and Ti2Ni incoherently precipitate in the B2
matrix upon cooling from 700°C to 87°C. The
B2 matrix transforms completely to B19 upon
further cooling to 22°C, aided by the simultaneous
Ti2Cu/B19 epitaxies (Figs. 3 and 4, Table 1,
and table S3). The B19/Ti2Cu epitaxy provides an
internal stress pattern, which stabilizes the B19
phase at low temperatures. During stress cycling,
the equivalent epitaxy alternatingly stabilizes the
B2 phase. At each temperature and stress, the
transforming phases attain equilibrium by forming
a compatible morphology directed by the
internal epitaxy-generated stress distribution.
Complete transformation is attained at each cycle
because the epitaxial stresses are reversible. Hence,
we propose that the epitaxially promoted completion
of the B2↔B19 phase transformation creates
the low-fatigue state of the Ti54Ni34Cu12 films.
The Ti2Cu precipitates act like sentinels, assuring
that the B2↔B19 transformation proceeds toward
completion at each cycle. The transformation
will return the film to a stress state and morphology
that are compatible with the pristine state.
The decrease of the anisotropic peak broadening
of the epitaxy-effected XRD peaks indicates
trainability.
This proposal must be revisited in light of the
favorable values of the quantitative compatibility
criteria calculated from the lattice parameters of
both alloys Ti51Ni36Cu13 and Ti54Ni34Cu12 (Table 2).
These values approach the ideal triplet (l = 1,
CCI = CCII = 0) and suggest good reversibility
even in polycrystals (29), although they are inferior
to those for Zn45Au30Cu25 (19). The values
for sample 2 are closer to the ideal than those for
sample 1, which is in accord with the vastly better
fatigue characteristics of sample 2. The question
then arises whether this large difference of
the fatigue life results from the observed epitaxy
or that of the two triplets listed in Table 2.
We observe that despite their reversibility, the
phase transformations in SMAs are of first order
(nucleation- and growth-controlled). We suggest
that the epitaxy leads to reversible nucleation,
whereas the low cofactors promote reversible
near-equilibrium growth so that the combination
of both mechanisms yields the observed ultralow
fatigue. In the limit of CCI and CCII→0, no
energy will be stored in the product phase in the
form of twin boundaries. This creates a strongly
reproducible, and therefore repeatably transformable,
equilibrium state.
Given the fatigue-controlling importance of
the dual epitaxy of Ti2Cu in TiNiCu-based (SMA)
films, it is natural to search for other alloying
elements that have the potential to play a similar
role. Following this lead, we can assume that
structurally related Ti2Ag precipitates will act
very similar to Ti2Cu. Because TiNiAg SMAs display
transformation characteristics comparable
with that of TiNiCu (30), they could be promising
candidates for biocompatible ultralow fatigue
SMA films. Elastocaloric cooling requires bulk
materials, which is a difficult but, in principle,
solvable challenge. More generally, we expect
similar behavior in phase-transforming materials
that contain dual-epitaxial precipitates.
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ACKNOWLEDGMENTS
The work at the University of Kiel was supported by the Deutsche
Forschungsgemeinschaft (DFG) via the Priority Program 1599.
L.K. and J.S. appreciate the assistance of V. Duppel (Max Planck
Institute for solid state research) for recording the electron
diffraction patterns, B. V. Lotsch for enabling electron microscopy,
and C. Szillus for TEM sample preparation. The work at the
University of Maryland was supported by grant DOE
DESC0005448; use of the Advanced Photon Source - Argonne
National Laboratory was supported by the U.S. Department of
Energy (DOE) Office of Science, under contract DE-AC02-
06CH11357. M.W. and W.G. thank P. Zavalij for his guidance with
the Rietveld refinement and J. Steiner for the compatibility
calculations. The synchrotron diffraction data are available from
the corresponding author. Other data are available in the main text
and the supplementary materials.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/348/6238/1004/suppl/DC1
Materials and Methods
Figs. S1 to S9
Tables S1 to S4
Reference (32)
12 September 2014; accepted 14 April 2015
10.1126/science.1261164
MEMORY
Engram cells retain memory under
retrograde amnesia
Tomás J. Ryan,1,2
* Dheeraj S. Roy,1
* Michele Pignatelli,1
*
Autumn Arons,1,2
Susumu Tonegawa1,2
†
Memory consolidation is the process by which a newly formed and unstable memory
transforms into a stable long-term memory. It is unknown whether the process of memory
consolidation occurs exclusively through the stabilization of memory engrams. By using
learning-dependent cell labeling, we identified an increase of synaptic strength and
dendritic spine density specifically in consolidated memory engram cells. Although
these properties are lacking in engram cells under protein synthesis inhibitor–induced
amnesia, direct optogenetic activation of these cells results in memory retrieval, and
this correlates with retained engram cell–specific connectivity. We propose that a specific
pattern of connectivity of engram cells may be crucial for memory information storage and
that strengthened synapses in these cells critically contribute to the
memory retrieval process.
M
emory consolidation is the phenomenon
by which a newly formed memory
transitions from a fragile state to a stable,
long-term state (1–3). The defining
feature of consolidation is a finite time
window that begins immediately after learning,
during which a memory is susceptible to disruptions,
such as protein synthesis inhibition (4–6),
resulting in retrograde amnesia. The stabilization
of synaptic potentiation is the dominant cellular
model of memory consolidation (7–10) because
protein synthesis inhibitors disrupt late-phase
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RESEARCH | REPORTS
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long-term potentiation of in vitro slice preparations
(11–13). Although much is known about the
cellular mechanisms of memory consolidation,
it remains unknown whether these processes
occur in memory engram cells. It may be possible
to characterize cellular consolidation and
empirically separate mnemonic properties in
retrograde amnesia by directly probing and manipulating
memory engram cells in the brain.
The term memory engram originally referred to
the hypothetical learned information stored in
the brain, which must be reactivated for recall
(14, 15). Recently, several groups demonstrated
that specific hippocampal cells that are activated
during memory encoding are both sufficient (16–18)
and necessary (19, 20) for driving future recall
of a contextual fear memory and thus represent a
component of a distributed memory engram (21).
Here, we applied this engram technology to the
issue of cellular consolidation and retrograde
amnesia.
We used the previously established method
for tagging the hippocampal dentate gyrus (DG)
component of a contextual memory engram with
mCherry (supplementary materials, materials and
methods, and fig. S1) (16, 22). To disrupt consolidation,
we systemically injected the protein synthesis
inhibitor anisomycin (ANI) or saline (SAL)
as a control immediately after contextual fear
conditioning (CFC) (Fig. 1A). The presynaptic
neurons of the entorhinal cortex (EC) were constitutively
labeled with ChR2 expressed from an
AAV8-CaMKIIa-ChR2-EYFP virus (Fig. 1B). Voltage
clamp recordings of paired engram (mCherry+
)
and nonengram (mCherry–
) DG cells were conducted
simultaneously with optogenetic stimulation
of ChR2+
perforant path (PP) axons (Fig. 1,
C and D). mCherry+
cells of the SAL group showed
significantly greater synaptic strength than did
paired mCherry–
cells, whereas in the ANI group,
mCherry+
and mCherry–
cells were of comparable
synaptic strength (Fig. 1E). Calculation of AMPA/
N-methyl-D-aspartate (NMDA) receptor current ratios
(23) showed that at 24 hours after training,
mCherry+
engram cells displayed potentiated
synapses relative to paired mCherry–
non-engram
1008 29 MAY 2015 • VOL 348 ISSUE 6238 sciencemag.org SCIENCE
Fig. 1. Synaptic plasticity and connectivity of engram cells. (A) Mice
taken off doxycycline (DOX) 24 hours before CFC and dispatched 24 hours
after training. SAL or ANI was administered immediately after training.
(B) AAV8-CaMKIIa-ChR2-EYFP and AAV9-TRE-mCherry viruses injected
into the entorhinal cortex and dentate gyrus, respectively, of c-Fos-tTA mice.
(C) Paired recordings of engram (red) and nonengram (gray) DG cells during
optogenetic stimulation of ChR2+
PP axons. (D) Representative image of a pair
of recorded biocytin-labeled engram (mCherry+
) and nonengram (mCherry–
)
DG cells. ChR2+
PP axons are in green. (E) (Top) Example traces of AMPA and
NMDA receptor–dependent postsynaptic currents in mCherry+
and mCherry–
cells, evoked by means of light activation of ChR2+
PP axons. (Bottom) EPSC
amplitudes and AMPA/NMDA current ratios of mCherry+
and mCherry–
cells
of the two groups are displayed as means (columns) and individual paired
data points (gray lines). Paired t test; *P < 0.05, **P < 0.001. SAL group
compared with the ANI group, unpaired t test; *P < 0.05. (F) (Left) Representative
confocal images of biocytin-filled dendritic fragments derived from
SAL and ANI groups for ChR2+
and ChR2–
cells (arrow heads indicate dendritic
spines). (Right) Average dendritic spine density showing an increase occurring
exclusively in ChR2+
fragments. Data are represented as mean T SEM. Unpaired
t tests **P < 0.01, ***P < 0.001. (G) Engram connectivity. (Top left) AAV9TRE-ChR2-EYFP
and AAV9-TRE-mCherry viruses, injected into the DG and
CA3, respectively, of c-Fos-tTA mice. (Bottom left) Example of mCherry+
(1)
and mCherry–
(2) biocytin-filled CA3 pyramidal cells. ChR2+
mossy fibers
(MF) are in green. (Top right) mCherry+
cell but not mCherry–
cell displayed
excitatory postsynaptic potentials in response to optogenetic stimulation of
MF. (Bottom right) Probability of connection of DG ChR2+
engram axons and
CA3 mCherry+
and mCherry–
cells. Error bars are approximated by using
binomial distribution. Fisher’s exact test; *P < 0.05.
1
RIKEN-MIT Center for Neural Circuit Genetics at the Picower
Institute for Learning and Memory, Department of Biology and
Department of Brain and Cognitive Sciences, Massachusetts
Institute of Technology, Cambridge, MA 02139, USA. 2
Howard
Hughes Medical Institute, Massachusetts Institute of
Technology, Cambridge, MA 02139, USA.
*These authors contributed equally to this work. †Corresponding
author. E-mail: tonegawa@mit.edu
RESEARCH | REPORTS
cells in the SAL group (Fig. 1E). However, no such
difference between mCherry+
and mCherry–
was
observed in the ANI group. In addition, mCherry+
engram cells of the SAL group showed significantly
greater AMPA/NMDA current ratios than
those of mCherry+
engram cells of the ANI group.
Analysis of spontaneous excitatory postsynaptic
currents (EPSCs) of engram and non-engram
cells of both SAL and ANI groups showed the
same pattern (fig. S2).
We also quantified dendritic spine density for
DG engram cells labeled with an AAV9-TREChR2-EYFP
virus. Spine density of ChR2+
cells
was significantly higher than corresponding
ChR2–
cells in the SAL group (Fig. 1F and fig. S3),
but spine densities of ChR2+
and ChR2–
cells of
the ANI group were similar (supplementary materials,
materials and methods). Spine density of
ChR2+
cells of the SAL group was significantly
higher than that of ANI ChR2+
cells (Fig. 1F), but
ChR2–
cell spine density was comparable. This result
was confirmed with analysis of the membrane
capacitance (fig. S4G). ChR2 expression did not
affect intrinsic properties of DG cells in vitro (fig.
S5, A to E). Direct bath application of ANI did not
affect intrinsic cellular properties in vitro (fig.
S5F), although it mildly reduced synaptic currents
acutely (fig. S5, G to I). When ANI was injected
into c-Fos-tTA animals 24 hours after CFC and
engram labeling, engram cell–specific increases
in dendritc spine density and synaptic strength
were undisturbed (fig. S6). We also examined engram
cells labeled by means of a context-only experience
(17) and found equivalent engram-cell
increases in spine density and synaptic strength
(fig. S7) as those labeled by means of CFC.
DG cells receive information from EC and relay
it to CA3 via the mossy fiber pathway. We labeled
DG engram cells using an AAV9-TRE-ChR2-EYFP
virus and simultaneously labeled CA3 engram
cells using an AAV9-TRE-mCherry virus (Fig. 1G).
Connection probability was assessed 24 hours
after CFC by stimulating DG ChR2+
cell terminals
optogenetically and recording excitatory postsynaptic
potentials in CA3 mCherry+
and mCherry–
cells in ex vivo preparations. CA3 mCherry+
engram
cells showed a significantly higher probability
of connection than did mCherry–
cells with
DG ChR2+
engram cells, demonstrating preferential
engram cell–to–engram cell connectivity. This
form of engram pathway–specific connectivity
was unaffected by posttraining administration
of ANI (Fig. 1G).
We next tested the behavioral effect of optogenetically
stimulating engram cells in amnesic
mice (Fig. 2A). During CFC training in context
B, both SAL and ANI groups responded to the
unconditioned stimuli at equivalent levels (fig. S8).
One day after training, the SAL group displayed
robust freezing behavior to the conditioned stimulus
of context B, whereas the ANI group showed
substantially less freezing behavior (Fig. 2C). Two
days after training, mice were placed into the
distinct context A for a 12-min test session consisting
of four 3-min epochs of blue light on or
off. During this test session, neither group showed
freezing behavior during light-off epochs, but both
froze significantly during light-on epochs (Fig. 2D).
Remarkably, no difference in the levels of lightinduced
freezing behavior was observed between
groups. Three days after training, the mice were
again tested in context B in order to assay the
conditioned response, and retrograde amnesia for
the conditioning context was still clearly evident
(Fig. 2E). Subjects treated with SAL or ANI after
the labeling of a neutral contextual engram (no
shock) did not show freezing behavior in response
to light stimulation of engram cells
(Fig. 2D). We replicated the DG retrograde amnesia
experiment using an alternative widely used
protein synthesis inhibitor, cycloheximide (CHM)
SCIENCE sciencemag.org 29 MAY 2015 • VOL 348 ISSUE 6238 1009
Fig. 2. Optogenetic stimulation of DG engram cells restores fear memory in retrograde amnesia.
(A) Behavioral schedule. Beige shading signifies that subjects are on DOX, precluding ChR2 expression.
Mice are taken off DOX 24 to 30 hours before CFC in context B. SAL or ANI was injected into the mice
after training. (B) Habituation to context A with light-off and light-on epochs. Blue light stimulation of
the DG did not cause freezing behavior in naïve, unlabeled mice of the pre-SAL (n = 10 mice) or preANI
(n = 8 mice) groups. (C) Memory recall in context B 1 day after training (test 1). ANI group displayed
significantly less freezing than SAL group (P < 0.005). No-shock groups with SAL (n = 4 mice) or ANI
(n = 4 mice) did not display freezing upon reexposure to context B. (D) Memory recall in context A
2 days after training (engram activation) with light-off and light-on epochs. Freezing for the two light-off
and light-on epochs are further averaged in the inset. Significant freezing due to light stimulation was
observed in both the SAL (P < 0.01) and ANI groups (P < 0.05). Freezing levels did not differ between
groups. SAL and ANI-treated no-shock control groups did not freeze in response to light stimulation of
context B engram cells. (E) Memory recall in context B 3 days after training (test 2). ANI group displayed
significantly less freezing than SAL group (P < 0.05). (F and G) Images showing DG sections from
c-Fos-tTA mice 24 hours after SAL or ANI treatment. (H) ChR2-EYFP cell counts from DG sections
of SAL (n = 3 mice) and ANI (n = 4 mice) groups. (I) In vivo anesthetized recordings (supplementary
materials, materials and methods). (J and K) Light pulses induced spikes in DG neurons recorded from
head-fixed anesthetized c-Fos-tTA mice 24 hours after treatment with either SAL or ANI. Data are
presented as mean T SEM.
RESEARCH | REPORTS
(fig. S9). We examined whether ANI administration
immediately after CFC altered the activitydependent
synthesis of ChR2-EYFP in DG cells
and found that this was not the case (Fig. 2, F to
H). Nevertheless, the dosage of ANI used in this
study did inhibit protein synthesis in the DG, as
shown with Arc+
cell counting (fig. S10). Thus,
the dosage of ANI used was sufficient to induce
amnesia but was insufficient to impair c-FostTA–driven
synthesis of virally delivered ChR2EYFP
in DG cells. Extracellular recordings from
SAL- and ANI-treated mice confirmed the cell
counting results (Fig. 2, I to K). In line with fig.
S6 and previous reports (24), ANI injection 24
hours after CFC did not cause retrograde amnesia
(fig. S11). To provide a negative control for
light-induced memory retrieval in amnesia, we
disrupted memory encoding by activating hM4Di
DREADDs receptors (25) downstream of the DG,
in hippocampal CA1, during CFC, and found that
subsequent DG engram activation did not elicit
memory retrieval (fig. S12).
The recovery from amnesia through the direct
light activation of ANI-treated DG engram
cells was unexpected because these cells showed
neither synaptic potentiation nor increased dendritic
spine density. We conducted additional
behavioral experiments in order to confirm and
characterize the phenomenon. First, we investigated
whether recovery from amnesia can be
demonstrated by means of light-induced optogenetic
place avoidance test (OptoPA); this would
be a measure of an active fear memory recall
(supplementary materials, materials and methods)
(18), rather than a passive fear response
monitored with freezing. SAL and ANI groups
displayed equivalent levels of avoidance of the
target zone in response to light activation of the
DG engram (Fig. 3A). Second, in our previous
study we showed that an application of the standard
protocol (20 Hz) for activation of the CA1
engram was not effective for memory recall (17).
However, we found that a 4-Hz protocol applied
to the CA1 engram of the SAL and ANI groups
elicited similar recovery from amnesia (Fig. 3B).
Third, we used tone fear conditioning (TFC) and
manipulated the fear engram in lateral amygdala
(LA) (26) and found light-induced recovery of
memory from amnesia. Fourth, we asked whether
amnesia caused by disruption of reconsolidation
of a contextual fear memory (27, 28) can
also be recovered through the light-activation of
DG engram cells, which was found to be the
case (Fig. 4A). We applied the memory inception
method (supplementary materials, materials
and methods) (17, 29) to DG engram cells
and found that both SAL and ANI groups showed
freezing behavior that was specific to the original
context A, demonstrating that light-activated context
A engrams formed in the presence of ANI
can function as a conditioned stimulus (CS) in a
context-specific manner (Fig. 4B). Last, we tested
the longevity of CFC amnesic engrams for memory
recovery by means of light activation and
found that memory recall could be observed
8 days after training (fig. S13).
1010 29 MAY 2015 • VOL 348 ISSUE 6238 sciencemag.org SCIENCE
Fig. 3. Recovery of memory from amnesia under a variety of conditions.
(A) DG engram activation and optogenetic place avoidance (OptoPA). During
habituation, neither group displayed significant avoidance of target zone. For
natural recall, the ANI group (n = 10 mice) displayed significantly less freezing
than SAL group (n = 12 mice) in context B (P < 0.005). SAL and ANI groups
displayed similar levels of OptoPA. (B) CA1 engram activation and CFC.
1 day after CFC (test 1), ANI group (n = 9 mice) displayed significantly less
freezing than that of SAL group (n = 10 mice) in context B (P < 0.01). Two
days after training (engram activation), light-activation of CA1 engrams
elicited freezing in both SAL (P < 0.01) and ANI groups (P < 0.001). Three
days after training (test 2), ANI group froze less than did SAL group in context B
(P < 0.01). (C) LA engram activation and TFC. The behavioral schedule was
identical to that in Fig. 3B, except that context tests were replaced with tone tests
in context C (supplementary materials, materials and methods). (Left) Example
image of ChR2-mCherry labeling of LA neurons. Of DAPI cells, 2% were labeled
with ChR2. (Right) One day after training (test 1), ANI group (n = 9 mice) displayed
significantly less freezing to tone than did SAL group (n = 9 mice) (P < 0.05).Two
days after training (engram activation), significant light-induced freezing was observed
for both SAL (P < 0.005) and ANI groups (P < 0.005). Three days after
training (test 2), ANI group froze less to tone than did SAL group (P < 0.05).
RESEARCH | REPORTS
Interactions between the hippocampus and
amygdala are crucial for contextual fear memory
encoding and retrieval (18). c-Fos expression increases
in the hippocampus and amygdala upon
exposure of an animal to conditioned stimuli
(30, 31). These previous observations open up the
possibility of obtaining cellular-level evidence to
support the behavioral-level finding that the
recovery from amnesia can be accomplished with
direct light activation of ANI-treated DG engram
cells. Thus, we compared the effects of natural recall
and light-induced recall on amygdala c-Fos+
cell counts in amnesic mice (Fig. 5, A to C). c-Fos+
cell counts (Fig. 5B) were significantly lower in
basolateral amygdala (BLA) and central amygdala
(CeA) of ANI-treated mice as compared with SAL
mice when natural recall cues were delivered, showing
that amygdala activity correlates with fear
memory expression (Fig. 5C). In contrast, lightinduced
activation of the contextual engram cells
resulted in equivalent amygdala c-Fos+
counts in
SAL and ANI groups (Fig. 5C), which supports
the optogenetic behavioral data.
Next, we modified this protocol in order to
include labeling of CA3 and BLA engram cells
with mCherry and examined the effects of lightinduced
activation of DG engram cells on the
overlap of mCherry+
engram cells and c-Fos+
recall-activated cells in CA3 and BLA (Fig. 5D).
The purpose of this experiment was to investigate
whether there is preferential connectivity between
the upstream engram cells in DG and the downstream
engram cells in CA3 or BLA. Natural recall
cues resulted in above-chance c-Fos+
/mCherry+
overlap in both CA3 and BLA, which supports
the physiological connectivity data (Fig. 5, E to
K). c-Fos+
/mCherry+
overlap was significantly reduced
in the ANI group as compared with the
SAL group but was still higher than chance levels,
presumably reflecting incomplete amnesic
effects of ANI (Fig. 5K). Light-activation of DG engram
cells resulted in equivalent c-Fos+
/mCherry+
overlap as natural cue-induced recall, and this was
unaffected by post-CFC ANI treatment. These
data suggest that there is preferential and protein
synthesis–independent functional connectivity between
DG and CA3 engram cells, which supports
the physiological data (Fig. 1G), and that
this connectivity also applies between DG and
BLA engram cells.
We previously showed that DG cells activated
during CFC training and labeled with ChR2 via
the promoter of an immediate early gene (IEG)
can evoke a freezing response when they are
reactivated optogenetically 1 to 2 days later (16),
and this has since been achieved in the cortex
(21). We have also shown that these DG cells, if
light-activated while receiving an unconditioned
stimulus (US), can serve as a surrogate contextspecific
CS to create a false CS-US association
(17, 18), and that activation of DG or amygdala
engram cells can induce place preference (18).
Furthermore, recent studies showed that optogenetic
inhibition of these cells in DG, CA3, or
CA1 impairs expression of a CFC memory (19, 20).
Together, these findings show that engram cells
activated through CFC training are both sufficient
and necessary to evoke memory recall, satisfying
two crucial attributes in defining a component of
a contextual fear memory engram (15). What has
been left to be demonstrated, however, is that
these DG cells undergo enduring physical changes
as an experience is encoded and its memory is
consolidated. Although synaptic potentiation
has long been suspected as a fundamental mechanism
for memory and as a crucial component of
the enduring physical changes induced by experience,
this has not been directly demonstrated,
SCIENCE sciencemag.org 29 MAY 2015 • VOL 348 ISSUE 6238 1011
Fig. 4. Reconsolidation and memory updating. (A) DG engram activation
and CFC reconsolidation. ANI (n = 11 mice) and SAL (n = 11 mice) groups
showed similar levels of ChR2 labeling. Both groups showed light-induced
freezing behavior 1 day after training (engram activation 1), pre-SAL (P <
0.001), pre-ANI (P < 0.02). Two days after training (test 1), the fear memory
was reactivated by exposure to context B, and SAL or ANI was injected.Three
days after training (test 2), the ANI group froze significantly less than did the
SAL group to context B (P < 0.01). Four days after training (engram activation
2), significant light-induced freezing was observed for the SAL (P < 0.001) and
ANI (P < 0.003) groups. (B) DG inception (supplementary materials,
materials and methods) after contextual memory amnesia. Context-only
engram was labeled for target context A, followed by injection of SAL (n = 11
mice) or ANI (n = 11 mice). Amnesia was demonstrated in the ANI group
through decreased ChR2+
/c-Fos+
colabeling after context A reexposure 1 day
after labeling. After fear inception, neither SAL nor ANI groups displayed
freezing behavior in context B, whereas both groups displayed significant
freezing in context A, with no significant difference between groups. No-light
inception SAL (n = 7 mice) and ANI (n = 6 mice) controls displayed no freezing
to context A or B. Statistical comparisons are performed by using unpaired
t tests; ***P < 0.001. Data are presented as mean T SEM.
RESEARCH | REPORTS
until the current study, as a property of the engram
cells. Our data have directly linked the
optogenetically and behaviorally defined memory
engram cells to synaptic plasticity.
On the basis of a large volume of previous
studies, (1–3, 7, 8, 32–34), a concept has emerged
in which retrograde amnesia arises from consolidation
failure as a result of disrupting the
process that converts a fragile memory engram,
formed during the encoding phase, into a stable
engram with persistently augmented synaptic
strength and spine density. Indeed, our current
study has demonstrated that amnesic engram
cells in the DG 1 day after CFC training display
low levels of synaptic strength and spine density
that are indistinguishable from nonengram
cells of the same DG. This correlated with a lack
of memory recall elicited by contextual cues.
1012 29 MAY 2015 • VOL 348 ISSUE 6238 sciencemag.org SCIENCE
Fig. 5. Amygdala activation and functional connectivity
in amnesia through light activation of
DG engram. (A) Schedule for cell-counting experiments.
Mice were either given a natural recall session
in context B or a light-induced recall session in
context A. Mice were perfused 1 hour after recall.
(B) Representative image showing c-Fos expression
in the BLA and CeA. (C) c-Fos+
cell counts in
the BLA and CeA of mice after natural or lightinduced
recall (n = 3 or 4 mice per group). (D)
Schedule for cell-counting experiments. c-Fos-tTA
mice with AAV9-TRE-ChR2-EYFP injected into the
DG and AAV9-TRE-mCherry injected into both CA3
and BLA were fear-conditioned off DOX and 1 day
later were given a natural recall session in context
B or a light-induced recall session in context A.
Mice were perfused 1 hour after recall. (E to G)
Representative images showing mCherry engram
cell labeling, c-Fos expression, and mCherry+
/c-Fos+
overlap in CA3. (H to J) Representative images
showing mCherry engram cell labeling, c-Fos expression,
and mCherry/c-Fos overlap in BLA. (K)
c-Fos+
/mCherry+
overlap cell counts in CA3 and
BLA of mice after natural or light-induced recall
(n = 3 or 4 mice per group). Chance levels were
estimated at 0.76 (CA3) and 0.42 (BLA). Data
are presented as mean T SEM. Statistical comparison
are performed by using unpaired t tests;
*P < 0.05, **P < 0.01.
RESEARCH | REPORTS
However, direct activation of DG engram cells of
the ANI group elicited as much freezing behavior
as did the activation of these cells of the SAL
group. This unexpected finding is supported by
a set of additional cellular and behavioral experiments.
Whereas amygdala engram cell reactivation
upon exposure to the conditioned context is
significantly lower in the ANI group as compared
with the SAL group, optogenetic activation of DG
engram cells results in normal reactivation of
downstream CA3 and BLA engram cells (Fig. 5).
At the behavioral level, the amnesia rescue was
observed under a variety of different conditions
in which one or more parameters were altered
(Figs. 2 and 3 and figs. S9 and S13). Thus, our
overall findings indicate that memory engrams
survive a posttraining administration of protein
synthesis inhibitors during the consolidation
window and that the memory remains retrievable
by means of ChR2-mediated direct engram
activation even after retrograde amnesia is induced.
The drive initiated with light-activation
of one component of a distributed memory engram
(such as that in the DG) is sufficient to reactivate
engrams in downstream regions (such
as that in CA3 and BLA) that would also be affected
by the systemic injection of a protein synthesis
inhibitor (ANI).
These findings suggest that although a rapid
increase of synaptic strength is likely to be crucial
during the encoding phase, the augmented
synaptic strength is not a crucial component of
the stored memory (35–37). This perspective is
consistent with a recent study showing that an
artificial memory could be reversibly disrupted
by depression of synaptic strength (38). On the
other hand, persistent and specific connectivity
of engram cells that we find between DG engram
cells and downstream CA3 or BLA engram
cells in both SAL and ANI groups may represent
a fundamental mechanism of memory information
storage (39). Our findings also suggest that
the primary role of augmented synaptic strength
during and after the consolidation phase may be
to provide natural recall cues with efficient access
to the soma of engram cells for their reactivation
and, hence, recall.
The integrative memory engram-based approach
used here for parsing memory and amnesia
into encoding, consolidation, and retrieval
aspects may be of wider use to other experimental
and clinical cases of amnesia, such as Alzheimer’s
disease (40).
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ACKNOWLEDGMENTS
We thank X. Liu and B. Roth for sharing reagents; X. Zhou,
Y. Wang, W. Yu, S. Huang, and T. O’Connor for technical assistance;
J. Z. Young for proofreading; and other members of the Tonegawa
Laboratory for their comments and support. This work was supported
by the RIKEN Brain Science Institute, Howard Hughes Medical
Institute, and the JPB Foundation (to S.T.). pAAV-TRE-ChR2-EYFP,
pAAV-TRE-ChR2-mCherry, and pAAV-TRE-mCherry were developed
by X.L., in the group of S.T., at the Massachusetts Institute of
Technology; therefore, a materials transfer agreement (MTA) is
required to obtain these virus plasmids.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/348/6238/1007/suppl/DC1
Materials and Methods
Figs. S1 to S13
Reference (41)
22 December 2014; accepted 30 April 2015
10.1126/science.aaa5542
COGNITIVE NEUROSCIENCE
Unlearning implicit social biases
during sleep
Xiaoqing Hu,1,2
James W. Antony,1,3
Jessica D. Creery,1
Iliana M. Vargas,1
Galen V. Bodenhausen,1
Ken A. Paller1
*
Although people may endorse egalitarianism and tolerance, social biases can remain
operative and drive harmful actions in an unconscious manner. Here, we investigated
training to reduce implicit racial and gender bias. Forty participants processed
counterstereotype information paired with one sound for each type of bias. Biases were
reduced immediately after training. During subsequent slow-wave sleep, one sound was
unobtrusively presented to each participant, repeatedly, to reactivate one type of training.
Corresponding bias reductions were fortified in comparison with the social bias not
externally reactivated during sleep. This advantage remained 1 week later, the magnitude
of which was associated with time in slow-wave and rapid-eye-movement sleep after
training. We conclude that memory reactivation during sleep enhances counterstereotype
training and that maintaining a bias reduction is sleep-dependent.
S
ocial interactions are often fraught with
bias. Our preconceptions about other people
can influence many types of behavior.
For example, documented policing errors
have repeatedly shown the potential harm
of racial profiling (1). In experiments that used a
first-person-shooter videogame, both White and
Black participants were more likely to shoot Black
than White individuals, even when they held a
harmless object rather than a gun (2). When hiring
potential research assistants, both male and female
faculty members were more likely to hire male
than equally qualified female candidates (3).
Although the tendency for people to endorse
racist or sexist attitudes explicitly has decreased
in recent years (4), social biases may nevertheless
influence people’s behavior in an implicit or unconscious
manner, regardless of their intentions
or efforts to avoid bias (5). Ample evidence indicates
that implicit biases can drive discriminatory
behaviors and exacerbate intergroup conflict
(5–8). For instance, implicit racial biases decrease
investments given to racial out-group members
SCIENCE sciencemag.org 29 MAY 2015 • VOL 348 ISSUE 6238 1013
1
Department of Psychology, Northwestern University,
Evanston, IL 60208, USA. 2
Department of Psychology,
University of Texas at Austin, Austin, TX 78712, USA.
3
Princeton Neuroscience Institute, Princeton University,
Princeton, NJ 08544, USA.
*Corresponding author. E-mail: kap@northwestern.edu
RESEARCH | REPORTS
DOI: 10.1126/science.aaa5542
, 1007 (2015);348Science
et al.Tomás J. Ryan
Engram cells retain memory under retrograde amnesia
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