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Stability of recent and remote contextual fear memory
Paul W. Frankland, Hoi-Ki Ding, Eiki Takahashi, et al.
Learn. Mem. 2006 13: 451-457
Access the most recent version at doi:10.1101/lm.183406

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Research

Stability of recent and remote contextual
fear memory
Paul W. Frankland,1,2,3,6 Hoi-Ki Ding,1,2 Eiki Takahashi,3,4 Akinobu Suzuki,5Satoshi Kida,5 and Alcino J. Silva3
1

Program in Neuroscience and Mental Health, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8; 2Department of
Physiology, University of Toronto, Ontario, Canada M5S 1A8; 3Departments of Neurobiology, Psychiatry and Psychology andBrain Research Institute, University of California–Los Angeles, California 90095-1761, USA; 4Tsukuba Research Laboratory, Eisai
Co. Ltd., Tsukuba, Ibaraki 300-2635, Japan; 5Departments of Agricultural Chemistry and Bioscience, Faculty of Applied Science,
Tokyo University of Agriculture, Tokyo 156-8502, Japan
Following initial encoding, memories undergo a prolonged period of reorganization. While such reorganization may
occur in many different memory systems, its purpose is not clear. Previously, we have shown that recall of recent
contextual fear memories engages the dorsal hippocampus (dHPC). In contrast, recall of remote contextual fear
memories engages a number of different cortical regions, including the anterior cingulate cortex (ACC). To examine
whether this reorganization leads to greater memory stability, we examined reconsolidation of 1 d-old (recent) and
36 d-old (remote) contextual fear memory in mice. We infused the protein synthesis inhibitor, anisomycin (ANI),
into either the dHPC or ACC immediately following retrieval of either a recent or remote contextual fear memory.
In the dHPC, ANI infusions disrupted subsequent expression of recent, but not remote, contextual fear memory.
Similar infusions into the ACC had no effect on either recent or remote contextual fear memories, whereas
systemically applied ANI blocked subsequent remote memory expression when long re-exposure durations wereused. Together, these data suggest that as memories mature they become increasingly stable. Furthermore, thedissociation between the effects of systemically and centrally administered ANI on remote memory suggests thatstability is due, in part, to the distributed nature of remote contextual fear memories.

Memories are not formed instantaneously. Instead, following initial encoding, memories appear to undergo a prolonged period ofreorganization (McClelland et al. 1995; Squire and Alvarez 1995;
Dudai 2004; Wiltgen et al. 2004; Frankland and Bontempi 2005).
The result of this reorganization is that circuits underlying encoding or initial storage may differ from those required for permanent storage. This type of reorganization has most often beenstudied in cortico-hippocampal circuits (Squire et al. 2004;
Frankland and Bontempi 2005). However, time-dependent reorganization is observed across many different memory systems(Shadmehr and Holcomb 1997; Tronel and Sara 2002; Bahar et al.
2004) and in many different species, including invertebrates (McBride et al. 1999; Menzel 2001; Keller et al. 2005). While thesestudies indicate that reorganization might be a somewhat ubiquitous process during memory maturation, its exact purpose isstill not clear. Because behavioral expression of memory doesn’tnecessarily change over time, one possibility is that reorganization leads to greater memory stability (Dudai and Eisenberg 2004;
Alberini 2005; Stickgold and Walker 2005).
One way to probe memory stability is to apply consolidation
blockers (e.g., protein synthesis inhibitors) during or immediately following memory retrieval. Such manipulations interferewith the restabilization (or reconsolidation) of memories, leading
to amnesia (Nader 2003). While reconsolidation has now been
demonstrated across species and memory paradigms (Nader
2003; Dudai and Eisenberg 2004), post-reactivation protein synthesis blockade does not always lead to amnesia (Vianna et al.
2001; Kraus et al. 2002; Biedenkapp and Rudy 2004; Cammarota
6

Corresponding author.
E-mail paul.frankland@sickkids.ca; fax (416) 813-6846.
Article is online at http://www.learnmem.org/cgi/doi/10.1101/lm.183406.

et al. 2004; Pedreira et al. 2004). One important variable appears
to be the age of the memory. Recent studies in rodents (Milekic
and Alberini 2002; Suzuki et al. 2004), as well as medaka fish
(Eisenberg and Dudai 2004), have shown that as memories mature they become increasingly immune to disruption by proteinsynthesis blockers. This suggests that there might be a trade-off
between plasticity and stability as memories age.
This age-stability relationship has so far only been investigated using systemically applied consolidation blockers (Milekic
and Alberini 2002; Eisenberg and Dudai 2004; Suzuki et al. 2004).
However, we have recently mapped brain regions involved in therecall of recent and remote contextual fear memories in mice
(Frankland et al. 2004). These studies showed that the dHPC wasstrongly activated only following recall of the recent (day-old)fear memory. In contrast, a number of different cortical regions(including the ACC) were strongly activated only following recallof the remote (month-old) fear memory. Here we target theseregions to probe the stability of recent and remote contextual
fear memories. Using identical apparatus and procedures as inour mapping studies (Frankland et al. 2004), we examined theeffects of post-reactivation blockade of protein synthesis on subsequent expression of recent and remote contextual fear
memory.

Results
ANI disrupts Fos induction following PTZ treatment
We first evaluated whether our ANI manipulations disrupted protein synthesis. To do this we examined whether ANI attenuates
induction of the activity-regulated gene, c-fos, produced by thechemical convulsant pentylenetetrazol (PTZ). In the first study,

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These data indicate that infusions of ANI
into the dHPC disrupt reconsolidationof recent, but not remote, contextual
fear memories (delay ⳯ drug interaction: F(1,32) = 4.99, P < 0.05) (Fig. 3C).
This effect on recent memory was contingent on memory reactivation. Whenthe reactivation was omitted, similar
ANI infusions had no effect on subsequent expression of that memory tested
1 d later (F(1,15) = 0.69, P > 0.05) (Fig. 3D).
To explore the generality of this effect on recent contextual fear memories,
we also trained another set of mice witha single footshock and re-exposed themto the conditioning chamber 1-d later.
Figure 1. ANI infusions into the ACC attenuate Fos expression induced by systemic injections of PTZ.
As before, we found that ANI infusions(A) Representative photomicrographs of ACC in mice receiving either PBS or ANI infusions into theACC. PTZ induction of Fos is reduced by pretreatment with ANI. (B) Quantification of Fos expression
disrupted reconsolidation of a recent
in mice pretreated with PBS and ANI (*P < 0.05; see text for details). Number of Fos-positive nuclei percontextual fear memory (F(1,11) = 5.76,
100 ⳯ 100-µm field are shown.
P < 0.05) (Fig. 3E), demonstrating that
ANI blocks reconsolidation of recent
contextual fear memories regardless of whether mice are weakly
mice received intra-ACC infusions of PBS or ANI. Thirty minutes
or strongly fear conditioned. Histological analyses revealed that
following this pretreatment, mice received PTZ (50 mg/kg, ip).
This dose of PTZ produced clonic convulsions (i.e., sudden
twitching of head or jerky movement of body) in the majority of
mice, and strong Fos expression throughout the brain (including
the ACC). Importantly, Fos expression in the ACC was reducedby over 50% in mice pretreated with ANI, consistent with theidea that ANI attenuates protein synthesis (t(13) = 5.94, P < 0.05)(Fig. 1). This reduction in Fos expression was evident in a roughly
circular region 1.0–1.5 mm in diameter, below the tip of theinjector. This indicates that infusions were largely restricted to
the ACC, with limited diffusion to neighboring structures.
In the second study, mice were pretreated with systemic
injections of PBS or ANI. Again, PTZ treatment induced strong
Fos expression and this expression was reduced by ANI pretreatment in the ACC (PBS: 13.94 Ⳳ 0.89; ANI: 6.21 Ⳳ 0.89[t(8) = 6.21, P < 0.05]), as well as other cortical regions, including
the retrosplenial (PBS: 9.14 Ⳳ 1.08; ANI: 0.50 Ⳳ 0.14[t(12) = 7.95, P < 0.05]) and perirhinal (PBS: 5.04 Ⳳ 1.12; ANI:
1.16 Ⳳ 0.27 [t(12) = 3.38, P < 0.05]) cortex (Fig. 2). Together thesestudies establish that the ANI manipulations used in the behavioral studies disrupt protein synthesis, measured here as a reduction in Fos expression induced by the chemical convulsant, PTZ.

Post-reactivation blockade of protein synthesisin the dHPC disrupts subsequent expression of recent,but not remote, contextual fear memory
In this first series of behavioral experiments, mice were trainedwith three footshocks. Then, either 1-d (recent) or 36-d (remote)later mice were placed back into the context for 2.5 min (reexposure). Immediately following this context re-exposure, micereceived infusions of ANI into the dHPC. One day following this,they were tested.
We found that the destabilizing effects of blocking protein
synthesis in the dHPC following memory recall depended on theage of the memory (Fig. 3). All groups of mice exhibited similarlevels of freezing when re-exposed to the conditioning context(Recent: F(1,14) = 0.06, P > 0.05; Remote: F(1,18) = 0.16, P > 0.05).
However, intra-dHPC infusions of ANI following the retrieval ofa 1-d-old (recent) contextual fear memory blocked expression ofthat memory tested one day later (F(1,14) = 11.34, P < 0.05) (Fig.
3A). In contrast, similar infusions following the retrieval of 36d-old (remote) contextual fear memory had no effect on subsequent memory expression (F(1,18) = 0.44, P > 0.05) (Fig. 3B).

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Figure 2. Systemic ANI injections attenuate cortex-wide Fos expression
induced by systemic injections of PTZ. Representative photomicrographsof ACC (top), retrosplenial cortex (middle), and perirhinal cortex (bottom)
in mice pretreated with either PBS or ANI. In each of these cortical regions, Fos expression is reduced in mice pretreated with ANI.

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Reconsolidation of recent and remote memory

hippocampus, intra-ACC infusions of
ANI following the retrieval of either recent (F(1,15) = 0.51, P > 0.05) or remote(F(1,18) = 0.14, P > 0.05) contextual fear
memory had no effect on subsequent
memory expression (Fig. 4A,B). This result is confirmed by the absence of a significant delay ⳯ drug interaction(F(1,33) = 0.36, P > 0.05) (Fig. 4C). Therefore, whereas inactivation of the ACC
blocks retrieval of 36-d-old (remote)contextual fear memories (Frankland etal. 2004), blocking protein synthesis following retrieval does not affect subsequent stability of that memory. Histological analyses revealed that the tips ofthe guide cannulae were correctly located just dorsal to the ACC in these andall following experiments.

Intra-ACC blockade of protein
synthesis fails to disrupt
reconsolidation of remotecontextual fear memory following
extended context re-exposure
The duration of re-exposure is an important variable in reconsolidation experiments. Previous studies have establishedthat longer duration re-exposures tend
to have greater destabilizing effects on
memory than shorter ones (Pedreira andMaldonado 2003; Suzuki et al. 2004). We
therefore tested whether intra-ACC infusions of ANI block reconsolidation whenlonger re-exposure durations were used
(Fig. 5A). Mice were trained as above and then 36 d later reexposed to the conditioning context for either 10 or 15 min.
These re-exposures should be maximally destabilizing since
longer duration re-exposures produce significant extinction (Suzuki et al. 2004). Indeed, while all groups of mice exhibited similar levels of freezing during the re-exposure phase (10 min:F(1,17) = 3.60, P > 0.05; 15 min: F(1,17) = 0.05, P > 0.05), overall

Figure 3. Post-reactivation blockade of protein synthesis in dHPC. Intra-dHPC infusions of ANI following context re-exposure disrupted subsequent expression of recent (A), but not remote (B), contextual fear memory. The percent time spent freezing is shown during the training (TR), re-exposure(RE), and test phases (TE) of the experiment. A summary of the test freezing scores for these mice isshown in C. The destabilizing effects of intra-dHPC ANI on recent contextual fear memory are contingent on memory reactivation. When context re-exposure was omitted, ANI infusions into the dHPC
had no effect on subsequent memory expression (D). Intra-dHPC infusions of ANI also block reconsolidation of a weaker contextual fear memory. In this experiment, mice were trained with one, rather
than three shocks (E). Significant differences are marked by an asterisk (*P < 0.05, see text for details).

the tips of the guide cannulae were correctly located just dorsal tothe dHPC in these experiments.

Post-reactivation blockade of protein synthesis in the
ACC has no effect on subsequent expression of recent
or remote contextual fear memory
We previously found that the ACC is activated following recall of remote contextual fear memory. Further experiments showing that (1) this activity isabsent in ␣-CaMKII+/ⳮ mice that are unable to form remote memories, and (2)localized inactivation of this region specifically blocks remote memory, suggested that the ACC plays a crucial role
in remote memory recall (Frankland etal. 2004). Therefore, we next examined
the impact of blocking protein synthesisin the ACC on the stability of both recent and remote contextual fear memories (Fig. 4). Again, mice were trainedwith three footshocks, and 1 or 36 d later
placed back in the conditioning context
for 2.5 min. During this re-exposure
phase, all groups of mice exhibited similar levels of freezing (Recent:F(1,15) = 0.11, P > 0.05; Remote:F(1,18) = 0.23, P > 0.05). In contrast to the

Figure 4. Post-reactivation blockade of protein synthesis in the ACC. Intra-ACC infusions of ANI
following context re-exposure did not disrupt subsequent expression of recent (A), or remote (B),contextual fear memory. The percent time spent freezing is shown during the training (TR), reexposure (RE), and test phases (TE) of the experiment. A summary of the test freezing scores for thesemice is shown in C.

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to disrupt subsequent expression of remote contextual fear memories, evenwhen longer duration re-exposures areused.

Systemic ANI injections disrupt
reconsolidation of remotecontextual fear memories following
extended duration
context re-exposures
One reason why reactivated remote contextual memories may be insensitive tothe amnesic effects of ANI is because
they are supported by distributed cortical networks (McClelland et al. 1995;
Frankland and Bontempi 2005). It is possible that a more global, cortex-wide disruption of protein synthesis would block
the reconsolidation of remote contextual fear memories. To test this, we examined the effects of systemically administered ANI on remote contextual
fear memory. In the first series of experiments (Fig. 5B), mice were trained asabove and 36 d later re-exposed to theconditioning context for either 10 or 15
min. Prior to this re-exposure, mice received injections of ANI (150 mg/kg,
i.p.) or PBS. Both ANI- and PBS-treatedmice exhibited similar levels of freezing
when re-exposed to the conditioning
context (10 min: F(1,18) = 0.18, P > 0.05;
15 min: F(1,18) = 0.04, P > 0.05). However, only ANI injections prior to the 15min re-exposure disrupted subsequent
memory tested 1 d later (F(1,18) = 6.16,
P < 0.05). Similar infusions prior to the
10 min re-exposure had no effect on
subsequent memory expression(F(1,18) = 0.30, P > 0.05).
In the previous central injection experiments, ANI was infused after, rather
than before context re-exposure. Therefore, we ran an additional experiment
where mice received systemic injectionsof ANI or PBS immediately after a 15Figure 5. Effects of context re-exposure duration on reconsolidation of remote contextual fear
min re-exposure. Mice in the ANI andmemory. For each experiment, the percent time spent freezing is shown during the training (TR),PBS groups exhibited similar levels of
re-exposure (RE), and test phases (TE) of the experiment. (A) Intra-ACC infusions of ANI had no effectfreezing during the re-exposure phaseon subsequent expression of remote contextual fear memory using either 10- or 15-min context
re-exposures. These test data are summarized in the graph on the far right. (B) Systemic injections of(F(1,18) = 1.42, P > 0.05). However, conANI blocked reconsolidation of remote contextual fear memory only when the longer (15-min) context
sistent with our earlier results, systemic
re-exposure was used. These test data are summarized in the graph on the far right. (C) Systemic
ANI treatment disrupted subsequent
injection of ANI immediately following (rather than preceding) a 15-min re-exposure also blocks
memory tested 1 d later (F(1,18) = 29.73,
reconsolidation (left). The destabilizing effects of ANI on remote contextual fear memory are continP < 0.05) (Fig. 5C). Finally, we tested
gent on memory reactivation. When context re-exposure was omitted, systemic ANI injections had nowhether this effect was contingent on
effect on subsequent memory expression (right). Significant differences are marked by an asterisk(*P < 0.05, see text for details).
memory reactivation. When this reactivation was omitted, similar ANI injections had no effect on subsequent exfreezing levels were lower in mice re-exposed for 15 min, sugpression of that memory tested 1 d later (F(1,15) = 0.05, P > 0.05)
gesting that this longer duration re-exposure may start to pro(Fig. 5C).
duce some extinction. In either case, intra-ACC infusions of ANI
These data indicate that systemic ANI injections disrupt refollowing either the 10-min (F(1,17) = 0.16, P > 0.05) or 15-minconsolidation of remote contextual fear memories, but only
(F(1,17) = 1.98, P > 0.05) re-exposure had no effect on subsequent
when longer duration re-exposures are used. The effects of syscontextual fear memory expression tested 1-d later. These results
temically administered ANI contrast with those of intra-ACC inindicate that post-reactivation infusions of ANI into the ACC fail
fused ANI. Under identical conditions, local blockade of protein

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Reconsolidation of recent and remote memory

synthesis in the ACC had no effect on subsequent memory expression, suggesting that a more global disruption of protein synthesis is necessary to destabilize remote memory. This is different
from the situation for recent contextual fear memories. Both local (dHPC) (present study; Suzuki et al. 2004) or systemic (Suzuki
et al. 2004) blockade of protein synthesis disrupts reconsolidation of recent contextual fear memories.

Discussion
We used a contextual fear-conditioning paradigm in mice to examine how time-dependent reorganization might affect memory
stability. Two features of contextual fear conditioning make it
especially suited for this analysis. First, contextual fear conditioning has been used extensively to study behavioral and moleculardeterminants of reconsolidation in mice (Kida et al. 2002;
Biedenkapp and Rudy 2004; Fischer et al. 2004; Lattal and Abel
2004; Suzuki et al. 2004; von Hertzen and Giese 2005), as well asother species (Debiec et al. 2002; Pedreira and Maldonado 2003;
Biedenkapp and Rudy 2004; Lee et al. 2004; Pedreira et al. 2004;
Merlo et al. 2005). Second, imaging and lesion experiments indicate that the circuits supporting contextual fear undergo significant reorganization in the month following learning (Anagnostaras et al. 1999; Shimizu et al. 2000; Frankland et al. 2001,
2004). The current experiments indicate that blocking protein
synthesis in the dHPC disrupts recent, but not remote contextual
fear memories. Similar infusions into the ACC had no effect oneither recent or remote contextual fear memories, whereas systemically applied ANI blocked subsequent remote memory expression when long re-exposure durations were used. Together,
these data suggest that as memories mature they become increasingly stable. Furthermore, the dissociation between the effects ofsystemically and centrally administered ANI on remote memory
suggests that stability is due, in part, to the distributed nature of
remote contextual fear memories.
The effects of intra-dHPC infusions of ANI on the stability ofrecent and remote contextual fear memory parallels the timedependent role the hippocampus plays in the storage of thesetypes of memories. Using identical apparatus, behavioral procedures, and retention delays, we previously showed that the hippocampus was activated following recall of recent, but not remote contextual fear memory (Frankland et al. 2004). Here wefound that blocking protein synthesis in the dHPC-disrupted reconsolidation of recent contextual fear memories, regardless ofwhether mice had been trained using a weak (one-shock) or
strong (three-shock) protocol. However, we found that similar
infusions had no effect on remote contextual fear memories,
consistent with the lack of involvement of the dHPC in processing contextual fear memories at later time points (Hall et al.
2001; Frankland et al. 2004; von Hertzen and Giese 2005). Thiswas still the case even when a longer re-exposure (10-min) wasused (data not shown). This lack of effect on remote contextual
fear memory contrasts with a previous study in rats (Debiec et al.
2002). In this study, intrahippocampal infusions of ANI blockedreconsolidation of both recent (1-d-old) and remote (i.e., 45-dold) contextual fear memories. This discrepancy may reflect species differences between rats and mice. Alternatively, it is possiblethat infusion of a larger volume or dose would be more effective
in destabilizing remote memory in our study. However, our dataclearly demonstrate a time-dependent stability gradient, with recent memories more sensitive to intra-dHPC infusions of ANI
compared with remote memories.
We previously found that the ACC was activated following
recall of remote (but not recent) contextual fear memories, andthat inactivation of the ACC specifically disrupts recall of thesememories (Frankland et al. 2004). Here we found that post-

retrieval blockade of protein synthesis in the ACC does not destabilize remote contextual fear memories. This lack of effect on
remote memories suggests two possibilities. First, the ACC may
only be necessary for retrieval (and not storage) of remote contextual fear memories. Such a situation would suggest a divisionof labor in the cortex with the ACC coordinating retrieval ofmemories that are stored in more posterior cortical regions(Frankland and Bontempi 2005). If this were the case, then postreactivation disruption of protein synthesis in more posteriorcortical regions might disrupt the reconsolidation of remote contextual fear memories.
A second possibility is that the distributed nature of remotememory protects against the destabilizing effects of locally applied protein synthesis inhibitors. Using cellular imaging approaches, we have previously shown that recall of remote contextual fear memory is associated with activation of multiple cortical regions (Frankland et al. 2004), suggesting that remotecontextual fear memories are supported by a broadly distributed
network. In the present series of experiments, we found that only
systemically applied ANI disrupted subsequent expression of remote contextual fear memory, albeit only when a longer duration context re-exposure was used. Therefore, whereas local
blockade of protein synthesis in the dHPC was sufficient to destabilize a recent contextual fear memory, a more global disruption of protein synthesis appears to be necessary to destabilize
remote contextual fear memory.
Together, these experiments have started to explore the relationship between memory age and memory stability. Consistent with previous reports (Milekic and Alberini 2002; Pedreiraand Maldonado 2003; Suzuki et al. 2004), our data indicate thatas memories mature they become increasingly immune to thedestabilizing effects of protein-synthesis blockers. Gradual reorganization of contextual fear memories in distributed cortical (orcortico-hippocampal) networks (McClelland et al. 1995; Nadel
and Moscovitch 1997; Squire et al. 2004; Wiltgen et al. 2004;
Frankland and Bontempi 2005) may underlie this increased stability.

Materials and Methods
Animals
Male C57Bl/6 mice were used in these experiments. For the local
infusion experiments, these mice were purchased from Taconic
Farms. For the systemic injection experiments, they were purchased from Charles River. Mice were maintained on a 12-h
light/12-h dark cycle, and had free access to food and water. Mice
were at least 8 wk of age at the start of experiments, and all
behavioral procedures were conducted during the light phase of
the cycle. Experiments were conducted blind to the treatment
condition of the mouse.

Surgery
Under chloral hydrate anesthesia and using standard stereotaxic
procedures, stainless-steel guide cannulae (22-gauge) were implanted into the dHPC (ⳮ2.0 mm, Ⳳ2.0 mm, ⳮ2.0 mm) or ACC(+0.8 mm, 0 mm, ⳮ1.0 mm). Mice were allowed to recover for atleast 1 wk following surgery. Following this, they were handled
for three consecutive days prior to the commencement of contextual fear conditioning.

Drugs and infusion procedures
The protein synthesis inhibitor, Anisomycin (ANI; Sigma), wasused in these experiments. For the local infusion studies, ANI(100 µg/µL) was dissolved in phosphate-buffered saline (PBS) andthe pH-adusted to 7.0–7.4. ANI was infused immediately following the re-exposure phase of the experiment. Mice were briefly
anesthetized with halothane to facilitate insertion of the injec-

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tion cannula. Infusions into the dHPC (0.75 µL) or ACC (0.50 µL)
were made at a rate of 0.2 µL/min. The injection cannula (26gauge) was left in place for 2 min following the infusion. Thisdose of locally infused ANI inhibits >90% of protein synthesis forat least 4 h (Rosenblum et al. 1993). For the systemic injection
experiments, ANI (150 mg/kg, i.p.) was injected 30 min prior to
the re-exposure phase of the experiment. At this dose, ANI inhibits >90% of protein synthesis in the brain in the first 2 hfollowing injection (Flood et al. 1973).

Effect of ANI manipulations on c-fos expression
To indirectly examine the impact of ANI on protein synthesis
inhibition, separate groups of mice received infusions of either
PBS (n = 8) or ANI (n = 7) into the ACC, as described above.
Thirty minutes following this, mice were administered thechemical convulsant pentylenetetrazol (PTZ; 50 mg/kg, ip), andthen 30 min later perfused transcardially. Brains were subsequently prepared for immunocytochemistry using anti-Fos primary rabbit polyclonal antibody (1:20000), as previously described (Frankland et al. 2004). Staining was revealed using theavidin-biotin peroxidase method (ABC kit). Quantitative analysis
was performed using a NIH image-processing system and brain
regions were anatomically defined according to the Paxinos andFranklin atlas (Paxinos and Franklin 2000). Immunoreactive neurons were counted by an experimenter unaware of the treatment
condition.
In a second experiment, mice received systemic injections ofeither PBS or ANI, as described above. Thirty minutes later they
were administered PTZ (50 mg/kg, ip), and then 30 min later
perfused transcardially. Brains were subsequently prepared for
Fos immunocytochemistry as above. The advantage of using PTZ
in these experiments is that it induces high levels of Fos expression. This makes it easier to detect any reductions in Fos expression produced by pretreatment with ANI.

Contextual fear-conditioning procedures
The apparatus and general procedures for contextual fear conditioning have previously been described (Anagnostaras et al. 2000;
Frankland et al. 2004; Suzuki et al. 2004). Mice were trained andtested in a conditioning chamber (32 cm ⳯ 25 cm ⳯ 25 cm),containing a stainless-steel shock-grid floor. For the systemic injection experiments, a similar, but smaller (17.5 cm ⳯ 17.5 cm ⳯
15 cm), conditioning chamber was used.
Each experiment consisted of three phases: Training, reexposure, and testing. During each of these phases, freezing behavior (defined as a complete absence of movement, except for
respiration) (Fanselow 1980) was measured using automated procedures (Anagnostaras et al. 2000).
In the first series of experiments, during training, mice wereplaced in the conditioning chamber for 5 min. After 2 min they
were presented with three unsignaled footshocks (2-sec duration,
0.75 mA, 1 min apart). During the re-exposure phase, mice wereplaced back into the conditioning context for 2.5 min either 1 or
36 d later. Immediately following this re-exposure, mice received
infusions of PBS or ANI into the dHPC or ACC, respectively. One
day later, contextual fear memory was assessed in a 2-min test.
Mouse numbers were as follows: dHPC recent (PBS n = 9; ANIn = 7); dHPC remote (PBS n = 9; ANI n = 11); ACC recent (PBSn = 7; ANI n = 10); ACC remote (PBS n = 13; ANI n = 7).
In the second series of experiments, we first examined theeffects of omitting the re-exposure. Accordingly, mice weretrained as above with three footshocks. One day later they received infusions of PBS (n = 9) or ANI (n = 8) into the dHPC, butwere not re-exposed to the conditioning chamber. The following
day, contextual fear memory was assessed in a 2-min test. In asecond experiment, we tested whether ANI disrupts reconsolidation of a weaker contextual fear memory. In this experiment,
mice were trained with a single footshock (a 0.75 mA shock,delivered after 2 min of a 3-min training session) and re-exposedto the conditioning chamber for 2.5 min the following day. Immediately following this re-exposure, mice received infusions of456

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either PBS (n = 5) or ANI (n = 8) into the dHPC. One day later,
contextual fear memory was assessed in a 2-min test.
In the third series of experiments, we examined the effects
of using longer duration context re-exposures on memory stability. Again, mice were trained with three footshocks. Thirty-six
days later, they were re-exposed to the conditioning context foreither 10 or 15 min. For the local infusion experiments, micereceived infusions of PBS (10 min, n = 9; 15 min, n = 13) or ANI(10 min, n = 10; 15 min, n = 6) into the ACC immediately following this re-exposure. For the systemic injection experiments,
mice received PBS (10 min, n = 10; 15 min, n = 10) or ANI (10min, n = 10; 15 min, n = 10) injections 30 min before the reexposure. One day later, contextual memory was assessed in a
2-min test.
In addition, we also tested whether systemically administered ANI would have the same effects if injected immediately
following the re-exposure. In this experiment, mice were trained
as before with three footshocks. Then, 36 d later they were reexposed to the conditioning context for 15 min. Immediately
following this re-exposure mice received PBS (n = 10) or ANI(n = 10) injections and contextual fear memory was then assessedin a 2-min test the following day. In a separate group of mice, we
examined the effects of omitting the re-exposure. Accordingly,
mice were trained as above with three footshocks. Thirty-six days
later they received systemic injections of PBS (n = 10) or ANI(n = 10), but were not re-exposed to the conditioning chamber.
The following day, contextual fear memory was assessed in a
2-min test.

Histology
At the completion of the experiments, mice were overdosed withchloral hydrate and perfused transcardially with saline, followed
by 4% formaldehyde solution to fix the brain tissue. Brains were
removed, post-fixed in 4% formaldehyde solution, and cut into
50-µm coronal sections on a cryostat. Sections were mounted onslides, stained with neutral red, and cannula placements were
examined under a light microscope.

Statistical analyses
Separate ANOVAs were performed to determine the effects of ANI
on freezing in the re-exposure and retention tests in each of theconditions (e.g., dHPC/recent, dHPC/remote, etc). In addition,
two-way ANOVAs (drug [ANI, PBS] ⳯ delay [recent, remote])
were used to evaluate whether these treatments differentially affected recent vs. remote memory. t-tests were used to analyze theeffects of ANI treatment on Fos expression.

Acknowledgments
This work was supported by grants from National Alliance for
Research on Schizophrenia and Depression, the Canadian Institutes of Health Research (P.W.F.), the NIH (A.J.S.), and the Ministry of Education, Science and Culture, Japan (S.K.). We thank
Rui Costa, Sheena Josselyn, and Nohjin Kee for comments on
earlier drafts of this manuscript.

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Received December 30, 2005; accepted in revised form May 8, 2006.

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