402 • The Journal of Neuroscience, January 14, 2009 • 29(2):402– 413

Behavioral/Systems/Cognitive

Brain Region-Specific Gene Expression Activation Requiredfor Reconsolidation and Extinction of Contextual Fear
MemoryNori Mamiya,1 Hotaka Fukushima,1 Akinobu Suzuki,1 Zensai Matsuyama,1 Seiichi Homma,1 Paul W. Frankland,2 andSatoshi Kida1,3
1

Department of Bioscience, Faculty of Applied Bioscience, Tokyo University of Agriculture, Tokyo 156-8502, Japan, 2Program in Neurosciences and Mental
Health, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada M5G 1X8, and 3Core Research for Evolutional Science and Technology,
Japan Science and Technology Agency, Saitama 332-0012, Japan

During fear conditioning, animals learn an association between a previously neutral or conditioned stimulus (CS) and an aversive or
unconditioned stimulus (US). Subsequent reexposure to the CS alone triggers two competing processes. Brief reexposure to the CS
initiates reconsolidation processes that serve to stabilize or maintain the original CS–US memory. In contrast, more prolonged reexposure to the CS leads to the formation of an inhibitory extinction (CS–no US) memory. Previous studies have established that bothreconsolidation and extinction require gene expression. Consistent with this, here we first show that genetic disruption of cAMPresponsive element-binding protein (CREB)-mediated transcription blocks both reconsolidation and long-term extinction of contextual
fear memory. We next asked whether reconsolidation and extinction engage CREB-mediated transcription in distinct brain regions.
Accordingly, we used immunohistochemical approaches to characterize the activation of the transcription factor CREB [as well as theexpression of the CREB-dependent gene Arc (activity-regulated cytoskeleton-associated protein)] after brief versus prolonged reexposure to a previously conditioned context. After brief reexposure, we observed significant activation of CREB-mediated gene expression in
the hippocampus and amygdala. In contrast, after the prolonged reexposure, we observed significant activation of CREB-mediated gene
expression in the amygdala and prefrontal cortex. Finally, we showed that blocking protein synthesis in either the hippocampus or the
amygdala blocked reconsolidation of contextual fear memory, whereas similar blockade in the amygdala and prefrontal cortex prevented
the formation of extinction memory. These experiments establish that reactivated contextual fear memories undergo CREB-dependent
reconsolidation or extinction in distinct brain regions.
Key words: reconsolidation; extinction; CREB; hippocampus; amygdala; mPFC

Introduction
To generate long-term memory, short-term memory undergoes
a gene expression-dependent stabilization process known as
memory consolidation (Flexner et al., 1965; Davis and Squire,
1984; McGaugh, 2000). Although it was previously thought that
this consolidation occurs just once, there is growing evidence thatmemory retrieval is a dynamic process that can either reinforce oralter memory (Misanin et al., 1968; Schneider and Sherman,
1968; Lewis, 1979; Mactutus et al., 1979; Przybyslawski and Sara,
1997; Nader et al., 2000; Sara, 2000).
The fate of a retrieved memory appears to depend, in part, on
the nature of the retrieval episode. In fear-conditioning paraReceived Sept. 26, 2008; revised Nov. 10, 2008; accepted Nov. 19, 2008.
S.K. was supported by Grants-in-Aid for Scientific Research, High Technology Research and Priority Areas–
Molecular Brain Science (18022038 and 22022039) from the Ministry of Education, Culture, Sports, Science, andTechnology, Japan; by Core Research for Evolutional Science and Technology, Japan; and by the Research Grant for
Nervous and Mental Disorders from the Ministry of Health, Labour, and Welfare, Japan.
Correspondence should be addressed to Satoshi Kida, Department of Bioscience, Faculty of Applied Bioscience,
Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan. E-mail: kida@nodai.ac.jp.
DOI:10.1523/JNEUROSCI.4639-08.2009
Copyright © 2009 Society for Neuroscience 0270-6474/09/290402-12$15.00/0

digms, for example, blocking protein synthesis before or immediately after brief reexposure to the conditioning stimulus (CS)alone disrupts the subsequent expression of the fear memory(Nader et al., 2000; Debiec et al., 2002; Kida et al., 2002; Suzuki etal., 2004). Such experiments have led to the idea that the reactivated fear memory is destabilized and then restabilized through agene expression-dependent reconsolidation process (Nader et al.,
2000; Taubenfeld et al., 2001; Debiec et al., 2002; Kida et al.,2002). In contrast, more prolonged reexposure to the CS leads toextinction of the fear memory (Pavlov, 1927; Rescorla, 2001; Myers and Davis, 2002). Similar to reconsolidation, this retrievalinitiated process appears to depend on new gene expression because blockade of protein synthesis before prolonged reexposure
blocks the formation of an extinction memory [i.e., a new CS–nounconditioned stimulus (US) inhibitory memory that competes
with the original CS–US memory trace] (Berman and Dudai,
2001; Vianna et al., 2001).
Together, these studies indicate that retrieval of a fear memory
initiates two processes, reconsolidation and extinction, that havedistinct temporal signatures (Eisenberg et al., 2003; Pedreira andMaldonado, 2003; Suzuki et al., 2004). Recent studies have also

Mamiya et al. • CREB Activation in Reconsolidation and Extinction

begun to suggest that these two processes are partially dissociable
at the neurochemical level. For example, we showed that administration of either L-type voltage-gated calcium channels or CB1
receptor antagonists block extinction of contextual fear memory
but have no effect on reconsolidation (Suzuki et al., 2004). In thecurrent study, we ask whether reconsolidation and extinction are
also dissociable at the anatomical level. We first showed that activation of cAMP-responsive element-binding protein (CREB)mediated transcription is necessary for both reconsolidation andlong-term extinction of contextual fear memory. Next, we used
immunohistochemical approaches to characterize the activationof the transcription factor CREB [as well as the expression of the
CREB-dependent gene activity-regulated cytoskeletonassociated protein (Arc)] after brief versus prolonged context
reexposure. After brief reexposure, we observed significant activation of CREB-mediated gene expression in the hippocampus
and amygdala. In contrast, after the prolonged reexposure, we
observed significant activation of CREB-mediated gene expression in the amygdala and prefrontal cortex. These results suggestthat reconsolidation and extinction engage protein synthesisdependent processes in distinct brain regions. Consistent with
this, we finally showed that blocking protein synthesis in either
the hippocampus or amygdala blocked reconsolidation of contextual fear memory, whereas similar blockade in the amygdala
and prefrontal cortex prevented the formation of extinction
memory.

Materials and Methods
Mice. All experiments were conducted according to the Guide for the Careand Use of Laboratory Animals, Japan Neuroscience Society and the Guide
for the Tokyo University of Agriculture. Male C57BL/6 mice were obtained
from Charles River. Transgenic mice expressing an inducible CREB repressor (CREB IR mice) have been backcrossed to C57BL/6 (Kida et al.,
2002; Suzuki et al., 2008). Mice were housed in cages of five or six,
maintained on a 12 h light/dark cycle, and allowed ad libitum access tofood and water. Mice were at least 8 weeks of age when tested. Testing was
performed during the light phase of the cycle. All experiments were conducted blind to the treatment condition of the mouse.
Contextual fear conditioning test. Mice were trained and tested in conditioning chambers (17.5 ⫻ 17.5 ⫻ 15 cm) that had a stainless steel grid
floor through which footshocks could be delivered (Suzuki et al., 2004,
2008). Training consisted of placing the mice in the chamber and delivering an unsignaled footshock (2 s duration, 0.4 mA) 148 s later. Mice
were returned to the homecage 30 s after the footshock (training).
For the first experiment, we examined the effects of disrupting CREB
function on memory reconsolidation and extinction using transgenic
mice that express an inducible CREB repressor (CREB IR) in the forebrain, where a dominant-negative CREB protein is fused with the ligand
binding domain of a mutant estrogen receptor (ER). Previous studies
have shown that systemic injection of tamoxifen, the artificial ligand for
ER, into these transgenic mice inhibits CREB activity in the forebrain(Kida et al., 2002). Twenty-four hours after the training session, CREB IR
and wild-type (WT) mice were placed back in the training context for 3
or 30 min, during which time freezing behavior was assessed (reexposure) as the percentage of time mice spent freezing (defined as a complete
lack of movement, except for respiration). Mice were administered an
intraperitoneal injection of 16 mg/kg 4-hydroxytamoxifen (TAM;
Sigma), which was dissolved in 10 ml of peanut oil (Sigma) or vehicle(VEH; a similar volume of peanut oil) 6 h before the reexposure (Kida etal., 2002). Twenty-four hours after the reexposure session, mice wereonce again placed in the training context (5 min), and freezing behavior
was assessed (test). Freezing behavior was measured automatically(O’Hara & Co.) (Anagnostaras et al., 2001).
For the second experiment (phosphorylated and total CREB immunocytochemistry), we examined the brain regions that are activated after
reexposure to the CS (see Fig. 2). Before the commencement of contex-

J. Neurosci., January 14, 2009 • 29(2):402– 413 • 403

tual fear conditioning, mice were handled individually for 2 min each day
for 1 week. Mice were divided into eight groups: (1) Recon-33 and -60,
and Ext-33 and -60 groups; four (conditioned) groups of mice weretrained as described above and 24 h later were reexposed to the context
for either 3 min (Recon groups) or 30 min (Ext groups). The animals
were then anesthetized with Nembutal (750 mg/kg, i.p.) 3 min (Ext-33),
30 min (Recon-33, Ext-60), or 57 min (Recon-60) after the reexposure;(2) no US-33 and -60 groups; two (unconditioned) groups received a
training session in the absence of footshock and 24 h later were reexposedto the context for either 3 min (no US-33) or 30 min (no US-60). The
animals were then anesthetized, as above, 30 min after reexposure; and(3) no association-33 and -60 groups; the other two (unconditioned)
groups were delivered a footshock 5 h after being placed in the conditioned chamber and returned to the homecage 30 s after the footshock.
Twenty-four hours later, mice were reexposed to the context for either 3min (no association-33) or 30 min (no association-60) and anesthetized,
as above, 30 min after the reexposure. It is important to note that, in thisexperiment, the total length of time from the beginning of the reexposure
to the anesthetization is 33 min (Recon-33, Ext-33, no US-33, noassociation-33) or 60 min (Recon-60, Ext-60, no US-60, no association60). The Ext-33 and Recon-60 groups were used to compare the effects ofthe duration of reexposure on CREB activation with the Recon-33 andExt-60 groups, respectively, using the identical time course from the
beginning of the reexposure.
For the third experiment (Arc immunocytochemistry), mice weretrained as above in the presence or absence of a footshock and 24 h later
were reexposed to the context for 3 or 30 min. The animals were anesthetized 90 min after the reexposure.
For the fourth experiment, we examined the effects of inhibition ofprotein synthesis in the medial prefrontal cortex (mPFC), hippocampus,
and amygdala on memory reconsolidation and extinction. A protein
synthesis inhibitor, anisomycin (ANI; Sigma), was dissolved in VEH
solution, containing 3 drops of Tween 80 in 2.5 ml of 7.5% dimethylsulfoxide in artificial CSF, and adjusted to pH 7.4 with NaOH. Mice weretrained as above and 24 h later received intracranial injections of ANI(62.5 ␮g) immediately after the reexposure phase of 3 or 30 min. Twentyfour hours after the reexposure session, mice were once again placed in
the training context (5 min), and freezing behavior was assessed. Infusions into each brain region (0.5 ␮l) were made at a rate of 0.25 ␮l/min.
The injection cannula was left in place for 2 min after the infusion. Thisdose of locally infused ANI inhibits ⬎90% of protein synthesis for at least
4 h (Rosenblum et al., 1993).
Immunocytochemistry. After the anesthetization, mice were perfusedwith PBS/0.1 mM sodium fluoride (NaF) containing 4% paraformaldehyde. Brains were then removed, fixed overnight, transferred to 30%
sucrose, and stored at 4°C. Coronal sections (30 ␮m) were cut on acryostat. After cutting the brain, sections were washed and preincubated
in 3% H2O2 in methanol for 1 h, followed by incubation in blocking
solution (PBS/0.1 mM NaF plus 1% goat serum albumin, 1 mg/ml BSA,
and 0.05% Triton X-100) for 3 h. Consecutive sections were incubatedwith a polyclonal rabbit primary antibody for anti-phospho-CREB(S133; 1:1000; Upstate Biotechnology), anti-CREB (1:1000; Upstate Biotechnology), or anti-Arc (1:1000; Santa Cruz Biotechnology) in theblocking solution overnight. To measure CREB activation, consecutive
sections were alternately incubated with anti-phospho-CREB or antiCREB antibodies. Subsequently, sections were washed with PBS/0.1 mM
NaF and incubated for 3 h at room temperature with biotinylated goatanti-rabbit IgG (SAB-PO kit; Nichirei Biosciences), followed by 1 h atroom temperature in the streptavidin– biotin–peroxidase complex(SAB-PO kit). Structures were anatomically defined according to theatlas of Franklin and Paxinos (1997). Quantification of phosphorylated
CREB (pCREB)-, CREB-, or Arc-positive cells in sections (100 ⫻ 100␮m) of mPFC (bregma between 2.10 and 1.98 mm), amygdala (bregma
between ⫺1.22 and ⫺1.34 mm), and dorsal hippocampus (bregma between ⫺1.46 and ⫺1.82 mm) was analyzed with a computerized image
analysis system, as described previously (Winroof version 5.5 software;
Mitani Corporation) (Frankland et al., 2006; Suzuki et al., 2008). Immunoreactive neurons were counted bilaterally with a fixed sample window

Mamiya et al. • CREB Activation in Reconsolidation and Extinction

404 • J. Neurosci., January 14, 2009 • 29(2):402– 413

across at least three sections by an experimenter
blind to the treatment condition.
CREB activation (relative pCREB/CREB levels) was calculated by normalizing the numberof pCREB-positive cells to the total number of
CREB-positive cells. Data of pCREB/CREB levels and Arc expression for each group were expressed as a percentage of the averaged values in
the no US-33 control group (Kee et al., 2007).
Surgery for microinfusion of drug. Surgeries
were performed as described previously(Frankland et al., 2006; Suzuki et al., 2008). Under Nembutal anesthesia and using standard
stereotaxic procedures, stainless steel guidecannulae (22 gauge) were implanted into themPFC (2.7 mm, ⫾0 mm, ⫺1.6 mm), the dorsal
hippocampus (⫺1.8 mm, ⫾1.8 mm, ⫺1.9mm), or amygdala (⫺1.3 mm, ⫾3.3 mm, ⫺4.4mm). Mice were allowed to recover for at least 1
week after surgery. After this, they were handled
for 1 week before the commencement of contextual fear conditioning.
Data analysis. Data were analyzed withANOVA. One-way and post hoc Newman–
Keuls comparisons were used to analyze the effects of treatments, drugs, and genotypes onfreezing responses. One- or two-way ANOVA
followed by post hoc Newman–Keuls and post
hoc Bonferroni’s comparisons, respectively,
were used to analyze the effects of groups,
times, and reexposure on CREB activation and
Arc expression. Planned comparisons using apaired t test were used to analyze the differencesof freezing scores from the contextual conditioning of each group between the first 5 min
reexposure, during the 30 min reexposure, andtest. Comparisons of within-session extinction
using a paired t test and repeated ANOVA wereused to analyze the effects of drug and genotype(Suzuki et al., 2004, 2008). All values in the text
and figure legends are means ⫾ SEM.

Results

Figure 1. Effects of disrupting CREB function on memory reconsolidation and extinction. A, C, Experimental design used withdata presented below. A, B, Effects of disrupting CREB function on reconsolidation. A, Freezing score during 3 min reexposure(WT/VEH, n ⫽ 13; CREB IR/VEH, n ⫽ 11; WT/TAM, n ⫽ 13; CREB IR/TAM, n ⫽ 11). B, Freezing score during test session. C, D,
Effects of disrupting CREB function on extinction. C, Freezing score in 5 min blocks during 30 min reexposure (WT/VEH, n ⫽ 12;
CREB IR/VEH, n ⫽ 15; WT/TAM, n ⫽ 14; CREB IR/TAM, n ⫽ 15). D, Freezing score during test session were shown. E, Summary of
the relationship between the duration of reexposure and freezing score at test session (B, D). Error bars are SEM. *p ⬍ 0.05, versus
WT/VEH, WT/TAM, and CREB IR/VEH.

The relationship between reconsolidationand extinction of fear memory has been
investigated (Eisenberg et al., 2003; Pedreira and Maldonado, 2003; Suzuki et al.,2004). Our previous study demonstrated
that inhibition of protein synthesis after a short reexposure (3min) to the context disrupts the reactivated contextual fear memory, whereas a prolonged reexposure (30 min) followed by inhibition of protein synthesis blocked long-term extinction without
disrupting the fear memory (Suzuki et al., 2004). These observations indicate that short and long reexposure triggers reconsolidation or extinction phases, respectively. In this study, to ask
whether reconsolidation and extinction phases are dissociable atthe anatomical level, we investigated brain regions regulating thereconsolidation and extinction phases.
Roles of CREB-mediated transcription in reconsolidationand extinction
Previous studies have shown that CREB-mediated transcription
is required for the consolidation and reconsolidation of contextual fear memory (Kida et al., 2002). However, little is known
about whether transcriptional activation by CREB is required formemory extinction. Therefore, we first examined the roles of
CREB-mediated transcription in the reconsolidation and extinc-tion of contextual fear memory and then tried to investigate thebrain regions where CREB is activated in the reconsolidation andextinction phases.
To examine the effects of loss of function of CREB on reconsolidation and extinction, we used CREB IR transgenic mice (Kidaet al., 2002). In these experiments, CREB IR and WT littermate
controls were trained with a single footshock and 24 h later were
reexposed to the context for 3 or 30 min. Six hours before thereexposure, mice received a systemic injection of TAM or VEH toinduce the inhibition of CREB activity (Kida et al., 2002).
In the reconsolidation experiment, all groups showed comparable levels of freezing during the 3 min reexposure (Fig. 1 A)
(F(3,44) ⫽ 0.15; p ⬎ 0.05). When the contextual fear memory wastested 24 h later, one-way ANOVA revealed a significant effect ofgroup (Fig. 1 B) (F(3,44) ⫽ 2.83; p ⬍ 0.05). The post hoc Newman–
Keuls test showed that TAM-injected CREB IR mice significantly
froze less compared with the other groups (Fig. 1 B) ( p ⬍ 0.05).
Consistent with previous observations, inhibition of CREB activity disrupted the reactivated fear memory, indicating that theMamiya et al. • CREB Activation in Reconsolidation and Extinction

J. Neurosci., January 14, 2009 • 29(2):402– 413 • 405

calcium/calmodulin-dependent protein
kinase IV (Shaywitz and Greenberg, 1999;
Mayr and Montminy, 2001). This phosphorylation is an essential step in the activation of CREB-mediated transcription(Gonzalez and Montminy, 1989; Ginty etal., 1994; Bito et al., 1996; Silva et al.,1998). Therefore, phosphorylation of
CREB at serine 133 has been used to identify brain regions activated in gene
expression-dependent memory processes(Viola et al., 2000; Hall et al., 200l).
Therefore, to ask whether reconsolidation and extinction phases are dissociable
at the anatomical level, we next performed
immunohistochemical analysis to identify
brain regions where CREB is activated in
the reconsolidation and extinction phases.
Reconsolidation of fear memory has beenshown to be mediated by the amygdala(Nader et al., 2000; Duvarci and Nader,
2004; Tronel et al., 2005; Lee et al., 2006)
and hippocampus (Debiec et al., 2002;
Frankland et al., 2006; Suzuki et al., 2008).
In contrast, extinction of the fear memory
Figure 2. Experimental design. The experimental design to investigate changes in CREB activation in mPFC, hippocampus, and is thought to depend on the mPFC (Moramygdala induced by reconsolidation or extinction phases is shown.
gan et al., 1993; Morgan and LeDoux,
1995, 1999; Morrow et al., 1999; Quirk etcontextual fear memory requires CREB-dependent memory foral., 2000, 2006; Santini et al., 2004) and amygdala (Lee et al., 2001;
reconsolidation (Kida et al., 2002; Suzuki et al., 2008).
Lu et al., 2001; Walker et al., 2002; Lee et al., 2006; Myers andWe next examined the effects of disrupting CREB function on
Davis, 2007). From these findings, we focused our anatomical
memory extinction. Overall freezing levels did not differ acrossanalyses on the mPFC, amygdala, and hippocampus.
groups during the 30 min reexposure (F(3,52) ⫽ 2.42; p ⬎ 0.05)
CREB activation was assessed as the ratio of pCREB to total
but did decrease over time in all groups (F(5,260) ⫽ 74.2; p ⬍ 0.05)
CREB levels in four conditioned and four unconditioned groups(Fig. 1C). Measurements 24 h after this 30 min reexposure con(Fig. 2). The conditioned groups of mice were trained with afirmed that the extinction of conditioned freezing was persistentfootshock and 24 h later were re-exposed to the context for 3 minin VEH-treated WT mice (first 5 min in the reexposure session vs(Recon groups) or 30 min (Ext groups). Recon groups were astest; t(22) ⫽ 3.03; p ⬍ 0.05), indicating that long-term extinction
sessed for CREB activation 30 min (Recon-33 group) or 57 minoccurred in these experimental conditions. In contrast, extinc(Recon-60 group) after the reexposure, whereas Ext groups weretion tested 24 h after the 30 min reexposure was blocked by theassessed for CREB activation 3 min (Ext-33 group) or 30 min
inhibition of CREB function. One-way ANOVA across four(Ext-60 group) after the reexposure. Unconditioned groups weregroups revealed a significant effect of group (Fig. 1 D) (F(3,52) ⫽treated similarly, except either that they did not receive a foot5.32; p ⬍ 0.05). TAM-injected CREB IR mice showed significantly
shock (no US groups) or that they stayed in the conditioned
more freezing compared with the other groups (Fig. 1 D) ( p ⬍chamber for 5 h before receiving the footshock (no association
0.05). Thus, these results indicate that CREB is required for longgroups) and were assessed for CREB activation 30 min after theterm extinction.
reexposure for 3 min (no US-33 and no association-33 groups) or
The effects of blocking CREB function on memory reconsoli30 min (no US-60 and no association-60 groups). It is importantdation and extinction are summarized in Figure 1 E. These results
to note that the conditioned groups froze at equal levels during
indicate that activation of CREB-mediated transcription is rethe 3 min reexposure and for the first 5 min of the 30 min reexquired for both reconsolidation and long-term extinction. It is
posure (Recon-33, 56.4 ⫾ 5.2%; Recon-60, 58.3 ⫾ 3.4%; Ext-33,
worth noting that although genetic inhibition of CREB activity
57.6 ⫾ 4.5%; Ext-60, 59.2 ⫾ 4.1%), indicating that the contextual
blocked the formation of the new extinction memory, it presumfear memory was reactivated. Longer (30 min) reexposure reduced
ably had no effect on the expression of the original fear memory.
freezing over time within the session in both the Ext-33 and Ext-60
This observation is consistent with previous findings using sysgroups (to 24.9 ⫾ 5.9 and 25.1 ⫾ 3.1%, respectively), indicating that
temic injection of ANI (Suzuki et al., 2004, 2008) and (1) conwithin-session extinction occurred (overall freezing: F(1.125) ⫽ 0.12,
firms that reconsolidation and extinction of contextual fearp ⬎ 0.05; decrease over time: F(5,125 ⫽ 28.45, p ⬍ 0.05). In contrast,
memory have distinct temporal signatures and (2) suggests thatthe unconditioned groups showed significantly lower freezing durthese two processes interact.
ing the 3 min and the first 3 min of the long reexposure comparedwith the conditioned groups (data not shown).
CREB activation in distinct brain regions in reconsolidationand extinction phases
mPFC
The transcription factor CREB is activated through phosphoryWe first analyzed CREB activation in the mPFC [including thelation at serine 133 by CREB kinases such as protein kinase A andprelimbic (PL) and infralimbic (IL) regions]. Our experiments

406 • J. Neurosci., January 14, 2009 • 29(2):402– 413

Mamiya et al. • CREB Activation in Reconsolidation and Extinction

Figure 3. Effects of the reexposure duration on CREB activation in the mPFC. A, Representative PL pCREB-positive cell immunohistochemistry obtained from indicated mice. Scare bar, 100 ␮m.
B, C, Effects of the 3 min reexposure duration on CREB activation in PL (B) and IL (C) regions of mPFC (Recon-33, n ⫽ 11; no US-33, n ⫽ 10; no association-33, n ⫽ 9; Recon-60, n ⫽ 9). D, E, Effectsof the 30 min reexposure duration on CREB activation in PL (D) and IL (E) regions of mPFC (Ext-33, n ⫽ 10; no US-60, n ⫽ 15; no association-60, n ⫽ 14; Ext-60, n ⫽ 17). *p ⬍ 0.05, compared withthe other groups. F, G, Comparison of CREB activation after 3 and 30 min reexposure in PL (F ) and IL (G) regions of mPFC in the conditioned group. Error bars are SEM. CREB activation (relative
pCREB/CREB levels) was calculated by normalizing the number of pCREB-positive cells to the total CREB positive cells. Data of CREB activation for each group were expressed as the percentage of theaveraged values in the no US-33 control group.

revealed that CREB was activated in the extinction but not thereconsolidation phase. One-way ANOVA across the 3 min reexposure (reconsolidation) groups (Recon-33, Recon-60, no US33, and no association-33) revealed no significant effect of groupin the PL and IL regions (PL: F(3,35) ⫽ 0.17, p ⬎ 0.05, IL: F(3,35) ⫽0.12, p ⬎ 0.05), indicating that CREB was not activated in eitherof these mPFC regions in the reconsolidation phase (Fig. 3 B, C).
In contrast, one-way ANOVA across the 30 min reexposure (extinction) groups (Ext-33, Ext-60, no US-60, and no association60) revealed significant effects of group in the PL and IL regions(Fig. 3 D, E) (PL: F(3,52) ⫽ 5.99, p ⬍ 0.05; IL: F(3,52) ⫽ 6.25, p ⬍0.05). The post hoc Newman–Keuls test showed a significantly
higher level of pCREB in both mPFC regions of the Ext-60 groups
compared with the other groups (Fig. 3 D, E) ( p ⬍ 0.05). This
result indicates that CREB was significantly activated in the PL
and IL regions of the conditioned groups after the 30 min, but not
3 min, reexposure. We next compared the time course of CREB
activation from the beginning of the reexposure (using the identical time schedule) among the conditioned groups (Recon-33,
Recon-60, Ext-33, and Ext-60). Consistent with the above results,
a two-way ANOVA with reexposure (3 vs 30 min) and time (fromthe beginning of reexposure; 33 vs 60 min) revealed significant

Mamiya et al. • CREB Activation in Reconsolidation and Extinction

J. Neurosci., January 14, 2009 • 29(2):402– 413 • 407

Figure 4. Effects of the reexposure duration on CREB activation in the hippocampus. A, Representative CA1 pCREB-positive cell immunohistochemistry obtained from indicated mice. Scare bar,
100 ␮m. A–D, Effects of the 3 min reexposure duration on CREB activation in CA1 (B), CA3 (C) and DG (D) regions of hippocampus (Recon-33, n ⫽ 11; no US-33, n ⫽ 10; no association-33, n ⫽ 9;
Recon-60, n ⫽ 9). *p ⬍ 0.05, compared with the unconditioned groups (no US-33 and no association-33). E–G, Effects of the 30 min reexposure duration on CREB activation in CA1 (E), CA3 (F ) andDG (G) regions of hippocampus (Ext-33, n ⫽ 10; no US-60, n ⫽ 15; no association-60, n ⫽ 14; Ext-60, n ⫽ 17). H–J, Comparison of CREB activation after 3 and 30 min reexposure in CA1 (H ), CA3(I ), and DG (J ) regions of hippocampus in the conditioned group. Error bars are SEM. CREB activation was calculated by normalizing the number of pCREB-positive cells to the total CREB-positive cells.
Data of CREB activation for each group were expressed as the percentage of the averaged values in the no US-33 control group.

reexposure versus time interactions (Fig. 3 F, G) (reexposure vstime interaction; PL: F(1,43) ⫽ 11.25, p ⬍ 0.05; IL: F(1,43) ⫽ 6.08,
p ⬍ 0.05). The post hoc Bonferroni’s test showed significantly
higher activation of CREB in the Ext-60 group compared with theother groups ( p ⬍ 0.05), indicating that CREB was activated inboth mPFC regions 30 min after the 30 min reexposure (Fig.
3 F, G). Thus, these observations indicate that CREB is activatedin the PL and IL prefrontal regions when contextual fear memory
is extinguished but not reconsolidated.
Hippocampus
We next analyzed the hippocampus. In contrast to the results forthe mPFC, hippocampal CREB is activated in the reconsolidation
but not extinction phase. One-way ANOVA across the 3 min
reexposure groups revealed significant effects of group in the CA1
and CA3 but not the dentate gyrus (DG) regions of the hippocampus (Fig. 4 B–D) (CA1: F(3,35) ⫽ 4.74, p ⬍ 0.05; CA3: F(3,35)⫽ 9.25, p ⬍ 0.05; DG: F(3,35) ⫽ 0.80, p ⬎ 0.05). Post hoc analyses
showed a significant higher level of pCREB in the CA1 and CA3
regions of the Recon-33 and Recon-60 groups compared with theother control groups ( p ⬍ 0.05), indicating that CREB is activated in the hippocampal CA1 and CA3 regions of the conditioned groups after a 3 min reexposure (Fig. 4 B–D). In contrast,
no CREB activation was observed in these three regions of the
hippocampus in the 30 min reexposure groups (CA1: F(3,52) ⫽0.24, p ⬎ 0.05; CA3: F(3,52) ⫽ 0.29, p ⬎ 0.05; DG: F(3,52) ⫽ 0.52,
p ⬎ 0.05), indicating that CREB is not activated in the hippocampus in the extinction phase (Fig. 4 E–G). Consistent with this, a
two-way ANOVA across the conditioned groups followed by thepost hoc Bonferroni’s test showed significantly higher activationof CREB in the CA1 and CA3 but not DG region of the Recongroups (Recon-33 and -60) compared with the Ext groups (Fig.
4 H–J ) (reexposure vs time interaction; CA1: F(1,43) ⫽ 0.03, p ⬎0.05; CA3: F(1,43) ⫽ 0.09, p ⬎ 0.05; DG: F(1,43) ⫽ 0.82, p ⬎ 0.05).
These observations indicate that CREB is activated in the CA1
and CA3 regions of the hippocampus when contextual fear memory is reconsolidated but not extinguished. Additionally, it is important to point out that CREB activation was not observed in the
hippocampus in the extinction phase, although contextual fear

408 • J. Neurosci., January 14, 2009 • 29(2):402– 413

Mamiya et al. • CREB Activation in Reconsolidation and Extinction

Figure 5. Effects of the reexposure duration on CREB activation in the amygdala. A, Representative BLA pCREB-positive cell immunohistochemistry obtained from indicated mice. Scare bar, 100
␮m. B–D, Effects of the 3 min reexposure duration on CREB activation in LA (B), BLA (C), and Ce (D) regions of amygdala (Recon-33, n ⫽ 11; no US-33, n ⫽ 10; no association-33, n ⫽ 9; Recon-60,
n ⫽ 9). *p ⬍ 0.05, compared with the unconditioned groups (no US-33 and no association-33). E–G, Effects of the 30 min reexposure duration on CREB activation in LA (E), BLA (F ) and Ce (G) regionsof amygdala (Ext-33, n ⫽ 10; no US-60, n ⫽ 15; no association-60, n ⫽ 14; Ext-60, n ⫽ 17). *p ⬍ 0.05, compared with the other groups. H–J, Comparison of CREB activation after 3 and 30 min
reexposure in LA (H ), BLA (I ), and Ce (J ) regions of amygdala in the conditioned group. Error bars are SEM. CREB activation was calculated by normalizing the number of pCREB-positive cells to thetotal CREB-positive cells. Data of CREB activation for each group were expressed as the percentage of the averaged values in the no US-33 control group.

memory was reactivated during the reexposure. This observation
indicates that although short reexposure that triggers memory
reconsolidation induces CREB activation in the hippocampus,
this activation is suppressed when longer context reexposure that
produces within-session extinction is used.
Amygdala
We finally analyzed the levels of pCREB in the amygdala. CREB
was activated in the lateral (LA), basolateral (BLA), and central(Ce) regions of the amygdala in both the reconsolidation andextinction phases. One-way ANOVA across the 3 min or 30 min
reexposure groups revealed significant effects of group in thethree regions of the amygdala (Fig. 5B–G) (LA: 3 min, F(3,35) ⫽9.87, p ⬍ 0.05; 30 min, F(3,52) ⫽ 7.26, p ⬍ 0.05; BLA: 3 min, F(3,35)⫽ 9.17, p ⬍ 0.05; 30 min, F(3,52) ⫽ 7.20, p ⬍ 0.05; Ce: 3 min, F(3,35)⫽ 4.80, p ⬍ 0.05; 30 min, F(3,52) ⫽ 8.48, p ⬍ 0.05). The level ofpCREB was significantly increased in the three amygdaloid regions of the Recon-33, Recon-60, and Ext-60 groups comparedwith the other 3 or 30 min, respectively, reexposure control
groups (Fig. 5B–G) ( p ⬎ 0.05). These results indicated that no

CREB activation was observed 3 min after the 30 min reexposurein the conditioned extinction groups. Consistent with this, a twoway ANOVA across the conditioned groups revealed significant
reexposure versus time interactions in these three regions of theamygdala (Fig. 5H–J ) (reexposure vs time interaction; LA: F(1,43)⫽ 7.60, p ⬍ 0.05; BLA: F(1,43) ⫽ 6.60, p ⬍ 0.05; Ce: F(1,43) ⫽ 5.71,
p ⬍ 0.05). The post hoc Bonferroni’s test showed that the pCREB
levels were significantly increased in the three amygdaloid regions of the Recon-33, Recon-60, and Ext-60 groups comparedwith the Ext-33 group (Fig. 5H–J ) ( p ⬍ 0.05). Collectively, theseobservations indicate that CREB was activated in the amygdala
when the fear memory was being reconsolidated and extinguished but showed a different time course from the beginning of
the reexposure to the context between the reconsolidation andextinction phases. Extinction conditioned groups showed activation of CREB but also showed a delay of CREB activation compared with the reconsolidation group, although the contextual
fear memory was reactivated in both groups. These differences inthe time course of CREB activation between the reconsolidation

Mamiya et al. • CREB Activation in Reconsolidation and Extinction

J. Neurosci., January 14, 2009 • 29(2):402– 413 • 409

Ninety minutes after the reexposure, mice
were assessed for Arc expression using immunohistochemistry. After the 3 min reexposure, conditioned groups showed asignificant upregulation of Arc expression
in the CA1 and CA3 regions of the hippocampus and the LA, BLA, and Ce regions of the amygdala (Fig. 6 A) (CA1:
F(1,17) ⫽ 13.80, p ⬍ 0.05; CA3: F(1,17) ⫽10.40, p ⬍ 0.05; LA: F(1,17) ⫽ 12.16, p ⬍0.05; BLA: F(1,17) ⫽ 14.95, p ⬍ 0.05; Ce:
F(1,17) ⫽ 5.24, p ⬍ 0.05). No changes wereobserved in the DG region of the hippocampus, nor in the PL and IL regions of
the mPFC when compared with the unconditioned control groups (Fig. 6 A)
(DG: F(1,17) ⫽ 0.46, p ⬎ 0.05; PL: F(1,17) ⫽0.07, p ⬎ 0.05; IL: F(1,17) ⫽ 0.01, p ⬎ 0.05).
In contrast, after the 30 min reexposure, conditioned groups showed a significant upregulation of Arc expression in thetwo mPFC regions and the three amygdaloid regions, but not the three hippocampal regions, compared with the unconditioned control groups (Fig. 6 B) (PL: F(1,20)⫽ 16.56, p ⬍ 0.05; IL: F(1,20) ⫽ 12.68, p ⬍0.05; LA: F(1,20) ⫽ 25.41, p ⬍ 0.05; BLA:
Figure 6. Effects of the duration of reexposure on Arc expression in the mPFC, amygdala, and hippocampus. A, B, Experimental F(1,20) ⫽ 12.81, p ⬍ 0.05; Ce: F(1,20) ⫽ 4.43,
design used with data presented below. A, Effects of the 3 min reexposure duration on Arc expression in the mPFC, amygdala, and p ⬍ 0.05; CA1: F(1,20) ⫽ 0.01, p ⬎ 0.05;
hippocampus (reconsolidation, n ⫽ 10; control, n ⫽ 9). *p ⬍ 0.05, compared with the control group. B, Effects of the 30 min CA3: F(1,20) ⫽ 0.03, p ⬎ 0.05; DG: F(1,20) ⫽reexposure duration on Arc expression in mPFC, amygdala, and hippocampus (extinction, n ⫽ 11; control, n ⫽ 11). *p ⬍ 0.05,
0.01, p ⬎ 0.05). These results are consiscompared with the control group. Error bars are SEM. Data of Arc expression for each group were expressed as the percentage oftent with the observations in Figures 3–5
the averaged values in the control group. Scale bar, 100 ␮m.
and indicate that the short reexposure thattriggered reconsolidation induced CREBand extinction groups indicates that CREB activation triggered
mediated gene expression in the amygdala/hippocampus,
by a 30 min reexposure is not simply attributable to the reactivawhereas the long reexposure that triggered extinction induced
tion of the fear memory and, furthermore, that the mechanisms
expression in the amygdala/mPFC. Furthermore, similar to thefor the regulation of CREB activation differ in the reconsolidaresults of the CREB activation (Fig. 4), we found that hippocamtion and extinction phases.
pal Arc is upregulated in the reconsolidation but not extinction
As a control experiment, we examined CREB activation in thephases, suggesting that these two processes may interact at thevisual cortex. In contrast to the results in Figures 3–5, no CREB
molecular level in the hippocampus.
activation was observed in the conditioned groups after the 3
and 30 min reexposure (supplemental Fig. 1, available at www.
Roles of protein synthesis in the mPFC, hippocampus, andjneurosci.org as supplemental material).
amygdala in memory reconsolidation and extinction
In summary, our anatomical analyses suggest that activation
The above analyses suggest that short context reexposure thatof CREB in the hippocampus and amygdala contributes to thetriggers reconsolidation induces activation of CREB-mediatedreconsolidation of the contextual fear memory, whereas this acgene expression in the amygdala and hippocampus. In contrast,
tivation in the amygdala and prefrontal cortex contributes to thelong context reexposure that triggers extinction induces CREB
extinction.
activation in the amygdala and mPFC (Figs. 3–5). These results
raise the possibility that new gene expression in distinct brain
Upregulation of Arc expression in distinct brain regions inregions is required for reconsolidation and extinction of contexthe reconsolidation and extinction phases
tual fear memory, respectively. To further test this possibility, we
To ask whether the activation of CREB observed in Figures 3–5
examined the effects of protein synthesis inhibition in the mPFC,
are reflected by the expression of a CREB-target gene, we meaamygdala, or hippocampus on reconsolidation and extinction ofsured the expression of the immediate-early gene Arc in the recontextual fear memory. We performed a similar experiment asconsolidation and extinction phases. Arc is a CREB-dependent
in Figure 1, except that WT mice received an infusion of thegene (Kawashima et al., 2008), the expression of which is reguprotein synthesis inhibitor ANI or VEH immediately after the 3
lated by neuronal activity. Previous studies have shown that it isor 30 min reexposure to the context (Fig. 7A). Cannula tip placeupregulated after contextual fear memory formation and rements are shown in the supplementary data, and only mice withtrieval (Guzowski et al., 1999; Zhang et al., 2005; Kee et al., 2007).
cannula tips within the boundaries of the mPFC, hippocampus orMice were trained in the presence (conditioned group) or abamygdala were included in the data analysis (supplemental Fig. 2,
sence (unconditioned control group) of a single footshock andavailable at www.jneurosci.org as supplemental material). Dur24 h later were placed back in the training context for 3 or 30 min.
ing the 3 min reexposure, the VEH and ANI groups showedMamiya et al. • CREB Activation in Reconsolidation and Extinction

410 • J. Neurosci., January 14, 2009 • 29(2):402– 413⫽ 26.07, p ⬍ 0.05; hippocampus: overall freezing, F(1.90) ⫽ 0.08,
p ⬎ 0.05; decrease over time, F(5,90) ⫽ 26.61, p ⬍ 0.05; amygdala:overall freezing, F(1.75) ⫽ 0.43, p ⬎ 0.05; decrease over time, F(5,75)⫽ 30.92, p ⬍ 0.05). Measurements 24 h after this 30 min reexposure indicated that long-term extinction occurred in the VEHtreated mice (first 5 min in reexposure session vs test; mPFC: t(16)⫽ 5.03, p ⬍ 0.05; hippocampus: t(18) ⫽ 3.75, p ⬍ 0.05; amygdala:t(13) ⫽ 4.27, p ⬍ 0.05). Infusion of ANI into the mPFC or amygdala immediately after the 30 min reexposure impaired the longterm extinction of the contextual fear memory tested 24 h later(mPFC: F(1,16) ⫽ 9.24, p ⬍ 0.05; amygdala: F(1,15) ⫽ 9.25, p ⬍0.05); ANI-treated mice exhibited significantly higher freezing
levels compared with VEH-treated mice (Fig. 7C) ( p ⬍ 0.05). In
contrast, infusion of ANI into the hippocampus did not alter thelevel of freezing tested 24 h later (F(1,18) ⫽ 0.08; p ⬎ 0.05) (Fig.
7C). These results indicated that protein synthesis in the mPFC
and amygdala but not the hippocampus is required for long-term
extinction of contextual fear memory.
Together, these observations support our results from the biochemical experiments that CREB-mediated gene expression is
induced in distinct regions in the reconsolidation and extinction
phases and suggest that the hippocampus, amygdala, and mPFC
play distinct roles in the reconsolidation and extinction of thecontextual fear memory through region-specific regulation ofgene expression.

Discussion
Figure 7. Effects of protein synthesis inhibition in the mPFC, hippocampus, or amygdala onreconsolidation and extinction. A, Experimental design used with data. Mice were infused VEH
or ANI into the mPFC, hippocampus, or amygdala immediately after 3 or 30 min reexposure. B,
Effects of protein synthesis inhibition in mPFC, hippocampus, or amygdala on reconsolidation.
Freezing scores during the test session are shown (mPFC: VEH, n ⫽ 10; ANI, n ⫽ 11; hippocampus: VEH, n ⫽ 12; ANI, n ⫽ 12; amygdala: VEH, n ⫽ 9; ANI, n ⫽ 9). C, Effects of protein
synthesis inhibition in mPFC, hippocampus, or amygdala on extinction. Freezing scores during
test session are shown (mPFC: VEH, n ⫽ 9; ANI, n ⫽ 9; hippocampus: VEH, n ⫽ 10; ANI, n ⫽ 10;
amygdala: VEH, n ⫽ 8; ANI, n ⫽ 9). During reexposure, freezing scores in 5 min blocks arepresented. Error bars are SEM. *p ⬍ 0.05 versus VEH-treated groups.

comparable levels of freezing ( p ⬎ 0.05). Infusion of ANI into themPFC immediately after the 3 min reexposure did not alter thelevel of freezing tested 24 h later (F(1,19) ⫽ 0.17; p ⬎ 0.05); ANItreated mice exhibited comparable freezing levels compared with
VEH-treated mice (Fig. 7B). In contrast, an infusion of ANI into
the hippocampus or amygdala immediately after the 3 min reexposure impaired the expression of the contextual fear memory
tested 24 h later (hippocampus: F(1,23) ⫽ 13.84, p ⬍ 0.05; amygdala: F(1,16) ⫽ 5.32, p ⬍ 0.05); ANI-treated mice exhibited significantly lower freezing levels compared with VEH-treated mice
(Fig. 7B) ( p ⬍ 0.05). These results are consistent with previous
findings and confirmed that hippocampal protein synthesis isrequired for reconsolidation of the contextual fear memory (Debiec et al., 2002; Frankland et al., 2006; Suzuki et al., 2008). The
novel result is that we found that reconsolidation of contextual
fear memory requires new protein synthesis in the amygdala.
Together with our anatomical analyses (Figs. 3– 6), our results
indicate that reconsolidation of contextual fear memory requires
new gene expression in the hippocampus and amygdala but notin the mPFC.
During the 30 min reexposure, comparable within-session extinction was observed between the VEH and ANI groups (mPFC:
overall freezing, F(1.80) ⫽ 3.56, p ⬎ 0.05; decrease over time, F(5,80)

Our previous behavioral studies demonstrated that memory reactivation triggers two processes: reconsolidation and extinction(Suzuki et al., 2004). These two processes have opposing actions
on the fate of the retrieved memory: reconsolidation serves tostabilize or strengthen the memory, whereas extinction involves
the formation of an inhibitory CS–no US association that competes with the original memory. Using a contextual fearconditioning paradigm, in the present study we first establishedthat both these processes depend on CREB-mediated transcription (Fig. 1). Using immunohistochemical and targeted pharmacological approaches, our studies next indicated that these competing processes are supported by partially dissociable brain
regions. After brief reactivation, new proteins are required in the
hippocampus/amygdala for reconsolidation of contextual fear
memories (Fig. 7B). In contrast, after prolonged reactivation,
new proteins are required in the amygdala/mPFC for long-term
extinction (Fig. 7C).
A critical biochemical feature of reconsolidation and longterm extinction of contextual fear memory is a requirement forgene expression (Suzuki et al., 2004). We previously demonstrated that CREB-mediated transcription is required for the reconsolidation of the contextual fear memory (Kida et al., 2002).
In this study, genetic suppression of CREB function also blocked
long-term extinction, indicating that long-term extinction undergoes a transcription-dependent process (Fig. 1C,D). To investigate the brain regions activated in the reconsolidation and extinction phases, we analyzed the brain regions where CREB is
activated by measuring levels of pCREB after either brief (3 min)or prolonged (30 min) reexposure to the conditioning context.
Furthermore, to determine whether our observations of CREB
activation are reflected by the expression of CREB-dependent,
neuronal activity-dependent genes, we measured the expression
levels of Arc.
When a shorter context reexposure was used, we found that
CREB-mediated gene expression (CREB phosphorylation and
Arc induction) was induced in the CA1 and CA3 regions of theMamiya et al. • CREB Activation in Reconsolidation and Extinction

hippocampus and the LA, BLA, and Ce regions of the amygdala(Figs. 3– 6). These data suggest that reconsolidation engages
CREB-dependent processes in the hippocampus and amygdala(but not the mPFC). These observations are consistent with previous findings showing that retrieval of contextual fear memory
induces c-fos and Zif268 expression in the CA1 of hippocampus
but not the PL and IL of the mPFC (Frankland et al., 2004).
Similarly, another study showed that this retrieval induces Zif268
expression in the LA, BLA, and Ce regions of the amygdala andthe CA1 but not the DG of the hippocampus (Lee et al., 2001).
Consistent with previous observations (Debiec et al., 2002; Suzuki et al., 2008), we showed that hippocampal inhibition of protein synthesis disrupts subsequent expression of the contextual
fear memory (Fig. 7B). More importantly, we finally demonstrated that protein synthesis inhibition in the amygdala also disrupted the reactivated contextual fear memory (Fig. 7B). Theseobservations indicate that reconsolidation of the contextual fear
memory depends on new gene expression in the hippocampus
and amygdala.
After prolonged reexposure to the context, we found that
CREB-mediated gene expression was induced in the amygdala
and the mPFC (Figs. 3, 5, 6). Consistent with these changes in
CREB phosphorylation and Arc expression, we found that longterm extinction was blocked by protein synthesis inhibition in themPFC and amygdala but not in the hippocampus (Fig. 7C).
These observations suggested that long-term extinction of contextual fear memory depends on new gene expression in themPFC and amygdala. Recent studies have shown that the amygdala plays a significant role in the acquisition of tone fear extinction, whereas the mPFC is implicated in its consolidation (Quirk
et al., 2000; Santini et al., 2004). Indeed, consolidation of tone
fear extinction requires protein synthesis and NMDA receptordependent bursting in the mPFC (Santini et al., 2004; BurgosRobles et al., 2007). Together with our findings, these data suggest that the amygdala and mPFC might play critical roles in theextinction of both contextual and tone fear memories.
Our observations that CREB activation and Arc expression are
induced in the hippocampus/amygdala and amygdala/mPFC in
the reconsolidation and extinction phases, respectively, indicatesthat gene expression is regulated in distinct ways after brief versus
prolonged context reexposure (Figs. 3– 6). In particular, theseobservations extend our previous behavioral observations that
reexposure to the CS triggers two distinct time-dependent processes, reconsolidation and extinction, and indicate that reconsolidation and extinction are dissociable at the anatomical level.
Previous studies have established that protein synthesis inhibition after a brief reexposure to the CS disrupts the subsequent
expression of the reactivated fear memory (Nader et al., 2000;
Debiec et al., 2002; Kida et al., 2002). In contrast, blocking protein synthesis after prolonged reexposure blocks long-term extinction but does not disrupt the original fear memory (Eisenberg
et al., 2003; Pedreira and Maldonado, 2003; Suzuki et al., 2004).
Whereas it is not unexpected that new proteins are necessary forthe formation of a long-term extinction memory (Santini et al.,2004), it is surprising that the original fear memory is left intact,
although it has been reactivated under the condition in which
protein synthesis was inhibited (Suzuki et al., 2004). This observation indicates that reconsolidation and extinction do not proceed independently (but see Duvarci et al., 2006). That is, thesetwo processes must interact in some way.
Importantly, the present series of experiments identify the
hippocampus and the amygdala as candidate anatomical loci forthis interaction between the reconsolidation and extinction

J. Neurosci., January 14, 2009 • 29(2):402– 413 • 411

phases. First, we observed that CREB-mediated gene expression
is induced in the hippocampus after short, but not longer, reexposure to the CS (Fig. 4). This suggests that gene expression in the
hippocampus undergoes distinct regulation in the reconsolidation and extinction phases. Our observation that CREBmediated gene expression was not induced in the extinction
phase even though contextual fear was reactivated indicates thatsuch induction must be suppressed when within-session extinction occurs. Second, we observed differences in the time course of
amygdaloid CREB activation in the reconsolidation and extinction phases (Fig. 5), indicating that amygdaloid CREB activation
is differentially regulated in these two phases and, furthermore,that amygdaloid CREB activation observed in the extinction
phase is not simply caused by fear memory reactivation. Furthermore, amygdaloid inhibition of protein synthesis after prolonged
reexposure blocked long-term extinction without affecting thesubsequent expression of the original fear memory, whereas thisinhibition after brief reexposure disrupted the fear memory (Fig.
7). Therefore, we conclude that the interaction between the reconsolidation and extinction phases occurs in both the hippocampus and the amygdala at the molecular level. However, wecannot exclude another possibility: the CS onset initiates memory reactivation, but CS offset is the critical signal that determines
the fate of memory by engaging reconsolidation or extinction
processes (Pedreira et al., 2004, Pérez-Cuesta et al. 2007).
Our anatomical observations that acquisition of withinsession extinction of the contextual fear inhibited CREBmediated gene expression in the hippocampus corresponds withour behavioral observations that the acquisition of extinction
prevents the disruption of the contextual fear memory by the
inhibition of protein synthesis or CREB activity (Figs. 1, 7). Because previous studies demonstrated that destabilization and restabilization of reactivated contextual fear memories are hippocampus dependent (Debiec et al., 2002; Suzuki et al., 2008), it
is possible that contextual fear extinction modulates the stabilityof the reactivated contextual fear memory by regulating hippocampal function. Therefore, it will be important to evaluate
whether acquisition of contextual fear extinction prevents thedestabilization of reactivated memory. A second possibility is thatthe originally contextual fear memory is destabilized and thenrestabilized (inactivated) in the extinction phase in a gene
expression-independent manner (such as redistribution of preexisting proteins).
Restabilization of the reactivated contextual fear memory requires new gene expression in the hippocampus (Debiec et al.,2002). In contrast, we observed that inhibition of protein synthesis in the hippocampus did not affect long-term extinction (Fig.
7C), suggesting that the new protein synthesis in the hippocampus does not contribute to the consolidation of extinction memory. These contrasting observations indicate that the hippocampus plays distinct roles in reconsolidation and extinction ofcontextual fear memory. However, recent studies indicated critical roles of the hippocampus in the extinction of contextual fear.
Indeed, cdk5 signaling, activation of the ubiquitin-proteasomal
pathway, activation of CB1, and actin rearrangement in the hippocampus have been shown to be required for extinction of contextual fear memory (Fischer et al., 2004; Sananbenesi et al., 2007;de Oliveira Alvares et al., 2008; Lee et al., 2008). Therefore, it ispossible that the hippocampus regulates contextual fear extinction through activation of these pathways without activating gene
expression. Suppression of gene expression in the hippocampus
might be a critical step that leads to long-term extinction. Addi-

412 • J. Neurosci., January 14, 2009 • 29(2):402– 413

tional studies are required to investigate the regulatory roles of
the hippocampus in memory extinction.
We observed that CREB activation and Arc expression were
induced in the amygdala in both the reconsolidation and extinction phases (Figs. 5, 6). In agreement with this, previous studies
demonstrated that extinction learning of tone fear induces expression of c-fos in the amygdala (Herry and Mons, 2004; Herryet al., 2008). However, a recent study demonstrated that retrievalof the contextual fear memory reactivates BLA neurons that are
activated during contextual fear conditioning, whereas extinction learning prevents their activation (Reijmers et al., 2007).
Together with these findings, our observation that the timecourse of amygdaloid CREB activation differs in the reconsolidation and extinction phases raises the possibility that distinct populations of amygdaloid neurons are activated in these phases. A
recent study identified two distinct populations of BLA neurons(fear and extinction neurons), the activity of which is reversely
correlated with low and high fear behavior (Herry et al., 2008).
Therefore, it will be important to examine whether identical ordifferent population of neurons are activated in the reconsolidation and extinction phases of contextual fear memory.

References
Anagnostaras SG, Gale GD, Fanselow MS (2001) Hippocampus and contextual fear conditioning: recent controversies and advances. Hippocampus
11:8 –17.
Berman DE, Dudai Y (2001) Memory extinction, learning anew, and learning the new: dissociation in the molecular machinery of learning in cortex.
Science 291:2417–2419.
Bito H, Deisseroth K, Tsien RW (1996) CREB phosphorylation and dephosphorylation: a Ca 2⫹- and stimulus duration-dependent switch for hippocampal gene expression. Cell 87:1203–1214.
Burgos-Robles A, Vidal-Gonzalez I, Santini E, Quirk GJ (2007) Consolidation of fear extinction requires NMDA receptor-dependent bursting in
the ventromedial prefrontal cortex. Neuron 53:871– 880.
Davis HP, Squire LR (1984) Protein synthesis and memory. Psychol Bull
96:518 –559.
Debiec J, LeDoux JE, Nader K (2002) Cellular and systems reconsolidationin the hippocampus. Neuron 36:527–538.
de Oliveira Alvares L, Pasqualini Genro B, Diehl F, Molina VA, Quillfeldt JA(2008) Opposite action of hippocampal CB1 receptors in memory reconsolidation and extinction. Neuroscience 154:1648 –1655.
Duvarci J, Nader K (2004) Characterization of fear memory reconsolidation. J Neurosci 24:9269 –9275.
Duvarci J, Mamou CB, Nader K (2006) Extinction is not a sufficient condition to prevent fear memories from undergoing reconsolidation in thebasolateral amygdala. Eur J Neurosci 24:249 –260.
Eisenberg M, Kobilo T, Berman DE, Dudai Y (2003) Stability of retrieved
memory: inverse correlation with trace dominance. Science
301:1102–1104.
Fischer A, Sananbenesi F, Schrick C, Spiess J, Rdulovic J (2004) Distinct
roles of hippocampal de novo protein synthesis and actin rearrangement
in extinction of contextual fear. J Neurosci 24:1962–1966.
Flexner LB, Flexner JB, Stellar E (1965) Memory and cerebral protein synthesis in mice as affected by graded amounts of puromycin. Exp Neurol
13:264 –272.
Frankland PW, Bontempi B, Talton LE, Kaczmarek L, Silva AJ (2004) The
involvement of the anterior cingulated cortex in remote contextual fear
memory. Science 304:881– 883.
Frankland PW, Ding HK, Takahashi E, Suzuki A, Kida S, Silva AJ (2006)
Stability of recent and remote contextual fear memory. Learn Mem
13:451– 457.
Franklin KB, Paxinos G (1997) The mouse brain in stereotaxic coordinates.
San Diego: Elesevier Academic.
Ginty DD, Bonni A, Greenberg ME (1994) Nerve growth factor activates a
Ras-dependent protein kinase that stimulates c-fos transcription via
phosphorylation of CREB. Cell 77:713–725.
Gonzalez GA, Montminy MR (1989) Cyclic AMP stimulates somatostatin

Mamiya et al. • CREB Activation in Reconsolidation and Extinction
gene transcription by phosphorylation of CREB at serine 133. Cell
59:675– 680.
Guzowski JF, McNaughton BL, Barnes CA, Worley PE (1999)
Environment-specific expression of the immediate-early gene Arc in hippocampal neuronal ensembles. Nat Neurosci 2:1120 –1124.
Hall J, Thomas KL, Everitt BJ (2001) Fear memory retrieval induces CREB
phosphorylation and Fos expression within the amygdala. Eur J Neurosci
13:1453–1458.
Herry C, Mons N (2004) Resistance to extinction is associated with impaired immediate early gene induction in medial prefrontal cortex andamygdala. Eur J Neurosci 20:781–790.
Herry C, Ciocchi S, Senn V, Demmou L, Muller C, Luthi A (2008) Switchingon and off fear by distinct neuronal circuits. Nature 454:600 – 606.
Kawashima T, Okuno H, Nonaka M, Adachi-Morishima A, Kyo N, Okamura
M, Takemoto-Kimura S, Worley PF, Bito H (2008) A synaptic activityresponsive element in the Arc/Arg3.1 promoter essential for synapse-tonucleus signaling in activated neurons. Proc Natl Acad Sci U S A. Advance online publication. Retrieved December 31, 2008. doi:10.1073/
pnas.0806518106.
Kee N, Teixeira CM, Wang AH, Frankland PW (2007) Preferential incorporation of adult-generated granule cells into spatial memory networks inthe dentate gyrus. Nat Neurosci 10:355–362.
Kida S, Josselyn SA, deOrtiz SP, Kogan JH, Chevere I, Masushige S, Silva AJ(2002) CREB required for the stability of new and reactivated fear memories. Nat Neurosci 5:348 –355.
Lee JL, Thomas KL, Everitt BJ (2001) Cellular imaging of zif268 expression
in the hippocampus and amygdala during contextual and cued fear memory retrieval: selective activation of hippocampal CA1 neurons during therecall of contextual memories. J Neurosci 21:2186 –2193.
Lee JL, Milton AL, Everitt BJ (2006) Reconsolidation and extinction of conditioned fear: inhibition and potentiation. J Neurosci 26:10051–10056.
Lee SH, Choi JH, Lee N, Lee HR, Kim JI, Yu NK, Choi SL, Lee SH, Kim H,
Kaang BK (2008) Synaptic protein degradation underlies destabilization of retrieved fear memory. Science 319:1253–1256.
Lewis DJ (1979) Psychobiology of active and inactive memory. Psychol Bull
86:1054 –1083.
Lu KT, Walker DL, Davis M (2001) Mitogen-activated protein kinase cascade in the basolateral nucleus of amygdala is involved in extinction offear-potentiated startle. J Neurosci 21:RC162(1–5).
Mactutus CF, Riccio DC, Ferek JM (1979) Retrograde amnesia for old (reactivated) memory: some anomalous characteristics. Science
204:1319 –1320.
Mayr B, Montminy M (2001) Transcriptional regulation by thephosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol
2:599 – 609.
McGaugh JL (2000) Memory—a century of consolidation. Science
287:248 –251.
Misanin JR, Miller RR, Lewis DJ (1968) Retrograde amnesia produced byelectroconvulsive shock after reactivation of a consolidated memory
trace. Science 160:554 –555.
Morgan MA, LeDoux JE (1995) Differential contribution of dorsal and ventral medial prefrontal cortex to the acquisition and extinction of conditioned fear in rats. Behav Neurosci 109:681– 688.
Morgan MA, LeDoux JE (1999) Contribution of ventrolateral prefrontal
cortex to the acquisition and extinction of conditioned fear in rats. Neurobiol Learn Mem 72:244 –251.
Morgan MA, Romanski LM, LeDoux JE (1993) Extinction of emotional
learning: contribution of medial prefrontal cortex. Neurosci Lett
163:109 –113.
Morrow BA, Elsworth JD, Rasmusson AM, Roth RH (1999) The role ofmesoprefrontal dopamine neurons in the acquisition and expression of
conditioned fear in the rat. Neuroscience 92:553–564.
Myers KM, Davis M (2002) Behavioral and neural analysis of extinction.
Neuron 36:567–584.
Myers KM, Davis M (2007) Mechanisms of fear extinction. Mol Psychiatry
12:120 –150.
Nader K, Schafe GE, LeDoux JE (2000) Fear memories require protein synthesis in the amygdala for reconsolidation after retrieval. Nature
406:722–726.
Pavlov IP (1927) Conditioned reflexes. London: Oxford UP.
Pedreira ME, Maldonado H (2003) Protein synthesis subserves reconsolidation or extinction depending on reminder duration. Neuron 38:863– 869.

Mamiya et al. • CREB Activation in Reconsolidation and Extinction
Pedreira ME, Pérez-Cuesta LM, Maldonado H (2004) Mismatch between
what is expected and what actually occurs triggers memory reconsolidation or extinction. Learn Mem 11:579 –585.
Pérez-Cuesta LM, Hepp Y, Pedreira ME, Maldonado H (2007) Memory is
not extinguished along with CS presentation but within a few seconds
after CS-offset. Learn Mem 14:101–108.
Przybyslawski J, Sara SJ (1997) Reconsolidation of memory after its reactivation. Behav Brain Res 84:241–246.
Quirk GJ, Russo GK, Barron JL, Lebron K (2000) The role of ventromedial
prefrontal cortex in the recovery of extinguished fear. J Neurosci
20:6225– 6231.
Quirk GJ, Gracia R, Gonzalez-Lima F (2006) Prefrontal mechanisms in extinction of conditioned fear. Biol Psychiatry 60:337–343.
Reijmers LG, Perkins BL, Matsuo N, Mayford M (2007) Localization of astable neuronal correlate of associative memory. Science 317:1230 –1233.
Rescorla RA (2001) Experimental extinction. In: Handbook of contemporary learning theories (Mowrer RR, Klein S, eds), pp 119 –154. Mahwah,
NJ: Erlbaum.
Rosenblum K, Meiri N, Dudai Y (1993) Taste memory: the role of protein
synthesis in gustatory cortex. Behav Neural Biol 59:49 –56.
Sananbenesi F, Fischer A, Wang X, Schrick C, Neve R, Radulovic J, Tsai LH(2007) A hippocampal Cdk5 pathway regulates extinction of contextual
fear. Nat Neurosci 10:1012–1019.
Santini E, Ge H, Ren K, deOrtiz SP, Quirk GJ (2004) Consolidation of fear
extinction reuires protein synthesis in the medial prefrontal cortex. J Neurosci 24:5704 –5710.
Sara SJ (2000) Retrieval and reconsolidation: toward a neurobiology of remembering. Learn Mem 7:73– 84.
Schneider AM, Sherman W (1968) Amnesia: a function of the temporal
relation of footshock to electroconvulsive shock. Science 159:219 –221.
Shaywitz AJ, Greenberg ME (1999) CREB: a stimulus-induced transcription

J. Neurosci., January 14, 2009 • 29(2):402– 413 • 413
factor activated by a diverse array of extracellular signals. Annu Rev Biochem 68:821– 861.
Silva AJ, Kogan JH, Frankland PW, Kida S (1998) CREB and memory. Annu
Rev Neurosci 21:127–148.
Suzuki A, Josselyn SA, Frankland PW, Masushige S, Silva AJ, Kida S (2004)
Memory reconsolidation and extinction have distinct temporal and biochemical signatures. J Neurosci 24:4787– 4795.
Suzuki A, Mukawa T, Tsukagoshi A, Frankland PW, Kida S (2008) Activation of LVGCCs and CB1 receptors required for destabilization of reactivated contextual fear memories. Learn Mem 15:426 – 433.
Taubenfeld SM, Milekic MH, Monti B, Alberini CM (2001) The consolidation of new but not reactivated memory requires hippocampal C/EBPbeta. Nat Neurosci 4:813– 818.
Tronel S, Milekic MH, Alberini CM (2005) Linking new information to areactivated memory requires consolidation and not reconsolidation
mechanisms. PLoS Biol 3:1630 –1638.
Vianna MR, Szapiro G, McGaugh JL, Medina JH, Izquierdo I (2001) Retrieval of memory for fear-motivated training initiates extinction requiring protein synthesis in the rat hippocampus. Proc Natl Acad Sci U S A
98:12251–12254.
Viola H, Furman M, Izquierdo LA, Alonso M, Barros DM, de Souza MM,
Izquierdo I, Medina JH (2000) Phosphorylated cAMP response
element-binding protein as a marker of memory processing in rat hippocampus: effect of novelty. J Neurosci 20:RC112(1–5).
Walker DL, Ressler KJ, Lu KT, Davis M (2002) Facilitation of conditioned
fear extinction by systemic administration or intra-amygdala infusions ofD-cycloserine as assessed with fear-potentiated startle in rats. J Neurosci
22:2343–2351.
Zhang WP, Guzowski JF, Thomas SA (2005) Mapping neuronal activationand the influence of adrenergic signal during contextual memory retrieval. Learn Mem 12:239 –247.