Coherent amygdalocortical theta promotes fear
memory consolidation during paradoxical sleep
Daniela Popaa,b,1, Sevil Duvarcib,1, Andrei T. Popescub, Clément Lénaa, and Denis Paréb,2
aInstitut de Biologie de l’École Normale Supérieure (IBENS), Centre National de la Recherche Scientifique UMR8197, Institut National de la Santé et de la
Recherche Médicale U1024, 75005 Paris, France; andbCenter for Molecular and Behavioral Neuroscience, Rutgers State University, Newark, NJ 07102
Edited by James L. McGaugh, University of California, Irvine, CA, and approved February 25, 2010 (received for review November 13, 2009)
Brain activity in sleep plays a crucial role in memory consolidation,
an offline process that determines the long-term strength of
memory traces. Consolidation efficacy differs across individuals,
unknown. Here, we studied how interindividual variability in fear
memory consolidation relates to neural activity in brain structures
that participate in Pavlovian fear learning. From the end of training
to testing 24 h later, some rats showed increased and others
decreased conditioned fear responses. We found that overnight
bidirectional changes in fear memory were selectively correlated
with modifications in theta coherence between the amygdala,
medial prefrontal cortex, and hippocampus during paradoxical
system may influence interindividual differences in memory con-
solidation of aversive experiences.
amygdala|fear conditioning|prefrontal cortex|hippocampus|Granger
(1, 2), and neuronal activity taking place during sleep is thought
memories determines their long-term retention, and interindividual
variations in memory performance have been related to various
factors affecting consolidation, including neuromodulatory trans-
mission (8), levels of circulating stress hormones (9), or genetic
factors (10). Several strongly interconnected brain structures are
involved in the formation andmaintenance of emotional memories,
including the hippocampus (Hi), medial prefrontal cortex (mPFC),
and basolateral amygdala (BLA) (11–13). The latter structure in
particular was shown to mediate the facilitating effects of emotions
on memory consolidation (14). Indeed, we form more vivid and
enduring memories for emotionally arousing experiences (15). Via
the release of peripheral stress hormones (16), emotional arousal
causes long-lasting increases in the firing rate of BLA neurons (17),
interfere with memory for events that took place shortly before, in
many learning tasks (18). Importantly, in most learning paradigms,
the same interventions performed shortly before testing long-term
memory recall have no effect, indicating that the BLA can facilitate
the consolidation of memories in other brain structures (14, 18).
Potentially related to the facilitation of emotional memories by
BLA activity, various lines of evidence implicate posttraining
paradoxical sleep (PS) in memory consolidation (6, 19). These
include PS reactivation of brain areas implicated in prior learning
(20) and spontaneous replay of waking activity patterns during PS
(21). In fact, it was suggested that the distinct pattern of neuronal
activity occurring during PS favors memory consolidation (4, 22)
and that emotional memories are particularly susceptible to this
effect (5, 23). Indeed, after fear conditioning, the responsiveness
of amygdala and thalamic neurons to conditioned stimuli is
enhanced during PS (24). Moreover, aversive events increase PS
amounts (25, 26), whereas PS deprivation after training impairs
consolidation of aversive memories (27). Therefore, the present
study investigated whether interindividual variations in the con-
ecently formed memories undergo a period of consolidation
solidation of emotional memories arerelated toBLA, mPFC, and
Hi interactions during PS.
We focused on the consolidation of classically conditioned fear
responses to auditory cues because this form of learning was
shown to cause synaptic plasticity in a widely distributed network
of structures, including different amygdala nuclei (BLA and
central amygdala) (28–31), multiple stages of the auditory
pathways (32, 33), Hi (34), and mPFC (35). Thus, auditory fear
conditioning constitutes an ideal model to study the role of sleep
activity in system-level memory consolidation.
In our experimental paradigm (Fig. 1A), rats (n = 12) were
subjected to a classic fear conditioning protocol in which an
auditory conditioned stimulus (CS) coterminated with a noxious
unconditioned stimulus (US). Before and after fear conditioning,
spontaneous unit activity and local field potentials (LFPs) were
recorded during waking and sleep in the BLA, mPFC, and Hi.
Twenty-four hours later, recall of the fear memory was tested by
presenting the CS in a different context (Fig. S1).
From the last CSofthe training session to the recall test the next
day, there were marked variations in the retention of the con-
ditioned fear response (CR), with some rats showing inflation and
others reduction in thetime spentfreezing during theCS(Fig. 1B).
Importantly, there was no correlation between the change in CR
seen from training to recall and freezing levels during tone habitu-
the end of training. We therefore tested whether interindividual
variations in consolidation, measured as the difference in freezing
between the last CS of the fear conditioning session vs. the first five
neuronal activity during sleep. Because theta oscillations in the Hi-
BLA (36) and mPFC (23, 37) network have been implicated in
memory, and because neuronal activity (38) and theta oscillations
areprominentduringPS intheHi (39), BLA(22), and mPFC (40),
we examined whether fluctuations in PS theta activity correlated
with variations in memory consolidation.
Theta oscillations were present in Hi, BLA, and mPFC LFPs
during PS (Fig. S2), with rhythmic theta-related unit activity at
thesesites(40–42)(Fig.S3),indicatingthat phase-locked synaptic
activity likely contributes to local theta LFP generation in these
structures. The amplitude of the theta oscillations in BLA and
mPFC was weakly correlated with that of hippocampal theta
n = 12), indicating that volume conduction between these struc-
Author contributions: D. Popa, C.L., and D. Paré designed research; D. Popa and S.D.
performed research; D. Popa, S.D., A.T.P., and C.L. analyzed data; and D. Popa, C.L.,
and D. Paré wrote the paper.
The authors declare no conflict of interest.
1D. Popa and S.D. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
| April 6, 2010
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Theta oscillations at the three sites were coherent with one
spectra (Fig. 2A). However, coherent theta occurred only inter-
mittently during PS, with interleaved bouts of correlated and
uncorrelated activity coinciding with similarly high-amplitude
hippocampal theta (Fig. S2). Remarkably, interindividual varia-
tions in fear memory consolidation strongly correlated with
changes in theta coherence from pre- to posttraining PS episodes
(Fig. 2B, n = 11) for BLA-mPFC (r = 0.81, P = 0.003) and BLA-
Hi, (r = 0.67, P = 0.025) but not for Hi-mPFC (r = −0.46, P =
0.16). In contrast, such correlations were not found in other fre-
quency bands or vigilance states (see Table S1, although a stat-
istical trend was found for the correlation between consolidation
and changes of delta slow-wave sleep coherence in the BLA-Hi)
nor with changes in LFP power. Although there was no relation-
ship between consolidation and the latency (36.1 ± 2.4 min) of
a correlation with the duration of posttraining PS episodes
(duration: r = 0.53, P = 0.09). This result is consistent with earlier
reports of increased PS after learning (19).
Moreover, for all pairs of recording sites, changes in theta LFP
coherence from pre- to posttraining PS were not correlated with
freezing during the last CS of the training session (BLA-Hi: r =
0.15, P = 0.29; BLA-mPFC: r = 0.21, P = 0.47; Hi-mPFC: r =
0.25, P = 0.46). Overall, these results indicate that the changes in
theta coherence from pre- to posttraining PS episodes were not
related to baseline anxiety levels, to the strength of the CS-US
Freezing (% time)
Freezing (% time)
Memory recall test
(first 5 trials)
Pavlovian fear conditioning. (A) Time course of percentage time spent
freezing (mean ± SEM) to the CS in rats (n = 12) during habituation, fear
conditioning (day 1), and memory recall test (day 2). (B) Overnight changes
in individual performance are bidirectional and are not determined by the
level of freezing at the end of the conditioning.
Interindividual variations in the efficacy of memory consolidation in
Change in LFP coherence after conditioning
Changes in PS θ-coherence predict change in freezing
PS after cond.
PS before cond.
BLA - mPFC
Hi - mPFC
Δ θ-band coherence
Δ % freezing
BLA - Hi
BLA - mPFC
BLA - Hi
r = 0.67
P = 0.02
r = 0.81
P = 0.003
Hi - mPFC
r = 0.46
P = 0.16
epochs correlate with interindividual variations in the efficacy of memory
consolidation. (A) Examples of pre- to posttraining shifts in LFP coherence
spectra in amygdala-Hi-cortex network during PS. (B) Linear regression of
theyandxaxesindicateincreases infreezinglevelsandtheta coherencefrom
training to testing, respectively. Each point corresponds to one animal (n =
11). The plain and dotted curves correspond to the 95% tolerance and con-
fidence bands of the linear regression, respectively. The correlation in B (Left)
remained significant even after omitting the two most extreme animals.
Changes in LFP theta coherence from pre- to postconditioning PS
Spectral Granger causality during PS
Hi - BLAmPFC - BLAHi - mPFC
Preferential θ-band directional interactions
during PS predict consolidation
Hi→BLAHi→BLABLA→mPFC Hi→mPFCBLA→mPFC Hi→mPFC
Change in % time freezing
(A) Examples of spectral Granger causality between pairs of structures. Each
curve corresponds to a pair of channels (e.g., Hi → BLA), and peaks indicate
the frequencies for which the largest fraction of the power of the second
channel may be attributed to a causal influence of activity in the first
channel. (B) Overnight change in freezing (mean ± SEM) is predicted by the
preferential directional theta interactions in the Hi-mPFC-BLA network
during PS after (Right) but not before (Left) conditioning. Words “yes” and
“no” indicate whether the preferential direction of interaction in the group
corresponds to the label indicated above. The average change in freezing
from training to testing is indicated by the dashed line (mean) and gray
band (mean ± SEM).
Granger causality analysis in the Hi-mPFC-BLA network during PS.
Popa et al.PNAS
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| vol. 107
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association at the end of training, or to the timing of PS epochs,
and are thus specifically correlated with the efficacy of the con-
solidation process (Table S2).
network, we used Granger causality analysis (43, 44) (Fig. 3), a
method that measures whether one time series is causal to another
by testing whether past values of the former help predict future
domain (43) and has been used to study the directionality of inter-
actionsbetweencorticalareas forspecific frequency bands (45,46).
When present,peaks inthe theta bandinthecausality spectra (Fig.
3A) predominantly indicated an influence of Hi theta over mPFC
andBLA theta beforeandafter conditioning,indicatinga rhythmic
entrainment of these structures by the Hi (Fig. S4). To examine
whether the directionality of theta interactions was associated with
interindividual differences inthe efficacy of memory consolidation,
we compared the performance of the rats exhibiting predominant
theta interactions in causality spectra with the performance of the
other rats (Fig. 3B). This analysis revealed that the directionality of
theta interactions among the three structures after (Fig. 3B, Right)
but not before (Fig. 3B, Left) training was differentially related to
the efficacy of the consolidation process (Table S3). Indeed, in the
than the opposite direction displayed a significantly higher efficacy
test, P = 0.024; n = 12). In contrast, the directionality of Hi-mPFC
P = 0.79).
exhibits synchronized theta activity during CS presentations after
conditioning (36), and it was proposed that this activity supports
increased coherence was observed during CS presentations, its
could not be disentangled. Subsequent work revealed that this
amygdalohippocampal theta synchrony is not a mere reflection of
fear expression because it was selectively observed during the
recall of long-term but not short-term fear memories (48). The
present study lends further support to this view. Indeed, despite a
theta coordination occur in PS, involve the mPFC, and are tightly
correlated with interindividual variations in efficacy of the con-
solidation process. Our study notably suggests that the BLA-to-
mPFC pathway (11, 49) is strongly involved in consolidation, an
effect mirroring the role of the mPFC-to-BLA pathway in fear
memory expression and extinction (49). Finally, our study indi-
cates that the increased PS duration observed in earlier reports
(19) might facilitate memory consolidation by allowing for pro-
longed theta-band interactions in limbic networks.
Although previous studies emphasized that conditioned fear
memories are stored in the amygdala (29, 30), our results instead
imply that consolidation of this form of memory involves coordi-
nated interactions in a distributed network of structures. This is
consistent with previous data indicating that fear conditioning
stages of the auditory pathways (32, 33), Hi (34), and mPFC (35).
Variations in consolidation of emotional memories had been
previously described as a function of circulating hormones or
neuromodulators, which are thought to affect the intracellular
processes of plasticity (1). Our study provides a complementary
view by indicating a direct link between consolidation efficacy and
neuronal activity in a limbic network. Therefore, our results sug-
gest that theta coordination of the fear network during PS par-
ticipates in the consolidation of Pavlovian fear memories.
Materials and Methods
Procedures were conducted in accordance with National Institutes of Health’s
Guide for the Care and Use of Laboratory Animals and were approved by the
Rutgers University Animal Care and Use Committee. Adult male Sprague-
Dawley rats (Charles River) were housed individually with ad libitum access
to food and water and maintained on a 12-h light/dark cycle. Rats were
anesthetized with a mixture of isoflurane and O2and administered atropine
methyl nitrate to reduce secretions and aid breathing. In aseptic conditions,
rats were mounted in a stereotaxic apparatus with nonpuncture ear bars. A
local anesthetic (bupivacaine, s.c.) was injected in the region to be incised.
The scalp was incised, small burr holes were made in the skull above the BLA,
mPFC, and Hi, and bundles of eight microwires were inserted in each of
these structures under stereotaxic guidance [from the bregma in mm: BLA:
anteroposterior (AP) −3.3, mediolateral (ML) 5.0, dorsoventral (DV) 8.7;
mPFC: AP +2.7, ML 0.5, DV 4.6; Hi: AP −5.6, ML 5.0, DV 8.0, and AP −3.3, ML
1.8, DV 2.7]. Electromyographic recordings were performed by means of
wires inserted in the neck muscles. The rats were allowed 1 week to recover
from the surgery.
and B). For fear conditioning (context A), rats were placed in a conditioning
chamber with a metal grid floor (Coulbourn Instruments) that was enclosed
single house light. For testing recall, the chamber contained a black Plexiglas
(0.5 mA, 1 s). Before and after fear conditioning, spontaneous unit and LFP
of vigilance (in one rat, no PS was obtained before conditioning). Recall was
tested 24 h later in context B with multiple CS presentations. Behavior was
recorded by a video camera and scored offline. Time spent freezing (immo-
bility, with the exception of breathing) was measured.
At the end of behavioral experiments, the animals were given an overdose
of pentobarbital (100 mg kg−1, i.p.) and perfused intracardially with 0.9%
saline, followed by paraformaldehyde (4%). The brains were then removed,
stored in paraformaldehyde (4%), sectioned at a thickness of 100 μm, and
the sections counterstained with cresyl violet to assess the position of the
recording sites. This report only includes data from recording sites histo-
logically confirmed to be in the structures of interest.
ACKNOWLEDGMENTS. This work was supported by National Institute of
Mental Health Grant RO1 MH073610 (to D. Paré) and by the Institut National
de la Santé et de la Recherche Médicale (C.L.).
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