Selective Erasure of a Fear Memory
Jin-Hee Han, et al.
Science 323, 1492 (2009);
DOI: 10.1126/science.1164139
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The following resources related to this article are available online at www.sciencemag.org(this information is current as of May 3, 2010 ):treated mice, Foxp3+ T cells still lost Foxp3 expression but did not gain expression of CXCR5,
CD40L, and PD-1 (Fig. 4C). Consistent withthese observations, blocking CD40-CD40L interactions resulted in a reduction of GC formation in
PPs and of B220– IgA+ plasma cells in the LP(fig. S5, A and B). Thus, the differentiation of
Foxp3+ T cells into TFH cells requires B cells andCD40 expression, presumably by either B cells
or DCs or both.
Foxp3+ T cells were converted into TFH cells
only in PPs; neither TFH cells nor GCs could be
detected in spleen or LNs of CD3e–/– mice adoptively transferred with Foxp3+ T cells (Fig. 4, D
and E, and fig. S6). Furthermore, immunization
with sheep red blood cells (SRBCs) in the presenceof bacterial components failed to induce GCs in
spleen of CD3e–/– mice that received Foxp3+ T
cells. In contrast, control mice and CD3e–/– micethat had received Foxp3– T cells generated GCs
in the spleen after SRBC immunization (Fig. 4, D
and E). Thus, the precursors of PP TFH cells areenriched in the Foxp3+ T cell population, whereas other T cells, like Foxp3– T cells, can differentiate into TFH cells in the spleen on experimental
systemic immunization.
Our studies demonstrate that Foxp3+ T cells
in PPs can differentiate efficiently into cells withcharacteristics of TFH cells, which then participate in the induction of GCs and IgA synthesis in
the gut. How can we then explain the preferential
generation of PP TFH cells from the peripheral
Foxp3+ T cell population? Two possible scenarios
can be conceived. First, TFH cell differentiation
per se may require a Foxp3-dependent molecular
program. This is unlikely, however, because adoptively transferred Foxp3– Tcells efficiently generated

TFH cells in the spleen on immunization with SRBCs
and because scurfy T cells, which contain amutated Foxp3 gene, could generate TFH-phenotype
cells in PPs, albeit less efficiently than control T
cells (fig. S7). A more likely scenario is that PP
TFH cell differentiation may be controlled by thesame signals that promote Foxp3 expression in
gut T cells (27–30), such as antigen recognition
through the T cell receptor (TCR). Consistent
with this idea, adoptive transfer of ovalbuminspecific OT-II TCR transgenic RAG-2–/– CD4+ T
cells into CD3e–/– hosts led to generation of
Foxp3+ T cells in the gut LP and TFH cells in the
PPs if mice were fed ovalbumin (fig. S8). These
data suggest that, depending on the environment,
TCR stimulation induces either IL-10–producing“suppressor” or IL-21–producing “helper” T cells(fig. S9). Despite the presence of the same
antigens that previously induced Foxp3 expression, the IL-6–, IL-21–, and activated B cell–richenvironment of PPs results in many Foxp3+ T
cells differentiating into TFH cells. These studies
have implications for how the suppression of inflammatory reactions and induction of IgA synthesis occur in the gut.
References and Notes
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(2003).

Selective Erasure of a Fear Memory
Jin-Hee Han,1,2,3 Steven A. Kushner,1,4 Adelaide P. Yiu,1,2 Hwa-Lin (Liz) Hsiang,1,2Thorsten Buch,5 Ari Waisman,6 Bruno Bontempi,7 Rachael L. Neve,8Paul W. Frankland,1,2,3 Sheena A. Josselyn1,2,3*
Memories are thought to be encoded by sparsely distributed groups of neurons. However,identifying the precise neurons supporting a given memory (the memory trace) has been along-standing challenge. We have shown previously that lateral amygdala (LA) neurons withincreased cyclic adenosine monophosphate response element–binding protein (CREB) are
preferentially activated by fear memory expression, which suggests that they are selectively
recruited into the memory trace. We used an inducible diphtheria-toxin strategy to specificallyablate these neurons. Selectively deleting neurons overexpressing CREB (but nota similar portion of random LA neurons) after learning blocked expression of that fear
memory. The resulting memory loss was robust and persistent, which suggests that the memory
was permanently erased. These results establish a causal link between a specific neuronal
subpopulation and memory expression, thereby identifying critical neurons within
the memory trace.
nsembles of neurons are thought to serveas the physical representation of memory(the memory trace) (1). However, identifying the precise neurons that constitute a memory trace is challenging because these neuronalensembles are likely sparsely distributed (2).

E

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Previous studies detected neurons whose activity is correlated with memory encoding, expression, or both (3–7). However, correlative studies
do not address whether these neurons are essential components of the memory trace. A direct
test of this requires specifically disrupting only

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9. S. Floess et al., PLoS Biol. 5, e38 (2007).
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K. M. Murphy, Immunity 24, 677 (2006).
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J. Exp. Med. 204, 1765 (2007).
30. C. M. Sun et al., J. Exp. Med. 204, 1775 (2007).
31. We thank Y. Doi, A. Yagisawa, T. Miyao, and H. Fujimoto
for technical assistance; B. Malissen and K. Itoh for mice;
D. Littman and A. Coutinho for inspiring discussions;
I. Taniuchi for discussion and for critically reading of
the manuscript. This work is supported in part by
Grants-in-Aid for Scientific Research in Priority Areas(S.F., S.H.) and for Young Scientists (A) (S.H.), and by
the Uehara Memorial Foundation (S.H.). S.H. and S.F.
contributed as senior authors.

Supporting Online Materialwww.sciencemag.org/cgi/content/full/323/5920/1488/DC1
Materials and Methods
Figs. S1 to S9References
1 December 2008; accepted 22 January 200910.1126/science.1169152

those activated neurons and determining whether
subsequent memory expression is blocked. Establishing this causal role has been difficult, because of the limited ability of current techniques
to target a specific subset of neurons within abrain region.
To target neurons whose activity is correlatedwith memory, we took advantage of our recent
findings that LA neurons with relatively increased
levels of the transcription factor CREB werepreferentially activated by auditory fear memory
1
Program in Neurosciences and Mental Health, Hospital for
Sick Children, 555 University Avenue, Toronto, ON, M5G
1X8, Canada. 2Institute of Medical Sciences, University ofToronto, Toronto, ON, M5G 1X8, Canada. 3Department of
Physiology, University of Toronto, Toronto, ON, M5G 1X8,
Canada. 4Department of Psychiatry, Erasmus University Medical Center, 3015 CE Rotterdam, Netherlands. 5Departmentof Pathology, University of Zurich, CH-8057 Zurich, Switzerland.
6
I.Medizinische Klinik und Poliklinik, Johannes GutenbergUniversität Mainz, 55131 Mainz, Germany. 7Centre deNeurosciences Intégratives et Cognitives, CNRS UMR 5228
and University of Bordeaux 1, 33405 Talence, France. 8Department of Brain and Cognitive Sciences, Massachusetts Instituteof Technology, Cambridge, MA 02139, USA.

*To whom correspondence should be addressed. E-mail:sheena.josselyn@sickkids.ca

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training or testing (8). To manipulate CREB levels
in a subpopulation (roughly 15%) of LA neurons,a region critical for auditory fear memory (9–12),
we used replication-defective herpes simplex
viral (HSV) vectors (13). Neurons overexpressing CREB (with CREB vector) were three times
as likely to be activated as their noninfectedneighbors after fear memory training or testingin wild-type (WT) mice and 10 times as likely
to be activated in CREB-deficient mice. Conversely, WT neurons with a dominant-negative
CREB vector were 1/12 as likely as their neighbors to be activated by fear training or testing.
These findings suggest that neurons with relatively higher CREB function are preferentially
recruited into the fear memory trace and that

Fig. 1. Selective ablation of LA neurons. (A) iDTR mice express DTR under control of a floxed STOP
cassette (no DTR expression, indicated by white). CREB-cre or Cntrl-cre microinjected into LA(green). Cre removes STOP cassette, which allows DTR expression (pink). DT (blue triangles) induces
apoptosis (red) only in cells that have undergone recombination. (B) (Left) Low GFP (with cre
vector, green) and high activated caspase-3 (aCas3, red) levels in experimental (iDTR/CREB-cre/DT,
iDTR/Cntrl-cre/DT), but not control (WT/CREB-cre/DT), mice. DAPI (4′,6′-diamidino-2-phenylindole)–
stained nuclei (blue). Scale bar, 100 mm. (Right) High activated caspase-3 levels in experimental[iDTR/CREB-cre/DT (n = 24), iDTR/Cntrl-cre/DT (n = 17)], but not control [WT/CREB-cre/DT (n = 4)],mice (F2,42 = 6.44, P < 0.001). (C) (Left) High TUNEL (red) levels in experimental (iDTR/CREB-cre/DT, iDTR/Cntrl-cre/DT), but not control (iDTR/CREB-cre/PBS), mice. Scale bar, 100 mm. (Right, top)Morphological indicators of apoptosis in experimental mice. Scale bar, 50 mm. (Right, bottom) HighTUNEL levels in LA of experimental mice [iDTR/CREB-cre/DT (n = 5), iDTR/Cntrl-cre/DT (n = 6)
versus control (n = 5); F2,13 = 22.31, P < 0.001].
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posttraining ablation of just these neurons
should disrupt expression of the establishedfear memory.
To ablate neurons overexpressing CREB in thepresent study, we used transgenic mice in whichcell death may be induced in a temporally andspatially restricted manner. Apoptosis is induced
after diphtheria toxin (DT) binds to the DT receptor (DTR) (14). Because mice do not express
functional DTRs (15, 16), we used transgenic micethat express simian DTR in a Cre-recombinase(cre)–inducible manner (iDTR mice) (15). A loxPflanked STOP cassette that normally silences
DTR expression is excised by cre, which allows
constitutive expression of DTR. Injection of DT
any time thereafter induces apoptosis only incells expressing DTR.
We restricted DT-induced ablation to neurons overexpressing CREB by inserting cDNA
encoding cre recombinase into our CREB vector(CREB-cre). DTR expression only occurs in neurons expressing cre, which allowed us to persistently tag infected neurons for subsequent ablation(Fig. 1A and fig. S1). As a control, we used Cntrlcre vector to induce apoptosis in a similar portionof LA neurons that are not preferentially activated by fear testing. To examine cell death, weused two markers of apoptosis, caspase-3 activation and terminal deoxynucleotidyl transferase–
mediated deoxyuridine triphosphate nick endlabeling (TUNEL) (17). We microinjected CREBcre, Cntrl-cre, and CREB alone (without cre)
vectors into the LA of iDTR transgenic and WT
littermate mice, then administered DT or vehicle. We observed substantial cell death only in
LA neurons of iDTR mice microinjected with
CREB-cre or Cntrl-cre vectors and administered DT (experimental groups, Fig. 1, B andC, and fig. S2) (15). Negligible apoptosis wasobserved in regions outside the LA (figs. S3and S4) or in control groups that lacked a key
component (iDTR, cre or DT) (Fig. 1, B and
C). Note that Cntrl-cre and CREB-cre vectors
produced equal levels of cell death (Fig. 1, B
and C); however, the efficiency of cell death
was not complete (see SOM text). Therefore,
this system allows temporally specific ablationof tagged neurons.
To verify that LA neurons overexpressing
CREB are selectivity activated by fear memory
testing with our modified vector, we microinjected WT mice with CREB-cre or Cntrl-cre
vector before auditory fear training. To visualize
neurons specifically activated by memory expression, we examined Arc (activity-regulated
cytoskeleton-associated protein; Arg3.1) RNA(18). Neuronal activity induces a rapid, but transient, burst of Arc RNA that is quickly transported
to the cytoplasm, which allows nuclear-localized
Arc RNA to serve as a molecular signature of a
recently (5 to 15 min previously) active neuron(18). Five minutes after testing, we removed brains
and examined Arc (activated by fear testing) andgreen fluorescent protein (GFP) (with vector)
RNA. Neurons with CREB-cre vector preferentially

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expressed Arc after memory testing; neurons with
CREB-cre were three times as likely to be activated by fear memory testing as their noninfectedneighbors. In contrast, neurons with Cntrl-cre
vector and their noninfected neighbors wereequally likely to be activated by memory testing(Fig. 2).
We microinjected CREB-cre vector into the
LA of iDTR mice before weak auditory feartraining. Cntrl-cre vector was used to ablate asimilar portion of random LA neurons (i.e., notpreferentially activated by fear memory testing).
We assessed memory before (test 1) and after(test 2) inducing cell death in tagged neurons
by administering DT. The CREB-cre vector
enhanced fear memory following weak training (Fig. 3, test 1), consistent with previous
results (8, 19–22). Selectively deleting neurons with CREB-cre vector completely reversed
this enhancement (test 2). Note that CREBenhanced memory was not blocked if either
cre or DT was omitted, consistent with the absence of apoptosis in these control groups. The
reversal of CREB-enhanced memory was notdue to memory extinction with repeated testing
because the control groups froze robustly on test
2. Therefore, increasing CREB in a subpopulation of LA neurons enhances a weak memoryand specifically ablating just these neurons reverses this enhancement.
Although increasing CREB in a subpopulation of LA neurons does not further enhancea strong memory, neurons overexpressing CREB
are, nevertheless, preferentially activated by fear
memory expression (8). This suggests that CREB
levels dictate which neurons are recruited intoa memory trace, even in the absence of behavioral change. To examine the effects of ablatingneurons overexpressing CREB on a strong
memory, we trained mice that received CREBcre or Cntrl-cre microinjections using an intense
protocol. Strong training produced robust auditory fear memory in both groups before DT
administration (Fig. 4A). After DT, only CREBcre mice showed a loss of auditory fear memory.
To investigate whether memory in mice microinjected with CREB-cre is particularly susceptible to the ablation of a small number of neurons,
we microinjected both Cntrl-cre and CREB (nocre) vectors, which allowed us to delete only
Cntrl-cre neurons after training. Deleting thissmall portion of neurons (that were not overexpressing CREB) had no effect on memory (fig.
S5). Therefore, memory loss was specific; it was
not determined by the absolute number of deleted LA neurons but by whether these deleted
neurons overexpressed CREB at the time oftraining.
Administering DT after a fear memory test
blocked expression of both CREB-enhanced
memory produced by weak training and robust
memory produced by strong training in iDTR
mice microinjected with CREB-cre (but not
Cntrl-cre) vector. Fear memory testing reactivates memory and may trigger a second wave of

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consolidation (reconsolidation) that, similar toinitial consolidation, requires protein synthesis(23, 24). Because DT induces cell death by inhibiting protein synthesis, it is possible that impaired reconsolidation contributes to the memory
loss. To assess this, we trained mice but omitted
the memory reactivation induced by test 1. Consistent with our previous results, fear memory
was blocked in CREB-cre, but not Cntrl-cre,mice (Fig. 4B). Therefore, memory loss was independent of memory reactivation, which ruledout the possibility that blocking reconsolidationaccounts for the memory disruption.
If neurons overexpressing CREB during training are critically involved in the subsequent memory trace, then deleting them should permanently
block memory expression. To examine the persistence of memory loss, we trained mice, administered DT, and assessed memory 2, 5, and 12days later. Memory loss in CREB-cre mice waslong-lasting, whereas memory remained robustin Cntrl-cre mice (Fig. 4C). Therefore, we foundno evidence of memory recovery in mice inwhich neurons overexpressing CREB were deleted, which suggested that memory was nottransiently suppressed. To rule out the possibility that the memory loss was due to a nonspecific impairment in LA function, we showed that
CREB-cre mice could relearn (Fig. 4C). Similarly, pretraining deletion of neurons overexpressing CREB did not impair the acquisition, or
stability, of a conditioned fear memory (fig. S6).
Deleting neurons overexpressing CREB does notaffect subsequent learning, presumably because
the high portion of remaining (noninfected) neurons are sufficient to encode a new memory.
Finally, ablating CREB-overexpressing neurons did not block expression of a memory acquired before surgery (fig. S8). Together, thesefindings indicate that ablating neurons thatwere overexpressing CREB at the time of memory encoding blocks memory for that particularlearning event, highlighting the specificity ofmemory loss.

Fig. 2. Neurons overexpressing CREB preferentially activated by fear memory testing.
(A) Double-labeled nuclei in
LA of CREB-cre, but not Cntrlcre, mice. Nuclei (blue), GFP+
(with CREB-cre or Cntrl-cre
vector, green), Arc+ (pink), or
double-labeled nuclei (GFP+
and Arc+; arrows). Scale bar,
20 mm. (B) In CREB-cre mice, Arc was preferentially localized in infected (GFP+), rather than noninfected (GFP–), neurons. In Cntrl-cre mice, Arc was equally distributed in infected and noninfected neurons(Vector × GFP/Arc colocalization interaction, F1,5 = 18.74, P < 0.05).

Fig. 3. Overexpressing CREB in LA neurons enhances memory induced by weak training; subsequent
ablation of these neurons reverses this enhancement. (Top) CREB-cre microinjection enhanced memory
after weak training [test 1, CREB-cre/DT (n = 8), Cntrl-cre/DT (n = 9), P < 0.001]. DT administration
reversed this memory enhancement (test 2, P < 0.001). CREB-enhanced memory was not blocked on
test 2 if either cre [CREB/DT (n = 7), P > 0.05] or DT [CREB-cre/PBS (n = 6), P > 0.05] was omitted.
Group × Test interaction F3,26 = 13.90, P < 0.001. (Bottom) Schematic of LA neurons after DT or PBS.
Blue, DAPI-labeled neuronal nuclei; pink, neurons activated by memory; and white, ablated neurons.

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REPORTSOur results show that neurons with increased
CREB levels at the time of fear learning are critical to the stability of that memory, because selectively ablating these neurons after training

blocks expression of this fear memory. This indicates that these neurons themselves are essential for memory expression in the days after fear
conditioning; they are not simply creating a local

environment that promotes memory formation(such as releasing trophic factors) (25). Deleting
neurons whose activity is related to memory expression (overexpressing CREB at the time oftraining) produced memory loss, whereas ablating a similar number of random neurons (expressing Cntrl vector or overexpressing CREB
well before or after training) did not. The observed amnesia was specific, robust, persistentand not due to a disruption in either reconsolidation or overall LA function. Together, these results suggest that ablating neurons overexpressing
CREB permanently erases the fear memory (seesupporting online material text). Fear learning
may generate a broad memory trace that encompasses more LA neurons than affected by ourtreatment or multiple memory traces throughout
the brain (26, 27). However, deleting just neurons overexpressing CREB at the time of training produces amnesia, which suggests that theseneurons play an essential role within what is
likely a broader fear neuronal network. These
results establish a causal link between a defined
subpopulation of neurons and expression of afear memory and, thereby, identify a key component of the memory trace.
References and Notes

Fig. 4. Specifically ablating LA neurons overexpressing CREB, but not a similar portion of random
neurons, blocks expression of strong memory. (A) Strong training produced equally robust and specificauditory fear memory (low baseline and high tone freezing) in CREB-cre (n = 6) and Cntrl-cre (n = 6)
mice on test 1. Although Cntrl-cre mice showed robust memory after DT administration (test 2), CREBcre mice showed impaired memory (low baseline and low tone freezing) (Vector × Test × Timesignificant three-way interaction, F1,10 = 5.55, P < 0.05). (B) Memory loss was not due to disruption ofreconsolidation. Cntrl-cre (n = 5) mice showed robust auditory fear memory (low baseline but high tone
freezing), whereas CREB-cre (n = 6) mice showed a loss of auditory fear memory (low baseline and tone
freezing) (Vector × Time interaction, F1,10 = 6.20, P < 0.05). (C) Memory loss was persistent in CREB-cre
mice, but they could relearn. Over repeated tests (tests 1 to 3), CREB-cre mice (n = 10) showed stable,
low tone freezing, whereas Cntrl-cre mice (n = 7) showed robust tone freezing [Vector × Test analysis ofvariance (ANOVA), significant effect of Vector F1,15 = 67.81, P < 0.05 only]. (Right) After retraining
CREB-cre mice showed an increase in tone freezing (F1,15 = 1.7, P > 0.05).
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grant (06-NEURO-027-03) to B.B., and Restracomp
Fellowships from the Hospital for Sick Children to
A.P.Y. and J.H.H.

Supporting Online Materialwww.sciencemag.org/cgi/content/full/323/5920/1492/DC1
Materials and Methods

Human Substantia Nigra
Neurons Encode Unexpected
Financial Rewards
Kareem A. Zaghloul,1* Justin A. Blanco,2 Christoph T. Weidemann,3 Kathryn McGill,1Jurg L. Jaggi,1 Gordon H. Baltuch,1 Michael J. Kahana3*
The brain’s sensitivity to unexpected outcomes plays a fundamental role in an organism’s ability
to adapt and learn new behaviors. Emerging research suggests that midbrain dopaminergic
neurons encode these unexpected outcomes. We used microelectrode recordings during deep brain
stimulation surgery to study neuronal activity in the human substantia nigra (SN) while patientswith Parkinson’s disease engaged in a probabilistic learning task motivated by virtual financial
rewards. Based on a model of the participants’ expected reward, we divided trial outcomes into
expected and unexpected gains and losses. SN neurons exhibited significantly higher firing rates
after unexpected gains than unexpected losses. No such differences were observed after
expected gains and losses. This result provides critical support for the hypothesized role of the
SN in human reinforcement learning.
heories of conditioning and reinforcement learning postulate that unexpected
rewards play an important role in allowing an organism to adapt and learn new behaviors (1, 2). Research on nonhuman primatessuggests that midbrain dopaminergic neurons
projecting from the ventral tegmental area andthe pars compacta region of the SN encode
unexpected reward signals that drive learning(3–6). These dopaminergic neurons are phasi-

T

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Figs. S1 to S9References
Movie S1

cally activated in response to unexpected rewards
and depressed after the unexpected omission of
reward (7–9), and they are major inputs to alarger basal ganglia circuit that has been implicated in reinforcement learning across species(10–15).
The response of these neurons to rewards
has not been directly measured in humans. We
recorded neuronal activity in human SN whilepatients undergoing deep brain stimulation (DBS)

4 August 2008; accepted 15 January 200910.1126/science.1164139

surgery for Parkinson’s disease performed a probability learning task. Patients with Parkinson’s
disease show impaired learning from positive andnegative feedback in cognitive tasks (16–18),
probably because of the degenerative nature of
their disease and the decreased number of dopaminergic neurons capable of mounting phasicchanges in activity in response to reward signals(17–19). We sought to capture remaining viabledopaminergic SN cells in our patients and determine whether they exhibit responses modulated by reward expectation.
We used microelectrode recordings to measure
intraoperative activity of SN in 10 Parkinson’s
patients (6 men, 4 women, mean age of 61 years)
undergoing DBS surgery of the subthalamic nucleus (STN) while they engaged in a probability learning task. We rewarded participants inthe task with virtual financial gains to motivate
learning. We identified SN by anatomic location and its unique firing pattern (Fig. 1A) (20).
The learning task involved choosing between a
red and a blue deck of cards presented on a computer screen (Fig. 1B). We informed partici1
Department of Neurosurgery, University of Pennsylvania,
Philadelphia, PA 19104, USA. 2Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
3
Department of Psychology, University of Pennsylvania, Philadelphia, PA 19104, USA.

*To whom correspondence should be addressed. E-mail:zaghlouk@uphs.upenn.edu (K.A.Z.); kahana@psych.upenn.
edu (M.J.K.)

Fig. 1. (A) Intraoperative plan for DBS
surgery with targeting of the STN. Microelectrodes are advanced along a
tract through the anterior thalamic
nuclei (Th), zona incerta (ZI), STN, andinto the SN to record neural activity.
Each anatomical region is identified
by surgical navigation maps overlayedwith a standard brain atlas (top) andby its unique firing pattern and microelectrode position (bottom). Depth measurements on the right of the screen
begin 15 mm above the pre-operatively
identified target, the inferior border ofSTN. In this example, the microelectrodetip lays 0.19 mm below the target. A,
anterior; P, posterior. (B) Probability
learning task. Participants are presented
with two decks of cards on a computer
screen. They are instructed to repeatedly
draw cards from either deck to determine which deck yields the higher
reward probability. Participants are given up to four seconds for each draw. After each draw, positive or negative feedback is presented for two seconds. Decks
are then immediately presented on the screen for the next choice.

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28. We thank T. Pekar, J. Park, and J. Guo for technicalassistance. Supported by Canadian Institutes of HealthResearch (MOP74650, MOP77561) and EJLB Foundation
grants to S.A.J. and P.W.F., National Alliance for
Research on Schizophrenia and Depression fellowship to
J.H.H., travel fellowship (France-Canada Research
Foundation), Agence Nationale de la Recherche (France)