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Tracking the Fear Engram: The Lateral Amygdala Is an Essential Locus of Fear Memory Storage Brief Communication
Tracking the Fear Engram: The Lateral Amygdala Is an
Essential Locus of Fear Memory Storage
Glenn E. Schafe,
1
Vale´rie Doye`re,
2,3
and Joseph E. LeDoux
3
1
Department of Psychology and Interdepartmental Neuroscience Program, Yale University, New Haven, Connecticut 06520,
2
Laboratoire de Neurobiologic
de lApprentissage, de la Me´moire, et de la Communication, Unite´ Mixte de Recherche 8620, Centre National de la Recherche Scientifique-Universite´
Paris-Sud, 91405 Orsay, France, and
3
Center for Neural Science, New York University, New York, New York 10003
Although it is believed that different types of memories are localized in discreet regions of the brain, concrete experimental evidence of the
existence of such engrams is often elusive. Despite being one of the best characterized memory systems of the brain, the question of where
fear memories are localized in the brain remains a hotly debated issue. Here, we combine site-specific behavioral pharmacology with
multisite electrophysiological recording techniques to show that the lateral nucleus of the amygdala, long thought to be critical for the
acquisition of fear memories, is also an essential locus of fear memory storage.
Key words: fear conditioning; memory consolidation; amygdala; synaptic plasticity; rat; MAP kinase
Introduction
The fear-learning system of the brain has generated enormous
interest, attributable in part to its attractiveness as a neurobiolog-
ical model of learning and memory and also to its potential clin-
ical significance (Davis, 1992; LeDoux, 2000). Despite years of
progress, however, there is still no universal consensus regarding
the locus of fear memory storage (Cahill et al., 1999; Fanselow
and LeDoux, 1999; Pare´, 2002).
Most work on the neural system of fear learning has involved
studies of auditory pavlovian fear conditioning (Fanselow and
LeDoux, 1999; LeDoux, 2000), a learning paradigm in which an
emotionally neutral auditory conditioned stimulus (CS) comes
to elicit fear after it is paired with an aversive unconditioned
stimulus (US). This research has led to the view that the lateral
nucleus of the amygdala (LA) is a key site of plasticity underlying
fear learning (LeDoux, 2000; Blair et al., 2001; Maren and Quirk,
2004). Several independent lines of evidence have supported this
view. These have included electrophysiological recording studies
showing that CS and US pathways converge onto single cells in
the LA and that paired presentations of the CS and US enhance
the response to the CS in LA cells (Romanski et al., 1993; Quirk et
al., 1995; Rogan et al., 1997), lesion studies showing that damage
to or inactivation of the LA prevents fear conditioning (LeDoux,
2000), and pharmacological studies showing that blockade of
intracellular signaling cascades in the LA that are essential for
either short- or long-term synaptic plasticity impairs short-term
memory (STM) and long-term memory (LTM) of fear condi-
tioning, respectively (Rodrigues et al., 2004).
An alternative view argues that the LA is not a site of fear
memory storage and offers alternative interpretations of each of
the aforementioned findings (Cahill et al., 1999). For example, it
has been pointed out that the LA is not unique but rather one of
many regions of the wider fear network to exhibit neurophysio-
logical changes during and after fear conditioning. Indeed, audi-
tory fear conditioning induces alterations in the activity of neu-
rons not only in the LA but also in the auditory cortex (Bakin and
Weinberger, 1990; Edeline and Weinberger 1993) and the audi-
tory thalamus (Gabriel et al., 1975; Lennartz and Weinberger,
1992). These findings suggest the possibility that the neurophys-
iological changes observed in the LA during and after fear condi-
tioning might merely be a reflection of plastic changes that have
occurred in these upstream regions rather than representing a
neural signature of memory storage in the LA itself (Cahill et al.,
1999). Furthermore, rather than indicating that the LA is a site of
storage of fear memories, it has been suggested that memory
deficits observed after lesion and pharmacological manipulations
may instead indicate that the LA is essential for triggering or
modulating the strength of plasticity and memory storage in
other regions of the wider fear network (Cahill et al., 1999).
It has become clear that lesion, electrophysiological, or phar-
macological methods alone are not sufficient to identify the locus
of fear memory storage. In the present study, we have therefore
taken a new approach to tracking the fear engram by combining
a site-specific pharmacological manipulation of the LA that tar-
gets memory consolidation processes with simultaneous multi-
site electrophysiological recordings in different regions of the fear
memory system.
Materials and Methods
Subjects. Adult male Sprague Dawley rats (Hilltop Laboratories, Philadel-
phia, PA) were housed individually with ad libitum food and water on a
Received July 1, 2005; revised September 17, 2005; accepted September 18, 2005.
This work was supported by National Institute of Mental Health (NIMH) Grants MH46516, MH00956, MH39774,
MH11902, and MH570161 (J.E.L.), Centre National de la Recherche Scientifique/NSF Cooperative Grant 17089 (V.D.
and J.E.L.), NIMH Grant MH62519 (G.E.S.), and by a grant from the W. M. Keck Foundation to New York University.
We thank Jeffrey Erlich, Elizabeth Bauer, Tad Blair, and Jean-Marc Edeline for helpful discussions about this manu-
script and Yu Zhou for assistance with making electrodes.
Correspondence should be addressed to Dr. Glenn E. Schafe, Department of Psychology, Yale University, 2 Hill-
house Avenue, Box 208205, New Haven, CT 06520. E-mail: glenn.schafe@yale.edu.
DOI:10.1523/JNEUROSCI.3307-05.2005
Copyright © 2005 Society for Neuroscience 0270-6474/05/2510010-06$15.00/0
10010 The Journal of Neuroscience, October 26, 2005 25(43):10010 10015 12 h light/dark cycle. All procedures were approved by the New York
University Animal Care and Use Committee.
Surgery. Rats were anesthetized with Nembutal (45 mg/kg, i.p.) and
implanted bilaterally with cannulas (26 gauge; Plastics One, Roanoke,
VA) aimed at the LA [anteroposterior (AP),
3.2; mediolateral (ML),
5.2; dorsoventral (DV),
8.0] and a bipolar stimulation electrode (250
m; Rhodes Medical, Summerland, CA) aimed at the left auditory thal-
amus [medial geniculate nucleus/posterior intralaminar nucleus (MGm/
PIN); AP,
5.4; ML,
3.0; DV,
6.6]. Attached to the left cannula was
an insulated stainless steel recording wire (1 M ) that extended
0.5
mm from the base of the infusion cannula. The final depth was deter-
mined using stimulation of the MGm/PIN as a guide (Doye`re et al.,
2003). Two cortical silver balls, placed contralaterally, served as a refer-
ence and ground, and dental cement was used to anchor the electrodes
and connecting device to the skull. Rats were administered
buprenorphine-HCl (0.02 mg/kg) for analgesia and given at least 1 week
to recover.
Electrophysiological and behavioral procedures. Rats were habituated to
the conditioning chamber and to cable connection and given 2 d of
pre-exposure to three 20 s CSs (1 kHz, 50 ms tone pips, 80 dB, at 1 Hz),
with a variable intertrial interval (ITI) of 160 s, on average. On the fourth
day, rats were infused bilaterally with either 1,4-diamino-2,3-dicyano-
1,4-bis(2-aminophenylthio)-butadiene (U0126) (1 g; 0.5 l; n
8) or
an equivalent volume of 50% DMSO vehicle (n
5). This dose of U0126
was chosen based on findings showing that it reliably impairs fear mem-
ory consolidation and the activation of extracellular signal-regulated ki-
nase (ERK)/mitogen-activated protein kinase (MAPK) in the LA (Schafe
et al., 2000).
Thirty minutes after infusion, rats received five CSUS pairings (ITI,
130 s), each CS terminating with a 0.5 s, 1 mA footshock (US). Reten-
tion of fear memory was then tested in a distinct chamber both at 3 h
(STM; three CSs) and at 24 h (LTM; 10 CSs). During pre-exposure and
retention tests, auditory-evoked field potentials (AEFPs) were recorded
from both the LA and MGm/PIN (through the tip of the stimulation
electrode). For technical reasons, we were able to record simultaneously
from both the LA and MGm/PIN in a subset of rats (vehicle, n
3;
U0126, n
6).
Data analysis. The rats freezing behavior was recorded onto videotape
during all sessions for off-line scoring. AEFPs were recorded and ana-
lyzed using DataWave and averaged across all tone-pip presentations
within a trial and collapsed across trials. The amplitude of the short-
latency negative component was measured for each potential ( 1215
ms for the LA;
79 ms for the MGm/PIN). We assessed retention of
beh