Please cite this article in press as: Dı́az-Mataix et al.
, Detection of a Temporal Error Triggers Reconsolidation of Amygdala-Dependent
Memories, Current Biology (2013), http://dx.doi.org/10.1016/j.cub.2013.01.053
Current Biology 23, 1–6, March 18, 2013 ª2013 Elsevier Ltd All rights reserved
Detection of a Temporal Error
Triggers Reconsolidation
of Amygdala-Dependent Memories
Lorenzo Dı́az-Mataix,1,* Raquel Chacon Ruiz Martinez,1,2
Glenn E. Schafe,3 Joseph E. LeDoux,1,4
and Valérie Doyère1,5,6,*
1Center for Neural Science, New York University, New York,
NY 10003, USA
2Surgery Department, Medical School, University of São
Paulo, LIM 26 HC-FMUSP, 01246 São Paulo, Brazil
3Department of Psychology, Yale University, New Haven,
CT 06520, USA
4Nathan Kline Institute for Psychiatric Research, Orangeburg,
NY 10962, USA
5Université Paris-Sud, Centre de Neurosciences Paris-Sud,
UMR 8195, 91405 Orsay, France
6CNRS, 91405 Orsay, France
Summary
Updating memories is critical for adaptive behaviors, but the
rules and mechanisms governing that process are still not
well defined. During a limited time window, the reactivation
of consolidated aversive memories triggers memory lability
and induces a plasticity-dependent reconsolidation process
in the lateral nucleus of amygdala (LA) [1–5]. However,
whether new information is necessary for initiating reconso-
lidation is not known. Here we show that changing the
temporal relationship between the conditioned stimulus
(CS) and unconditioned stimulus (US) during reactivation
is sufficient to trigger synaptic plasticity and reconsolida-
tion of an aversive memory in the LA. These findings demon-
strate that time is a core part of the CS-US association and
that new information must be presented during reactivation
in order to trigger LA-dependent reconsolidation processes.
In sum, this study provides new basic knowledge about the
precise rules governing memory reconsolidation of aversive
memories that might be used to treat traumatic memories.
Results and Discussion
Traumatic fear memories are strong and persistent and form
the basis of several pathological disorders, including posttrau-
matic stress disorder (PTSD) and anxiety disorders. The
search for procedures that may render these memories sensi-
tive to pharmacological or behavioral treatments is thus crit-
ical. It is known that after memories have been consolidated
into a long-term state, they can enter a new labile state when
reactivated prior to being reconsolidated. During this lability
window, it is believed that memories are updated and new
elements are incorporated [4]. However, the exact rules gov-
erning the updating processes during reconsolidation are not
yet understood. One proposal emerging from the literature is
that reconsolidation processes are initiated when additional
learning is invoked during the reactivation procedure, forcing
the original memory to be updated and reconsolidated [6, 7].
*Correspondence: llorensdiazmataix@gmail.com (L.D.-M.), valerie.doyere@
u-psud.fr (V.D.)
http://dx.doi.org/10.1016/j.cub.2013.01.053
Report
Pavlovian threat (fear) conditioning has been used exten-
sively to study emotional learning and memory both in animals
and humans [8], and the reconsolidation of auditory fear condi-
tioning has been shown to be highly selective and dependent
on plasticity mechanisms in the lateral nucleus of amygdala
(LA) [9, 10]. Interestingly, only weaker fear memories have
been observed to be susceptible to reconsolidation; recently
formed stronger fear memories appear to be less susceptible
to reconsolidation interference even when using a conditioned
stimulus-unconditioned stimulus (CS-US) trial for reactivation
[5, 11]. This is potentially problematic for the use of reconsoli-
dation blockade as part of a therapy for traumatic memories
because these memories by definition involve strong aversive
experiences.
In Pavlovian conditioning, the subject not only learns that
the CS predicts the arrival of the US but also learns when the
US is expected to arrive [12]. Time is a critical element in asso-
ciative learning [13, 14]. Here, we asked whether a change in
CS-US time interval is necessary and sufficient to trigger an
update of an aversive memory and its reconsolidation in an
amygdala-dependent manner. To do so, we designed a
temporal auditory fear-conditioning protocol in which a 60 s
tone CS is associated with a foot-shock US delivered 30 s after
the tone onset (US@30; see Figure S1 available online). This
design permits the presentation of a single training trial in a
reactivation procedure that alters only the temporal relation-
ship between the CS and US while equating the total number
and duration of stimuli presented (context, CS, and US).
We first verified that intra-LA infusion of a protein synthesis
inhibitor immediately after reactivation is insufficient to
interfere with the reconsolidation of a recently formed strong
fear memory [5]. Rats were given ten CS-US@30 pairings fol-
lowed 24 hr later by a reactivation trial consisting of a single
CS-US@30 pairing identical to the training condition. As ex-
pected, rats given intra-LA infusion of anisomycin following
the reactivation trial showed equivalent levels of freezing
during the reactivation trial [t(14) = 0.729, nonsignificant
(n.s.)] and during the postreactivation long-term memory
(PR-LTM) test 24 hr later [t(14) = 0.135, n.s.; Figure 1A] relative
to vehicle-infused controls. Thus, in agreement with Wang
et al. [5], recently formed stronger fear memories are less
susceptible to reconsolidation interference using a protein
synthesis inhibitor. In contrast, when the CS-US time interval
was reduced from 30 to 10 s during the memory reactivation
trial, rats infused with anisomycin showed significantly
reduced freezing during the PR-LTM test relative to vehicle-
infused controls [PR-LTM: t(12) = 8.403, p < 0.001; reactivation:
t(12) = 0.488, n.s.; Figure 1B]. This suggests that the detection
of a difference in the CS-US interval between training and reac-
tivation is sufficient to induce reconsolidation of a stronger
aversive memory. Importantly, both anisomycin and vehicle
groups showed equivalent levels of freezing during a test of
postreactivation short-term memory [PR-STM: t(10) = 0.040,
n.s.; reactivation: t(10) = 0.785, n.s.; Figure 1C], suggesting
that the impairment observed during PR-LTM was due to the
disruption of reconsolidation processes and not due to
damage to the amygdala. Furthermore, when anisomycin
was infused into the central nucleus of amygdala (CeA), no
Please cite this article in press as: Dı́az-Mataix et al., Detection of a Temporal Error Triggers Reconsolidation of Amygdala-Dependent
Memories, Current Biology (2013), http://dx.doi.org/10.1016/j.cub.2013.01.053
Current Biology Vol 23 No 6
2

Figure 1. Changing the CS-US Time Interval during Reactivation of Strong Aversive Memories Triggers an LA-Dependent Reconsolidation Process
Each panel shows schematic of the experimental design (top) and percentage of freezing (mean 6 SEM) to the CS during reactivation (React) and postreac-
tivation long-term memory test (PR-LTM) in rats infused with vehicle (white bars) or anisomycin (black bars) (bottom). All four experiments consisted of
training with ten trials of 60 s tone paired with a US foot shock delivered 30 s after tone onset (CS60 2 US@30). Freezing during reactivation was equivalent
between vehicle and anisomycin rats in all four experiments. *p < 0.05. LA, lateral nucleus of amygdala; CeA, central nucleus of amygdala.
(A) Rats reactivated with the same CS-US time interval as the one learned during training (CS60 2 US@30) and given intra-LA anisomycin did not show an
impairment of memory during PR-LTM.
(B and C) Rats infused with anisomycin in the LA and reactivated with a CS-US interval shifted to 10 s (CS60 2 US@10) showed an impairment during PR-LTM
(B), but not when memory was tested 3 hr (postreactivation short-term memory; PR-STM) after reactivation (C).
(D) The infusion of anisomycin in the CeA did not induce an impairment of memory during LTM in the rats reactivated with a shifted CS-US interval.

reconsolidation impairment was observed [PR-LTM: t(9) =
0.271, n.s.; reactivation: t(9) = 0.347, n.s.; Figure 1D]. Thus, re-
consolidation of a fear memory following a temporal shift
depends upon protein synthesis in the LA, but not in the
CeA, as previously reported using standard fear conditioning
and reactivation procedures [15]. In agreement with this
observation, we observed that a change in CS-US interval
triggers plasticity mechanisms in the LA, but not the CeA, as
measured by retrieval-induced expression of the immediate
early gene zif-268, a marker of synaptic plasticity that has
been implicated in fear memory reconsolidation [16, 17]
(Figure 2). The number of zif-268-positive cells was signifi-
cantly higher in the LA in rats reactivated with a change in
the CS-US time interval relative to rats reactivated with the
initial CS-US time interval and those in the nonreactivated
group [F(2,21) = 6.011, p < 0.01; Figures 2C–2F]. No significant
differences were observed among the three groups for
the number of zif-268-positive cells in the CeA [F(2,21) =
2.892, n.s.; Figures 2G–2J].
The aforementioned findings demonstrate not only that
a change in the CS-US interval can return strong aversive
memories to a labile state but also that the CS-US interval itself
has been learned during the conditioning session. If a temporal
discrepancy is the critical parameter that triggers reconsolida-
tion, then increasing the CS-US interval should be equally
effective as decreasing it. In our next experiment, we therefore
trained rats with a 60 s tone CS paired with a US foot shock
delivered 10 s after the tone onset (US@10). Twenty-four hours
later, rats received intra-LA infusion of anisomycin or vehicle
immediately after a CS-US reactivation trial with a US foot
shock delivered 30 s after tone onset. During the PR-LTM
test, rats infused with anisomycin showed a significant
decrease in the level of freezing compared to vehicle-infused
rats [PR-LTM: t(13) = 4.487, p < 0.001; reactivation: t(13) =
0.169, n.s.; Figure 3A]. Therefore, a change in CS-US time
interval, either shorter or longer, triggers an amygdala-depen-
dent reconsolidation process. Remarkably, the temporal
pattern of freezing during PR-LTM seemed to differ among
the three experimental groups: no shift-US@30, shift-US@10,
and shift-US@30. Although no statistical comparison can be
made between experimental groups because they belong to
independent experiments (Figures 3B–3D), we can note that
 Please cite this article in press as: Dı́az-Mataix et al., Detection of a Temporal Error Triggers Reconsolidation of Amygdala-Dependent
Memories, Current Biology (2013), http://dx.doi.org/10.1016/j.cub.2013.01.053
Error Detection Triggers Memory Reconsolidation
3

Figure 2. Memory Reactivation Induces Synaptic Plasticity in LA Only when the CS-US Time Interval Is Shifted
(A) Schematic of the experimental design.
(B) Photomicrograph showing the amygdala nuclei analyzed for zif immunoreactivity. The broken lines delineate the nuclei borders. LA, lateral nucleus of
amygdala; CeA, central nucleus of amygdala; opt, optic tract. Scale bar represents 200 mm.
(C–E and G–I) Photomicrographs of transverse zif-stained sections from representative cases illustrating animals placed in the context but not presented
with any CS-US (no react; C), no shift (D), and shift animals (E) in the LA or in the CeA (G–I) at 2.8 mm posterior to bregma. The broken lines delineate the nuclei
borders. Scale bars represent 200 mm.
(F and J) The average of zif-648-positive cells per square millimeter (mean 6 SEM) across three anterior-posterior levels. Only the rats whose memory was
reactivated with a CS-US time interval different than the one used during training showed an increase in the expression of zif-648-positive cells in the LA (F),
but not in the CeA (J). *p < 0.05 by Newman-Keuls post hoc test.

whereas vehicle-treated shifted animals tend to express
freezing at an equivalent level throughout the CS duration,
the anisomycin-treated shifted rats show different patterns
depending on their shifted conditions. In particular, anisomy-
cin-treated rats shifted to a shorter CS-US interval show
a higher level of freezing at the beginning of the CS and a
progressive decay as time elapses during the CS, indicating
a stronger contribution of the fear related to the new CS-US
interval. Thus, although further experiments are needed to
specifically address this issue, the present data suggest that
when a change in the temporal parameters during reactivation
is detected, the new temporal relationship between the CS
and the US during reactivation is acquired in a single trial
and possibly in a manner independent of the LA.
We next asked whether a strong fear memory established
with fewer training trials is susceptible to reconsolidation inter-
ference with anisomycin in the absence of new information.
Rats were conditioned with two pairings of the CS with the
US@30, followed by reactivation with a single CS-US trial with
no change in the CS-US interval. Both anisomycin- and
vehicle-infused rats showed similar levels of freezing during
reactivation [t(10) = 0.537, n.s.] and during PR-LTM [t(10) =
0.365, n.s.; Figure 4A]. In contrast, when the CS-US interval
was reduced to 10 s during the reactivation trial, anisomycin-
treated animals showed impaired PR-LTM compared to
vehicle rats [PR-LTM: t(11) = 2.231, p < 0.05; reactivation:
t(11) = 0.031, n.s.; Figure 4B]. Similar findings were observed
when a single CS-US pairing was used during training [no-shift
condition: PR-LTM: t(14) = 1.523, n.s.; reactivation: t(14) =
0.019, n.s., Figure 4C; shift condition: PR-LTM: t(9) = 2.294,
p < 0.05; reactivation: t(9) = 0.443, n.s., Figure 4D]. Thus, fear
memories appear to require new information during reactiva-
tion for the memory to become destabilized. Furthermore,
these findings suggest that the CS-US time interval in auditory
fear conditioning, like the CS-US association, can be learned in
a single trial; the learning of the timing (see Figure 3) thus does
not depend on the prior learning of the association. These find-
ings extend those of a previous study [18] by showing that an
interval as short as 30 s can be learned after a single CS-US
training trial in an auditory fear-conditioning paradigm and
that the precision of the US timing at the outset of learning is
at least 20 s, because a difference between 10 and 30 s was
detected. This adds empirical support to the concept of
temporal maps as prerequisite for associative learning [14].
Please cite this article in press as: Dı́az-Mataix et al., Detection of a Temporal Error Triggers Reconsolidation of Amygdala-Dependent
Memories, Current Biology (2013), http://dx.doi.org/10.1016/j.cub.2013.01.053
Current Biology Vol 23 No 6
4

Figure 3. Time Is a Critical Parameter of the CS-US Association that Triggers the Update of Strong Fear Memory
Schematic of the experimental design for each experiment is shown at the top of each panel.
(A) Percentage of freezing (mean 6 SEM) to the CS during reactivation (React) with a longer CS-US interval (US@30) than the one learned during training
(US@10) and during PR-LTM test in rats infused in the LA with vehicle (white bars) or anisomycin (black bars) (bottom). Freezing during reactivation was equiv-
alent between groups. Rats given intra-LA anisomycin showed an impairment of memory during PR-LTM.
(B–D) Temporal pattern of freezing during PR-LTM tests (mean 6 SEM in 3 s bins), for rats nonshifted (C), shifted with a US delivered later (B), or shifted
with a US delivered earlier (D) during reactivation. In all experiments, there was a significant effect of time [B: F(19,280) = 4.62; C: F(19,260) = 2.91;
D: F(19,240) = 4.45; #p < 0.0001]. Only when a change in CS-US interval was imposed during reactivation, the anisomycin produced a significant reduc-
tion of freezing [B: F(1,260) = 234.7; C: F(1,280) = 2.24, n.s.; D: F(1,240) = 1019.6; *p < 0.0001]. When anisomycin was infused after reactivation with
a shifted CS-US time interval from 30 to 10 s, the temporal pattern of freezing was different from vehicle controls [D: time 3 group interaction
F(19,240) = 3.50; +p < 0.0001].

Conclusions expectation [20, 21], changing the temporal relationship

In sum, our results demonstrate that when a change in the
temporal relationship between CS and US is detected, an
update of the previously acquired aversive memory and its re-
consolidation is triggered in an amygdala-dependent manner.
Our results also demonstrate unequivocally, and for the first
time with amygdala-dependent memories, that when the asso-
ciation is well learned, changing the temporal association
architecture is sufficient to trigger reconsolidation even of
recently acquired strong aversive memories [5]. In contrast, if
nothing novel is added and no additional learning is elicited,
the aversive memory trace is not rendered labile [6, 7, 19]. In
contrast to the findings of Duvarci and Nader [11], in our study
the freezing level reached its maximum after a single training
trial, indicating that the CS-US association was fully learned;
the additional CS-US trial during reactivation with no change
in the temporal structure was therefore not sufficient to trigger
additional learning, as in Wang et al. [5]. Given that learning
the time interval and learning the association may be tightly in-
tertwined, and because time is a critical element of the US
between CS and US appears to elicit an update of temporal
expectancy rules (e.g., modifying the previously consolidated
temporal association) and therefore may be the most powerful
tool to trigger reconsolidation.
Our results also demonstrate that changes in the temporal
relationship between CS and US trigger synaptic plasticity
and reconsolidation processes in the LA. Neurophysiological
studies in human and nonhuman primates, as well as in
rodents, have suggested that the amygdala may be involved
in the detection of prediction error, both in appetitive and
aversive Pavlovian situations. In effect, the amygdala shows
anticipatory neurophysiological activity [22, 23, 24], as well
as reactivity to surprising temporal irregularities or unex-
pected events [25, 26]. Whether temporal processing (timing,
CS-US interval storage, and comparison between experienced
and expected US value) is computed in the LA is not known.
Our results strongly suggest that aversive prediction error
detection—whether processed in part in the amygdala itself
or only transmitted from upstream neural structures—is
Please cite this article in press as: Dı́az-Mataix et al., Detection of a Temporal Error Triggers Reconsolidation of Amygdala-Dependent
Memories, Current Biology (2013), http://dx.doi.org/10.1016/j.cub.2013.01.053
Error Detection Triggers Memory Reconsolidation
5

Figure 4. The CS-US Time Interval Is Learned at the Same Time as the CS-US Association
Each panel shows schematic of the experimental design (top) and percentage of freezing (mean 6 SEM) to the CS during reactivation (React) and PR-LTM
test in rats infused in the LA with vehicle (white bars) or anisomycin (black bars) (bottom). Freezing during reactivation was equivalent between vehicle and
anisomycin rats in all experiments. *p < 0.05.
(A and B) Rats trained with two CS-US pairings. Rats reactivated with the same CS-US interval as the one learned during training (US@30) and given intra-LA
anisomycin did not show an impairment of memory during PR-LTM (A); in contrast, when memory was reactivated with a different CS-US time interval
(US@10), anisomycin-infused rats showed an impairment of memory (B).
(C and D) The same effect was observed after one-trial training.

a fundamental mechanism in triggering reconsolidation of
aversive memories in the amygdala. Collectively, our findings
provide precise boundary rules for effective destabilization
of strong aversive memories.
Experimental Procedures
Behavioral Experiments
Adult Sprague-Dawley rats provided by Hilltop Lab Animals and weighing
250–300 g at the beginning of the experiments were used. All procedures
were in accordance with the NIH Guide for the Care and Use of Experimental
Animals and were approved by the New York University Animal Care and
Use Committee.
After recovering from surgical implantation of cannulae (see Supple-
mental Experimental Procedures for details and Figures S2 and S3 for
cannulae placements), rats were fear conditioned with a novel protocol
that allowed us to change the time of arrival of the US. The CS was a 60 s,
5 kHz, 80 dB SPL sine wave tone. The US (1 s, 1 mA foot shock) was deliv-
ered 30 s (CS60 2 US@30) or 10 s (CS60 2 US@10) after the onset of the tone,
depending on the experiment (see Figure S1). Memory reactivation session
took place 24 hr after fear conditioning by presenting one reinforced trial.
The US was delivered either at the same time after the tone onset as during
conditioning (no-shift groups) or at a different time (shift groups). Immedi-
ately after, the rats received an infusion of anisomycin or vehicle in the LA
or in the CeA, depending on the experiment. The memory-retention tests
were performed either 3 hr (PR-STM) or 24 hr (PR-LTM) after reactivation
and involved five tone-alone presentations in a modified context (see
Figure S4 for contextual freezing). Freezing was used to measure the condi-
tional emotional fear response.
Immunohistochemistry
In separate groups of animals 90 min after the reactivation session, brains
were taken, cut, and processed for zif-268 immunohistochemistry (see
Supplemental Information).
Statistical Analysis
ANOVA and post hoc tests were performed using GraphPad Prism 5.0 soft-
ware. The significance level was set at a = 5%.
Supplemental Information
Supplemental Information includes four figures and Supplemental Experi-
mental Procedures and can be found with this article online at http://dx.
doi.org/10.1016/j.cub.2013.01.053.
Acknowledgments
We thank Claudia Farb for her excellent assistance with histology and
Begoña Brotons for her help in the design of the graphical abstract.
R.C.R.M. was the recipient of grants CAPES #2350/09-2 and FAPESP #11/
08575-7 and 12/06825-9 from the government of Brazil. V.D. was supported
by Agence Nationale de la Recherche grants. V.D. and J.E.L. were recipients
Please cite this article in press as: Dı́az-Mataix et al., Detection of a Temporal Error Triggers Reconsolidation of Amygdala-Dependent
Memories, Current Biology (2013), http://dx.doi.org/10.1016/j.cub.2013.01.053
Current Biology Vol 23 No 6
6

of collaborative grants from CNRS and the Partner University Fund. J.E.L. 24. Bucci, D.J., and Macleod, J.E. (2007). Changes in neural activity associ-
was the recipient of NIH grants R01 MH038774 and R01 MH46516 funding ated with a surprising change in the predictive validity of a conditioned
this research. stimulus. Eur. J. Neurosci. 26, 2669–2676.
25. Herry, C., Bach, D.R., Esposito, F., Di Salle, F., Perrig, W.J., Scheffler, K.,
Received: October 10, 2012 Lüthi, A., and Seifritz, E. (2007). Processing of temporal unpredictability
Revised: December 23, 2012 in human and animal amygdala. J. Neurosci. 27, 5958–5966.
Accepted: January 18, 2013 26. Metereau, E., and Dreher, J.C. (2013). Cerebral correlates of salient
Published: February 28, 2013 prediction error for different rewards and punishments. Cereb. Cortex
23, 477–487.

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Supplemental Information

Detection of a Temporal Error

Triggers Reconsolidation

of Amygdala-Dependent Memories
Lorenzo Díaz-Mataix, Raquel Chacon Ruiz Martinez, Glenn E. Schafe, Joseph E. LeDoux,
and Valérie Doyère

Supplemental Inventory
Figure S1: Behavioral protocol. Related to Figures 1, 2, 3, and 4.
Explains in a graphical way the experimental design of all the experiments of the main
manuscript.

Figure S2: Cannulae placements in animals infused with artificial cerebrospinal fluid. Related to
Figures 1, 3, and 4.
Shows the brain location of the cannulae tip in the animals treated with artificial cerebrospinal
fluid for all the behavioral experiments showed in the main manuscript.

Figure S3: Cannulae placements in animals infused with anisomycin. Related to Figures 1, 3,
and 4.
Shows the brain location of the cannulae tip in the animals treated with anisomycin for all the
behavioral experiments showed in the main manuscript.

Figure S4: Contextual fear is not affected by the blockade of auditory cued fear reconsolidation
induced by the anisomycin and the change in the US expected time of arrival. Related to Figures
1, 3, and 4.
Shows freezing to the context during the memory reactivation and test sessions of all the animals
included in the behavioral experiments of the main text.

We have not cited the supplemental figures in the main figures legend to make easier the reading
of the manuscript since most of the supplementary figures refer to most of the main figures.

Supplemental Experimental Procedures

Describes in detail the methods (including subjects, surgery drug infusions, histology, behavioral
apparatus and stimuli, fear conditioning and memory procedures, immunohistochemistry, and
statistical analysis) that are briefly explained in the main manuscript and are essential for the
reproducibility of the experiments.
Figure S1. Behavioral Protocol, Related to Figures 1, 2, 3, and 4
Two days before fear conditioning, all rats were exposed to the conditioning context during 1
hour each day for habituation to the context (Day -2 and Day -1). On Day 0 rats were placed in
the conditioning chambers and conditioning trials were delivered (10 or 2 or 1 tone-footshock
pairings). The conditioned stimulus (CS) was a 60-sec, 5kHz, 80 dB SPL sine wave tone. The
unconditioned stimulus (US) was a 1second footshock with an intensity of 1mA, delivered 30
seconds (for experiments showed in figure 1, 2 and 4) or 10 seconds after the onset of the
conditioned stimulus (for experiment showed in figure 3). 24 hours after conditioning (Day 1)
rats were placed in the conditioning boxes and memory was reactivated with the presentation of
the same CS-US time pattern (no shift groups) or a different CS-US time pattern (shift groups) as
during conditioning. Immediately after reactivation vehicle or anisomycin was infused into the
lateral or the central nucleus of the amygdala. Depending on the experiment, fear memory was
tested in a modified context either 3 hours (postreactivation short-term memory test, PR-STM),
or 24 hours (postreactivation long-term memory, PR-LTM) after drug infusion with the
presentation of 5 tones.
Figure S2. Cannulae Placements in Animals Infused with Artificial Cerebrospinal Fluid.
Related to Figures 1, 3, and 4
Black squares indicate the location of the injecting cannula tip. 1A – experiment described in
Figure 1A; 1B - experiment described in Figure 1B; 1C – experiment described in Figure 1C; 1D
– experiment described in Figure 1D; 3A – experiment described in Figure 3A. 4A – experiment
described in Figure 4A; 4B – experiment described in Figure 4B; 4C – experiment described in
Figure 4C; 4D – experiment described in Figure 4D. All sections depicted at 3.3 posterior to
bregma in the left (L) or right (R) hemisphere of the rat brain.
Figure S3. Cannulae Placements in Animals Infused with Anisomycin. Related to Figures 1,
3, and 4
Black circles indicate the location of the injecting cannula tip. 1A – experiment described in
Figure 1A; 1B - experiment described in Figure 1B; 1C – experiment described in Figure 1C; 1D
– experiment described in Figure 1D; 3A – experiment described in Figure 3A. 4A – experiment
described in Figure 4A; 4B – experiment described in Figure 4B; 4C – experiment described in
Figure 4C; 4D – experiment described in Figure 4D. All sections depicted at 3.3 posterior to
bregma in the left (L) or right (R) hemisphere of the rat brain.
Figure S4. Contextual Fear Is Not Affected by the Blockade of Auditory Cued Fear Reconsolidation Induced by Anisomycin
and the Change in the US Expected Time of Arrival. Related to Figures 1, 3, and 4
Schematic of the experimental design and percentage of freezing (mean ± s.e.m.) to the context during reactivation (React, minute
immediately prior the CS-US reactivation trial) and postreactivation long-term memory test (PR-LTM, minute immediately prior the
first CS during the memory test) in rats infused with vehicle (white bars) or anisomycin (black bars).
(A) No difference of contextual freezing was observed during reactivation or PR-LTM between vehicle or anisomycin intra-LA
infused animals when rats where conditioned with 10 CS-US@30 pairings and memory was retrieve 24 hours later with the same CS-
US time pattern (reactivation: t(14)=0.993, n.s.; PR-LTM: t(14)=0.338, n.s.).
(B and C) When animals where trained with 10 CS-US@30 pairings and memory was reactivated with a shifted CS-US presentation
(US@10), intra-LA anisomycin did not induced an impairment of the contextual memory in rats tested for long-term memory
(reactivation: t(12)=0.600, n.s.; PR-LTM: t(12)=0.057, n.s.) (B), or in rats tested for short-term memory (reactivation: t(10)=0.103,
n.s.; PR-LTM: t(10)=0.221, n.s.) (C).
(D) In the same experimental conditions (conditioning with 10 CS-US@30 pairing and reactivation with 1 CS-US@10 presentation),
when anisomycin was infused in the central nucleus of the amygdala (CeA) contextual freezing was not affected (reactivation:
t(9)=0.112, n.s.; PR-LTM: t(9)=0.271, n.s.).
(E) The same lack of effect on the contextual freezing in intra-LA infused anisomycin rats when the animals were conditioned with 10
CS-US@10 and memory was reactivated with an exposure to a 1 CS-US@30 (reactivation: t(13)=0.186, n.s.; PR-LTM: t(13)=0.633,
n.s.).
(F and G) When rats were conditioning with the presentation of 2 CS-US@30 and intra-LA vehicle or anisomycin was infused neither
the rats whose memory was reactivated with the no shift CS-US (US@30) nor the rats whose memory was reactivated with a shifted
CS-US presentation (US@10) showed a difference in the contextual freezing between groups (No shift: reactivation: t(10)=0.296, n.s.;
PR-LTM: t(10)=1.020, n.s. Shift: reactivation: t(11)=0.520, n.s.; PR-LTM: t(11)=1.378, n.s.).
(H and I) Contextual freezing was not affected by the intra-LA infusion of anisomycin in rats trained with 1 CS-US@30 and reactivated
with the presentation of 1 non shifted CS-US (US@30) (reactivation: t(14)=0.414, n.s.; PR-LTM: t(14)=0.153, n.s.) (H) or reactivated
with 1 shifted presentation of the CS-US (US@10) (reactivation: t(9)=0.526, n.s.; PR-LTM: t(9)=1.001, n.s.) (I).
Supplemental Experimental Procedures
Subjects
A total of 144 Sprague-Dawley rats (Hilltop Lab Animals, Scottdale, PA), weighing 250-300g at
the beginning of the procedures, were fear conditioned. Rats were housed individually in plastic
Nalgene cages and maintained on a 12/12 hr light/dark cycle. Food and water were provided ad
libitum. All procedures were in accordance with the NIH Guide for the Care and Use of
Experimental Animals, and were approved by the New York University Animal Care and Use
Committee.

Surgery
Surgical procedures were conducted under Nembutal anesthesia (45 mg/kg; i.p.), rats were
implanted bilaterally with 26-gauge stainless guide cannulae (PlasticsOne, Roanoke, VA) aimed
at the lateral nuclei of the amygdala (LA) or the central nuclei of the amygdala (CeA). All
coordinates were taken from Paxinos and Watson (2005) [SR1]. Coordinates for intra-LA were:
3.0 mm posterior to bregma, 5.3 mm lateral to the midline and 8.0 mm ventral to the skull
surface. Coordinates for intra-CeA cannulae were: 2.8 mm posterior to bregma, 4.2 mm lateral to
the midline and 6.5 mm ventral to the skull. The guide cannulae were fixed to screws in the skull
using acrylic dental cement. A dummy cannula was inserted into each guide cannula to prevent
clogging.
Postsurgical analgesics (2 mg/kg ketoprofen) were given daily for 3 days after all
surgeries. Rats had at least one week to recover before the start of behavioral procedures.

Drug Infusions
Using a 33-gauge injector cannulae and an infusion pump (Sage Instruments, Freedom, CA),
anisomycin or an equivalent volume of artificial cerebrospinal fluid (ACSF) were injected
bilaterally into the LA or Ce at a rate of 0.25µl/min. Following drug infusion, injector cannulae
were left in place for an additional minute to allow diffusion of the drug away from the cannula
tip. Anisomycin (Sigma, St. Louis, MO) was dissolved in equimolar HCL, diluted with artificial
cerebrospinal fluid (ARCF), and adjusted to pH 7.4 with NaOH. The drug concentration was
125µg/µl. The volume of anisomycin (or ACSF) infused intra-LA or CeA was 0.5 µl on each
side

Histology
At the termination of the experiment, rats were euthanized by an overdose of chloral hydrate
(600 mg/kg) and perfused with 10% buffered formalin. Their brains were sectioned at 50μm
thickness. The sections were stained using Cresyl violet and examined with light microscopy for
cannula penetration. After histological verification, only animals that had both cannulae into the
LA (or CeA) were included in the present report (See: Figure S2 and 3).

Behavioral Apparatus and Stimuli

Rats underwent habituation, fear conditioning, fear reactivation and fear memory tests in one of
four identical chambers constructed of aluminum and Plexiglas walls (Rat Test Cage, Coulbourn
Instruments, Allentown, PA), with metal stainless steel rod flooring that was attached to a shock
generator (Model H13-15; Coulbourn Instruments). The chambers were lit with a single house
light, and each chamber was enclosed within a sound-isolation cubicle (Model H10-24A;
Coulbourn Instruments). An infrared digital camera, mounted on top of each chamber, allowed
DVD taping during behavioral procedures for later behavioral scoring. Stimulus presentation was
controlled through a computer equipped with Graphic State 2 software (Coulbourn Instruments).
Chamber grid floors, trays and walls were thoroughly cleaned with water and dried between
sessions. The house light was turned on before putting the rat in the chamber, and thus there was
no session start signal. Rats were allowed to freely explore the chamber before each behavioral
procedure for variable amount of time depending on the sessions (4 min for fear conditioning, 5
min for reactivation, and 3 min for test sessions).

Fear Conditioning and Memory Procedures

Fear Conditioning Procedure

Two days before fear conditioning, all rats were exposed to the conditioning context during 1
hour each day for habituation to the context (Day 1 and Day 2). Habituation and conditioning
was conducted in groups of four rats at a time, each in a different chamber (see above). On Day 3
rats were placed in the conditioning chambers and conditioning trials were delivered (10 or 2 or
1 tone-footshock pairings). The conditioned stimulus (CS) was a 60-sec, 5 kHz, 80 dB SPL sine
wave tone. The unconditioned stimulus (US) was a 1second footshock with an intensity of 1mA,
delivered 30 (CS60-US@30) or 10 (CS60-US@10) seconds after the onset of the conditioned
stimulus depending on the experiment (see Figure S1). Mean inter-trial interval was 5 min (2-7
min range). Freezing, a measure of conditioned fear, was continuously recorded during the
conditioning session. After conditioning, rats were returned to their home cages and to the
colony room.

Memory Reactivation
Memory reactivation session took place 24 h after fear conditioning. A single stimulus consisting
in a single tone-footshock pairing was then presented. The US was delivered either at the same
time after the tone onset as during conditioning (no shift groups) or at a different time after the
tone onset as during conditioning (shift groups) (see Figure S1). Immediately after exposure to
the stimulus, the rats received an infusion of anisomycin or vehicle in the lateral amygdala or in
the central nucleus of the amygdala depending on the experiment.

Postreactivation Memory Retention Tests

To measure postreactivation short-term memory (PR-STM) the retention test was given 3 h after
reactivation. To measure postreactivation long-term memory (PR-LTM) the retention test was
given approximately 24 h after drug infusions. Rats were tested for either PR-STM, or PR-LTM
in order to avoid extinction effects due to PR-STM testing on LTM freezing. The memory
retention test involved presentation of 5 conditioned stimuli not paired with the unconditioned
stimulus in a modified context with a blackboard covering the electrified grid floor and with
peppermint scent. An average of the five scores for each CS for each rat was used for the
statistical analysis.

Measurement of Freezing Behavior

Freezing was used to measure the conditional emotional fear response, and was defined as the
cessation of all movement with the exception of respiration-related movement and non-awake or
rest body posture. Freezing was taped in a DVD and later scored offline with a digital stopwatch
by recording the total time spent freezing during every 60-sec tone in testing sessions. For the
reactivation sessions, freezing was measured for the period before presenting the US (9 or 29
seconds depending on the experiment) to prevent potential US-induced behavior to be included
in the analysis. In all sessions the pre-CS freezing was measured during the minute prior to the
first CS presentation. In all experiments freezing was scored blind with respect to the treatment
and time-shift group.
In addition, for the experiments with strong conditioning (10 CS-US pairings during
training), freezing during the CS was scored in bins of 3 seconds using a digital stopwatch and a
homemade software (G. Dutrieux, Dr. Manuel Vázquez).
In order to have a measure of the contextual fear, freezing was scored during the minute
immediately before the reactivation trial and during the minute immediately before of the first
CS of the memory retention tests for each rat (see Figure S4).

Immunohistochemistry

Perfusion
Ninety minutes after the end of reactivation session, the animals were deeply anesthetized with
chloral hydrate (150 mg/kg) and perfused transcardially with a solution of 4% paraformaldehyde
in 0.1 M phosphate buffer at pH 7.4. The brains were removed and left in PFA for 3 hours and
then transferred to a solution of 30% sucrose in 0.1 M phosphate buffer at 4 ºC overnight. The
brains were then frozen and five series of 40 µm sections were cut with a sliding microtome
along the frontal plane.

zif-268 Immunoreactivity
One series of sections for each of the three groups (no-reactivation, trained with CS60-US@30 or
CS60-US@10 and non-reactivated; shift, trained with CS60-US@30 and reactivated with CS60-
US@10; and no-shift, trained with CS60-US@10 and reactivated with CS60-US@10) was processed
for zif-268 immunohistochemistry. First, the sections were incubated for at least 1 h with
blocking solution [0.1% Triton in phosphate-buffered saline (PBS) and 1% bovine serum
albumin (BSA, Sigma)]. Then the sections were labeled overnight at 4 °C with the following
primary antibody diluted in blocking solution: monoclonal antibody EGR-1 raised in rabbit
(44D5, Cell Signaling, Ref 11/2009) at a dilution of 1:4000. The primary antiserum was
localized using a variation of the avidin–biotin complex system (ABC). In brief, sections were
incubated for 90 min at room temperature in a solution of biotinylated goat anti-rabbit IgG
(Vector Laboratories, Burlingame, CA, USA, Vectastain), and then placed in the mixed avidin–
biotin horseradish peroxidase (HRP) complex solution (ABC Elite Kit PK6101; Vector
Laboratories) for the same period of time. The peroxidase complex was then incubated for five
minutes in a chromogen solution containing 0.02% 3,30 diaminobenzidine tetrahydrochloride
(DAB, Sigma, St Louis, MO, USA) with hydrogen peroxide (1:3000). The DAB reaction was
halted by extensive washing in phosphate-buffered saline (PBS; pH 7.4). Sections were mounted
on gelatin-coated slides, dehydrated and coverslipped with DPX (Sigma). An adjacent series was
also stained with thionin to serve as a reference series for cytoarchitectonic purposes.

Quantification of zif-268-Labeled Cells

For the no-reactivation (n=8), shift (n=8) and no-shift (n=8) groups, the number of zif-268-
immunoreactive neurons were evaluated by an observer without knowledge of the animal’s
experimental condition and were generated for selected amygdaloid areas by using the 10X
objective of a Nikon Eclipse 80i microscope equipped with a Nikon Digital Camera DXM1200F.
For the labeling quantification, we first delineated the borders of the region of interest, as defined
in adjoining Nissl-stained sections, and zif-labeled cells were counted therein. The density of zif
labeling was determined by dividing the number of zif-268-immunoreactive cells by the area of
the region of interest. The parcellations of the amygdaloid regions followed The Rat Brain in
Stereotaxic Coordinates (Paxinos and Watson, 2005) and counts were taken at 2.6, 2.8, and
3.0mm posterior to Bregma. Both cell counting and area measurements were done with the aid of
a computer program (Image-J, News Version 1.44b).

Statistical Analysis
Data were analyzed by using unpaired t-test assuming equal variance after performing the
unpaired F-test for variance. For the immunohistochemistry experiment one-way ANOVA was
used. Two factor ANOVA was used for the time-pattern figure (figure 3) with CS test as a
within-subject factor and the treatment as between-subject factors. Significant effects were
analyzed using a single interaction and a post hoc Newman-Keuls significant difference test
where appropriate. The significance level was set at α=5%.

Supplemental References
1. Paxinos G., Watson C. (2005). The Rat Brain in Stereotaxic Coordinates (San Diego:
Academic Press).