Spine growth in the anterior cingulate cortex isnecessary for the consolidation of contextual
fear memory
Gisella Vetere a,b,1, Leonardo Restivo a,1, Christina J. Cole a,c, P. Joel Ross a, Martine Ammassari-Teule b,d,
Sheena A. Josselyn a,c,e, and Paul W. Frankland a,c,e,2aProgram in Neurosciences and Mental Health, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8; bIstituto di Ricerca a Carattere Clinico e Scientiﬁco
Fondazione Santa Lucia, 00143 Rome, Italy; dCell Biology and Neurobiology Institute, National Research Council, 00143 Rome, Italy; cDepartment of
Physiology and eInstitute of Medical Science, University of Toronto, Toronto, ON, Canada M5S 1A8
Edited* by Larry R. Squire, Veterans Affairs Medical Center, San Diego, CA, and approved March 23, 2011 (received for review November 1, 2010)

Remodeling of cortical connectivity is thought to allow initiallyhippocampus-dependent memories to be expressed independentlyof the hippocampus at remote time points. Consistent with this,consolidation of a contextual fear memory is associated with dendritic spine growth in neurons of the anterior cingulate cortex(aCC). To directly test whether such cortical structural remodeling isnecessary for memory consolidation, we disrupted spine growth in
the aCC at different times following contextual fear conditioningin mice. We took advantage of previous studies showing that thetranscription factor myocyte enhancer factor 2 (MEF2) negatively
regulates spinogenesis both in vitro and in vivo. We found that
increasing MEF2-dependent transcription in the aCC during a critical
posttraining window (but not at later time points) blocked both theconsolidation-associated dendritic spine growth and subsequent
memory expression. Together, these data strengthen the causal link
between cortical structural remodeling and memory consolidation
and, further, identify MEF2 as a key regulator of these processes.
structural plasticity| remote memory | viral vectorIn experimental animals, damage to the hippocampus disproportionately impacts recently acquired memories, with relative
sparing of remote memories (1–8). Such observations have led tothe idea that the hippocampus is transiently required for memory
expression, with remote memory expression being exclusively
dependent on the cortex (9). According to one model (10), posttraining hippocampal activity coordinates reactivation of memory
traces in the cortex. This reactivation leads to the remodeling ofcortical connections, allowing the memory to eventually be expressed independently of the hippocampus. A recent study in mice(5) provided correlative evidence for posttraining remodeling of
neurons in the anterior cingulate cortex (aCC), a subregion of theprefrontal cortex that plays an essential role in remote memory
expression (11). Increases in dendritic spine density on layer 2/3
pyramidal aCC neurons were observed 1 mo, but not 1 d, following contextual fear conditioning (5). As layer 2/3 pyramidal
neurons send and receive long-range cortical connections (12),such changes may contribute to increased functional connectivity
between the aCC and other cortical areas important for remotememory expression (13, 14). However, whether this increase inaCC spine density is necessary for memory consolidation is notknown. To test this, it would be necessary to evaluate whether
preventing posttraining spinogenesis, speciﬁcally in this region,
disrupts memory consolidation.
The transcription factor myocyte enhancer factor 2 (MEF2)
negatively regulates spinogenesis in an activity-dependent manner and therefore provides a tool to address this question. For
example, increasing MEF2 function decreases the number ofdendritic spines and excitatory synapses in vitro (15) and blocks
increases in spine density normally observed following repeatedcocaine administration in rat medium spiny nucleus accumbens
neurons in vivo (16). Accordingly, in our experiments, we used

8456–8460 | PNAS | May 17, 2011 | vol. 108 | no. 20

a viral vector-based strategy to increase MEF2 function in theaCC at speciﬁc times following contextual fear conditioning. We
found that increasing MEF2-dependent transcription in layer 2/3
aCC neurons during the ﬁrst (but not seventh) posttraining week
blocks both consolidation-associated structural changes in layer
2/3 aCC neurons and memory consolidation.
Results
Contextual Fear Memory Consolidation Is Associated with TimeDependent Spine Growth in the aCC. We ﬁrst asked whether con-solidation of a memory was accompanied by an increase in spine
density in aCC pyramidal neurons. We used a contextual fear
paradigm in which mice learn to associate a context with anaversive stimulus, such as a mild footshock (SI Materials andMethods). When placed back in the context, mice exhibit a rangeof species-typical fear reactions, including freezing [the absenceof all movement except breathing (1)]. Contextual fear conditioning offers a number of advantages that make it particularly
suitable for studying the role of structural plasticity in the aCC inmemory consolidation. First, training occurs in a single session(rather than over several days). Second, this training produces
a durable memory [that can last as long as a lifetime (17)] andcan be quantiﬁed using automated procedures (18). Third,
lesions of the hippocampus impair expression of recent, but not
remote, contextual fear memories (1, 3, 4, 19). Conversely, inactivation of the aCC impairs the expression of remote, but notrecent, contextual fear memories (11). In particular, thesestudies provide direct evidence that the circuits supporting thesememories reorganize over time, and that remote memory expression depends, in part, on the aCC (9).
Mice were trained in contextual fear conditioning and tested
either 1, 8, 42, or 49 d later (Fig. 1A). Freezing was equivalent at
all retention delays, indicating that this training protocol produces
robust, durable levels of conditioned fear (Fig. 1B). Consistent
with our previous report (5), we observed a time-dependent increase in spine density on apical dendrites of layer 2/3 aCC neurons (Fig. 1 C–F). This increase was limited to layer 2/3 pyramidal
neurons [which have long-range cortical connections (12)], assimilar changes were not observed in layer 5 aCC neurons [which
do not have long-range cortical connections (12)] (Fig. S1). Furthermore, increases in spine density of layer 2/3 aCC pyramidal

Author contributions: G.V., L.R., M.A.-T., S.A.J., and P.W.F. designed research; G.V., L.R.,
C.J.C., and P.J.R. performed research; G.V. and L.R. analyzed data; and S.A.J. and P.W.F.
wrote the paper.
The authors declare no conﬂict of interest.
*This Direct Submission article had a prearranged editor.
1

G.V. and L.R. contributed equally to this work.

2

To whom correspondence should be addressed. E-mail: paul.frankland@sickkids.ca.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1016275108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1016275108

Fig. 1. Time-dependent increases in dendritic spine density in aCC pyramidal neurons following contextual fear conditioning. (A) Experimental design. (B)Conditioned freezing in trained (T) mice was similar at all retention delays, and always greater than in control (C) mice (P < 0.001, by ANOVA). (C) Photomicrograph showing examples of Golgi-impregnated pyramidal neurons in the aCC. (Scale bar, 500 μm.) (D) Golgi-impregnated apical dendritic segments inthe aCC in trained and control mice at different retention delays. (Scale bar, 5 μm.) (E) Cumulative frequency of spine density on apical dendrites in layer 2/3
aCC pyramidal neurons. Spine density was greater in trained (closed circles) versus control (open circles) mice 8, 42, and 49 d following training (P < 0.05, by
Kolmogorov–Smirnov test). (F) Spine density increased with retention delay in layer 2/3 aCC pyramidal neurons [P < 0.001, by ANOVA (signiﬁcant delay
effect); spine density in trained mice was normalized with respect to controls]. These and subsequent graphs show means ± SEM.

Using a Viral Vector to Acutely Increase MEF2 Function in the aCC. To
increase MEF2-dependent transcription, we expressed MEF2VP16, a version of MEF2 in which the DNA binding and dimerization domains are fused to the transcriptional activation
domain of the viral protein VP16 (15). MEF2-VP16 binds MEF2
sites within the promoter region of target genes and leads to theirconstitutive transcription (15). Accordingly, we veriﬁed that thisMEF2-VP16 construct speciﬁcally increases MEF2-recognition
element (MRE)-dependent [but not cAMP-response element(CRE)-dependent] transcription in a luciferase reporter assay incultured cells (Fig. 2A).
To ensure that MEF2 is endogenously expressed in the aCC,
we next characterized the expression of MEF2 proteins in theadult mouse brain using an antibody that recognizes the major
Vetere et al.

MEF2 isoforms expressed in the brain (MEF2A, MEF2C, andMEF2D). Consistent with previous data (24), we conﬁrmed that
MEF2 is highly expressed in the brain (Fig. 2B), including theaCC (Fig. 2C). Furthermore, MEF2 was colocalized with theneuronal marker NeuN (Fig. 2D), indicating that MEF2 expression is restricted to neurons.
To increase MEF2-dependent transcription only in the aCC
at speciﬁc times after training, we used a replication-defective
HSV vector-based approach (25). We chose HSV because, unlike other vectors, HSV is neurotropic (25) and MEF2 is expressed exclusively in neurons. To visualize infected cells, ourHSV coexpressed GFP. Microinfusion of the MEF2 (HSVMEF2-VP16-GFP) or control (HSV-GFP) vector led to robusttransgene expression in ∼5–7% of layer 2/3 pyramidal neurons
for up to 1 wk, declining thereafter (Fig. 2E and Fig. S2). Furthermore, up-regulation of MEF2-dependent transcription was
not associated with a secondary inﬂammatory response (e.g.,
activated microglia; Fig. S3).
Increasing MEF2 Function in the aCC Disrupts Memory Consolidation
in a Time-Dependent Manner. As spine density on apical dendritesof layer 2/3 aCC pyramidal neurons increased during the ﬁrst,
but not the seventh, week following contextual fear conditioning(Fig. 1F), we hypothesized that limiting spine growth shortly
following training (but not at later time points) would disrupt
memory consolidation. To test this, we trained mice in contextual fear conditioning and microinfused the MEF2 or control
vector into the aCC either 1 or 42 d later. Seven days following
microinfusion, contextual fear memory was assessed by placing

PNAS | May 17, 2011 | vol. 108 | no. 20 | 8457

NEUROSCIENCE

neurons were most pronounced during the ﬁrst posttraining week
(i.e., day 1–8; P < 0.05, by Fisher’s protected least signiﬁcant difference test) and plateaued thereafter (i.e., days 42–49; P > 0.05)(Fig. 1F). Because memory reactivation is thought to drive structural changes underlying reorganization of cortical networks (20–22), more pronounced increases in spine density shortly following
training are consistent with the idea that reactivation strength andfrequency decline over time (23). In our previous study, we showedthat such changes in spine density are independent of memory
recall (5). Therefore, together these data indicate that consolidation of a contextual fear memory is associated with posttraining
remodeling of aCC layer 2/3 neurons, and the time course of thesestructural changes parallels the emergent role of the aCC in theexpression of contextual fear memories (11).

Fig. 2. Use of a viral vector to acutely increase MEF2 function in the aCC at speciﬁc times after training. (A) MEF2-VP16 robustly and selectively increases
MEF2-dependent transcription in cultured cells. Cells were cotransfected with (Left) MRE-luciferase or (Right) CRE-luciferase reporter plasmids and a control(GFP) or MEF2-VP16 plasmid. MEF2-VP16 selectively increased MRE- (P < 0.01, by t test) but not CRE-dependent transcription. Importantly, control data showthat forskolin (FSK) increases CRE-mediated transcription (P < 0.001, by t test). Data are expressed as mean ± SEM. (B) Sagittal sections from the adult mousebrain stained for a nuclear marker (Hoechst; Top), a neuron-speciﬁc marker (NeuN; Middle), and MEF2 (Bottom), indicating that endogenous MEF2 proteins
are highly expressed in neurons throughout the adult brain. (Scale bar, 1 mm.) (C) Coronal sections stained for Hoechst, NeuN, and MEF2 in the aCC. (Scalebar, 200 μm.) (D) High magniﬁcation of layer 2/3 pyramidal neurons in the aCC showing that endogenous MEF2 is expressed only in NeuN+ cells. (Scale bar,
50 μm.) (E) Time course of transgene expression following microinfusion of MEF2 vector into the aCC. Robust, bilateral transgene expression was observed inlayer 2/3 pyramidal neurons in the aCC up to 1 wk postmicroinfusion, but not thereafter (see also Fig. S2 for quantiﬁcation of data). (Scale bar, 1 mm.)mice back in the context and measuring freezing behavior (Fig.
3A). Only mice with robust, bilateral transgene expression inaCC layer 2/3 pyramidal neurons were included in analyses (forexclusion criteria, see Fig. S4 and SI Materials and Methods). We
found that increasing MEF2-dependent transcription in the aCC
during the ﬁrst posttraining week reduced levels of contextual
fear. In contrast, increasing MEF2-dependent transcription in theaCC during the seventh posttraining week had no effect on subsequent memory expression. The differential effect of increasing
MEF2 function at recent versus remote time points was conﬁrmed
by a signiﬁcant group × delay interaction [F(1,33) = 5.59; P <0.05] (Fig. 3B). Importantly, spared conditioned freezing in the
remote group indicates that increasing MEF2-dependent transcription in the aCC does not impact motor function (e.g., affecting the ability to freeze) or emotion (e.g., changes in basal
levels of anxiety) that might nonspeciﬁcally reduce conditioned
8458 | www.pnas.org/cgi/doi/10.1073/pnas.1016275108

fear. In addition, mice microinfused with the MEF2 vector 1 dafter training exhibited reduced freezing when tested 48 d later(Fig. S5), indicating that the memory deﬁcits induced by increasing MEF2-dependent transcription were persistent and did
not recover over time. Together, these results are consistent with
the idea that increasing MEF2-dependent transcription in theaCC during a critical posttraining window inhibits the spinogenesisthat is required for memory consolidation.
Increasing MEF2 Function in the aCC Disrupts Spine Growth That
Accompanies Memory Consolidation in a Time-Dependent Manner.

To evaluate whether increasing MEF2-dependent transcription
interfered with training-induced spine growth during this critical,
early posttraining window, we next quantiﬁed spine density ininfected aCC pyramidal neurons in mice in the recent and remotegroups (Fig. 4 A–C). In the recent group, spine density was reduced in MEF2-infected neurons (relative to the control infected
Vetere et al.

Fig. 3. Increasing MEF2 function in the aCC following training disrupts
consolidation of a contextual fear memory in a time-dependent manner. (A)Experimental design. (B) Reduced freezing levels following MEF2 vector
microinfusion 1 d, but not 42 d, after training [P < 0.05, by ANOVA (significant group × delay interaction)].

neurons), indicating that increasing MEF2-dependent transcription in aCC neurons during the ﬁrst posttraining week blocks both
memory consolidation and associated increases in spine density.
In contrast, spine density was not altered in MEF2-infected neurons in either the remote group (presumably when reactivationlevels have declined signiﬁcantly) or in home-cage control mice
(Fig. S6). This indicates that increasing MEF2-dependent transcription does not simply suppress or eliminate spines under basal
conditions (15, 16). The dissociable effects of increasing MEF2
function on spine density support the idea that MEF2 regulates
reactivation-driven changes in spine density within a deﬁned
posttraining window.

Vetere et al.

Fig. 4. Increasing MEF2 function in the aCC following training disrupts
consolidation-associated changes in spine density in a time-dependent
manner. (A) Examples of apical dendritic segments from control and MEF2infected neurons at different retention delays. (Scale bar, 5 μm.) (B and C)
Cumulative frequency plots (B) and group data (C) showing spine density in
aCC neurons infected with MEF2 or control vectors either 1 or 42 d following
conditioning. Spine density was reduced in MEF2-infected neurons 1 d, but
not 42 d, after training (P < 0.01, by Kolmogorov–Smirnov test).

textual fear memories. Previous studies suggest that the aCC
plays an increasingly important role in the expression of contextual fear (as well as other, initially hippocampus-dependent)
memories over time (6, 9, 39–41). For example, the aCC is activated following remote, but not recent, contextual fear memory
recall (11). Also, inactivating the aCC disrupts the expression of
remote, but not recent, contextual fear memory (11). Although
structural remodeling likely occurs in other cortical regions,these data suggest that the aCC is an essential node within abroader network supporting expression of remote memory.
Such time-dependent changes in dendritic spine density in aCC
neurons may reﬂect consolidation-associated modiﬁcation offunctional connections between the aCC and other cortical (13,14) and subcortical [e.g., amygdala (42)] regions.
Memory reactivation is thought to drive structural changes
necessary for the consolidation of memories in cortical networks(20–22). Consistent with this model, interfering with memory
reactivation disrupts memory consolidation (20, 21). Similarly,
here we have shown that interfering with spine growth in theweek following training (but not at a later time point) disrupted
memory consolidation. The time-limited nature of these effects
is consistent with observations that (i) the most pronounced
increases in spine density occur during the ﬁrst posttraining
week, and (ii) memory reactivation strength and frequency decline over time (23).
PNAS | May 17, 2011 | vol. 108 | no. 20 | 8459

NEUROSCIENCE

Discussion
With time, the importance of the hippocampus in memory expression diminishes as a more permanent trace is established in
the cortex. This time-dependent reorganization process is thought
to depend on the remodeling of cortical connections allowing
the memory to be expressed independently of the hippocampus.
Here we tested whether inhibiting spine growth speciﬁcally inthe aCC at different times following training disrupted memory
consolidation. To inhibit spine growth, we used MEF2, a transcription factor that has been shown to negatively regulate spine
growth both in vitro and in vivo (15, 16). We found that increasing
MEF2-dependent transcription in aCC neurons immediately
following training (but not thereafter) blocked both memory
consolidation and associated increases in spine density. Together,
these data strengthen the causal link between structural remodeling in the aCC and memory consolidation and, further, identify
MEF2 as a key regulator of the molecular machinery mediatingthese changes.
Our ﬁnding that increasing MEF2-dependent transcription
inhibits spine growth normally associated with consolidation of afear memory is consistent with reports showing that several
MEF2 target genes encode proteins that weaken excitatory synaptic transmission. For instance, genome-wide analysis of developing neurons (26) identiﬁed MEF2 target genes that (i)decrease surface AMPAR expression [arc, JNK, SynGAP (27–31)],(ii) uncouple mGluR5 from its effector targets in the postsynaptic
density and reduce the amplitude of AMPAR- and NMDARmediated excitatory postsynaptic currents [Homer1a (32–34)], (iii)internalize N-cadherin, a synaptic adhesion molecule implicatedin synaptic transmission [PCDH (protocadherin) family members,
including protocadherins 9, 10, and 17 (35)], and (iv) decreaseneuronal excitability [voltage-gated K+ channels (including kcna1
and kcna4) (36–38)]. Therefore, increasing MEF2-dependenttranscription may orchestrate a coordinated increase in expressionof genes that suppress excitatory synapse function.
In our experiments, increasing MEF2 function just in the aCC
was sufﬁcient to interfere with the formation of enduring con-

Materials and Methods
Quantiﬁcation of Spine Density in aCC Pyramidal Neurons Following Contextual
Fear Conditioning. Mice were trained in contextual fear conditioning and thentested 1 (trained, n = 6; control, n = 6), 8 (trained, n = 5; control, n = 4), 42(trained, n = 11; control, n = 11), or 49 (trained, n = 6; control, n = 5) d later(for a detailed description of apparatus and behavioral procedures, see SI
Materials and Methods). Following testing, their brains were removed andprepared for Golgi-Cox staining. Golgi-impregnated neurons in layer 2/3 andlayer 5 of the aCC were ﬁrst identiﬁed using a light microscope. For eachneuron, spine density was quantiﬁed along ﬁve randomly selected segmentson secondary and tertiary branches of apical dendrites. Segments were atleast 20 μm in length. Only protuberances with a clear connection of thehead of the spine to the shaft of the dendrite were counted as spines (see SI
Materials and Methods for further details).

HSV Vectors. To locally increase MEF2 function in the aCC at different times
following fear conditioning, we used replication-defective HSV. The MEF2VP16 construct was subcloned into an HSV amplicon backbone (pHSV-prPUC).
Transgene expression was regulated by the constitutive promoter for the HSV
immediate-early gene IE 4/5. This vector also expressed GFP to allow infected
neurons to be visualized. A control vector similarly expressed GFP alone.
Microinfusion of the MEF2 (HSV-MEF2-VP16-GFP) or control (HSV-GFP) vector produced robust transgene expression in layer 2/3 aCC pyramidal neurons (for more details, see SI Materials and Methods).

Immunohistochemistry. To characterize endogenous MEF2 expression in theadult mouse brain, naïve mice were perfused transcardially with PBS andparaformaldehyde. Sections were cut and then incubated with primary
antibodies against MEF2 and neuron-speciﬁc nuclear protein (NeuN). Alexa488 goat anti-mouse and Alexa-568 goat anti-rabbit were used as secondary
antibodies (for detailed procedures, see SI Materials and Methods).

Examination of the Effects of Increasing MEF2 Function on Consolidation of aContextual Fear Memory. Mice were trained in contextual fear conditioning.
Either 1 or 42 d later, mice were microinfused with the control (1 d, n = 11; 42d, n = 14) or MEF2 (1 d, n = 11; 42 d, n = 17) vector into the aCC (for detailed
behavioral and surgical procedures, see SI Materials and Methods). One
week later, mice were placed back in the context and freezing was measured. Following testing, brains were removed and spine density of infected
neurons was quantiﬁed in layer 2/3 aCC neurons using native GFP ﬂuorescence. For each neuron, spine density was quantiﬁed along ﬁve randomly
selected segments on secondary and tertiary branches of apical dendrites(segments > 20 μm in length) (for detailed procedures, see SI Materials
and Methods).

Luciferase Reporter Assays. To verify that our MEF2-VP16 construct speciﬁcallyincreases MRE-dependent (but not CRE-dependent) transcription, we used
a luciferase reporter assay in Neuro2A cells. Cells were transiently transfectedwith either MEF2-VP16 or control (GFP) constructs and an MRE- or CRE-luciferase
reporter plasmid. Luciferase expression was assessed 48 h later (for detailed
procedures, see SI Materials and Methods).

ACKNOWLEDGMENTS. We thank Kaori Takehara-Nishiuchi for comments on
this manuscript and Rachael Neve for providing HSV constructs. This work
was supported by grants from the Canadian Institutes of Health Research(MOP-77561 and MOP-74650 to P.W.F. and S.A.J., respectively). G.V. received
support from the University of Tor Vergata, Rome. L.R. and C.J.C. received
support from The Hospital for Sick Children, and P.J.R. received support fromthe Ontario Ministry of Research and Innovation.

1. Kim JJ, Fanselow MS (1992) Modality-speciﬁc retrograde amnesia of fear. Science 256:
675–677.
2. Clark RE, Broadbent NJ, Zola SM, Squire LR (2002) Anterograde amnesia andtemporally graded retrograde amnesia for a nonspatial memory task after lesions ofhippocampus and subiculum. J Neurosci 22:4663–4669.
3. Anagnostaras SG, Maren S, Fanselow MS (1999) Temporally graded retrograde
amnesia of contextual fear after hippocampal damage in rats: Within-subjects
examination. J Neurosci 19:1106–1114.
4. Maren S, Aharonov G, Fanselow MS (1997) Neurotoxic lesions of the dorsal
hippocampus and Pavlovian fear conditioning in rats. Behav Brain Res 88:261–274.
5. Restivo L, Vetere G, Bontempi B, Ammassari-Teule M (2009) The formation of recent
and remote memory is associated with time-dependent formation of dendritic spines
in the hippocampus and anterior cingulate cortex. J Neurosci 29:8206–8214.
6. Maviel T, Durkin TP, Menzaghi F, Bontempi B (2004) Sites of neocorticalreorganization critical for remote spatial memory. Science 305:96–99.
7. Takehara K, Kawahara S, Kirino Y (2003) Time-dependent reorganization of the brain
components underlying memory retention in trace eyeblink conditioning. J Neurosci
23:9897–9905.
8. Kim JJ, Clark RE, Thompson RF (1995) Hippocampectomy impairs the memory ofrecently, but not remotely, acquired trace eyeblink conditioned responses. Behav
Neurosci 109:195–203.
9. Frankland PW, Bontempi B (2005) The organization of recent and remote memories.
Nat Rev Neurosci 6:119–130.
10. Squire LR, Alvarez P (1995) Retrograde amnesia and memory consolidation: A
neurobiological perspective. Curr Opin Neurobiol 5:169–177.
11. Frankland PW, Bontempi B, Talton LE, Kaczmarek L, Silva AJ (2004) The involvement of
the anterior cingulate cortex in remote contextual fear memory. Science 304:881–883.
12. González-Burgos G, Barrionuevo G, Lewis DA (2000) Horizontal synaptic connections inmonkey prefrontal cortex: An in vitro electrophysiological study. Cereb Cortex 10:82–92.
13. Takashima A, et al. (2009) Shift from hippocampal to neocortical centered retrieval
network with consolidation. J Neurosci 29:10087–10093.
14. Sacco T, Sacchetti B (2010) Role of secondary sensory cortices in emotional memorystorage and retrieval in rats. Science 329:649–656.
15. Flavell SW, et al. (2006) Activity-dependent regulation of MEF2 transcription factors
suppresses excitatory synapse number. Science 311:1008–1012.
16. Pulipparacharuvil S, et al. (2008) Cocaine regulates MEF2 to control synaptic andbehavioral plasticity. Neuron 59:621–633.
17. Gale GD, et al. (2004) Role of the basolateral amygdala in the storage of fear
memories across the adult lifetime of rats. J Neurosci 24:3810–3815.
18. Anagnostaras SG, Josselyn SA, Frankland PW, Silva AJ (2000) Computer-assisted
behavioral assessment of Pavlovian fear conditioning in mice. Learn Mem 7:58–72.
19. Debiec J, LeDoux JE, Nader K (2002) Cellular and systems reconsolidation in the
hippocampus. Neuron 36:527–538.
20. Ego-Stengel V, Wilson MA (2010) Disruption of ripple-associated hippocampal activity
during rest impairs spatial learning in the rat. Hippocampus 20:1–10.
21. Girardeau G, Benchenane K, Wiener SI, Buzsáki G, Zugaro MB (2009) Selective
suppression of hippocampal ripples impairs spatial memory. Nat Neurosci 12:1222–1223.
22. Holtmaat A, Svoboda K (2009) Experience-dependent structural synaptic plasticity inthe mammalian brain. Nat Rev Neurosci 10:647–658.

23. Kudrimoti HS, Barnes CA, McNaughton BL (1999) Reactivation of hippocampal cell assemblies:
Effects of behavioral state, experience, and EEG dynamics. J Neurosci 19:4090–4101.
24. Neely MD, et al. (2009) Localization of myocyte enhancer factor 2 in the rodent
forebrain: Regionally-speciﬁc cytoplasmic expression of MEF2A. Brain Res 1274:55–65.
25. Fink DJ, DeLuca NA, Goins WF, Glorioso JC (1996) Gene transfer to neurons using
herpes simplex virus-based vectors. Annu Rev Neurosci 19:265–287.
26. Flavell SW, et al. (2008) Genome-wide analysis of MEF2 transcriptional program
reveals synaptic target genes and neuronal activity-dependent polyadenylation site
selection. Neuron 60:1022–1038.
27. Shepherd JD, et al. (2006) Arc/Arg3.1 mediates homeostatic synaptic scaling of AMPA
receptors. Neuron 52:475–484.
28. Chowdhury S, et al. (2006) Arc/Arg3.1 interacts with the endocytic machinery to
regulate AMPA receptor trafﬁcking. Neuron 52:445–459.
29. Rumbaugh G, Adams JP, Kim JH, Huganir RL (2006) SynGAP regulates synaptic
strength and mitogen-activated protein kinases in cultured neurons. Proc Natl Acad
Sci USA 103:4344–4351.
30. Thomas GM, Lin DT, Nuriya M, Huganir RL (2008) Rapid and bi-directional regulationof AMPA receptor phosphorylation and trafﬁcking by JNK. EMBO J 27:361–372.
31. Zhu Y, et al. (2005) Rap2-JNK removes synaptic AMPA receptors during depotentiation. Neuron 46:905–916.
32. Sala C, et al. (2003) Inhibition of dendritic spine morphogenesis and synaptictransmission by activity-inducible protein Homer1a. J Neurosci 23:6327–6337.
33. Tu JC, et al. (1998) Homer binds a novel proline-rich motif and links group 1
metabotropic glutamate receptors with IP3 receptors. Neuron 21:717–726.
34. Xiao B, Tu JC, Worley PF (2000) Homer: A link between neural activity and glutamate
receptor function. Curr Opin Neurobiol 10:370–374.
35. Yasuda S, et al. (2007) Activity-induced protocadherin arcadlin regulates dendritic
spine number by triggering N-cadherin endocytosis via TAO2β and p38 MAP kinases.
Neuron 56:456–471.
36. Brew HM, et al. (2007) Seizures and reduced life span in mice lacking the potassiumchannel subunit Kv1.2, but hypoexcitability and enlarged Kv1 currents in auditory
neurons. J Neurophysiol 98:1501–1525.
37. Kopp-Scheinpﬂug C, Fuchs K, Lippe WR, Tempel BL, Rübsamen R (2003) Decreased
temporal precision of auditory signaling in Kcna1-null mice: An electrophysiologicalstudy in vivo. J Neurosci 23:9199–9207.
38. Luján R, et al. (2003) Immunohistochemical localization of the voltage-gatedpotassium channel subunit Kv1.4 in the central nervous system of the adult rat. J
Chem Neuroanat 26:209–224.
39. Miller CA, et al. (2010) Cortical DNA methylation maintains remote memory. Nat
Neurosci 13:664–666.
40. Teixeira CM, Pomedli SR, Maei HR, Kee N, Frankland PW (2006) Involvement of theanterior cingulate cortex in the expression of remote spatial memory. J Neurosci 26:
7555–7564.
41. Ding HK, Teixeira CM, Frankland PW (2008) Inactivation of the anterior cingulate
cortex blocks expression of remote, but not recent, conditioned taste aversion
memory. Learn Mem 15:290–293.
42. Malin EL, Ibrahim DY, Tu JW, McGaugh JL (2007) Involvement of the rostral anterior
cingulate cortex in consolidation of inhibitory avoidance memory: Interaction withthe basolateral amygdala. Neurobiol Learn Mem 87:295–302.

8460 | www.pnas.org/cgi/doi/10.1073/pnas.1016275108

Vetere et al.