F
(30,420)
⫽ 2.92; p ⬍ 0.001). Simple main effects
analysis revealed a main effect of genotype at bins 4 – 6 and 8 –16 (smallest F
(2,154)
⫽ 4.54;
p ⬍ 0.05). Post hoc Newman–Keuls comparisons identified differences between GluR1
⫺/⫺
mice and all other groups ( p values ⬍ 0.05). A two-way ANOVA analysis of the activity data
revealed a main effect of genotype (F
(2,28)
⫽ 6.65; p ⬍ 0.01), of activity time bin (F
(15,420)
⫽
2.21;p⬍ 0.01),andagenotypebyactivitytimebininteraction(F
(30,420)
⫽ 3.004;p⬍ 0.0001).
Analysis of the simple main effects followed by post hoc Newman–Keuls comparisons revealed
a main effect of genotype at bins 4 – 6 and 8 –16 (smallest F
(2,59)
⫽ 3.43; p ⬍ 0.05), with
GluR1
⫺/⫺
mice differing from all other groups ( p values ⬍ 0.05).
10954 • J. Neurosci., October 10, 2007 • 27(41):10947–10956 Humeau et al. • AMPA Receptor Subunits in Amygdala Function
specific memories (Reisel et al., 2002; Schmitt et al., 2003, 2004).
Thus, it is possible that a GluR1-dependent, hippocampal mem-
ory could play a role in fear conditioning tasks such as that used in
the present study in which acquisition is rapid, and occurs in just
one or two trials. Against this, it could be argued that because
GluR1 gene inactivation disrupts conditioned freezing not only
to context but also to a discrete CS, then the latter, at least, is more
likely to be the result of an amygdala dysfunction. This is based on
several lesion studies that have shown that hippocampal damage
produces deficits in conditioned freezing to context but not to a
discrete CS (Kim and Fanselow, 1992; Phillips and LeDoux, 1992;
Maren et al., 1997) (but see Richmond et al., 1999). In contrast,
lesions of the BLA reliably disrupt freezing responses to both
kinds of cues (Phillips and LeDoux, 1992). Targeted lesions of the
BA, the main amygdala target of hippocampal afferents and ori-
gin of projections to the hippocampus (Pitkanen et al., 2000),
revealed a selective impairment of contextual fear conditioning
(Calandreau et al., 2005) (but see Goosens and Maren, 2006). In
accordance with a role of the BA in contextual conditioning, we
found that GluR1
⫺/⫺
but not GluR3
⫺/⫺
mice are deficient in BA
LTP.
In summary, we conclude that activity-dependent synaptic
plasticity in distinct amygdala pathways depends on different
AMPA receptor subunit combinations. The absence of condi-
tioned freezing to the auditory CS and the experimental context
in GluR1
⫺/⫺
mice suggests a dominant role for GluR1-
dependent synaptic plasticity in amygdala-dependent emotional
learning.
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