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Activation of LVGCCs and CB1 receptors required fordestabilization of reactivated contextual fear memories
Akinobu Suzuki, Takuya Mukawa, Akinori Tsukagoshi, et al.
Learn. Mem. 2008 15: 426-433
Access the most recent version at doi:10.1101/lm.888808

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Research

Activation of LVGCCs and CB1 receptors requiredfor destabilization of reactivated contextual fear
memories
Akinobu Suzuki,1,2 Takuya Mukawa,1 Akinori Tsukagoshi,1 Paul W. Frankland,3
and Satoshi Kida1,2,4
1

Department of Bioscience, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku,
Tokyo 156-8502, Japan; 2Core Research for Evolutional Science and Technology, Japan Science and Technology Agency,
Saitama 332-0012, Japan; 3Program in Neurosciences and Mental Health, Hospital for Sick Children Research Institute,
Toronto, Ontario, Canada M5G1X8Previous studies have shown that inhibiting protein synthesis shortly after reactivation impairs the subsequent
expression of a previously consolidated fear memory. This has suggested that reactivation returns a memory to alabile state and that protein synthesis is required for the subsequent restabilization of memory. While the molecularmechanisms underlying the restabilization of reactivated memories are being uncovered, those underlying the initialdestabilization are not known at all. Using a contextual fear conditioning paradigm in mice, here we show that
LVGCCs or CB1 receptors in hippocampus are required for the initial destabilization of reactivated memory. Either
pharmacological blockade of hippocampal protein synthesis or genetic disruption of CREB-dependent transcription
disrupts memory restabilization following reactivation. However, these effects were completely blocked when mice
were treated with inhibitors of either LVGCCs or CB1 receptors, indicating that LVGCCs or CB1 receptors are
required for the initial destabilization of reactivated memory. In control experiments, we show that blockade of
LVGCCs or CB1 receptors does not interfere with the ability of ANI to block protein synthesis, or with the ability of
ANI to impair initial consolidation. These experiments begin to reveal mechanisms underlying the destabilization ofpreviously consolidated memories following reactivation and indicate the importance of activation of LVGCCs andCB1 in this process.

To generate long-term (stable) memory, short-term (labile)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 that memory retrieval is a dynamic processthat either reinforces or alters 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; Tronel
et al. 2005; Tronson et al. 2006). For example, in fear conditioning paradigms, inhibition of protein synthesis before or immediately following brief re-exposure to the conditioning stimulus(CS) disrupts the subsequent memory expression (Abel et al.
1997; Kida et al. 2002; Suzuki et al. 2004). This has suggested that
following reactivation, a fear memory is briefly transformed to alabile state and then restabilized through a gene expressiondependent reconsolidation process (Nader et al. 2000; Taubenfeld et al. 2001; Debiec et al. 2002; Kida et al. 2002). Recent
studies have begun to identify the molecular mechanisms underlying the restabilization of reactivated memory (Taubenfeld et al.
2001; Kida et al. 2002; Lee et al. 2004; Duvarci et al. 2005; Miller
and Marshall 2005; Tronson et al. 2006). However, those underlying the initial destabilization remain poorly understood (Alberini et al. 2006; but see Ben Mamou et al. 2006).
Two potential molecular substrates for destabilization havebeen identified in studies examining extinction of contextual
fear memory. More prolonged re-exposure to the CS (in the ab-sence of the US) leads to extinction of fear memory (Pavlov 1927;
Rescorla 2001; Myers and Davis 2002). Since extinction involves
the formation of a new CS–no US inhibitory memory, it is not
unexpected that blockade of protein synthesis prior to the extended re-exposure blocks the development of extinction (Berman and Dudai 2001; Vianna et al. 2001). However, less expected
is that this blockade of protein synthesis does not interfere withthe stability of the original memory trace (Eisenberg et al. 2003;
Pedreira and Maldonado 2003; Suzuki et al. 2004; but see Duvarci
et al. 2006). This suggests that at least some of the processes
engaged during extinction (such as activation of L-type voltagegated calcium channels [LVGCCs] and cannabinoid receptor 1[CB1], both of which are required for memory extinction) (Cainet al. 2002; Marsicano et al. 2002; Suzuki et al. 2004) may additionally regulate the stability of the original memory trace. We
explored the possibility that activation of LVGCCs and CB1 receptors are key steps in destabilization of reactivated memories.
We found that blockade of LVGCCs and CB1 receptors protects
memories from the amnestic effects of either blocking protein
synthesis or disrupting CREB function during fear memory reactivation.

Results
CB1 and LVGCCs are required for memory extinction
Previously we examined the relationship between re-exposure
duration and stability of contextual fear memory in mice (Suzuki
et al. 2004). We found that when mice were briefly re-exposed (3min) to the conditioning chamber, systemic administration of
ANI blocked subsequent expression of that memory. In contrast,
longer-duration re-exposures (30 min) led to extinction of con-

4
Corresponding author.
E-mail kida@nodai.ac.jp; fax 81-3-54772317.
Article is online at http://www.learnmem.org/cgi/doi/10.1101/lm.888808.

15:426–433 © 2008 Cold Spring Harbor Laboratory Press
ISSN 1072-0502/08; www.learnmem.org

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higher levels of freezing in mice treated with higher doses of Nim
or SR. In addition, there was a significant Drug1 ⳯ Dose interaction (F(2,132) = 5.03, P < 0.01), indicating that dose-dependent effects were only observed in Vehicle-treated groups (Fig. 1). Together these data confirm previous experiments (Suzuki et al.
2004) and show that extinction depends on activation of
LVGCCs, CB1 receptors, and protein synthesis.

CB1 and LVGCCs are required for destabilizationof reactivated memory
Blocking protein synthesis either before or after brief re-exposure
to context leads to a disruption of subsequent expression of contextual fear memory. This indicates that new protein synthesis isrequired for the restabilization of contextual fear memories after
memory reactivation (Debiec et al. 2002; Kida et al. 2002; Suzuki
et al. 2004). We next tested whether the blockade of LVGCCs andCB1 receptors would block the amnestic effects of ANI on a reactivated memory (Fig. 2). During training, mice received a single
footshock (0.4 mA). Twenty-four hours following training mice
were placed back into the context for 3 min. During this reexposure, overall freezing levels did not differ across groups (datanot shown) (all P’s > 0.05). As we have previously shown, administration of ANI immediately following this re-exposure impaired
the expression of contextual fear memory tested 24 h later(F(1,22) = 25.50, P < 0.001). In this test, ANI-treated mice exhibited significantly lower freezing levels compared to vehicletreated mice. Next we examined whether coadministration ofeither nimodipine (8–32 mg/kg) or SR141716A (1–10 mg/kg)
would block the amnestic effects of ANI on memory expression.
Notably, the disruption of reactivated fear memory by ANI wasreversed by coadministration of either nimodipine or SR141716A
in a dose-dependent manner: Mice that were coadministered
Nim/ANI or SR/ANI exhibited similar levels of contextual fear
memory compared to mice treated with either nimodipine orSR141716A alone. A factorial ANOVA with Drug1 (Vehicle VS
ANI), Drug 2 (nimodipine VS SR141716A), and Dose (low,middle, high) confirmed these observations. Most critically,
there was no significant Drug1 ⳯ Drug2 interaction(F(1,132) = 0.20, P = 0.65), indicating similar levels of freezing in
nimodipine and SR141716A regardless of whether they were

Figure 1. Effects of ANI and CB1 and LVGCCs antagonists on memory
extinction. Experimental design used with data presented below. Mice
were administered these drugs immediately after 30 min of re-exposure(n = 12 for all groups). (C) Control; (A) anisomycin; (Nimo) nimodipine;(SR) SR141716A.

textual fear memory, and extinction was blocked by administration of ANI, or pharmacological blockade of LVGCCs or CB1
receptors. In the first experiment, we verified that pharmacological blockade of either protein synthesis, LVGCCs, or CB1 block
extinction of contextual fear conditioning groups (Fig. 1). In ourprevious studies, these treatments were administered 30 min
prior to re-exposure (Suzuki et al. 2004). Here we examined theeffects of these same treatments given immediately following
re-exposure. The advantage of this design is that it allows us toeliminate the possibility that injections of these drugs affect thefear memory retrieval and within-session extinction. During
training, mice received a single footshock (0.4 mA). Twenty-fourhours following training, mice were placed back into the context
for 30 min. During this re-exposure, overall freezing levels did
not differ across groups (data not shown) (all P’s > 0.05), but did
decrease over time, indicating that within-session extinction occurred (F(9,1188) = 196.5, P < 0.001). One day later, mice wereplaced back in the same context for 5 min. Consistent with ourprevious experiments, pharmacological inhibition of protein
synthesis blocked long-term extinction of contextual fear conditioning (F(1,22) = 28.26, P < 0.001): Mice treated with ANI (150mg/kg) showed higher levels of freezing compared to vehicletreated mice in this test 1 d following the extinction session. Next
we examined whether coadministration of either nimodipine(LVGCCs antagonist; 8–32 mg/kg) or SR141716A (CB1 antagonist; 1–10 mg/kg) would interfere with this ANI-induced blockade of extinction. We found that groups of mice coadministered
Nim/ANI or SR/ANI showed similarly high levels of contextual
fear memory, indicating that these treatments did not interferewith the ability of ANI to block extinction. In contrast, nimodipine and SR141716A blocked extinction in a dose-dependent
manner, as previously observed (Suzuki et al. 2004). A factorial
ANOVA with Drug1 (Vehicle VS ANI), Drug 2 (nimodipine VS
SR141716A), and dose (low, middle, high) confirmed these observations: Most critically, there was no significant
Drug1 ⳯ Drug2 interaction (F(1,132) = 0.003, P = 0.95), indicating
similar levels of freezing in nimodipine- and SR141716A-treatedgroups regardless of whether they were coadministered ANI.
There were main effects of Drug2 (F(1,132) = 7.55, P < 0.01), reflecting generally higher levels of freezing in mice treated with
Nim, and Dose (F(2,132) = 6.31, P < 0.01), reflecting generallywww.learnmem.org

Figure 2. Effects of ANI and CB1 and LVGCCs antagonists on memory
reconsolidation. Experimental design used with data presented below.
Mice were administered these drugs immediately after 3 min of reexposure (n = 12 for all groups). (C) Control; (A) anisomycin; (Nimo)
nimodipine; (SR) SR141716A.

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Destabilization of reactivated fear memory

main effect of Drug1 (F(1,77) = 77.19, P < 0.001), indicating reduced levels of freezing in mice treated with ANI (Fig. 3). No
other main effects and interactions were significant. Therefore,
these data reveal an important dissociation: Nimodipine andSR141716A block the amnestic effects of ANI only following
memory reactivation and not during initial consolidation. Together they are consistent with a role for LVGCCs or CB1 receptors in memory destabilization following retrieval. Because the
combined drug treatments were associated with reduced freezing
levels, they further show that neither Nim/ANI nor SR/ANI induce nonspecific increases in anxiety and fear.

Blockade of CB1 and LVGCCs does not interferewith the ability of ANI to block protein synthesis
Since coadministration of either nimodipine or SR141716A failed
to prevent the memory consolidation impairment induced by
ANI, these data additionally suggest that these drugs do not interfere with the ability of ANI to block protein synthesis. We next
explored this issue in more detail by examining induction of theimmediate early gene, c-fos, produced by the chemical convulsant pentylenetetrazol (PTZ) (Fig. 4). This approach has previously been used to examine Drug ⳯ Drug interactions since thePTZ-induced expression of Fos is strong (Frankland et al. 2006).
Mice were pretreated with vehicle, nimodipine, or SR141716A.
Thirty minutes following this pretreatment, mice received an in-

Figure 3. Effects of CB1 and LVGCCs antagonists on memory consolidation. Experimental design used with data presented below. Mice wereadministered these drugs immediately after training (n = 10 for allgroups). (C) Control; (A) anisomycin; (Nimo) nimodipine; (SR)SR141716A.

coadministered ANI. There were main effects of Drug1(F(1,132) = 6.70, P < 0.05), reflecting modest reductions in freezing
in some groups of mice treated with ANI, and Dose
(F(2,132) = 4.91, P < 0.001), indicating that lower doses of nimodipine and SR141716A were typically associated with lower levelsof freezing (Fig. 2). Consistent with previous studies, these results
first indicate that new proteins are required for the restabilizationof memories following retrieval. Blocking protein synthesis immediately following retrieval disrupted expression of contextual
fear memory tested 24 h later. Perhaps most strikingly, theseeffects were completely prevented by blocking LVGCCs and CB1
receptors. This suggests that activation of LVGCCs and CB1 receptors may be necessary for destabilization of contextual fear
memory following retrieval.

Blockade of CB1 and LVGCCs does not interferewith the amnestic effects of ANI on initial consolidation
To explore this phenomenon more comprehensively, we next
examined the effects of administration of pharmacologicalblockade of protein synthesis, LVGCCs, or CB1 on the initialconsolidation of a contextual fear memory (Fig. 3). If nimodipine
and SR141716A prevent the destabilization of memories following retrieval, then we reasoned that these treatments should only
be effective at blocking the effects of ANI following memory reactivation and not following initial learning. Accordingly, mice
were trained with a single footshock (0.4 mA). As expected, micereceiving post-training injections of ANI exhibited significantly
reduced levels of freezing when tested 24 h later (F(1,22) = 31.56,
P < 0.001). Consistent with previous experiments, these results
indicate that de novo protein synthesis is essential for the formation of long-term contextual fear memories. To examine
whether the amnestic effects of ANI would be blocked by pharmacological blockade of LVGCCs or CB1, mice were injectedwith different combinations of ANI, nimodipine, or SR141716A
immediately following training. Mice that were coadministered
Nim/ANI or SR/ANI exhibited reduced levels of contextual fear
memory compared to mice treated with only either nimodipine
or SR141716A alone. A factorial ANOVA with Drug1 (Vehicle VS
ANI), Drug 2 (nimodipine VS SR141716A), and Dose (low,middle, high) confirmed these observations. There was only awww.learnmem.org

Figure 4. Effects of CB1 and LVGCCs antagonists on the inhibition of
the PTZ-induced c-fos protein expression by anisomycin. (A) Experimental design. (B–J) Representative hippocampal c-fos protein immunohistochemistry obtained from indicated mice. (K) Quantitative analyses of cfos expression in dentate gyrus (n = 4 for all groups). (VEH) Vehicle;(Nim) nimodipine; (SR) SR141716A; (ANI) anisomycin.

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freezing during the re-exposure (data not shown) (all P’s > 0.05).
When contextual fear memory was tested 24 h later, freezing
levels were reduced in CREBIR treated with TAM. Importantly,
these deficits were not due to injection of TAM alone, since freezing levels were normal in WT mice treated with TAM. Furthermore, these deficits were not due to expression of the CREB repressor alone. Freezing levels in the CREBIR treated with VEH
were normal. Therefore, consistent with our previous study,
these data indicate that normal CREB function is required for therestabilization of reactivated contextual fear memory. Most strikingly, these effects were completely blocked by treatment withnimodipine. CREBIR mice coinjected nimodipine with TAM
showed comparable levels of freezing compared with other control groups, indicating that blocking LVGCCs prevented the disruption of contextual fear memory by inhibition of CREB activity
during reactivation. Consistent with these observations, a factorial ANOVA revealed a significant three-way interaction between
Genotype (WT VS CREBIR), Drug1 (Vehicle VS TAM), and Drug 2(Vehicle VS nimodipine): (F(1,109) = 5.39, P < 0.05). There were noother significant main effects or interactions, and post hoc Newman-Keuls tests confirmed that freezing levels were significantly
reduced in CREBIR mice compared to all other groups (all;P < 0.05) (Fig. 5). Together with our earlier experiments, thesedata indicate that LVGCCs play a role in memory destabilization
following reactivation. Blockade of LVGCCs protect reactivatedmemories against the amnesic effects of protein synthesis inhibition and inhibition of CREB activity.

jection of saline, PTZ (50 mg/kg, i.p.) or a combined PTZ + ANI
injection (Fig. 4A). This dose of PTZ produced clonic convulsions(i.e., sudden twitching of head or jerky movement of body) in the
majority of mice. We found that PTZ induced Fos expression in
the dentate gyrus, and that this effect was blocked by coadministration of ANI. Most importantly, the ability of ANI to block
PTZ-induced Fos expression was not affected by pretreatment
with either nimodipine or SR141716A (Fig. 4B–J). These observations were supported by a factorial ANOVA with Drug1 (Vehicle,
PTZ VS PTZ/ANI) and Drug 2 (Vehicle, nimodipine VS
SR141716A) as factors. This analysis revealed significant effects ofDrug1 (F(2,27) = 164.32, P < 0.001), consistent with the elevatedlevels of Fos expression in the PTZ-treated mice. However, there
were no significant effects of Drug 2 (F(2,27) = 0.49, P = 0.62) nor
significant interaction between Drug1 and Drug2 (F(4,27) = 0.54,
P = 0.71). Post hoc Newman-Keuls test confirmed that robust Fos
expression was observed in the dentate gyrus in PTZ-treated micecompared to saline-treated mice (P < 0.001), and levels were similar in mice pretreated with vehicle, nimodipine, or SR141716A(P > 0.05) (Fig. 4K). Together with the previous experiment, thesedata strongly suggest that neither nimodipine nor SR141716A
interferes with the ability of ANI to block protein synthesis.

Blockade of LVGCCs protects reactivated memories
against the amnesic effects of inhibition of CREB
activity
We have previously shown that normal CREB function is required for the restabilization of contextual fear memory following reactivation (Kida et al. 2002). Therefore we next asked
whether the effects of disrupting CREB function might beblocked by pharmacological blockade of LVGCCs (Fig. 5). To disrupt CREB function, we used transgenic mice that express an
inducible CREB repressor (CREBIR) in forebrain, where a dominant-negative CREB protein is fused with the ligand-binding domain (LBD) of a mutant estrogen receptor (ER). Injection oftamoxifen (TAM), the artificial ligand for ER, into these transgenic mice inhibits CREB activity in forebrain. In these experiments, CREBIR and WT littermate controls were trained with asingle footshock and re-exposed to the context for 3 min 1 dlater. Six hours prior to the re-exposure, mice received systemic
injection of TAM or VEH. All groups showed comparable levels of

Activation of CB1 and LVGCCs in hippocampus isrequired for destabilization of reactivated memory
Protein synthesis in the hippocampus is critical for the restabilization of contextual fear memory after reactivation (Debiec et al.
2002; Frankland et al. 2006). We therefore next tested whether
activation of CB1 and LVGCCs in the hippocampus is required
for the destabilization of reactivated contextual fear memory(Fig. 6). We therefore performed a similar experiment as in Figure
2 except that drugs were infused directly into the hippocampus,
rather than delivered systemically. Mice received infusion of ANI
with either CB1 blocker SR141716A or LVGCCs blocker Verapamil immediately after the 3-min re-exposure to the context (Fig.
6A). Consistent with previous findings (Debiec et al. 2002;
Frankland et al. 2006), infusion of ANI into the hippocampus
immediately following the re-exposure impaired the expressionof contextual fear memory tested 24 h later (F(1,56) = 11.324,
P < 0.01). ANI-treated mice exhibited significantly lower freezing
levels compared to vehicle-treated mice. These results indicate
the essential roles of hippocampal protein synthesis in the restabilization of reactivated contextual fear memory, confirming previous findings. Next we examined whether coinfusion of either
verapamil (0.5 or 2 µg) or SR141716A (2 or 4 µg) would block theamnestic effects of ANI in the hippocampus. Consistent with ourexperiments with systemic injections, the disruption of reactivated memory by ANI was reversed by coinfusion of either verapamil or SR141716A into the hippocampus in a dosedependent manner. Mice that were coinfused VER/ANI or SR/ANI
into hippocampus exhibited similar levels of contextual fear
memory compared to mice treated with either verapamil or
SR141716A alone. A factorial ANOVA with Drug1 (Vehicle VS
ANI), Drug 2 (verapamil VS SR141716A), and Dose (low, high)confirmed these observations: Most critically, there was no significant Drug1 ⳯ Drug2 interaction (F(1,90) = 1.30, P = 0.26), indicating similar levels of freezing in verapamil and SR141716A
regardless of whether they were coadministered ANI (Fig. 6B).
These results indicate that new hippocampal proteins are required for the restabilization of memories following retrieval.

Figure 5. Effects of LVGCCs antagonists on disruption of reactivatedmemory by the inhibition of CREB activity. Experimental design used withdata presented below. WT and CREBIR mice were administered TAM or
Peanut Oil (PO) 6 h before re-exposure (3 min) and then administerednimodipine (Nim) or vehicle (VEH) immediately after re-exposure (WT/PO/VEH, n = 15; WT/PO/Nim, n = 17; WT/TAM/VEH, n = 17; WT/TAM/
Nim, n = 13; CREBIR/PO/VEH, n = 12, CREBIR/PO/Nim, n = 13; CREBIR/TAM/VEH, n = 13, CREBIR/TAM/Nim, n = 14).
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molecular studies have focused almost exclusively on the latter(Taubenfeld et al. 2001; Kida et al. 2002; Lee et al. 2004; Duvarci
et al. 2005; Miller and Marshall 2005; von Hertzen and Giese
2005; Tronson et al. 2006) with the exception of a recent reportshowing importance of glutamate receptors in destabilization ofreactivated cued fear memory (Ben Mamou et al. 2006). Thesestudies have shown that restabilization engages similar but distinct molecular process compared with initial consolidation. The
studies presented here begin to unravel the molecular mechanisms underlying destabilization of reactivated memories. We
show that activation of LVGCCs and CB1 receptors are required
for the destabilization of fear memories following reactivation.
Pharmacological blockade of these processes protected retrieved
memories from the amnestic effects of anisomycin. Our findings
indicate the existence of an active process inducing the destabilization of reactivated memory. This destabilization process
might play a critical role in opening a window to update orstrengthen original memory.
Memory reactivation initiates two competing processes: reconsolidation and extinction. Reconsolidation may serve tostrengthen or modify the existing memory trace, whereas extinction involves the formation of an inhibitory memory that competes with the original fear memory. Because the temporal dynamics of these two processes differ, which comes to gain control
over behavior depends, in part, on the duration of the retrieval
episode (operationally the re-exposure in these studies): Reconsolidation is the dominant process following shorter duration
re-exposures, whereas extinction is the dominant process following longer duration re-exposures (Debiec et al. 2002; Eisenberg etal. 2003; Nader 2003; Pedreira and Maldonado 2003; Suzuki et al.
2004). Previous studies have established that blocking protein
synthesis during brief re-exposure disrupts the original fear
memory, whereas blocking protein synthesis during longer reexposure blocks the formation of a new extinction memory (Eisenberg et al. 2003; Pedreira and Maldonado 2003; Suzuki et al.
2004). The requirement for protein synthesis in the formation ofa new extinction memory is perhaps not surprising. However,the fact that protein synthesis inhibition selectively affects theformation of the extinction memory and leaves the original
memory unaffected is surprising since both reconsolidation andextinction would be expected to be initiated under these conditions. These results suggest that the acquisition of a new extinction memory may transiently alter the stability of the original
memory, raising the possibility that molecular processes engaged
during extinction may also regulate the stability of the original
trace, rendering it immune to amnestic challenges such as theapplication of protein synthesis inhibition (but see Duvarci et al.
2006).
Accordingly, our results indicate that pharmacologicalblockade of LVGCCs or CB1 receptors has two consequences onreactivated contextual fear memories. First, following longer reexposure to the context, pharmacological blockade of LVGCCs
or CB1 blocks the formation of new extinction memory, as previously shown (Cain et al. 2002; Marsicano et al. 2002; Suzuki etal. 2004). Second, while pharmacological blockade of LVGCCs or
CB1 did not affect reconsolidation of the original fear memory
following shorter (3 min) context re-exposure, they protected
these memories against the amnestic effects of anisomycin. We
similarly showed that blocking LVGCCs also prevented the disruption of reactivated contextual fear memory by inhibition of
CREB activity, indicating that these blockers inhibit the disruption of reactivated memory by two independent pathways; inhibition of protein synthesis and CREB activity. Together, our results support the idea that blocking molecular processes involvedin fear extinction also regulates the stability of the original fear
memory. These effects are not simply due to nimodipine or

Figure 6. Effects of ANI and CB1 and LVGCCs antagonists on memory
reconsolidation in hippocampus. (A) Cannula tip placements from mice
infused with each drug. Coronal sections are adapted from Paxinos andFranklin (1997) (1.94 mm posterior to Bregma). (B) The experimental
design used with the data presented below. Mice were administeredthese drugs into dorsal hippocampus immediately after 3 min of reexposure (n = 10–30). (C) Control; (A) anisomycin; (VER) verapamil; (SR)SR141716A.

More importantly, inhibitory effects of protein synthesis inhibition in hippocampus on restabilization of reactivated contextual
fear memory were completely prevented by blocking LVGCCs
and CB1 receptors. This suggests that the stability of reactivatedmemory (destabilization and restabilization) is regulated in the
hippocampus and that activation of LVGCCs and CB1 receptors
is required for the destabilization of reactivated contextual
memory in hippocampus.

Discussion
Previous studies have shown that retrieval temporarily destabilizes fear memories, and that gene expression-dependent processes are required for subsequent restabilization (memory reconsolidation) (Nader et al. 2000; Taubenfeld et al. 2001; Debiec etal. 2002; Kida et al. 2002). While reconsolidation must encompass both a destabilization process and a restabilization process,www.learnmem.org

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The studies presented here show that LVGCCs and CB1 play
key roles in not only extinction but also destabilization of contextual fear memory. Understanding the circumstances underwhich maladaptive memories become plastic (and modifiable) isof clear clinical relevance. Since LVGCCs and CB1-dependent
processes are both important in weakening fear memories (destabilization and extinction of fear memory), they might serve as
useful therapeutic targets for the treatment of conditions such
PTSD and phobias.

SR141716A interfering with the ability of anisomycin to blockprotein synthesis. In a control experiment, we showed that coadministration of these drugs does not block the ability of anisomycin to inhibit c-fos expression induced by the chemical convulsant PTZ. Perhaps most importantly, these effects were specific to reactivated memories. Pharmacological blockade of
LVGCCs or CB1 receptors did not block the amnestic effects ofanisomycin on initial learning. This dissociation confirms thatthese treatments do not interfere with the ability of anisomycin
to block protein synthesis and that these treatments specificallyregulate the stability of reactivated fear memories. These findings
suggest that memory reactivation triggers the activation of
LVGCCs and CB1 receptor, leading to destabilization and/or extinction of contextual fear memory. Further studies are required
to examine the possibility that formation of extinction memory
leads to the destabilization of reactivated contextual fear
memory. In this case, the destabilized memory might undergo
restabilization without new protein synthesis (such as the redistribution or relocalization of pre-existing proteins).
Previous studies have shown that the restabilization of reactivated contextual fear memory requires new protein synthesis
in hippocampus (Debiec et al. 2002; Frankland et al. 2006). We
replicated these effects, showing that blocking hippocampal protein synthesis following reactivation disrupts the subsequent expression of a contextual fear memory. Most strikingly, blockadeof LVGCCs or CB1 function in hippocampus prevented this disruption of reactivated contextual fear memory by protein synthesis inhibition. This finding localizes the destabilization process to the hippocampus, indicating that following reactivation,
both destabilization and restabilization engage hippocampaldependent processes. Similar to this, a recent study has shownthat the amygdala critically regulates the destabilization and restabilization of reactivated cued fear memory (Ben Mamou et al.
2006). On the other hand, abundant evidence has identified critical roles for extra-hippocampal regions such as the PFC andamygdala in contextual fear memory extinction (Myers andDavis 2002; Quirk et al. 2006; Ji and Maren 2007). Our findings
further suggest that CB1 and LVGCCs play distinct roles in different brain regions. In the hippocampus, they mediate destabilization of contextual fear memory following reactivation,
whereas they mediate extinction in other brain regions (i.e., PFC
and amygdala).
Our results suggest that the activation of CB1-dependent
signaling plays a key role in destabilization of the reactivated fear
memory. Once destabilized, that memory would require newgene expression for subsequent restabilization. Interestingly,
some studies suggest that activation of CB1-dependent signaling
may facilitate destabilization to a degree where restabilization is
no longer possible. Infusion of CB1 agonists into the amygdala orinsular cortex following memory retrieval of cued fear or conditioned taste aversion, respectively, leads to subsequent deficits inexpression of that memory (Lin et al. 2006; Kobilo et al. 2007).
These observations support our conclusion that CB1 plays a rolein the regulation of memory stability following the retrieval, as
well as memory extinction.
How do CB1 and LVGCCs cooperatively regulate memory
processes such as extinction and the destabilization of reactivated memory? LVGCCs are known to be expressed in postsynapse of excitatory neurons in hippocampus and increase inpost-synaptic Ca2+ concentration in response to depolarization.
In contrast, endocannabinoids are released in response to theincrease in post-synaptic Ca2+ concentration and then act as a
retrograde signal activates to activate pre-synaptic CB1 receptors
leading to decreased neurotransmitter release in pre-synapse.
Therefore, it is possible that CB1 is activated via the activation of
post-synaptic LVGCCs.
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Materials and Methods
Mice
All experiments were conducted according to the Guide for theCare and Use of Laboratory Animals, Japan Neuroscience Society.
Male C57BL/6N mice were obtained from Charles River (Yokohama, Japan). Transgenic mice expressing inducible CREB repressor (CREBIR mice) have been maintained since 1998 by backcrossing to C57BL/6 (more than F15 generations) (Kida et al. 2002).
Mice were housed in cages of five or six, maintained on a 12-h
light/dark cycle, and allowed free access to food and water. Mice
were at least 8 wk of age when tested. Testing was carried out
during the light phase of the cycle. All experiments were conducted blind to the treatment condition of the mouse.

Surgery
Surgeries were performed as described previously (Frankland etal. 2006). Under nembutal anesthesia and using standard stereotaxic procedures, stainless-steel guide cannula (22-gauge) were
implanted into the dorsal hippocampus (ⳮ1.8 mm, Ⳳ1.8 mm,
ⳮ1.9 mm). Mice were allowed to recover for at least 1 wk following surgery. Following this, they were handled for three consecutive days prior to the commencement of contextual fear conditioning of the mice.

Drugs
Because the neural sites critically responsible for acquisition, extinction, and reconsolidation of fear memories may be different,
we used systemic rather than intracranial injections of a protein
synthesis inhibitor (anisomycin; ANI; Sigma), CB1 antagonist(SR141716A) (Rinaldi-Carmona, et al. 1994; Suzuki, et al. 2004),
and L-type voltage-gated calcium channels (LVGCCs) antagonist(nimodipine; Sigma). ANI (150 mg/kg, i.p.) was dissolved in PBS(1 drop of Tween 80 in 3 mL of 2.5% dimethylsulphoxide and
10% Cremophor in saline, pH adjusted to 7.0–7.4). At this dose,
ANI inhibits >90% of protein synthesis in the brain during thefirst 2 h following injection (Flood et al. 1973). SR141716A (1, 3,
or 10 mg/kg, i.p.) was dissolved in vehicle solution, containing 1
drop of Tween 80 in 3 mL of 2.5% dimethylsulphoxide and 10%
Cremophor in saline. Nimodipine (8, 16, or 32 mg/kg, i.p.) was
sonicated into 100% Cremophor EL (Sigma) and then diluted tomake the final vehicle 10% Cremophor in solution (1 drop of
Tween 80 in 3 mL of 2.5% dimethylsulphoxide). In these experiments, drug doses were determined according to previous reports
showing effective doses for the blockade of long-term extinction(Cain et al. 2002; Marsicano et al. 2002; Suzuki et al. 2004).
For local infusion study, ANI (125 µg/µL), SR141716A (4 or
8 µg/µL), and verapamil (LVGCCs antagonist; 1 or 4 µg/µL;
Sigma) was dissolved in vehicle solution, containing three dropsof Tween 80 in 2.5 mL of 7.5% dimethylsulphoxide in artificial
cerebrospinal fluid (ACSF) and adjusted to pH 7.4 with NaOH.
For experiments using the CREBIR mice, 4-hydroxytamoxifen (16 mg/ kg, i.p.; Sigma) was dissolved in 10 mL of peanut oil(Sigma) (Kida et al. 2002).

Contextual fear conditioning
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 as previously described (Suzuki et al. 2004). Training consisted of placing the431

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Destabilization of reactivated fear memory

References

mice in the chamber and delivering an unsignaled footshock(2-sec duration, 0.4 mA) 148 sec later. Mice were returned to thehomecage 30 sec after the footshock.
To examine the effects of drugs on memory reconsolidation
or extinction, mice were trained drug-free, and 24 h later placedback in the training context for 3 or 30 min during which freezing behavior was assessed (re-exposure) as the percentage of time
mice spent freezing. Mice were treated with vehicle or drugs(ANI, SR, and nimodipine) immediately following removal fromthe context. Twenty-four hours later, mice were once again
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2001).
In one series of experiments, we examined the effects ofdrug treatment on memory consolidation. In these experiments,
mice were treated with saline or drug immediately after the training. Memory was assessed 24 h later as the percentage of time
mice spent freezing.

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Immunocytochemistry
To indirectly examine the impact of CB1 and LVGCCs blocker onprotein synthesis inhibition by ANI, we examined the inductionof the activity-regulated gene c-fos by the chemical convulsant
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Data analysis
Data were analyzed with analysis of variance (ANOVA). A oneway or factorial ANOVA and post hoc Newman-Keuls comparisons were used to analyze the effects of drugs and genotypes.
Analyses of planned comparisons were performed using a oneway ANOVA and post hoc Newman-Keuls comparisons. All values in the text and figure legends are means Ⳳ SEM.

Acknowledgments
We thank Rui Costa for critical reading of the manuscript. S.K.
was supported by Grant-in-Aids for Scientific Research, HighTechnology Research and Priority Areas–Molecular Brain Science(18022038) from the Ministry of Education, Science and Culture
and Technology, Japan, Core Research for Evolutional Scienceand Technology, Japan, and the Research Grant for Nervous andMental Disorders from the Ministry of Health, Labour, and Welfare, Japan.
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Received December 19, 2007; accepted in revised form March 31, 2008.

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