REPORTS

The Involvement of the Anterior
Cingulate Cortex in Remote
Contextual Fear Memory
Paul W. Frankland,1,2,3* Bruno Bontempi,4* Lynn E. Talton,1Leszek Kaczmarek,1,5 Alcino J. Silva1†
Although the molecular, cellular, and systems mechanisms required for initialmemory processing have been intensively investigated, those underlying permanent memory storage remain elusive. We present neuroanatomical, pharmacological, and genetic results demonstrating that the anterior cingulate cortex playsa critical role in remote memory for contextual fear conditioning. Imaging ofactivity-dependent genes shows that the anterior cingulate is activated by remotememory and that this activation is impaired by a null ␣-CaMKII mutation thatblocks remote memory. Accordingly, reversible inactivation of this structure innormal mice disrupts remote memory without affecting recent memory.
The formation of new memories involves
protein synthesis– dependent changes in synaptic structure and plasticity in the hippocampus (1–3). However, these memories are notstored permanently in the hippocampus. In
humans and animals, damage to the hippocampus preferentially disrupts recently acquired memories while sparing remotely acquired memories (4–7); these effects indicatethat remote memories eventually become independent of the hippocampus. To study therole of extrahippocampal structures in remotememory, we used contextual fear conditioning. In contextual fear conditioning, mice
form an association between a distinctive
context and an aversive event that takes placein that context. When placed back into thecontext, mice exhibit a range of conditioned
fear responses, including freezing (5). Contextual fear conditioning is ideally suited forthe study of remote memory, because a single
training session produces robust lifelong
memory (8) that can be measured using automated procedures (9).
To identify extrahippocampal regions involved in processing remote contextual fear
memories, we tracked the expression of genes(zif268 and c-fos) modulated by neuronal activity (10). Separate groups of mice were trainedwith 0 or 5 footshocks and tested either 1 daylater (recent memory test) or 36 days later
Departments of Neurobiology, Psychiatry, Psychology and Brain Research Institute, UCLA, 695 CharlesYoung Drive South, Los Angeles, CA 90095–1761,USA. 2Department of Integrative Biology, Hospital for
Sick Children Research Institute, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. 3Department of Physiology, University of Toronto, Toronto,Ontario, Canada M5S 1A8. 4Laboratoire de Neurosciences Cognitives, CNRS UMR 5106, Avenue desFacultés, 33405 Talence, France. 5Nencki Institute,Pasteura 3, 02– 093, Warsaw, Poland.
1*These authors contributed equally to this work.
†To whom correspondence should be addressed. Email: silvaa@mednet.ucla.edu.

(remote memory test) (11). Although context
exposure that is not reinforced may itself produce lasting memories (12), we observed notime-dependent changes in cortical gene expression after recent or remote memory tests inthe control mice that were not shocked (fig. S1).
Therefore, we analyzed gene expression in theshocked mice normalized with respect to thesestable levels in controls. This allowed us toisolate changes in gene expression associatedwith contextual fear memory, and control forgene expression associated with motor activity,general arousal, and other nonspecific aspectsof the testing procedure (13, 14). We focused
our analyses on the anterior cingulate cortex(ACC), prelimbic (PL) and infralimbic (IL) regions of the medial prefrontal cortex, temporalcortex (TC), and visual cortex (VC), because of
the proposed role for the cortex in remote memory (15–20). Zif268 expression was elevated in
ACC after the remote (160.5 ⫾ 4.6%), but not
the recent (89.4 ⫾ 7.4%), memory tests (Fig.
1A), which suggests that this region is preferentially involved in processing remote contextual fear memories. After the remote, but notrecent, memory test, similar pronounced increases in gene expression were observed in IL,PL, and TC (Fig. 1A). The same temporally
graded pattern was observed in ACC, IL, PL,and TC with Fos expression (Fig. 1B), whichindicates that these results generalize to otheractivity-dependent genes, and are not specific toZif268. Note that the distinct patterns of gene
expression following recent and remote memory tests indicate that gene expression is not
simply a correlate of freezing (or other fearrelated) behaviors, because freezing levels weresimilar at both time points.
The results presented above suggest thatprocessing of remote contextual fear memory involves coordinated activation of multiple cortical regions. We thus examined
cortical activation in mutant mice (␣CaMKII⫹/⫺), which have specific deficits

in remote memory (16). Both the magnitude and the specificity of the contextual
fear memory phenotype in the
␣-CaMKII⫹/⫺ mice make them an ideal
tool to examine neural systems for remotememory. Concurrent with their wild-type(WT) littermates (11), ␣-CaMKII⫹/⫺ mice
were contextually conditioned and testedeither 1 or 36 days after training. Contextual fear was dramatically reduced in the␣-CaMKII⫹/⫺ mice at the longer retention
delay when we used either freezing oractivity suppression as a measure (16) (Fig.
2). The pronounced increase in Zif268
expression in ACC after the remote memory test was absent in the mutants (Fig.
1A). Rather, Zif268 expression was similar
in mutants after the recent (82.1 ⫾ 4.7%)
and remote memory tests (93.2 ⫾ 10.6%).
The increased Zif268 expression associatedwith remote memory was also blocked inIL, PL, and TC (Fig. 1A), which indicatedthat cortical activation associated with remote memory may be widely blocked in the␣-CaMKII⫹/⫺ mice. Fos expression associated with remote memory was similarly
blocked in ACC, IL, and PL in␣-CaMKII⫹/⫺ mice (Fig. 1B). It is thoughtthat consolidation occurs in a reactivationdependent manner, either during online(e.g., retrieval tests) or offline (e.g., sleep)
situations (19–22). Reactivation may leadto the gradual refinement of the memory
network and integration of that networkwith related, preexisting memories (19).
Therefore, to assay synaptic remodelingunderlying these processes during memory
recall, we examined expression of growthassociated protein 43 (GAP-43), a marker
of synaptogenesis (23), in ACC (11). GAP43 expression was elevated after the remotememory test in WT (recent 11.5 ⫾ 1.7,
remote 19.2 ⫾ 2.1; P ⬍ 0.05), but not
␣-CaMKII⫹/⫺ (recent 10.2 ⫾ 0.9, remote11.2 ⫾ 2.1; P ⬎ 0.05), mice. These results
indicate that the ␣-CaMKII⫹/⫺ mutation
may impede cortical reorganization necessary for consolidation.
The CA1 region of the hippocampus isstrongly activated during acquisition andrecall of contextual fear conditioning (13),which suggests that this region has a critical role in processing contextual fear memories. In WT mice, gene expression waselevated in CA1 after the recent (Zif268:126.1 ⫾ 3.9%; Fos: 174.2 ⫾ 6.3%), but not
remote (Zif268: 79.9 ⫾ 5.9%; Fos: 93.5 ⫾11.4%), memory test (Fig. 1). These datasuggest that consolidation involves thegradual disengagement of CA1 (13), coupled with progressive recruitment of cortical regions. Furthermore, because Zif268
expression was reduced below control levels after the remote memory test in WT
mice, activity in CA1 may be inhibitedwww.sciencemag.org SCIENCE VOL 304 7 MAY 2004

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REPORTSFig. 1. Expression of activity-dependent genes after recent or remotememory tests. In order to isolate changes associated with memory, gene
expression in the shocked groups is expressed as a percentage relative tocontrols that were not shocked. Changes in gene expression in different
brain regions are shown for WT (black circles) and ␣-CaMKII⫹/⫺ (opencircles) mice after recent or remote memory tests. (A) Zif268 expression
was elevated in WT, but not ␣-CaMKII⫹/⫺ mice, after the remote memory
test in ACC [Genotype ⫻ Delay interaction F(1,28) ⫽ 16.82, P ⬍ 0.05], IL[F(1,28) ⫽ 9.82, P ⬍ 0.05], PL [F(1,28) ⫽ 33.73, P ⬍ 0.05], and TC[F(1,28) ⫽ 15.52, P ⬍ 0.05], but not VC [F(1,28) ⫽ 0.52, P ⬎ 0.05]. In theCA1 region of the hippocampus, Zif268 expression was elevated after theduring processing of remote memories. Incontrast, Zif268 levels remained elevatedafter the remote memory test in
CaMKII⫹/⫺ mice, which do not express
behavioral memory at this time point.
Therefore, the absence of this inhibitory
feedback in the mutants may allow newencoding to occur.
Reduced cortical activation of Zif268 and
Fos in the ␣-CaMKII⫹/⫺ mice after remotememory tests most likely reflects differences in
the organization of memory in these mutants.
However, it is possible that the ␣-CaMKII⫹/⫺
mutation disrupts regulation of these activitydependent genes. We therefore conducted additional control experiments (11). First, we examined gene expression associated with training(Fig. 3A). Consistent with the observation that␣-CaMKII⫹/⫺ mice acquire contextual fear conditioning normally, we found that Zif268 andFos expression was similar in WT and␣-CaMKII⫹/⫺ mice across different cortical regions. Furthermore, gene expression in the cortex was similar in WT and ␣-CaMKII⫹/⫺ mice
after removal from their home cage (Fig. 3B)
and in controls that were not shocked (fig. S1).
Therefore, the regulation of Zif268 and Fos appears to be normal in the ␣-CaMKII⫹/⫺ mice
under conditions associated with either low orhigh levels of neural activation, which suggeststhat changes in gene expression observed after882

recent, but not remote, memory tests in WT mice. In contrast, Zif268
expression was elevated at both time points in ␣-CaMKII⫹/⫺ mice[F(1,28) ⫽ 25.93, P ⬍ 0.05]. (B) Changes in Fos expression were qualitatively similar to those in Zif268 for WT and ␣-CaMKII⫹/⫺ mice. Fos
expression was elevated in WT, but not ␣-CaMKII⫹/⫺ mice, after theremote memory test in ACC [F(1,28) ⫽ 14.03, P ⬍ 0.05], IL [F(1,28) ⫽9.69, P ⬍ 0.05], PL [F(1,28) ⫽ 46.37, P ⬍ 0.05], but not TC [F(1,28) ⫽1.83, P ⬎ 0.05] nor VC [F(1,28) ⫽ 1.39, P ⬎ 0.05]. In CA1, Fos expression
was elevated after the recent, but not remote, memory tests in WT mice.
In contrast, Fos expression was elevated at both time points in
␣-CaMKII⫹/⫺ mice [F(1,28) ⫽ 30.78, P ⬍ 0.05].

Fig. 2. Contextual fear memory in ␣-CaMKII⫹/⫺ mice(open circles) versus WTmice (black circles). To assess
memory in ␣-CaMKII⫹/⫺mice and WT mice, two behavioral indices of conditioned fear were measured in
the same mice: (A) freezing;(B) activity suppression (9).
Whereas WT mice exhibitedrobust levels of freezing andactivity suppression in both
the recent (1 day after training) and remote (36 days after training) retention tests,freezing [Genotype ⫻ Delay interaction, F(1,28) ⫽ 10.62, P ⬍ 0.05] and activity suppression[Genotype ⫻ Delay interaction F(1,28) ⫽ 9.69, P ⬍ 0.05] were markedly reduced at the longer
retention delay in the ␣-CaMKII⫹/⫺ mice.

remote memory tests are a consequence ofchanges in mnemonic processing in thesemutants.
The results presented above indicate that specific cortical sites are activated by remote memory processes and that this activation is absent inmice with remote memory deficits. To directly
test whether these cortical sites are required forremote memory, we examined the effects oftransient inactivation using lidocaine (11). Because remote memories are likely stored in distributed cortical networks (19, 24), they may beresistant to focal disruption (25). However, ex-ecutive structures like PL and ACC, which areboth robustly activated by remote contextual
memory, are thought to play an integrative rolein memory (26–28) and may therefore be amenable for targeted disruption. Lidocaine infusions into ACC disrupted contextual fear memory at remote (18 and 36 days), but not recent (1or 3 days) time points (Fig. 4A). Similar infusions into the neighboring PL had no effect oneither 1-day-old or 36-day-old contextual fear
memories (Fig. 4B). Although the imaging data
show that a broad cortical network is activatedby remote memory, these inactivation results

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REPORTS
pronounced cortical activation associated withremote memory was absent. These data suggestthat CaMKII is necessary for the maturationand elaboration of cortical circuits underlying
remote memory. In normal mice, imaging andinactivation experiments identified the ACC asa critical node in a broader cortical network
processing remote memory. During memory encoding, ACC is thought to play an integrative rolein cognitive control processes [e.g., attention, conflict monitoring (26, 27, 29)]. It remains to be
determined whether the ACC is a site for memorystorage per se or whether it mediates analogous
processes for integrating multiple cortical representations underlying remote memories.
References and Notes

Fig. 3. Regulation of Zif268 and Fos in the cortex is normal in ␣-CaMKII⫹/⫺ mice. The number ofZif268- and Fos-positive nuclei are shown for WT (black bars) and ␣-CaMKII⫹/⫺ (white bars) mice.
(A) Gene expression induced in the cortex after acquisition of contextual fear conditioning.
Training-associated expression of Zif268 or Fos was similar in WT and ␣-CaMKII⫹/⫺ mice in eachof these regions (P values ⬎ 0.05). (B) Gene expression in the cortex in the home cage condition.
There were no differences in Zif268 or Fos expression between WT and ␣-CaMKII⫹/⫺ mice in eachof these regions (P values ⬎ 0.05).
Fig. 4. Targeted pharmacological inactivation of ACC and PL.
(A) Lidocaine-induced inactivation of ACC disrupts retrieval ofremote, but not recent, contextual fear memories. Planned
comparisons indicated thatfreezing levels were reduced inlidocaine-infused mice in theretention tests on the 18th or
36th day, but not the 1st or 3rd
day after training (P values ⬍0.05). (B) Lidocaine-induced inactivation of PL did not disrupt
retrieval of contextual fear
memories. Planned comparisons indicated that freezing
levels in phosphate-bufferedsaline (PBS)– and lidocaine-infused mice were not different (P values ⬎ 0.05).

identify ACC (but not PL) as an essential node
within this network for processing remotememory.
Modeling, neuropsychological, and neurophysiological studies have suggested a central
role for hippocampal-cortical networks inmemory consolidation (19, 20, 22). Interactions
between the hippocampus and cortex following
initial learning lead to the gradual establishmentof enduring memories in distributed corticalnetworks that are independent of the hippocam-pus. Here, we used brain imaging to identify
cortical regions involved in processing fear
memories. Our data show that processing fear
memories involves the activation of multiple
association cortical regions, consistent with theproposal that enduring memories are stored in
distributed cortical networks. Cortical activation was greater after remote, rather than recent,memory tests, consistent with an increasingly
important role for the cortex over time. In micewith specific deficits in remote memory, the

1. S. J. Martin, P. D. Grimwood, R. G. Morris, Annu. Rev.
Neurosci. 23, 649 (2000).
2. E. R. Kandel, Science 294, 1030 (2001).
3. Y. Dudai, Annu. Rev. Psychol. 55, 51 (2004).
4. S. M. Zola-Morgan, L. R. Squire, Science 250, 288 (1990).
5. J. J. Kim, M. S. Fanselow, Science 256, 675 (1992).
6. S. G. Anagnostaras, S. Maren, M. S. Fanselow, J. Neurosci. 19, 1106 (1999).
7. J. R. Manns, R. O. Hopkins, L. R. Squire, Neuron 38,127 (2003).
8. M. S. Fanselow, G. D. Gale, Ann. N.Y. Acad. Sci. 985,
125 (2003).
9. S. G. Anagnostaras, S. A. Josselyn, P. W. Frankland,
A. J. Silva, Learn. Mem. 7, 58 (2000).
10. L. Kaczmarek, H. A. Robertson, Handbook of Chemical
Neuroanatomy (Elsevier, Amsterdam, 2002), p. 370.
11. Materials and methods are available as supporting
material in Science online.
12. S. G. Anagnostaras, G. D. Gale, M. S. Fanselow, Hippocampus 11, 8 (2001).
13. J. Hall, K. L. Thomas, B. J. Everitt, J. Neurosci. 21, 2186
(2001).
14. K. L. Thomas, J. Hall, B. J. Everitt, Eur. J. Neurosci. 16,
1789 (2002).
15. B. Bontempi, C. Laurent-Demir, C. Destrade, R. Jaffard, Nature 400, 671 (1999).
16. P. W. Frankland, C. O’Brien, M. Ohno, A. Kirkwood,
A. J. Silva, Nature 411, 309 (2001).
17. K. Takehara, S. Kawahara, Y. Kirino, J. Neurosci. 23,9897 (2003).
18. Z. Cui et al., Neuron 41, 781 (2004).
19. J. L. McClelland, B. L. McNaughton, R. C. O’Reilly,Psychol. Rev. 102, 419 (1995).
20. L. R. Squire, P. Alvarez, Curr. Opin. Neurobiol. 5, 169(1995).
21. K. Louie, M. A. Wilson, Neuron 29, 145 (2001).
22. K. L. Hoffman, B. L. McNaughton, Science 297, 2070
(2002).
23. L. I. Benowitz, A. Routtenberg, Trends Neurosci. 20,84 (1997).
24. D. Marr, Proc. R. Soc. London Ser. B 176, 161 (1970).
25. K. S. Lashley, Symp. Soc. Exp. Biol. 4, 454 (1950).
26. T. W. Robbins, Prog. Brain Res. 126, 469 (2000).
27. G. Bush, P. Luu, M. I. Posner, Trends Cogn. Sci. 4, 215(2000).
28. E. K. Miller, J. D. Cohen, Annu. Rev. Neurosci. 24, 167(2001).
29. C. S. Carter, M. M. Botvinick, J. D. Cohen, Rev. Neurosci. 10, 49 (1999).
30. We thank A. K. Anderson, J. F. Guzowski, S. A. Josselyn,
K. Nader, and B. J. Wiltgen for comments on the manuscript. This work was supported by the NIH (A.J.S.);
Canadian Institutes of Health Research, Research Chairin Cognitive Neurobiology (P.W.F.); CNRS (B.B.); andFulbright program (L.K.).
Supporting Online Materialwww.sciencemag.org/cgi/content/full/304/5672/881/
DC1
Materials and Methods
Fig. S1
References and Notes
17 December 2003; accepted 12 March 2004

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