a r t ic l e s

MEF2 negatively regulates learning-induced structural
plasticity and memory formationnpg© 2012 Nature America, Inc. All rights reserved.

Christina J Cole1,2,7, Valentina Mercaldo1–3,7, Leonardo Restivo1–3, Adelaide P Yiu1–3, Melanie J Sekeres1,2,
Jin-Hee Han1–3,6, Gisella Vetere1–4, Tetyana Pekar1, P Joel Ross1, Rachael L Neve5,
Paul W Frankland1–3 & Sheena A Josselyn1–3
Memory formation is thought to be mediated by dendritic-spine growth and restructuring. Myocyte enhancer factor 2 (MEF2)
restricts spine growth in vitro, suggesting that this transcription factor negatively regulates the spine remodeling necessary
for memory formation. Here we show that memory formation in adult mice was associated with changes in endogenous MEF2
levels and function. Locally and acutely increasing MEF2 function in the dentate gyrus blocked both learning-induced increases
in spine density and spatial-memory formation. Increasing MEF2 function in amygdala disrupted fear-memory formation.
We rescued MEF2-induced memory disruption by interfering with AMPA receptor endocytosis, suggesting that AMPA receptor
trafficking is a key mechanism underlying the effects of MEF2. In contrast, decreasing MEF2 function in dentate gyrus andamygdala facilitated the formation of spatial and fear memory, respectively. These bidirectional effects indicate that MEF2 is a
key regulator of plasticity and that relieving the suppressive effects of MEF2-mediated transcription permits memory formation.
Memory formation is thought to be mediated by increases in synaptic
efficacy1. As more than 90% of excitatory synapses occur on dendritic
spines2, the growth and restructuring of spines may serve as a physicalbasis for the long-term storage of information. Consistent with this,­several human cognitive disorders are characterized by abnormal dendritic spine density and morphology3. Long-term memory formation
also critically depends on transcription4. Disrupting the function of­several transcription factors, including nuclear factor κB (NF-κB),
cAMP response element binding protein (CREB), serum response
factor (SRF) and zif 268 impairs memory formation, in part, by interferingwith the structural plasticity required for long-term memory formation4.
In this way, these transcription factors support memory formation5.
In contrast, recent studies find that MEF2 restricts activitydependent dendritic spine growth6–8, suggesting that this transcription
factor may suppress memory formation. Specifically, these studies showthat increasing MEF2-mediated transcription decreases the numberof dendritic spines and excitatory synapses in hippocampal neurons
in vitro6 and blocks the increase in spine density normally observed
after repeated cocaine administration in rat medium spiny nucleus
accumbens neurons in vivo8. These findings suggest that MEF2mediated transcription negatively regulates the dendritic spine growthnecessary for memory formation and that relieving the inhibitory effects
of MEF2-mediated transcription may facilitate memory formation.
Here we examined the role of MEF2-mediated transcription onlearning-induced dendritic spine remodeling and memory formation in adult mice. Because MEF2 is critical for neuronal survival,

differentiation and development9, we used viral vectors to manipulate
MEF2 function in a temporally and spatially restricted way in theadult brain. To examine memory formation, we used two behavioral
tasks that produce different types of memory and rely on distinct
brain regions. First, we examined whether memory formation in miceis normally associated with changes in endogenous MEF2 levels andfunction. We observed that memory formation was associated withkey changes in endogenous MEF2 levels and function, which would
decrease MEF2-mediated transcription. Second, we assessed theeffects of acutely manipulating MEF2 function on learning-induced
spine remodeling and memory formation. We found that overexpressing MEF2 suppressed memory formation and that geneticallyrelieving MEF2-mediated suppression promoted memory formation.
Finally, although MEF2 may regulate the transcription of many downstream genes, we identified Arc, as a MEF2 target gene10 that is critical
for the memory-impairing effects of MEF2. Arc protein decreases thesurface expression of AMPA-type glutamate receptors (AMPARs) byenhancing their endocytosis11,12. We found that the MEF2-induced
disruption of memory formation was rescued by acutely interferingwith AMPAR endocytosis at the time of learning. Together, thesefindings suggest that increasing MEF2 function impairs memory
formation through an Arc-mediated decrease in AMPAR surface
expression. These results may be interesting as MEF2 itself, as well as
several of its target genes, are implicated in human cognitive or psychiatric disorders, including autism spectrum disorder13, Angelman
syndrome10,14 and a Rett-like syndrome with mental retardation15.

1Program in Neurosciences and Mental Health, Hospital for Sick Children, Toronto, Ontario, Canada. 2Department of Physiology, University of Toronto, Toronto,
Ontario, Canada. 3Institute of Medical Sciences (IMS), University of Toronto, Toronto, Ontario, Canada. 4Cell Biology and Neurobiology Institute, National ResearchCouncil of Italy, S. Lucia Foundation, Rome, Italy. 5Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. 6Present
address: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yuseong-gu, Daejeon, Korea. 7These authors contributed equally tothis work. Correspondence should be addressed to S.A.J. (sheena.josselyn@sickkids.ca).

Received 18 May; accepted 9 July; published online 12 August 2012; doi:10.1038/nn.3189

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Figure 1 Memory formation was associated with inhibitory phosphorylation of MEF2
*
*
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and a decrease in amount of MEF2 protein. (a) Immunofluorescence images showing
1.0
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endogenous MEF2 protein in adult mouse brain (left; scale bar, 250 µm), and in theNS
1.0
dentate gyrus (DG) and lateral amygdala (LA) (right; scale bar, 50 µm). (b) pMEF2(MEF2A phosphorylated at Ser408) in upper blade DG of water maze–trained
0.5
0.5(TR, n = 4) and home-cage (HC, n = 2) mice (top left panels; scale bar, 50 µm)
and quantification of pMEF2 (top right panel). pMEF2 in LA of mice trained in00
auditory conditioned fear (tone plus uncondititioned stimulus shock); T + US,
HC 90 min 6 h
MEF2A
MEF2D
n = 4) and in mice given control behavioral conditions (HC, n = 3; tone alone (T),90 min6h
n = 3; or immediate shock (IS), n = 4; bottom left panels; scale bar, 50 µm) andHC
TrainedTrainedquantification of pMEF2 (bottom right panel). (c) pMEF2 and total MEF2 protein
pMEF2
amounts from tissue extracts from DG and CA1 region of dorsal hippocampus inmice trained in the water maze (trained, n = 9) or maintained in the home cage
MEF2A
(HC, n = 9) (top) and immunoblot (bottom). Full blots are shown in Supplementary
Figure 6. (d) Relative pMEF2 amounts in DG and CA1 region of dorsal hippocampus
MEF2D
in mice 90 min and 6 h after context fear training (left). MEF2A and MEF2D
GAPDH(MEF2A-D) amounts 90 min and 6 h after fear conditioning relative to the amounts
in HC mice (right). For pMEF2: HC n = 4, 90 min after fear conditioning n = 4, 6 hafter fear conditioning n = 4; for MEF2A: HC n = 12, 90 min = 9, 6 h n = 9: MEF2D; HC n = 8, 90 min n = 7, 6 h n = 7. Immunoblots are shown on thebottom. Full immunoblot is shown in Supplementary Figure 6. *P < 0.05; NS, not significantly different. All error bars indicate s.e.m.
Optical density(pMEF2/total MEF2A)© 2012 Nature America, Inc. All rights reserved.

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RESULTS
Memory formation associated with decreased MEF2 function
MEF2 proteins are endogenously expressed throughout the adultbrain, including the dentate gyrus, a brain region critical for spatial
memory16, and the lateral amygdala, a region critical for the acquisition of conditioned fear memory17–19 (Fig. 1a). Whereas invertebrates
have a single mef2 gene, vertebrates, including mice and humans,
have four such genes (Mef2a–d and MEF2A–D, respectively)20. The
four MEF2 proteins have in common a conserved N terminus (whichmediates dimerization, DNA binding and co-factor interaction) but
have different C termini (which contain the transcriptional activation domain)20. MEF2A and MEF2D are the isoforms most highly
expressed in forebrain regions, including the hippocampus21.
As previous results show that MEF2-mediated transcription negatively regulates spine growth, we hypothesized that memories areformed when endogenous MEF2-mediated transcription is decreased.
Phosphorylation of MEF2 at a critical serine residue (Ser408 on
MEF2A, which corresponds to Ser444 on MEF2D) inhibits MEF2mediated transcription6. Accordingly, we examined whether strong
behavioral training that normally produces robust memory in adultwild-type mice is correlated with phosphorylation of endogenous
MEF2 at this key inhibitory site (here referred to as pMEF2). Micetrained in the water maze (using a protocol that induces robust spatial
memory) exhibited significantly higher pMEF2 staining6 in the dentate gyrus, particularly the upper blade of the dentate gyrus, compared
to untrained mice maintained in the home cage (here referred to as‘home-cage mice’) (P < 0.0001, Kolmogorov-Smirnov test; Fig. 1b).
This finding is consistent with previous results showing that theupper blade of the dentate gyrus is particularly ­important in spatial
memory22. Similarly, auditory fear–conditioned mice (trained withtone plus shock pairing) had significantly higher pMEF2 levels in
the lateral amygdala than mice taken directly from the home cage
(P < 0.0001) or trained with either tone alone (P < 0.0001) or an
immediate shock (P < 0.0001) (both of these control behavior procedures do not produce auditory fear memory23,24) (Fig. 1b). Using
western blots to quantify relative pMEF2 levels (pMEF2/total MEF2),
we observed similar results. Water maze–trained mice had significantly higher relative pMEF2 levels in the dentate gyrus and CA1
region of the dorsal hippocampus compared to home-cage control
mice (F1,16 = 5.63, P < 0.05, by one-way ANOVA; Fig. 1c). Watermaze training was also associated with a robust decrease in thelevels of endogenous MEF2 protein (F1,16 = 5.29, P < 0.05, by oneway ANOVA; Fig. 1c). Similarly, context fear conditioning was also
associated with an increase in relative pMEF2 levels (90 min, but
not 6 h, after training) and a decrease in endo­genous MEF2A andMEF2D protein (90 min and 6 h after training) compared to the casein home-cage mice (MEF2A, F2,27 = 29.88, P < 0.001; MEF2D, F2,19 =
4.74, P < 0.05; Fig. 1d). Together, these data suggest that training
which normally induces robust memory formation decreased MEF2mediated transcription through multiple mechanisms (including a
relatively short-lasting inhibitory phosphorylation of MEF2 and alonger-lasting decrease in the amount of endogenous MEF2 protein).

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a r t ic l e sb Surgery

WM

WM

WM
and probe

c

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1

*

2

*

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*
GFP
MEF2
MEF2∆
MEF2

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WM and
probe

8

9

10

1

*

*

20

*

GFP
MEF2
MEF2∆

These findings are consistent with the interpretation that degrading
endogenous MEF2 protein enables memory formation.
Regionally and temporally specific manipulation of MEF2
We found that memory formation was associated with a decrease
in MEF2-mediated transcription, leading us to predict that acutely
increasing MEF2-mediated transcription at the time of training
would disrupt memory formation. To test this, we locally and acutely
increased MEF2 function in adult mice using replication-defective
viral vectors. In the brain, endogenous MEF2 expression is limited
to neurons25. Therefore, we used herpes simplex viral (HSV) vectors
because HSV is neurotropic (Supplementary Fig. 1). To increase
MEF2-mediated transcription, we used HSV encoding MEF2-VP16,
a version of MEF2 in which the DNA-binding domain (DBD) anddimerization domain of MEF2 are fused to the transcriptional activation domain of virion protein 16 (VP16) (Supplementary Fig. 2a).
MEF2-VP16 binds MEF2 sites in the promoter region of MEF2 target
genes, leading to their constitutive transcription6. As a control, we
used HSV encoding MEF2∆DBD-VP16 (MEF2∆), a mutant version
of MEF2-VP16 that lacks the DBD6,8. Both MEF2 and MEF2∆ viral
vectors also expressed GFP, allowing visualization of infected neurons.
As an additional control, we used a vector expressing GFP alone(GFP vector). We observed no evidence of toxicity associated withthese vectors (Supplementary Fig. 2b,c).
To confirm that MEF2, but not MEF2∆, increased MEF2-dependenttranscription, we used luciferase reporter assays. Cultured cells
transfected with MEF2 plasmid exhibited higher MEF2 recognition element (MRE)-dependent transcription than cells transfectedwith MEF2∆ or GFP plasmid (Supplementary Fig. 2d). To examine
the specificity of this increase, we infected cultured hippocampal
neurons with HSV-MEF2 or control HSV-GFP and assessed both
MRE- and cAMP response element (CRE)-dependent transcription
under unstimulated or stimulated (4 h, 170 mM KCl) conditions.
MEF2 vector increased MRE-dependent but not CRE-dependent
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Percentage time in zonef

Percentage
time in target zone

Percentage
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WM

Figure 2 Increasing MEF2 function during
10
10
training impaired formation of spatial memory.
(a) Location of dentate gyrus (DG) in wildtype
0
0
MEF2∆
mice shown using neutral red stain (left).
T O
T O
T O
T O
T O
T O
0
Zone
Microinjection of MEF2 vector into DG
Zone
produces high transgene (GFP) expression
WM andWM andin the DG (right), shown using DAPI counterstain
WM WM probe 1 Surgery Probe 2Surgery
WM WM probe 1 Probe 2(right). Scale bar, 100 µm. (b) Experimental
Day 1
2
3
17
21
Day 0
1
2
3
4
21
protocol: on day 0, mice were given surgery during
GFP
MEF2
GFP
60
60
40
which vectors were microinjected into the DG.
**
MEF2
On days 2, 3 and 4, mice were trained in the water
30
40
40
maze (WM). On day 4, mice were given a probe
20
test in the WM. Percent time spent in target (T) than
20
20
other (O) zones by mice microinjected with GFP
10vector (n = 11), MEF2∆ vector (n = 8) and MEF2000
vector (n = 11). (c) Density plot from probe test from
Probe 1
Probe 2
Probe 1
Probe 2
T O
T O
Zone
mice microinjected with indicated vectors. During training,
platform was located on bottom right (dotted circles).
(d) Memory in mice microinjected with GFP (n = 11), MEF2 (n = 14) and MEF2∆ (n = 9) vectors 8 d before training. (e) Memory in mice microinjectedwith MEF2 vector before training and retested after transgene expression dissipated (left); probe 1 (MEF2 overexpression), probe 2 (no MEF2
overexpression). GFP vector (n = 8), MEF2 vector (n = 6). Memory in mice microinjected with MEF2 vector (n = 9) or GFP vector (n = 9) after training(right). (f) Spatial memory in mice microinjected with MEF2 vector (n = 6), GFP vector (n = 6) in the lateral amygdala. *P < 0.05; NS, not significantly
different. All error bars indicate s.e.m.

e

npg

3.5

Number of visits

Percentage time in zoneDayd Surgery

7.0

GFP

Percentage time in zoneatranscription (Supplementary Fig. 2e). Therefore, consistent withprevious reports6,8, our MEF2 vector specifically increased MEF2mediated transcription.
MEF2 overexpression blocked formation of spatial memory
To examine the effects of acutely increasing MEF2 function on formation of spatial memory, we microinjected MEF2, MEF2∆ or GFP
vectors into the upper blade of the dentate gyrus of mice before giving
them strong (six trials a day for 3 d) water-maze training26. Vector
microinjection typically induced robust bilateral transgene expression
specifically in the upper blade of the dentate gyrus, and we observed
no differences in the extent of transgene expression in mice micro­
injected with different vectors (Fig. 2a). After the completion of training, we assessed spatial memory in a probe test (in which we removed
the platform from the pool). Increasing MEF2 expression during
training blocked the formation of spatial memory; mice microinjectedwith control vectors (GFP and MEF2∆ vectors) spent more time in
the area of the pool where the platform was located during training(target zone) than in other areas of the pool (other zones), whereas
mice microinjected with the MEF2 vector did not show a preference
for the target zone (vector × zone F2,27 = 8.32, P < 0.001, by two-way
ANOVA; mice microinjected with GFP, MEF2∆ (not MEF2) vectors
searched selectively in the target zone, by post hoc test; confirmedby one-way ANOVA comparing target zone, F2,27 = 6.64, P < 0.05;
Fig. 2b,c). Mice microinjected with MEF2∆ vector exhibited strong
spatial memory (not different from mice microinjected with GFP
vector), indicating that the memory-disrupting effects resulting frommicroinjection of the MEF2 vector depended on the ability of MEF2
to bind DNA and activate transcription. Mice microinjected with the
MEF2 vector exhibited normal latency in finding the platform during training, swim speed and thigmotaxis (tendency to swim in pool
perimeter) (Supplementary Fig. 3a), indicating that increasing theamount of MEF2 did not impair motor function or swimming ability,
or increase anxiety.
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Figure 3 Increasing MEF2 function blocked spine growth associated with memory formation. (a) Experimental
protocol for mice microinjected with vector (surgery) and either trained in the water maze (trained) or maintainedin the home cage (HC). (b) Infected granule cell from upper blade dentate gyrus from mice microinjected with
MEF2 vector (left). Scale bar, 20 µm. Sample tracing (right). (c) Dendritic segments of infected granule cells
from mice microinjected with GFP, MEF2 or MEF2∆ vectors and trained in water maze or maintained in HC.
Scale bar, 5 µm. (d) Spine density (per 10 µm of dendrite) was averaged per mouse from the following groups:
HC-GFP (11 neurons from 5 mice), HC-MEF2 (15 neurons, 5 mice), HC-MEF2∆ (10 neurons, 5 mice), trainedGFP (14 neurons, 7 mice), trained-MEF2 (15 neurons, 8 mice), trained-MEF2∆ (14 neurons, 6 mice). Error
bars, s.e.m.; *P < 0.05.

Spine density (per 10 µm)

12
10
8
6
4
2
0
HC

Trainedduring MEF2 overexpression, exhibited normal expression of spatial
memory (vector × test F1,16 = 2.86, vector F1,16 = 0.02, test F1,16 = 4.22,
all P > 0.05, by two-way ANOVA). Therefore, MEF2 overexpression specifically disrupted formation of spatial memory rather thanexpression of spatial memory.
Finally, we examined whether the disruption of the formation of
spatial memory resulting from microinjection with the MEF2 vector
was anatomically specific. We microinjected the MEF2 vector into
the lateral amygdala, a brain region not directly implicated in theformation of spatial memory28, and observed normal formation of
spatial memory (significant effect of zone F1,21 = 47.99, P < 0.001
only, target zone time not different between vectors F1,21 = 0.007,
P > 0.05; Fig. 2f). Similarly, microinjection of MEF2 vector (i) outsideof the upper blade of the dentate gyrus (bilateral miss) or (ii) unilaterally (rather than bilaterally) into the upper blade of the dentate
gyrus (unilateral hits) (Supplementary Fig. 3c), also did not impair
formation of spatial memory. Together, these results indicate thatthe memory-disrupting effects of increasing MEF2 function are bothanatomically and temporally specific.

It is possible that that the memory impairment resulting frommicroinjection of the MEF2 vector was not due to an acute increase
in MEF2 function during training but to a permanent disruption ofhippocampal circuits. To examine this possibility, we took advantageof the relatively brief duration of transgene expression using these
HSV vectors (transgene expression typically peaks roughly 2–4 dafter microinjection and dissipates 8–12 d after microinjection27;
Supplementary Fig. 3b). Therefore, we microinjected vectors into
the upper blade of the dentate gyrus of mice 8 d (rather than 2 d)
before training them in the water maze (Fig. 2d). In this experiment,
we trained mice (that previously overexpressed MEF2) at a time when
they were no longer overexpressing MEF2. Mice microinjected with
the MEF2 vector 8 d before training subsequently exhibited strong
spatial memory, which did not differ from that in mice microinjectedwith control vectors (MEF2∆ and GFP vectors) (significant effectof zone only F1,31 = 23.89, P < 0.001, target zone times not different between groups F2,31 = 0.47, P > 0.05, by two-way ANOVA).
Therefore, increasing MEF2 function during (but not over a week
before) water-maze training disrupted formation of spatial memory.
This finding indicates that the effects of MEF2 overexpression on
memory formation were transient and not due to permanent disruption or lesion of hippocampal memory circuits.
In the experiment shown in Figure 1b, we microinjected the
MEF2 vector 2 d before training and tested spatial memory 4 d later,
at a time of MEF2 overexpression. To assess whether the poor performance on the probe trial observed in that experiment was due toincreased levels of MEF2 interfering with expression (rather than formation) of spatial memory, we conducted two parallel experiments
in which we disambiguated MEF2 overexpression from memory
testing. First, we trained and tested mice at a time of high MEF2
expression (as in Fig. 1b) but retested these mice 17 d later, whenviral expression of MEF2 had dissipated (Fig. 2e). Mice microinjected with MEF2 vector before training exhibited poor spatial
memory on both the first (conducted 4 d after microinjection; MEF2
overexpression) and second (conducted 21 d after microinjection,
no MEF2 overexpression) probe test compared to mice micro­
injected with the GFP vector (significant effect of vector F1,12 = 39.09,
P < 0.001, test F1,12 = 4.95, P < 0.05 only, by two-way ANOVA). Second,
we trained and tested naive mice, and 14 d later microinjected thesemice with MEF2 or GFP vector, 4 d before a probe test (Fig. 2e).
These mice, trained when they had normal MEF2 levels, but tested

MEF2 overexpression blocked increases in spine density
Previous studies show that increasing MEF2 function negatively
regulates activity-dependent spine growth6–8. Because spine growth
and remodeling may underlie memory formation, we asked whether
increasing MEF2 function in the upper blade of the dentate gyrus
disrupted learning-induced increases in spine density. We compared the spine density of neurons infected with MEF2 vector or
GFP vector in mice trained in the water maze (from Fig. 2b) or
maintained in the home cage (Fig. 3a). To visualize dendritic spines,
we used a diaminobenzidine (DAB)-coupled antibody to GFP
to detect the GFP expressed from each vector. This allowed us toassess the spine density across the entire neuron (rather than justdendritic segments) using bright-field conditions. GFP expression
from MEF2, MEF2∆ or GFP vectors did not differ (Supplementary
Fig. 2f,g), all infected cells exhibited dentate gyrus granule cell morpho­
logy (Fig. 3b), and the overall neuronal structure or dendritic arborization did not differ between groups (Supplementary Fig. 4a).
Consistent with previous reports29,30, we observed that training
which induces spatial memory also increased spine density in neurons infected with the control GFP vector; infected dentate gyrus
granule cells from mice microinjected with GFP vector and trained

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Figure 4 Increasing MEF2 function after training impaired memory
incubation. (a) Outline of experiment: naive mice were trained and tested
in the water maze (WM and probe 1), and 24 h later, microinjected with
GFP (n = 7) or MEF2 (n = 9) vector into upper blade of the dentate gyrus.
Mice remained in home cage (without additional training) for 4 d and thentested in probe 2. After this, spine density of infected granule cells wasanalyzed. (b–d) Spatial memory was analyzed by determining percent time
mice spent in target (T) zone versus other (O) zones during probe tests.
Incubation is shown by mice spending more time in T zone during probe
2 than probe 1. Error bars, s.e.m.; *P < 0.05. (e) Density plots depicting
where mice spent time during probes 1 and 2.

Spine
Probe 2 analysisc

Probe 150

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Percentage time in zonea0

Probe 1 Probe 2

in the water maze (GFP-trained mice) exhibited greater spine density
than infected dentate gyrus granule cells from mice microinjectedwith GFP vector and maintained in the home cage (GFP–home cagemice) (Fig. 3c,d). This training-induced increase in spine density
was blocked by overexpressing MEF2 (spine density of infected
neurons from MEF2-trained mice did not differ from infected neurons from GFP–home-cage mice and was lower than infected neurons from GFP-trained mice). MEF2 vector had no effect on spine
density in home-cage mice, consistent with reports that MEF2 specifically inhibits activity-dependent spine growth 6,8. Finally, spine
density in neurons with MEF2∆ vector did not differ from that inneurons with GFP vector, in either trained or home-cage mice. This
interpretation was supported by the results of a two-way ANOVA,
which showed a significant vector × condition interaction F2,30 =3.61, P < 0.05, and main effects of vector F2,30 = 4.99, P < 0.05 andcondition F1,30 = 6.44, P < 0.05. Post hoc Newman-Keuls comparisons revealed spine density in infected neurons from GFP-trained >GFP–home cage, P < 0.05, GFP-trained > MEF2-trained, P < 0.05,
MEF2-trained = GFP–home cage = MEF2–home cage, P > 0.05 and
MEF2–home cage = GFP–home cage, P > 0.05. Therefore, acutely
increasing MEF2 function prevented the spine growth normallyobserved with formation of spatial memory. This repression of spine
growth depended on both neural activity (lack of effect in home-cagemice) and MEF2-mediated transcription (lack of effect with MEF2∆
vector). As memory formation is thought to require spine remodel­
ing, this prevention of spine growth may account for the memorydisruptive effects of increasing MEF2 function. It is interesting tonote that introduction of the MEF2 vector resulted in not only inhibition of spine growth in MEF2-infected dentate gyrus cells but also
in neighboring non-infected dentate gyrus cells (Supplementary
Fig. 4b,c), suggesting that increasing MEF2 function inhibited spine
growth and memory formation in a circuit rather than in a cellautonomous manner.
MEF2 overexpression after training prevented memory incubation
Activation of hippocampal circuits during training is critical for theformation of spatial memory. Similarly, reactivation of hippocampal
circuits after training may be important for strengthening a pre­viously
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VOLUME 15 | NUMBER 9 | SEPTEMBER 2012acquired memory31,32. Although ‘activity replay’ has been most­thoroughly studied in CA1 and CA3 regions of the hippocampus,
similar post-training reverberation processes may also occur in thedentate gyrus33. In the above experiments, we found that increasing
dentate gyrus MEF2 function during training blocked the formationof spatial memory, perhaps by preventing spine growth. We next
examined the effects of similarly enhancing MEF2 function shortly
after water-maze training on post-training memory processing andrelated spine remodeling.
We trained naive mice in the water maze and microinjected MEF2
or GFP vector into the upper blade of the dentate gyrus 24 h after
an initial probe test (Fig. 4a). As expected, all mice had intact spatial memory according to this initial probe test (probe 1; Fig. 4b).
Then we microinjected these mice with MEF2 or GFP vectors andreturned them to their home cage for 4 d, without additional training(probe 2). In the second probe test, mice with GFP vector exhibitedenhanced spatial memory (spending more time in the target zone
during probe 2 than probe 1), similar to the case in memory incubation34. In contrast, mice with MEF2 vector did not exhibit enhanced
spatial memory in the second probe test (vector × test interaction
F1,14 = 13.39, P < 0.05, two-way ANOVA; mice microinjected with
GFP vector spent significantly more time in target zone rather thanother zone during probe 2 than probe 1, P < 0.05, whereas mice with
MEF2 vector did not show this increase during probe 2, P > 0.05,
post hoc test; (Fig. 4b–e). This suggests that overexpressing MEF2
prevented this incubation-like memory enhancement. We examined
the spine density of infected dentate granule cells in these mice (afterprobe 2). Consistent with the absence of memory enhancement, micewith MEF2 vector had lower spine density than mice with GFP vector (spine density (mean spine number per 10 µm dendrite length ±s.e.m.) for GFP vector, 12.76 ± 0.16; MEF2 vector, 10.77 ± 0.25; F1,10 =48.45, P < 0.001, one-way ANOVA). Together, these results suggestthat increasing MEF2 function interfered with a post-training pro­
cess that resembles memory incubation and requires spine growth.
MEF2 overexpression in amygdala blocked fear memory
Increasing MEF2 function in the upper blade of the dentate gyrus
during water-maze training impaired formation of spatial memory.
To assess whether the role of MEF2 was conserved across brain
region and type of memory, we similarly microinjected vectors into
the lateral amygdala before strong conditioned fear training (onetone plus shock pairing with 0.5-mA shock) and examined contextual and discrete tone fear memory 24 h and 48 h later, respectively(Fig. 5a,b). Overexpressing MEF2 in the lateral amygdala during training blocked the formation of long-term fear memory forcontext fear (F2,19 = 6.37, P < 0.05; Fig. 5c) and tone fear (F2,19 =4.50, P < 0.05; Fig. 5d). Mice with MEF2 vector froze less than micewith MEF2∆ or GFP vector in both fear memory tests. Consistent
with the role of the dentate gyrus in context fear memory but not1259

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Figure 5 Overexpressing MEF2 in the lateral
amygdala blocked long-term memory formation
for contextual and cued fear conditioning.
Context
Tone
Surgery
Training(a) Location of lateral amygdala (left);
test
test
stained with neutral red. Scale bar, 400 µm.
Transgene (GFP) expression (right; green)
1
2
34
Day: 0after microinjection of MEF2 vector into thelateral amygdala. Sample was counterstainedwith DAPI (blue). (b) Outline of experimental
protocol: mice were microinjected with vector
LA; context test
DG; tone test
LA; tone test
DG; context test
and 2 d later, trained for tone fear conditioning
60
60
60
60
*
*
(one tone plus shock pairing, 0.5-mA shock).
50
50
50
50
Mice were tested for context and tone-fear
40
40
40
40
*
memory 24 h and 48 h later, respectively.
30
30
30
30
(c) In the context test, mice with MEF2 vector
in the lateral amygdala (LA) (n = 8) spent less
20
20
20
20
time freezing than mice with control vectors
10
10
10
10
(GFP vector, n = 7; MEF2∆ vector, n = 7), which0000
did not differ from each other. (d) In the tone
test, mice with MEF2 vector froze less thanmice with either control vector. (e,f) Context
and tone fear memory with microinjection ofindicated vectors in the dentate gyrus (DG) before fear conditioning. Mice with MEF2 vector (n = 8) in upper blade of the DG froze less than mice with
GFP vector (n = 7) in context test. Groups froze equally during tone test. All error bars indicate mean ± s.e.m.; *P < 0.05.

tone fear memory, we also observed that mice microinjected with
MEF2 vector in the upper blade of the dentate gyrus exhibited
disrupted context fear memory (F1,10 = 19.31, P < 0.05; Fig. 5e) but
intact tone fear memory (F1,10 = 0.15, P > 0.05; Fig. 5f). In contrast
to the effects on long-term memory, micro­injecting MEF2 vector in
the lateral amygdala did not disrupt tone fear memory, as assessed
90 min after training (mean percentage time spent freezing ± s.e.m.
for mice with GFP vector, 31.34 ± 3.72, n = 7; and with MEF2 vector, 28.72 ± 4.18, n = 7; P > 0.05). Together, these findings show that
increasing MEF2 function impaired formation of a long-term but
not short-term memory. Because the disruptive effects of increasing MEF2 function on memory formation were not exclusive to aparticular brain region (dentate gyrus or lateral amygdala) or typeof memory (spatial or fear), these results indicate that MEF2 has aconserved role in the formation of memory.
Disrupting MEF2 function permitted formation of memory
We observed that formation of memory in normal, intact mice wasassociated with inhibitory phosphorylation of MEF2 and decreased
amounts of MEF2A and MEF2D protein, suggesting that endogenous
MEF2-mediated transcription normally constrains memory formation.
Therefore, we asked whether acutely decreasing MEF2 levels during
training would enhance memory formation. To disrupt MEF2-mediated

f

G
FP
M
EF
2

2

M
EF

G
FP

Percentage freeze

2

2∆

M
EF

M
EF

G
FP

G
FP
M
EF
2
M
EF
2∆

Percentage freezetranscription, we used several complementary strategies. First, we used
HSV to express either a dominant-negative form of MEF2 (dnMEF2;
Supplementary Fig. 2a) or short-hairpin RNA (shRNA) against
Mef2a and Mef2d. We verified that these shRNAs robustly decreased
levels of endogenous MEF2A and MEF2D proteins (Supplementary
Fig. 5a,b). Second, we used AAV9 to express different shRNAs that
had been previously used to knock down Mef2a and Mef2d in vitro andin vivo6,8,21. To examine whether disrupting MEF2 function enhanced
memory, we trained mice under weak training conditions that are notnormally sufficient to support robust memory formation.
First, we examined spatial memory by ‘weakly’ training mice in thewater maze (three trials a day for 3 d, half the number of training trials
used previously) after microinjection of vector into the upper bladeof the dentate gyrus. Consistent with previous results26, this weaktraining was not sufficient to support formation of spatial memory
in mice with control vectors (GFP or MEF2∆) (mice searched equallyin the target and other zones during the probe test). In contrast,
mice with disrupted MEF2 function (HSV-dnMEF2, HSV-shRNA to
Mef2a and Mef2d or AAV9-shRNA vectors) exhibited robust spatial
memory, searching selectively in the target zone (significant vector ×zone interaction F4,49 = 12.08, P < 0.001, post hoc tests confirmed that
only mice with HSV-dnMEF2, HSV-shRNA or AAV9-shRNA vector searched preferentially in target zone; mice with HSV-dnMEF2,

1260

a

b

Percentage freeze

Figure 6 Disrupting MEF2 function permitted robust memory formation
GFP
shRNA
GFP
MEF2∆
dnMEF2
AAV9 shRNAafter weak training. (a) Mice were microinjected with HSV vectors
dnMEF250expressing GFP, MEF2∆, dnMEF2 or shRNA against Mef2a and Mef2d or
shRNA
50
*
AAV9 expressing different shRNA against Mef2a and Mef2d into upper
AAV9 shRNA*
40
blade of the dentate gyrus before weak water-maze training (three trials
40
per day for 3 d). Weak training was not sufficient to support formation*
30
30
of spatial memory in mice with control vectors (GFP vector (n = 13) or*
MEF2∆ vector (n = 7)); mice spent similar time searching in target (T)
20
20
NS
NS
and other (O) zones. Weak training was sufficient to induce robust
formation of spatial memory in mice with decreased MEF2 function
10
10(HSV-dnMEF2 (n = 17), HSV-shRNA (n = 7) or AAV9-shRNA (n = 10)
vector). (b) Mice were microinjected with HSV vectors expressing
00
GFP, dnMEF2 or shRNA against Mef2a and Mef2d or AAV9 expressing
T O
T O
T O
T O
T O
shRNA against Mef2a and Mef2d into lateral amygdala before weak fear
conditioning training (one tone plus shock pairing, 0.3-mA shock). Mice microinjected with dnMEF2 vector (n = 9), HSV-shRNA (n = 6) or AAV9-shRNA(n = 9) froze more than mice with GFP vector (n = 14) during tone fear test. All error bars indicate s.e.m. *P < 0.05; NS, not significantly different.
Percentage time in zone

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a r t ic l e saUnstimulated (US)KCI + FSKNormalized Arc
fluorescence intensity

5

Figure 7 Memory disruption produced by increasing MEF2 rescued bydisrupting AMPAR endocytosis. (a) Amount of Arc mRNA (normalized
intensity of fluorescence in situ hybridization signal for probe against Arc
mRNA) in primary hippocampal neurons transfected with GFP plasmidor MEF2 plasmid, and unstimulated or stimulated with KCl + forskolin(FSK). Representative images of primary neurons expressing GFP plasmid(left) or MEF2 plasmid (right) showing staining for GFP (transfected withplasmid, GFP, green) and Arc mRNA (red) both in an unstimulated (US)condition and after KCl + FSK stimulation. Scale bars, 20 µm. (b) Amountof Arc protein (Arc/GAPDH optical intensity) in primary hippocampal
neurons infected with GFP vector or MEF2 vector, and unstimulated or
stimulated with KCl + FSK. Immunoblot (bottom); full immunoblot isshown in Supplementary Figure 6. (c) Mice microinjected with MEF2 or
GFP vector in lateral amygdala were administered TAT-GluA2 3Y or TAT
control peptide before auditory fear conditioning (n = 8 for all groups). Allerror bars indicate s.e.m.; *P < 0.05; NS, not significantly different.

*

*

3
2
1
0

GFP

MEF2

Arc mRNA Merge

MEF2 Arc mRNA Merge

KCI + FSK

US

GFP

4

*

npg

c Surgery

Unstimulated (US)KCI + FSK

*
3

*

*

2

40

NS
1
0

GFP

MEF2

TAT-peptide
injections Training

Tone
test

1h
Day:

shRNA

GFP
MEF2 shRNAUS KCI US KCI US KCI
Arc
GAPDH

Percentage freeze

Optical density (Arc/GAPDH)© 2012 Nature America, Inc. All rights reserved.

b

0

1

*

2

2

3
GFP

NS

MEF2

*

30
20
10
0

Control
GluA23YTAT peptide

HSV-shRNA or AAV9-shRNA vector spent more time in target zone
than mice with either GFP or MEF2∆ vector, which did not differ
from each other F4,49 = 10.24, P < 0.001; Fig. 6a). Thus, decreasing
MEF2 function via exogenous expression of dnMEF2 or via shRNAmediated knockdown was sufficient to produce strong spatial memory
after subthreshold training.
Second, we observed that decreasing MEF2 levels in the lateral
amygdala facilitated the formation of auditory fear memory. We
microinjected HSV expressing GFP, dnMEF2, shRNA against Mef2aand Mef2d or AAV9 expressing shRNA (as above) into the lateral
amygdala before weak fear training (one tone plus shock pairingwith 0.3-mA shock). As expected using these weak training conditions, mice with control vector (HSV-GFP) showed low fear memory
during the tone fear test. However, mice with disrupted MEF2 function(HSV-dnMEF2, HSV-shRNA or AAV9-shRNA) froze at high levels
during the tone fear test, showing robust fear memory after thisweak fear training (F3,34 = 4.79, P < 0.001, post hoc tests showed that
HSV-dnMEF2, HSV-shRNA or AAV9-shRNA groups froze morethan mice microinjected with GFP vector; Fig. 6b). Therefore, using
two paradigms that produce different types of memory, we foundthat weak training was sufficient to induce strong memory formation if, and only if, MEF2 function was disrupted. Together, theseresults indicate that MEF2 acts as an endogenous molecular brakeon memory formation. Relieving the MEF2-inhibitory constraint
facilitated memory formation.
Memory impairment rescued by targeting AMPAR endocytosis
Our results showed that increasing MEF2 function disrupted memory formation. We next explored a possible molecular mechanism
underlying this effect. MEF2 is a transcription factor that regulates
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the expression of several target genes10. We focused on the MEF2
target gene Arc6,10 because Arc protein is implicated in synaptic function and memory35. Consistent with previous findings, we observedthat our MEF2 vector robustly increased the amounts of Arc RNA(F3,129 = 14.94, P < 0.001; Fig. 7a) and protein (Fig. 7b). Previous
findings indicate that neuronal activity increases Arc expression at
activated dendritic regions35. We observed that viral expression ofshRNA against Mef2a and Mef2d also prevented the activity-induced
increase in the amount of Arc protein (significant vector × stimulation interaction F2,46 = 7.86, P < 0.001, vector F2,46 = 8.74, P < 0.001,
stimulation F1,46 = 20.67, P < 0.001; Fig. 7b).
Arc reduces the amplitude of synaptic currents mediated by
AMPARs by promoting their endocytosis11,12,36. AMPARs are composed of four subunit types (GluR1–4 or GluA1–4), which combine
to form tetramers37. In mature hippocampal pyramidal neurons, most
AMPARs consist of GluA1-GluA2 or GluA2-GluA3 heterodimers37.
Endocytosis of GluA2-containing AMPARs decreases synaptic efficacy and is critical for the expression of some forms of long-term
depression (a rapid form of synaptic plasticity characterized by adecrease in synaptic strength and dendritic spine size or density) 37,38
and homeostatic synaptic scaling (a slower form of plasticity in which
the total synaptic strength of a neuron is modified to regulate neuronal
excitability)39. Arc may preferentially enhance internalization of
GluA2-containing AMPARs36. Together, these findings suggest that
MEF2 may disrupt memory formation by increasing Arc expression, which would decrease surface expression of GluA2-containing
AMPAR. Therefore, we tested whether interfering with GluA2containing AMPAR endocytosis during training prevented thememory deficits induced by overexpressing MEF2.
To disrupt GluA2-containing AMPAR endocytosis, we used an interference peptide (GluA23Y) that blocks activity-dependent endocytosis
of GluA2-containing AMPARs40. Fusing GluA23Y peptide to a TAT
protein transduction domain allows this peptide to enter the brain;systemic injection of TAT-GluA23Y peptide produces robust behavioral
effects in animal models of addiction40. We microinjected mice with
MEF2 or GFP vectors in the lateral amygdala as above, and 60 min
before auditory fear training, systemically administered TAT-GluA23Y
or a control (TAT alone) peptide. Consistent with our previous findings (Fig. 5d), mice with MEF2 vector (and control peptide) exhibited
impaired formation of tone fear memory. However, administering
TAT-GluA23Y peptide before training was sufficient to rescue the
MEF2-induced memory deficit (Fig. 7c). That is, mice with MEF2
vector but injected with TAT-GluA23Y peptide froze at high levels during
the tone memory test (freezing levels did not differ from control micewith GFP vector; significant vector × TAT peptide interaction F1,28 =4.26, P < 0.05; post hoc tests showed that mice with MEF2 vector plus
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control peptide showed lower freezing than mice with GFP vector plus
control peptide, but that mice with MEF2 vector plus GluA23Y peptide
showed similar levels of freezing to mice with GFP vector plus control
peptide). Injection of TAT-GluA23Y peptide had no effect on memory
formation in mice with control GFP vector, in agreement with previous reports41. These findings are consistent with the interpretationthat MEF2 disrupts memory formation by increasing Arc expression,
which, in turn, decreases surface expression of AMPARs.
DISCUSSION
Based on previous findings that MEF2 negatively regulates spine
growth, we hypothesized that MEF2-mediated transcription normallyinhibits memory formation. We found that strong training (sufficient to induce either robust fear or formation of spatial memory) was
associated with phosphorylation of endogenous MEF2 at a site that
inhibits MEF2-mediated transcription as well as decreased levels of
MEF2A and MEF2D protein. These observations are consistent with
the notion that MEF2-mediated transcription negatively regulates
memory formation. We found that overexpressing MEF2 specificallyduring training blocked spatial and fear memory formation, in a brain
region–dependent manner. Increasing MEF2 function also prevented
the increase in spine density normally associated with formation of
spatial memory, suggesting that MEF2-mediated transcription constrains memory formation by interfering with the required underlying structural plasticity. In contrast, we also found that relieving
the suppressive effects of MEF2-mediated transcription (expressing
dnMEF2 or knocking down endogenous MEF2A and MEF2D) was
sufficient to produce robust spatial or fear memory under weak training conditions that were not normally sufficient to support memory
formation. Finally, we examined a potential mechanism mediating thememory deficits induced by overexpressing MEF2. Consistent withprevious results, we found that increasing MEF2 amounts increased
Arc expression. As Arc decreases AMPAR-mediated synaptic currents by promoting AMPAR endocytosis, we investigated whether
the memory deficits induced by MEF2 could be prevented by interfering with AMPAR endocytosis at the time of training. We foundthat administering a peptide that disrupts AMPAR endocytosis completely restored the memory deficit induced by MEF2 overexpression.
Together, our results identified Mef2 as a memory suppressor genethat may disrupt memory formation through the synaptic protein Arc
to decrease the surface expression of AMPAR.
We locally and acutely manipulated MEF2 function in the adultbrain, rather than chronically throughout development because
MEF2 is required for neuronal survival and differentiation9. For
instance, inducible deletion of Mef2c at an early developmental stage(floxed Mef2c mice crossed with mice in which Cre recombinase isdriven by the nestin promoter) produces transgenic mice with bothabnormal neuronal aggregation and synaptic transmission42. Similardeletion of Mef2c at a later developmental stage (Cre recombinase
driven by GFAP promoter) produces adult transgenic mice that may
have developmental neurological deficits (mice have reduced bodyweight, impaired beam walking and an abnormal clasp reflex) 7. Asmight be expected by decreasing MEF2 function, these transgenic
mice with a deletion of Mef2c show increased dendritic spine density
in dentate gyrus neurons. However, these mice also show disrupted
context fear memory, perhaps because of developmental neurological deficits. In contrast, transgenic mice that chronically express
MEF2-VP16 across many brain regions (driven by NSE promoter)show decreased dentate gyrus spine density but normal context-fear
memory7. Although these studies clearly demonstrate the importanceof MEF2 during development, they do not allow the role of MEF2

in development versus adult memory formation to be clearly distinguished. Therefore, to specifically examine the role of MEF2 in adultmemory formation, we manipulated MEF2 function in a spatiallyand temporally restricted manner. In addition to the importance of
MEF2 in development, our data show that MEF2 is also importantin adult memory formation.
We found that increasing MEF2 function in the upper blade of thedentate gyrus prevented formation of spatial memory. This memoryformation impairment was specific in several ways. First, the memory
disruption resulting from the introduction of our MEF2 vector
depended on MEF2-mediated transcription as similar microinjection ofa MEF2∆ vector (encoding MEF2 lacking a DBD, which cannot activatetranscription) did not impair memory formation. Second, formation of
spatial memory was impaired only when MEF2 function was increased
during water-maze training and not 1 week before or over 2 weeks
after training. These findings indicate that the effects of increasing
MEF2 function are reversible and that increasing MEF2 function did
not impair expression of a previously acquired memory. Third, theeffects of MEF2 on formation of spatial memory were anatomically specific. In fact, we observed a double dissociation between the effects ofincreasing MEF2 function in the dentate gyrus versus lateral amygdalaon the formation of different types of memory. Microinjecting MEF2
vector in the upper blade of the dentate gyrus impaired spatial memory
while leaving tone-fear memory intact, whereas microinjecting MEF2
vector into lateral amygdala disrupted the formation of fear memory
but not of spatial memory. Tests of spatial and fear memories differ
along several dimensions (including performance demands, sensory
features, content of learning, amount of training required for memory
formation, brain regions critically involved and other factors). The
finding that overexpressing MEF2 disrupted the formation of bothtypes of memory suggests that it is unlikely that MEF2 vector impaired
general behavioral performance. Rather, these data indicate that MEF2
has a general role in regulating memory formation that is conserved
across brain region and type of memory.
We observed that memory formation was accompanied by anincrease in dendritic spine density in hippocampal granule cells, consistent with previous data29,30. Spines, first described over 100 years
ago43, are hypothesized to serve as a physical substrate of long-term
memory1,3,43. MEF2 restricts the number of excitatory synapses
and spine growth across several preparations and species (including Caenorhabditis elegans excitatory neuromuscular junction synapses44, rat hippocampus neurons in vitro6, mouse hippocampus
neurons in vivo7 and rat nucleus accumbens neurons in vivo8).
Here we found that acutely overexpressing MEF2 prevented theincrease in spine density that normally accompanies formation of
spatial memory. Consistent with the activity-dependent mechanism
previously described in other systems, increasing MEF2 did notdecrease spine density in home cage control mice. Therefore, MEF2
has a conserved role in structural plasticity in response to diverse
neuronal activity.
Memory incubation, the enhancement of memory over time in theabsence of additional training, is observed across several behavioral
paradigms23,45. However, little is known about the mechanisms
underlying incubation. One possibility is that post-training reactivation of activity patterns that occurred during training31,32 contributes
to incubation by promoting spine growth and strengthening neuralcircuits. We examined this possibility by overexpressing MEF2 in theupper blade of the dentate gyrus 1 d after water-maze training. Micemicroinjected with control GFP vector exhibited an ­incubation-like
effect (enhanced spatial memory on a second probe test, conductedwithout additional training). In contrast, mice microinjected with

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a r t ic l e s
MEF2 vector after training did not exhibit enhanced memory on thesubsequent memory test, suggesting that increasing MEF2 function
prevented memory incubation. Moreover, the increase in spine densitythat accompanied the stronger memory in mice microinjected with
the GFP vector was prevented by increasing MEF2 function after
training. Therefore, the formation of a spatial memory (and the associated increase in spine density) was blocked if MEF2 function wasincreased during training, and a post-training process that strengthens memory and resembles memory incubation (and the associatedincrease in spine density) was blocked if MEF2 function was increased
shortly after training. These results agree with recent findings that
increasing MEF2 function in the cortex shortly after context feartraining similarly disrupts memory consolidation46 and suggest thatthe ongoing refinement of spines is important for memory strengthening and/or maintenance.
The formation of a memory is thought to critically depend on transcription and de novo protein synthesis47. Recently, however, it has
been suggested that protein degradation is also involved in synaptic
plasticity and memory formation48,49. We observed that formationof either a spatial or fear memory was associated with a decrease in
MEF2A and MEF2D protein levels. Although we believe we are thefirst to identify a role for MEF2 degradation in memory formation,
degradation of neural MEF2 by a caspase-dependent process has beenpreviously reported50.
Our results suggest that MEF2-mediated transcription normallyconstrains memory formation. We examined this by decreasing MEF2
function and training mice under weak (subthreshold) conditions.
Disrupting MEF2 function (either by expressing dnMEF2 or knocking
down endogenous protein by RNA interference) facilitated memory
formation across two different paradigms (water-maze and auditory
fear memory). These findings indicate that MEF2 has a bidirectional
effect on memory formation, with MEF2-mediated transcription
repressing memory formation.
MEF2 may suppress memory formation by activating the transcription of genes that encode proteins that weaken the excitatory ­synaptictransmission likely required for memory formation. For instance,
Arc is an activity-dependent MEF2 target gene10 whose protein productdecreases the surface expression of AMPAR11,12. We found that specifically disrupting GluA2-containing AMPAR endocytosis at the time oftraining rescued the memory deficits produced by overexpressing MEF2.
These results suggest that excessive internalization of AMPAR may be a
critical mechanism mediating the memory deficits produced by MEF2.
MEF2 and several MEF2 target genes are implicated in humancognitive and psychiatric disorders 10,13–15. Our results suggest that the cognitive deficits associated with these human disorders may be caused by a disruption of the MEF2-mediatedgene network that regulates structural plasticity and memory formation. Our findings, that acutely manipulating MEF2 function in
the adult brain produces robust effects on memory formation, raise
the intriguing possibility that these human cognitive disorders may
not result solely from untreatable developmental abnormalities but
might be due to chronic dysregulation of the MEF2 function necessary for normal memory formation. Furthermore, our finding that the
memory deficits produced by MEF2 were reversed by interfering with
AMPAR endocytosis leads to the speculation that impaired AMPAR
trafficking may contribute to this cognitive dysfunction and suggests
a potential therapeutic target.
Methods
Methods and any associated references are available in the online
version of the paper.
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Note: Supplementary information is available in the online version of the paper.
Acknowledgments
We thank A. DeCristofaro, R. Braybon and M. Yamamoto for excellent
technical assistance. This work was supported by grants from the Canadian
Institutes of Health Research (CIHR; MOP-74650 (S.A.J.), MOP-86762(P.W.F.)), EJLB Foundation (S.A.J.) and Natural Science and Engineering
Research Council (S.A.J.). C.J.C., A.P.Y. and M.J.S. were supported by
Restracomp Fellowships (Hospital for Sick Children), A.P.Y. received support
from the Alzheimer’s Society of Canada, M.J.S. received a CIHR Frederick
Banting and Charles Best Canada Graduate Scholarships Doctoral Award anda grant from the Faculty of Medicine at the University of Toronto, and C.J.C.
received an Ontario Graduate Scholarship.
AUTHOR CONTRIBUTIONS
S.A.J. and P.W.F. designed, directed and coordinated the study. C.J.C., V.M., L.R.
and T.P. conducted behavioral experiments . C.J.C., A.P.Y. and M.J.S. performed
the surgeries. C.J.C., V.M. and L.R. performed spine analysis. V.M., L.R. and
G.V. performed immunohistochemistry. V.M. and P.J.R. conducted the cell culture
experiments. C.J.C., V.M. and L.R. performed statistical analysis. J.-H.H. designed
several constructs. R.L.N. generated viral vectors, shRNA and commented
extensively on the design of experiments and use of viral vectors. S.A.J. and P.W.F.
wrote the manuscript, with assistance from C.J.C., V.M. and L.R.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/doifinder/10.1038/nn.3189.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.

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npg© 2012 Nature America, Inc. All rights reserved.

Mice. Male and female (2–4 months of age) F1 hybrid (C57Bl/6N Tac × 129S6/
SvEv Tac) mice were group-housed (3–5 per cage) on a 12-h light-dark cycle.
Experiments were conducted during light phase. Food and water were available
ad libitum. Procedures were approved by The Hospital for Sick Children Animal
Care and Use Committee in accordance with Canadian Council on Animal Careand US National Institutes of Health guidelines.
Immunohistochemistry. Male mice were handled daily (7 d), perfused withparaformaldehyde (4% PFA) and brains were sliced (50 µm, coronal). Sections
were counterstained with DAPI.
pMEF2 immunohistochemistry: water maze. Mice were trained in water maze(n = 4) or maintained in home cage (n = 2) and perfused 30 min after a probe test.
pMEF2 immunohistochemistry: tone fear conditioning. Mice were divided into
treatment groups: (i) tone plus unconditioned stimulus (shock), tone fear conditioning (n = 4), (ii) tone alone (n = 3), (iii) immediate shock (n = 4), and (iv) home
cage (n = 3). Tone plus unconditioned stimulus mice were placed in a conditioning chamber and 2 min later presented a tone (2,800 Hz, 85 dB, 30 s) that terminated with shock (0.5 mA, 2 s). Tone-alone mice were treated identically, except
no shock was delivered. Immediate shock mice received shock (0.5 mA) without
tone, 2 s after placement. Mice were perfused 30 min after training.
pMEF2 immunohistochemistry: quantification. A single image was acquiredfor dentate gyrus (optical slicing: 15 µm, Zeiss LSM710); image stack for lateral
amygdala (6 slices, 5-µm step). Lateral amygdala was analyzed on a z-projection(optical slicing, 15 µm). Regions of interest (upper blade of the dentate gyrus,
lateral amygdala) were delineated using DAPI (ImageJ). pMEF2 signal intensity from single neurons (DAPI+ nuclei with diameter > 6.23 µm) was calculated as percent of background intensity (pMEF2 intensity in glial cells (nucleus
diameter < 6.23 µm)) and plotted as cumulative percentage. NonparametricKolmogorov-Smirnov tests were followed by pair-wise comparisons (α = 0.01 for
multiple comparisons)51.
Western blot. Tissue was homogenized in cell lysis buffer and phosphate inhibitors as previously described52. Bands were visualized by exposure to film after
treatment with ECL Western Blotting Detection Reagents (GE Healthcare).
Optical intensity of bands associated with proteins of interest was measured
relative to their respective GAPDH bands on the same blot (Image J).
pMEF2 western blots: water maze. Dentate gyrus and CA1 region of dorsal hippo­
campus was isolated 30–90 min after probe test in water maze–trained (trained,
n = 9) or home-cage mice (n = 9). Blots were prepared as described above.
pMEF2 western blots: context fear conditioning. Mice were trained for context
fear conditioning (3× 0.5 mA shock; trained) or maintained in the home cage.
Dentate gyrus and CA1 region of dorsal hippocampus was isolated 90 min or 6 hafter training (pMEF2: home cage n = 4, 90 min n = 4, 6 h n = 4; MEF2A; home
cage n = 12, 90 min n = 9, 6 h n = 9; and MEF2D: home cage n = 8, 90 min n = 7,
6 h n = 7). Blots were prepared as described above.
Arc western blot on primary hippocampal neurons. Primary hippocampal
neurons (12–14 d in vitro (DIV)) were prepared from embryonic day (E)18–19
mice53 and infected with MEF2 vector, vector expressing shRNA to Mef2a and
Mef2d or GFP vector by incubating neurons (5 h) in culture medium with viralparticles. Twenty-four hours later, TTX (1 µM) and AP5 (100 µM) were added
1 h before KCl depolarization with 31% depolarization buffer (170 mM KCl,
2 mM CaCl2, 1 mM MgCl2 and 10 mM HEPES) to culture medium for 4 h.
Cells were homogenized in cell lysis buffer and western blot was performed.
Primary antibodies. Primary antibodies included antibodies to GAPDH(1:5,000, Cell Signaling 14C10), phospho-Ser408 MEF2A (1:1,000; provided
by M.E. Greenberg), Arc (1:1,000, Synaptic Systems 156003), MEF2 (1:1,000;
C-21; Santa Cruz), MEF2D (1:1,000; BD Bioscience 610774) and GFP (1:300,
Millipore A11122).
Luciferase reporter gene assays. Gli36 cells (5 × 104, provided by A. Epstein) wereseeded in 24-well plates, and co-transfected (Lipofectamine 2000, OptiMEM,
Invitrogen) with 100 ng MRE-reporter plasmid (pGL3-TATA-DesMEF; provided
by E. Olson), 50 ng transfection-normalization plasmid CMV-lacZ (provided
by J. McGlade) and 500 ng p1005+-derived plasmids expressing GFP alone,
MEF2-VP16 or MEF2∆-VP16. Forty-eight hours after transfection, cells weredoi:10.1038/nn.3189

lysed (Reporter Lysis Buffer, Promega). Luciferase expression was determinedusing Firefly Luciferase Assay System (Promega), lacZ expression determinedusing ortho-nitrophenyl-β-galactoside–based colorimetric β-galactosidase
assay. Luciferase expression was normalized to lacZ expression within eachexperiment. Transfections were performed in duplicate within each experiment,n = 6 for each group.
Primary hippocampal neurons (DIV 4, 24-well plates) were co-transfected (Lipofectamine 2000) with MRE- (500 ng) or CRE-reporter plasmid(500 ng) and TK-pRL vector (expressesing Renilla luciferase (250 ng; Promega)
to normalize transfection efficiency). Twenty-four h later, neurons were infectedwith GFP or MEF2 virus. Twenty-four hours later, TTX (1 µM) and AP5 (100 µM)
were added 1 h before KCl depolarization with 31% depolarization buffer(170 mM KCl, 2 mM CaCl2, 1 mM MgCl2 and 10 mM HEPES) for 4 h. Neurons
were lysed and lysate (30 µl) assayed using Dual Luciferase Assay kit (Promega).
Firefly and Renilla luceferase activity levels were quantified and raw data from
MRE- or CRE-reporter assay divided by TK-pRL luciferase activity. Data represent means from independent experiments, with internal duplicates or triplicatesfor each condition, n = 8 (MRE), n = 6 (CRE).
Arc mRNA fluorescence in situ hybridization (FISH). Hippocampal neurons (DIV
10–12, 24-well plates) were transfected with GFP or MEF2 plasmids (Lipofectamine
2000). Twenty-four hours later, TTX (1 µM) and AP5 (100 µM) were added
1 h before 31% KCl depolarization buffer and forskolin (FSK, 30 µM) for 2 h.
Neurons were fixed (4% PFA, 10 min), treated with acetic anhydride (10 min)and methanol/acetone solution (90 s). Coverslips were incubated with prehybridization buffer (Sigma, 1 h, 56 °C) and Arc-DIG riboprobe (100 ng) inhybridization buffer overnight (56 °C). Neurons were incubated 16–18 h laterwith 1% H2O2 (30 min). FISH signal was detected by incubation with HRP-DIG(1:300, Jackson ImmunoResearch, 1 h, room temperature), amplified (TSA-bio,
30 min), incubated in streptavidin–Alexa Fluor 568 (1:300, Invitrogen, 1 h) andthen incubated with blocking solution (30 min, room temperature). Images wereacquired (Zeiss LSM710); single transfected neurons were traced (GFP channel)
and FISH quantified within selected area (ImageJ).
HSV vectors. Plasmids encoding MEF2-VP16, MEF2∆-VP16 were provided
by M.E. Greenberg6, and plasmids encoding dnMEF2 were provided by
I. Skerjanc54 (Supplementary Fig. 2a). All constructs were subcloned into
bicistronic pHSV-p1005+ amplicons expressing GFP alone or with MEF2
constructs. tGFP-shRNAmir against Mef2a (oligo identifier V3LHS_319382;
Thermo Scientific) and MEF2D (V2LHS_200022) were subcloned from lentivirus encoding human GIPZs vectors (Thermo Scientific) into pHSV-p1005+
amplicons. Vectors were prepared according to published methods55,56.
Validation of shRNA-mediated Mef2 knockdown. MEF2A and MEF2D expression was assessed by immunofluorescence in hippocampal primary neurons (10–12 DIV) transfected with shRNA directed against Mef2a and Mef2d. Neurons were
fixed (4% PFA, 20 min) and coverslips incubated with antibodies. Fluorescenceintensity of staining for MEF2A and MEF2D proteins was quantified (ImageJ).
Vector-induced toxicity. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl­
tetrazolium bromide) assays53,57 were used to investigate potential viraltoxicity. Hippocampal neurons (DIV 12–14) were infected with vector (GFP,
MEF2, MEF2∆ or dnMEF2 vector) for 5 h. MTT solution (10% v/v culture
medium volume) was added 24–30 h later (3 h, 37 °C). At least four samples
for each condition were quantified. Spectrophotometer measurements werecompared to those for similar non-infected cultures (n = 4 independent cultures
for each group). As a control, we treated additional hippocampal neurons
with increasing levels of 0.1 M PBS (control, 2 × 1 min 0.1 M PBS, 2 × 3 min
0.1 M PBS, 2 × 6 min 0.1 M PBS) to verify neuronal viability (n = 6 independentcultures for each group).
AAV vector. Plasmids encoding shRNA to Mef2a and shRNA Mef2d were provided by M.E. Greenberg. Constructs were packaged into AAV9 vector (AAV9CAG-GFP-H1-MEF2A-RNAi, AAV9-CAG-GFP-H1-MEF2D-RNAi) by Virovek(http://www.virovek.com/).
Surgery. Mice were pretreated with atropine sulfate (0.1 mg/kg, intraperitoneal),
anesthetized with chloral hydrate (400 mg/kg, intraperitoneal) and placed instereotaxic frame. Holes were drilled above the dentate gyrus (AP = −2.0 mm,

nature NEUROSCIENCE

ML = ± 2.0 mm, DV = −2.0 mm from bregma) or lateral amygdala (AP =−1.4 mm, ML = ± 3.5 mm, DV = −5.0 mm)58. Bilateral microinjections of HSV(2.0 µl for dentate gyrus; 1.5 µl for lateral amygdala) were delivered over 20 min
through glass micropipettes. Volume of AAV9 vector microinjection was 0.3 µl.
Because transgene expression using HSV viral system peaks 3 d after surgery27,
we trained mice 2 d after surgery in water-maze experiments (except where specified). In fear conditioning experiments, mice were trained 2 d after HSV micro­
injection. Mice with AAV9 were trained between 10–21 d after microinjection.

npg© 2012 Nature America, Inc. All rights reserved.

Histology. Placement or extent of viral infection for each mouse was determinedusing GFP immunofluorescence by an examiner unaware of behavioral data. Fourdays after HSV microinjection, mice were perfused and mouse brains were sliced.
Only mice with strong bilateral GFP expression in target region (‘bilateral hit’)
were included in subsequent data analysis.
Water maze–based testing of spatial memory. Training and testing in the water
maze were conducted as previously described59,60. For strong training, micereceived two blocks of three trials per day, for 3 d. For weak training (Fig. 6),
mice received one block of three trials per day, for 3 d. Sixty min after final training trial, spatial memory was assessed in a probe test during which the platform
was removed from the pool and the mouse allowed to search for 60 s. Behavioral
data were acquired and analyzed using Actimetrics software. During training,
we analyzed escape latency, swim speed and thigmotaxic behavior (time micespent within 5 cm of pool wall). In the probe test, we quantified spatial memory
by measuring amount of time mice spent searching in target zone (20 cm radius,
centered on location of platform during training; 11% of pool surface) versus
average time spent in three other equivalent zones in other areas of pool.
Statistical analysis. We analyzed the time required to reach the platform (escapelatency) using a two-way analysis of variance (ANOVA) with between-group factor ‘vector’ and within-group factor ‘day’ (3 d). An ANOVA (vector) was used toanalyze overall swim speed and thigmotaxis averaged over training. For the probe
test, we first quantified spatial bias by comparing amount of time mice spent intarget zone versus average time spent in equivalent zones in ‘other’ three quadrants of the pool using an ANOVA (between-subjects variable vector, withinsubjects variable zone (target, others)). Next, we analyzed time spent in target
zone between groups using one-way ANOVA (vector). Significant effects were
further analyzed with Newman-Keuls post-hoc tests.
Fear memory testing. Mice were trained as above. Context fear testing occurred
24 h after training, and tone fear testing occurred 48 h after training. For context
fear testing, mice were placed in conditioning context and the amount of timespent freezing (cessation of all movement except for respiration) during 5-mintest was assessed (Actimetrics). For auditory (tone) fear testing, mice were placedin a new chamber and, 2 min later, tone was presented (1 min). Freezing before
and during tone was assessed.

nature NEUROSCIENCE

Dendritic spine analysis. Brain slices (50 µm, from random subset of mice,
Fig. 2c) were stained and imaged52. Neurons were chosen for tracing basedon morpho­logy (elliptical cell body, unipolar dendritic tree signifying granulecell), location (upper blade dentate gyrus), isolation and general appearance(no dendritic truncations). Reconstructed neurons were analyzed (Neurolucida
Explorer)52. Spine density (protrusions in direct contact with dendritic shaft) per
10 µm of dendrite was averaged per mouse. Neurons and mice in each group;
home cage, GFP (11 neurons from 5 mice); home cage, MEF2 (15 neurons,
5 mice); home cage, MEF2∆ (10 neurons, 5 mice); trained, GFP (14 neurons,
7 mice); trained, MEF2 (15 neurons, 8 mice); trained, MEF2∆ (14 neurons, 6 mice);
after training, GFP (incubation experiment, 13 neurons, 5 mice); and after
training, MEF2 (16 neurons, 7 mice).
To determine whether effects of increasing MEF2 function on spine growth
were a cell-autonomous or circuit property, we examined effects of MEF2 vector onneighboring ‘control non-infected’ neurons. We microinjected mice with two vectors, each expressing a different fluorescence marker (green or red). ‘Green’ vector
expressed either MEF2-GFP or GFP alone. ‘Red’ vector expressed tandem dimmer(td)Tomato alone (just used as a fluorescence marker to allow assessment of spine
density) (Supplementary Fig. 4b). Mice were trained in the water maze or not.
TAT-GluA23Y peptide. To disrupt GluA2-containing AMPAR endocytosis, weused an interference peptide derived from the C-terminal tail of GluA2 (GluA23Y,
869YKEGYNVYG877)40. TAT-GluA2 and TAT-only control protein (GenScript)
3Y
were dissolved in saline and administered (1.5 µmol/g, intraperitoneally) 1 h
before training.
51. Keppel, G. & Wickens, T.D. Design and Analysis: A Researcher’s Handbook
4th edn. (Prentice Hall, 2004).
52. Yiu, A.P., Rashid, A.J. & Josselyn, S.A. Increasing CREB function in the CA1 regionof dorsal hippocampus rescues the spatial memory deficits in a mouse model of
Alzheimer’s disease. Neuropsychopharmacology 36, 2169–2186 (2011).
53. Zalfa, F. et al. A new function for the fragile X mental retardation protein in regulationof PSD-95 mRNA stability. Nat. Neurosci. 10, 578–587 (2007).
54. Karamboulas, C. et al. Disruption of MEF2 activity in cardiomyoblasts inhibits
cardiomyogenesis. J. Cell Sci. 119, 4315–4321 (2006).
55. Russo, S.J. et al. Nuclear factor kappa B signaling regulates neuronal morphology
and cocaine reward. J. Neurosci. 29, 3529–3537 (2009).
56. Clark, M.S. et al. Overexpression of 5-HT1B receptor in dorsal raphe nucleus using
Herpes Simplex Virus gene transfer increases anxiety behavior after inescapablestress. J. Neurosci. 22, 4550–4562 (2002).
57. Miller, R.R. & McDevitt, C.A. A quantitative microwell assay for chondrocyte celladhesion. Anal. Biochem. 192, 380–383 (1991).
58. Paxinos, G. & Franklin, K.B.J. The Mouse Brain in Stereotaxic Coordinates,
2nd edn. (Academic Press, San Diego, 2001).
59. Teixeira, C.M., Pomedli, S.R., Maei, H.R., Kee, N. & Frankland, P.W. Involvementof the anterior cingulate cortex in the expression of remote spatial memory.
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doi:10.1038/nn.3189