Neuropharmacology 52 (2007) 215e227
www.elsevier.com/locate/neuropharm
Long-term potentiation in the amygdala: A cellular
mechanism of fear learning and memory
Torfi Sigurdsson a,*, Valérie Doyère a,b, Christopher K. Cain a, Joseph E. LeDoux a
a
Center for Neural Science, New York University, 4 Washington Place, Room 809, New York, NY 10003, USA
b
NAMC, CNRS-UMR8620, Univ. Paris-Sud, 91504 Orsay, France
Received 23 May 2006; received in revised form 27 June 2006; accepted 28 June 2006
Abstract
Much of the research on long-term potentiation (LTP) is motivated by the question of whether changes in synaptic strength similar to LTP
underlie learning and memory. Here we discuss findings from studies on fear conditioning, a form of associative learning whose neural circuitry
is relatively well understood, that may be particularly suited for addressing this question. We first review the evidence suggesting that fear conditioning is mediated by changes in synaptic strength at sensory inputs to the lateral nucleus of the amygdala. We then discuss several outstanding questions that will be important for future research on the role of synaptic plasticity in fear learning. The results gained from these studies
may shed light not only on fear conditioning, but may also help unravel more general cellular mechanisms of learning and memory.
Ó 2006 Elsevier Ltd. All rights reserved.
Keywords: Long-term potentiation; Amygdala; Fear conditioning; Memory; Learning; Plasticity
1. Introduction: A cellular hypothesis of fear conditioning
Long-term potentiation (LTP) has received a tremendous
amount of attention in the roughly 30 years since it was first described by Bliss and Lomo (1973). This interest has been fueled
to a large degree by the widely held hypothesis that learning
and memory is mediated by changes in the strength of synapses
in neural circuits. According to this hypothesis, neural activity
during learning gives rise to long-term changes in synaptic
strength, which allows memories to be stored and later retrieved (Martin et al., 2000). Several properties of LTP have
made it an attractive experimental model for studying such synaptic changes. First, LTP is a persistent increase in synaptic
strength, making it a suitable mechanism for long-term memory storage. Second, LTP is associative in that its induction typically requires coincident pre-and postsynaptic activity, making
it suitable for encoding associations in the external world
* Corresponding author. Tel.: þ1 212 998 3624; fax: þ1 212 995 4704.
E-mail address: [email protected] (T. Sigurdsson).
0028-3908/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuropharm.2006.06.022
(Hebb, 1949). Finally, LTP is input-specific, which is likely
to increase the storage capacity of neural circuits that are capable of LTP. Taken together, these properties lend face validity to
the hypothesis that LTP-like changes underlie learning and
memory (Bliss and Collingridge, 1993; Martin et al., 2000).
Much of the research on LTP has been aimed at elucidating
its physiological and molecular mechanisms, with a considerable degree of success (reviewed in Bliss and Collingridge,
1993; Malenka and Nicoll, 1999; Malenka and Bear, 2004).
However, demonstrating experimentally that LTP-like changes
underlie learning and memory has proven more elusive. This is
partly due to our incomplete understanding of the synaptic organization of many memory circuits in the brain, making it difficult to relate synaptic changes in these circuits to changes in
behavior. For example, while LTP is best understood at the cellular and molecular levels in the hippocampus, the relationship
of hippocampal circuits and synapses to the complex memory
functions of the hippocampus is poorly understood. This
suggests that simple forms of learning may be more suited to
the task of relating LTP to learning and memory (Barnes,
1995; Stevens, 1998; Martin et al., 2000; Kandel, 2001).
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T. Sigurdsson et al. / Neuropharmacology 52 (2007) 215e227
In this article, we will first describe how fear conditioning,
a simple form of associative learning whose underlying neural
circuitry is relatively well understood, is likely to prove useful
in the effort to relate LTP to learning and memory. We then
review the evidence supporting the hypothesis that fear conditioning is mediated by LTP-like synaptic changes at sensory
inputs to the lateral amygdala (LA). Finally, we discuss
some outstanding questions raised by these findings that will
be important for future research.
1.1. What is fear conditioning?
Fear conditioning is a form of associative learning in which
an animal learns to associate a stimulus with danger. In a typical fear conditioning experiment, an initially neutral conditioned stimulus (CS), usually a tone, is paired in time with
an aversive unconditioned stimulus (US), typically a mild
footshock. As a result, the CS comes to elicit defensive or
fear responses, such as freezing, when presented alone.
Thus, fear conditioning enables animals to predict and avoid
dangerous situations and is therefore crucial to survival. Not
surprisingly, fear conditioning is a highly conserved form of
learning that is observed in a variety of species ranging from
snails to humans (LeDoux, 1994).
Besides being of interest in its own right, especially as
a model of pathological emotional states, several features
make fear conditioning a particularly attractive model for
studying the neural mechanisms of learning and memory.
Fear conditioning is both rapidly acquired, often following
only a single CSeUS pairing, and produces very long-lasting
memories. Furthermore, the induction and expression of fear
conditioning is under the control of discrete stimuli, the CS
and the US, which can be easily controlled by the experimenter. Many different kinds of stimuli can serve as the CS,
such as lights, odors and tones, as well as the context in which
conditioning occurs. In this review, however, unless otherwise
noted we will restrict our discussion to auditory fear conditioning, which is the most commonly studied, and as a consequence the best understood.
et al., 1999; Pitkanen, 2000). The central nucleus, in turn,
projects out of the amygdala to areas of the brainstem and hypothalamus that control the expression of defensive behaviors,
hormonal secretions and autonomic responses (Fig. 1; LeDoux
et al., 1988; Davis, 2000; for a more detailed overview of
amygdala anatomy, see Pitkanen, 2000).
Consistent with the anatomical data, lesioning or functionally inactivating the LA prior to training has consistently
been observed to cause deficits in fear conditioning (LeDoux
et al., 1990b; Helmstetter and Bellgowan, 1994; Campeau
and Davis, 1995b; Muller et al., 1997; Wilensky et al., 1999;
Amorapanth et al., 2000; Goosens and Maren, 2001; Nader
et al., 2001), suggesting that the LA is intimately involved in
the formation and storage of conditioned fear memories
(LeDoux, 2000; Maren, 2001). Although our review will focus
on how this form of learning is mediated by local synaptic
changes within the LA, we would like to emphasize that it is
not our intention to argue that the LA is the only site of storage
of auditory fear memories. Other nuclei of the amygdala may
also be involved, such as the basal and central nuclei (Pare
et al., 2004; Anglada-Figueroa and Quirk, 2005), as well as
structures outside the amygdala such as the auditory thalamus
1.2. The neural circuitry of fear conditioning
It has been known for some time that the amygdala, a group
of nuclei situated in the temporal lobe, is a critical component
of the fear conditioning circuitry (LeDoux, 2000; Maren,
2001). Auditory and somatosensory information representing
the CS and US, respectively, reaches the lateral nucleus of
the amygdala (LA) from both thalamic (LeDoux et al., 1984,
1990a,b; Turner and Herkenham, 1991; Doron and LeDoux,
2000; Linke et al., 2000) and cortical (Mascagni et al.,
1993; Romanski and LeDoux, 1993a; Shi and Cassell, 1997;
McDonald, 1998) sources. Within the LA, individual neurons
respond to both auditory and somatosensory stimuli (Romanski et al., 1993b), suggesting convergence of CS and US inputs
at the cellular level. Sensory information from the LA is then
relayed to the central nucleus, both directly and indirectly via
the basal, accessory basal, and intercalated nuclei (Royer
Fig. 1. Neural circuits underlying auditory fear conditioning. Auditory fear
conditioning is a form of associative learning in which an initially neutral conditioned stimulus (CS), such as a tone, acquires emotional significance after
being paired in time with an aversive unconditioned stimulus (US), such as
footshock. The circuits mediating auditory fear conditioning in rats consist
of CS (auditory) and US (somatosensory) inputs that converge onto single neurons in the lateral nucleus of the amygdala (LA). The LA then projects to the
central nucleus of the amygdala (CE) both directly and by way of other amygdala regions (not shown). Outputs of the CE in turn control the expression of
fear responses (such as freezing behavior) and related autonomic nervous system (blood pressure and heart rate) and endocrine (pituitary-adrenal hormones)
responses, via projections to the brainstem and hypothalamus. CG, central
grey; LH, lateral hypothalamus; PVN, paraventricular hypothalamus.
Figure adapted with permission from Medina et al. (2002). We emphasize,
however, that this account of amygdala anatomy is simplified for the purposes
of the present discussion; for a more detailed overview, see Pitkanen (2000).
T. Sigurdsson et al. / Neuropharmacology 52 (2007) 215e227
and cortex (Quirk et al., 1997; Armony et al., 1998; Weinberger, 2004; Apergis-Schoute et al., 2005). Our focus on the
LA largely reflects the fact that it is the most extensively studied region of the amygdala and its role in fear conditioning is
the best understood.
1.3. A cellular hypothesis of fear conditioning
The relative simplicity of the fear conditioning circuitry
makes it possible to propose a specific hypothesis about how
synaptic plasticity might underlie this form of learning. According to this cellular hypothesis, fear conditioning is mediated by
an increase in the strength of synapses that transmit CS information to principal neurons in the LA (Rogan and LeDoux, 1995;
Rogan et al., 1997, 2001; LeDoux, 2000; Blair et al., 2001;
Maren, 2001). The hypothesis assumes that prior to conditioning, the CS inputs are relatively weak and as a result the CS is
unable to elicit fear responses. In contrast, the US inputs are
stronger and capable of eliciting robust responses in LA
neurons. Because CS and US inputs converge onto LA neurons
(Romanski et al., 1993b), during fear conditioning the CS inputs
are active during strong postsynaptic depolarization caused by
the US. As a result, the CS inputs become stronger, making
the CS more effective at driving LA neurons, which in turn
can drive downstream structures that control fear responses
such as the central nucleus (Blair et al., 2001).
The cellular hypothesis of fear conditioning makes several
testable predictions. First, it predicts that responses of LA neurons to a CS should increase during fear conditioning. Second,
auditory inputs onto neurons in the LA should be capable of associative LTP. Third, fear conditioning ought to cause LTP-like
changes at these synapses. And fourth, blocking synaptic plasticity in the LA should impair fear conditioning. We will now
review the evidence addressing each of these predictions.
2. Evidence supporting the cellular hypothesis of fear
conditioning
2.1. CS-evoked responses in the LA increase
during fear conditioning
If fear conditioning is mediated by an increase in the
strength of synapses that carry auditory (CS) information to
the LA, one would expect to observe an enhancement in the responses of LA neurons to a CS during the course of fear learning. To date, this prediction has been confirmed by a number of
studies that have recorded either field potential or single-unit
responses in the LA during fear conditioning (Quirk et al.,
1995; Rogan et al., 1997; Collins and Pare, 2000; Repa et al.,
2001; Goosens et al., 2003) or intracellular responses during
CSeUS pairings (using an odor CS) in anesthetized animals
(Rosenkranz and Grace, 2002). These studies have found that
neural responses increase in magnitude to a CS that is paired
with a US but not to a CS that is presented in the absence of
the US. This suggests that plasticity in the LA during fear
conditioning specifically encodes the CSeUS association.
Importantly, the expression of this plasticity is not contingent
217
on the behavioral expression of fear, ruling out the possibility
that the enhanced CS responses simply reflect the behavioral
state of the animal (Goosens et al., 2003).
Although these results are consistent with changes in the
synaptic strength of sensory inputs onto LA neurons, others
have suggested that enhanced CS responses may simply reflect
plasticity in structures upstream of the LA, such as the auditory thalamus (Cahill et al., 1999). However, several findings
argue against this possibility. First, plasticity of CS responses
develops more rapidly in the LA than in the auditory thalamus
(Repa, 2002) or auditory cortex (Quirk et al., 1997). Second,
local pharmacological manipulations in the LA that impair
fear conditioning have been shown to reduce the enhancement
of CS responses in the LA but not in the auditory thalamus
(Schafe et al., 2005). These results strongly suggest that plasticity of CS responses recorded in the LA reflects, at least in
part, local synaptic changes within the LA. Interestingly, lesions or functional inactivation of the amygdala prior to fear
conditioning interferes with the enhancement of CS responses
in the auditory thalamus (Poremba and Gabriel, 1997; Maren
et al., 2001) and cortex (Armony et al., 1998), further arguing
against the possibility that conditioning-induced plasticity in
the LA simply reflects changes at afferent structures.
2.2. LTP occurs at auditory input synapses in LA
Synapses that transmit auditory information to the LA are
capable of LTP whose basic features make it an attractive cellular model of fear learning and memory. LTP has been demonstrated following tetanic (high-frequency) stimulation of
auditory thalamic inputs in vivo in anesthetized animals (Clugnet and LeDoux, 1990; Rogan and LeDoux, 1995; Yaniv et al.,
2001) and at both thalamic and cortical inputs in awake freely
behaving animals (Doyère et al., 2003). LTP can also be induced
at the two inputs in vitro by tetanizing the internal and external
capsules, the putative sources of thalamic and cortical auditory
inputs to the LA (Chapman and Bellavance, 1992; Huang and
Gean, 1994; Watanabe et al., 1995; Huang and Kandel, 1998;
Weisskopf et al., 1999b).
LTP can also be induced in the LA in vitro by pairing
weak presynaptic stimulation with strong postsynaptic depolarization (Weisskopf and LeDoux, 1999a; Huang et al.,
2000; Tsvetkov et al., 2004; Humeau et al., 2005). This
‘pairing protocol’ is particularly relevant in the present context, because it can model the activity patterns that are thought
to give rise to fear conditioning. As we mentioned earlier,
coactivation of CS and US inputs onto LA neurons during
fear conditioning is hypothesized to cause strengthening of
the CS inputs through an associative mechanism (Blair
et al., 2001). In the pairing protocol, the weak presynaptic
stimulation can represent the CS, whereas the strong postsynaptic depolarization can represent the US. The observation that
this pairing protocol induces LTP at auditory inputs onto LA
neurons suggests that this may be a suitable mechanism for
encoding CSeUS associations.
Associative LTP can also be induced in the LA by simultaneously stimulating the thalamic and cortical auditory inputs.
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Simultaneous high-frequency stimulation in awake animals
induces LTP at both inputs that is different from LTP induced
by stimulation of either input alone (Doyère et al., 2003).
Another study found that LTP was induced at cortical inputs
in vitro if thalamic and cortical afferents were tetanized simultaneously, whereas tetanization of either input alone did not induce LTP. Interestingly, although this LTP was associative, its
induction was mediated entirely by presynaptic mechanisms
(Humeau et al., 2003). The functional significance of this
form of associative LTP is at present not clear.
In addition to being associative, LTP in the LA is inputspecific. That is, LTP is typically observed only at inputs
whose activation is paired with postsynaptic depolarization
(Weisskopf and LeDoux, 1999a; Tsvetkov et al., 2004;
Humeau et al., 2005) or activation of other inputs (Humeau
et al., 2003). This feature may be relevant to fear conditioning,
since it can restrict changes in synaptic strength to inputs that
represent the CS. In contrast, inputs that transmit information
about tones that are not paired with a US (and are not active
during strong postsynaptic depolarization) are not potentiated
by to this mechanism. Thus, input-specificity may limit the
generalization of fear responses. Finally, it is important to
note that LTP in the LA is long lasting. In awake animals
LTP has been observed to last for at least 6 days (Doyère
et al., 2003), indicating that it is a plausible mechanism for
the long-term storage of fear memories.
2.3. Fear conditioning induces synaptic changes at
auditory inputs to the LA
A number of studies have examined whether fear conditioning causes LTP-like synaptic changes at auditory inputs onto
LA neurons. Two experimental strategies have been used for
this purpose. The first approach has been to examine whether
LTP induction at auditory inputs to the LA enhances auditory-evoked responses in the LA in a manner similar to the enhancement of CS-evoked responses observed during auditory
fear conditioning. This approach has revealed that LTP induction does indeed enhance auditory-evoked field potentials
recorded in the LA (Rogan and LeDoux, 1995). Subsequent
experiments showed that fear conditioning caused a similar
enhancement in field potential responses to an auditory CS
that developed in conjunction with conditioned fear responses
(Rogan et al., 1997). These findings therefore suggested that
LTP occurred in the LA during fear conditioning and were at
the time considered the best evidence available linking LTP
to learning and memory (Barnes, 1995; Eichenbaum, 1995;
Stevens, 1998).
The second experimental strategy has been to examine
synaptic transmission at auditory inputs onto LA neurons in
vitro in brain slices from animals that have undergone fear
conditioning. Synaptic transmission is examined in these studies by stimulating the internal or the external capsules, while
recording postsynaptic responses in the LA. The first study of
this kind found that transmission at thalamic inputs to the LA
was enhanced in fear conditioned animals compared to
animals that were either naive or had received presentations
of the CS and US in an unpaired fashion (McKernan and
Shinnick-Gallagher, 1997). These results were the first to directly demonstrate that fear conditioning leads to an LTP-like
enhancement of synaptic transmission at auditory inputs to the
LA, which specifically encodes the association between the
CS and the US. Subsequently it was shown that transmission
at cortical inputs is also enhanced following fear conditioning
(Schroeder and Shinnick-Gallagher, 2005). Further supporting
evidence, although less direct, has come from studies showing
that fear conditioning inhibits the induction of LTP at cortical
inputs (Tsvetkov et al., 2002; Schroeder and ShinnickGallagher, 2004, 2005). This ‘occlusion’ of LTP is typically
interpreted as being due to the fact that synapses in fear conditioned animals have already undergone LTP-like changes in
synaptic strength and are therefore less capable of showing
additional LTP.
Several studies suggest that presynaptic changes contribute
to the enhancement in synaptic transmission following fear
conditioning. A decrease in the paired-pulse ratio, a widely
used measure of transmitter release, has been observed at
both thalamic (McKernan and Shinnick-Gallagher, 1997;
Zinebi et al., 2002) and cortical (Tsvetkov et al., 2002;
Schroeder and Shinnick-Gallagher, 2005) inputs after fear
conditioning. This suggests that the synaptic enhancement is
partly mediated by greater transmitter release from presynaptic terminals, probably due to an increase in release probability
(Tsvetkov et al., 2002). However, recent findings suggest that
postsynaptic mechanisms may also play a role. Fear conditioning has been shown to lead to the insertion of new AMPA receptors at thalamic input synapses onto LA neurons (Rumpel
et al., 2005) as well as an increase in the surface expression of
AMPA receptor subunits in the LA (Yeh et al., 2006). At present, it therefore seems likely that fear conditioning enhances
synaptic transmission in the LA through a combination of
pre- and postsynaptic modifications.
2.4. LTP in the LA and fear conditioning
share molecular mechanisms
Although the findings reviewed so far strongly suggest that
fear conditioning induces changes in synaptic strength at auditory inputs to the LA, they do not address the question of
whether such changes are necessary for fear learning to occur.
In other words, does blockade of synaptic plasticity in the LA
impair fear conditioning? This has been addressed by asking
whether pharmacological agents that block LTP in the LA
also impair fear conditioning when injected directly into the
LA. To date, this question has been answered in the affirmative
by a number of studies targeting a range of molecular processes. These include, for example, the activation of NMDA
receptors, which is involved in both the induction of LTP in
the LA (Huang and Kandel, 1998; Bauer et al., 2002) and
the acquisition of fear conditioning (Miserendino et al.,
1990; Campeau et al., 1992; Rodrigues et al., 2001). A number
of intracellular signaling pathways, as well as gene transcription and protein synthesis, have also been implicated specifically in the long-term maintenance of LTP (Huang et al.,
T. Sigurdsson et al. / Neuropharmacology 52 (2007) 215e227
2000) and the consolidation of long-term fear memories (for
a full review, see Schafe et al., 2001; Rodrigues et al.,
2004). Similar results have been obtained using genetic manipulations, although these typically do not allow the same
degree of anatomical specificity as pharmacological manipulations (for reviews, see Tsien, 2000; Silva, 2003; Tonegawa
et al., 2003). Nevertheless, the results from both genetic and
pharmacological studies together convincingly demonstrate
that LTP shares many of the molecular mechanisms of fear
conditioning, suggesting that LTP-like changes may be a necessary requirement for fear learning and memory.
One potential criticism of these studies is that the molecular
mechanisms of LTP may not underlie the natural synaptic
changes that occur as a result of learning. Addressing this criticism, a recent study showed that local inhibition of MAP kinase impairs both the formation of long-term fear memory and
long-term enhancement of CS responses in LA (Schafe et al.,
2005). This study also showed that the behavioral impairment
was correlated with the level of impairment of conditioned
neural responses. Furthermore, the enhancement of CS-evoked
responses in the auditory thalamus was not affected, suggesting that the impairment in LA plasticity was mediated by disruption of synaptic plasticity within this nucleus (see Fig. 2).
In another study, Rumpel et al. (2005) demonstrated that preventing the insertion of new AMPA receptors into LA neurons,
which normally occurs during fear conditioning, impaired fear
learning. Because insertion of AMPA receptors is expected to
enhance synaptic transmission, these results also strongly suggest that synaptic plasticity in the LA is a necessary requirement for fear conditioning. Interestingly, the authors were
also able to show that insertion of AMPA receptors only
needed to be prevented in 10e20% of LA cells in order to
cause impaired fear learning. These results therefore suggest
that conditioned fear memories are stored by synaptic changes
in a relatively distributed network within the LA.
219
2.5. The current standing of the cellular hypothesis
As the previous discussion makes clear, a large number of
studies suggest that an increase in synaptic strength at auditory
inputs to the lateral amygdala occurs as a result of fear conditioning and that these changes are necessary for fear conditioning to take place. These findings thus satisfy two of the criteria
proposed by Martin et al. (2000) to be necessary for establishing that synaptic changes underlie learning and memory: synaptic changes must be demonstrated to occur as a result of
learning (the ‘detectability’ criterion) and preventing such
changes from taking place must interfere with learning (the
‘anterograde alteration’ criterion). These two criteria have
also been satisfied for other simple forms of learning, in particular in the marine snail Aplysia (Kandel, 2001) which, along
with results from studies of fear conditioning, provide some
of the most convincing evidence to date that synaptic plasticity
underlies learning and memory.
Before concluding this section, it is worth mentioning that
Martin et al. (2000) described two additional criteria for establishing synaptic plasticity as a mechanism of memory formation: reversing the synaptic changes induced by learning
should disrupt the memory (‘retrograde alteration’ criterion)
and artificially inducing synaptic changes analogous to those
observed during learning should produce similar behavioral
changes (the ‘mimicry’ criterion). These two criteria have received much less attention than the ones described previously.
Retrograde alteration of conditioned fear memories would
require reversing the synaptic enhancement caused by fear conditioning. In principle, this could be achieved by delivering
low-frequency stimulation (LFS) to auditory afferents, which
has been shown to cause reversal of LTP (‘depotentiation’) in
the LA (Lin et al., 2003a). Interestingly, one study found that
when LFS was delivered to the LA in animals that had previously been fear conditioned, an apparent loss of conditioned
Fig. 2. Synaptic plasticity in the LA is required for auditory fear conditioning. (A) In this experiment, rats received injections of a MAP kinase inhibitor (U0126) or
vehicle into the LA prior to fear conditioning. In vehicle-treated animals (left traces) field potential responses to the auditory CS (arrows) were enhanced during
a long-term memory test (LTM) in both auditory thalamus (lower traces) and in the LA (upper traces). However, in animals injected with U0126 into the LA (right
traces), neural plasticity was impaired in the LA but not in the auditory thalamus. These results therefore suggest that the MAP kinase inhibitor impaired synaptic
plasticity locally in the LA. (B) The impairment in neural plasticity in the LA was correlated with impairments in the consolidation of auditory fear conditioning.
Animals that showed greater impairment in neural plasticity displayed less fear responses to the CS during a long-term memory test. This suggests that plasticity in
the LA is required for auditory fear learning and memory. Adapted with permission from Schafe et al. (2005).
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T. Sigurdsson et al. / Neuropharmacology 52 (2007) 215e227
fear was observed when animals were tested the next day (Lin
et al., 2003a). However, since the authors of this study did not
record neural responses in the LA it is difficult to determine
whether the LFS did in fact reverse the synaptic changes
brought about by fear conditioning. Further experiments are
needed to address this issue. The mimicry criterion has also received scant attention, although fear conditioning may be particularly well suited for addressing it (Stevens, 1998; Martin
et al., 2000). Doing so would involve asking whether LTP induction at auditory inputs to LA induces fear responses to
a neutral auditory stimulus. As mentioned previously, it has
been demonstrated that LTP at thalamic inputs causes an enhancement of auditory-evoked responses in the LA similar to
what is observed during fear conditioning (Rogan and LeDoux,
1995; Rogan et al., 1997). It remains to be seen, however,
whether such artificially induced synaptic changes are sufficient to produce behavioral responses similar to conditioned
fear. Whether or not they do so may depend on whether fear
conditioning is also mediated by synaptic plasticity in areas
outside the LA.
3. Outstanding questions for future research
Although considerable progress has been made in relating
synaptic plasticity in the amygdala to fear learning and memory, several questions concerning this relationship remain unanswered. Below we discuss three research questions that may
advance our understanding of how plasticity in the LA contributes to fear conditioning in the coming years. This list is not
meant to be exhaustive, but rather to highlight a few important
outstanding questions.
3.1. Are LTP and fear conditioning sensitive
to the same stimulus parameters?
In the previous section we described several converging
lines of evidence that fear conditioning is mediated by synaptic plasticity at sensory inputs to the LA. It has been argued
that the rules that govern synaptic plasticity should reflect to
some degree the rules that govern learning, although it is probably unrealistic to expect them to be completely isomorphic
(Martin et al., 2000). In this section we discuss how two of
the basic properties of fear conditioningdits dependence on
CSeUS contiguity and CSeUS contingencydmay be realized in the rules that govern LTP induction in the LA.
Temporal overlap between the CS and the US (or contiguity)
is one of the necessary requirements for fear conditioning,
although it is not a sufficient one (see ’blocking’; Kamin,
1969). Presenting the animal with a CS and US that overlap
in time leads to fear conditioning, whereas CS and US presentations that are explicitly unpaired in time do not (Rescorla,
1967). For this reason, unpaired CSeUS presentations are
commonly used as a control procedure for determining whether
the effects of fear conditioning (for example, on synaptic transmission) are due to CSeUS contiguity or other nonassociative
effects.
The contiguity requirement for fear conditioning has parallels in the ‘pairing protocols’ that are commonly used to induce associative LTP in the LA. As we described earlier,
these protocols induce LTP by pairing weak presynaptic stimulation with strong postsynaptic depolarization (Weisskopf
and LeDoux, 1999a; Huang et al., 2000; Tsvetkov et al.,
2004; Humeau et al., 2005). In the context of fear conditioning, presynaptic stimulation of sensory afferents can represent
activity caused by the CS whereas postsynaptic depolarization
represents the US. Consistent with the contiguity requirement
for behavioral learning, presynaptic stimulation and postsynaptic depolarization must occur together in time for LTP to
be induced (Weisskopf and LeDoux, 1999a; Tsvetkov et al.,
2004; Humeau et al., 2005). Thus, both fear conditioning
and associative LTP in the LA have a contiguity requirement,
further supporting the hypothesis that LTP mediates fear learning (Blair et al., 2001).
Although contiguity between the CS and US is necessary
for fear conditioning, it is not sufficient. Fear conditioning
also depends strongly on CSeUS contingencydthe degree
to which the CS predicts the occurrence of the US
(Rescorla, 1968). For instance, strong fear conditioning can
be induced with only a few temporal pairings of the CS and
US, but presentation of additional CSs (partial reinforcement)
or USs (degraded contingency) decreases the strength of the
CSeUS association (Rescorla, 1968; Singh and Banerji,
1986; Cain et al., 2005). In such cases, CSeUS contiguity is
preserved, but contingency is reduced.
Associative LTP in the LA has been shown to require stimulus contingencies that bear a striking parallel to the contingency requirements for fear conditioning (Bauer et al.,
2001). Using a pairing protocol, the authors demonstrated
associative LTP at thalamic inputs when weak presynaptic
stimulation (to mimic a CS presentation) was contiguous
with postsynaptic depolarization (to mimic a US presentation).
Interestingly, when contingency was degraded by adding unpaired postsynaptic depolarizations to the protocol, LTP was
severely impaired (Fig. 3). This degraded contingency effect
was observed if unpaired depolarizations were interleaved
with pairings, preceded pairings or followed pairings suggesting that single neurons in the LA can integrate information
about the relationship between presynaptic activity and postsynaptic depolarization across at least 5 min. This important
finding provides further evidence in support of the hypothesis
that LTP in the LA mediates fear conditioning. It will be interesting to examine whether a pairing protocol designed to
mimic partial reinforcement (additional episodes of presynaptic stimulation) will have a similar effect on associative LTP in
the LA.
These findings raise the important question of how information about contingency and contiguity might be integrated
within single LA neurons to cause changes in synaptic
strength. At first glance, one might suggest that contiguous
stimulations (pairings) induce associative LTP and unpaired
depolarizations reverse this plasticity through a depotentiationlike process. However, it is important to note that associative
LTP is input-specific and degrading contingency with unpaired
T. Sigurdsson et al. / Neuropharmacology 52 (2007) 215e227
221
Fig. 3. Fear conditioning and LTP in the lateral amygdala are sensitive to the same stimulus contingencies. (A) To test whether LTP in the LA, induced by pairing
presynaptic stimulation (EPSPs) with postsynaptic depolarization, is compromised when contingency is reduced, extra depolarizations were inserted between pairings while keeping EPSP and depolarization contiguity constant. (B) LTP was induced in the LA when EPSPs were paired with postsynaptic depolarization (circles), but not when pairings were interleaved with extra postsynaptic depolarizations (triangles). Adapted with permission from Bauer et al. (2001).
depolarizations produces a significant reduction in LTP even
when these depolarizations occur before the pairings. Thus,
contingency mechanisms appear to be cell-wide whereas
contiguity mechanisms are synapse-specific. Any mechanistic
explanation of contingency will also have to take into account
the vastly different timescales of these two processes. Whereas
contiguous interactions between presynaptic activity and
postsynaptic depolarization occur on the order of milliseconds
to induce LTP, unpaired depolarizations can affect LTP when
separated from a pairing by 10 s, and perhaps longer. Answers
to these questions will be critical for our understanding of fear
conditioning and associative learning in general, especially
considering that associative learning in real life situations often occurs with less than 100% contingency.
3.2. The role of inhibitory transmission
LTP and fear learning
Most studies on the cellular mechanisms of fear conditioning have examined synaptic transmission and plasticity at excitatory inputs onto pyramidal cells in the LA. This focus is
motivated by the hypothesis that fear conditioning is mediated
by synaptic plasticity at these synapses specifically (Blair et al.,
2001). However, approximately 25% of neurons in the LA
are inhibitory interneurons (McDonald and Augustine, 1993)
and both feedforward (Rainnie et al., 1991; Li et al., 1996b;
Woodson et al., 2000) and feedback (Smith et al., 2000; Szinyei
et al., 2000; Samson et al., 2003) inhibition plays a large role in
synaptic transmission in the LA. In this section, we first discuss
how inhibitory transmission may regulate LTP in the LA and
fear learning. We then review evidence suggesting that inhibitory interneurons in the LA are also capable of synaptic
plasticity and discuss possible functional implications of these
findings.
LTP in the LA has been shown to be more easily induced in
the absence of inhibitory transmission, suggesting that inhibition is an important gate on LTP induction (Watanabe et al.,
1995; Bissiere et al., 2003). In principle, this gating function
can be controlled by neuromodulators which have been shown
to affect inhibitory interneurons in the LA, such as serotonin
(Rainnie, 1999; Stutzmann and LeDoux, 1999), norepinephrine (Coyle and Duman, 2003; Braga et al., 2004),
glucocorticoids (Johnson et al., 2005), cannabinoids (Katona
et al., 2001; McDonald and Mascagni, 2001; Marsicano
et al., 2002) and dopamine (Brinley-Reed and McDonald,
1999; Bissiere et al., 2003; Marowsky et al., 2005). Of these,
dopamine has been shown to suppress feedforward inhibition
in the LA and facilitate the induction of LTP (Bissiere et al.,
2003). In contrast, gastrin-releasing peptide (GRP) excites
inhibitory interneurons and constrains the induction of LTP
at excitatory inputs in the LA; mice lacking the GRP receptor
display less inhibition and enhanced LTP (Shumyatsky et al.,
2002). Both of these modulators affect fear conditioning in
a manner consistent with their effects on LTP: dopamine receptor antagonists impair fear conditioning (Guarraci et al.,
1999, 2000; Greba and Kokkonidis, 2000; Greba et al.,
2001) and conditioned neural responses (Rosenkranz and
Grace, 2002) whereas GRP receptor knockout mice show
enhanced fear learning (Shumyatsky et al., 2002). Thus,
neuromodulators and peptides, by affecting inhibitory
transmission, can modulate LTP in the LA and fear conditioning. A better understanding of this gating function will be
important for refining cellular models of fear learning.
In addition to gating excitatory LTP, LA interneurons are
also capable of synaptic plasticity. To date, only two studies
have investigated LTP in LA interneurons and it appears that
both input synapses (excitatory synapses onto interneurons)
and output synapses (inhibitory synapses onto pyramidal cells)
can undergo LTP (Fig. 4; Mahanty and Sah, 1998; Bauer and
LeDoux, 2004). LTP of input synapses was induced in both
studies in vitro by high-frequency stimulation of auditory inputs, which are known to make glutamatergic synapses onto
GABAergic interneurons (Smith et al., 2000; Szinyei et al.,
2000; Woodson et al., 2000). Postsynaptic calcium influx is required for induction of this LTP although the calcium source
remains a matter of debate. One study found that calciumpermeable AMPA receptors were required for LTP induction
(Mahanty and Sah, 1998) whereas the other study found that
NMDA receptors were necessary (Bauer and LeDoux,
2004). LTP of excitatory inputs onto interneurons is also
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T. Sigurdsson et al. / Neuropharmacology 52 (2007) 215e227
Fig. 4. Heterosynaptic LTP of inhibitory interneurons in the LA. (A) High-frequency stimulation was delivered in vitro to thalamic inputs onto LA interneurons.
This induced LTP not only at thalamic inputs (left), but also at cortical inputs (right). This suggests that LTP of inhibitory interneurons in the LA is heterosynaptic.
(B) LTP was associated with decreased paired-pulse facilitation, suggesting that it is mediated by an increase in presynaptic transmitter release. Adapted with
permission from Bauer and LeDoux (2004).
somewhat unique in that it is not input-specific: high-frequency stimulation of either the thalamic or cortical inputs
leads to LTP at both inputs (Fig. 4; Bauer and LeDoux, 2004).
LTP of inhibitory synapses can also be induced by delivering high-frequency stimulation to GABAergic inputs onto pyramidal cells (Bauer and LeDoux, 2004; GABAergic inputs
can be isolated by stimulating within the LA in the absence
of excitatory transmission). Interestingly, although this LTP
is dependent on postsynaptic calcium, it is independent of
AMPA or NMDA receptor activity. The molecular induction
and expression mechanisms for LTP of GABAergic transmission in the LA are currently unknown.
Although the study of inhibitory LTP in the LA is still in
its infancy, its existence raises questions about its potential
function. One possibility, inspired by the finding that inhibitory LTP is heterosynaptic, is that this LTP increases the
signal-to-noise ratio of CS processing during fear conditioning
(Bauer and LeDoux, 2004). Another interesting possibility is
that inhibitory LTP in the LA may be important for fear extinction (Bauer and LeDoux, 2004; Barad, 2005), a form of
learning that is known to depend on the LA (Davis, 2002;
Marsicano et al., 2002; Myers and Davis, 2002; Lin et al.,
2003b; Chhatwal et al., 2005). Extinction occurs when a CS
is repeatedly presented in the absence of the aversive US,
causing fear responses to the CS to diminish. The synaptic
changes underlying fear conditioning are generally believed
to be preserved with extinction but the expression of these
changes may be suppressed by new inhibitory learning
(for review see Myers and Davis, 2002). The possibility that
this may be mediated by plasticity of inhibitory transmission
is supported by several findings. First, extinction learning
and expression are blocked by an inverse agonist of the
GABAA-receptor (Harris and Westbrook, 1998). Second,
extinction training increases the expression of gephryin, a
GABAA-receptor clustering protein, in the LA (Chhatwal
et al., 2005). Third, disruption of cannabinoid signaling in
the LA impairs both extinction and plasticity of inhibitory
transmission. Further research is clearly needed to understand
how inhibitory plasticity in the LA relates to extinction and
other behavioral processes.
3.3. Differences between thalamic and cortical inputs
As we have already mentioned, the LA receives two sets of
auditory inputs that originate in the auditory thalamus and cortex. These come primarily from the posterior intralaminar nucleus of the thalamus (PIN; LeDoux et al., 1984, 1990a; Turner
and Herkenham, 1991; Doron and LeDoux, 2000; Linke et al.,
2000) and cortical auditory association area TE3 (Mascagni
et al., 1993; Romanski and LeDoux, 1993a; Shi and Cassell,
1997; McDonald, 1998). Within the LA, the two inputs innervate overlapping regions (LeDoux et al., 1991) and many LA
neurons respond to stimulation of either auditory thalamus or
cortex (Li et al., 1996a). Although we have reviewed evidence
suggesting that synaptic changes at auditory inputs to the LA
mediate fear conditioning, one outstanding question is whether
long-term changes at the thalamic and cortical inputs make distinct contributions to this process.
Several studies suggest that LTP induction differs considerably at the two inputs. The magnitude of LTP induced by highfrequency stimulation is greater at cortical inputs in awake
animals (Fig. 5; Doyère et al., 2003) as well as in anesthetized
animals across a range of induction frequencies and protocols
(Sigurdsson et al., 2006). Although this does not necessarily
mean that the thalamic inputs are less plastic than cortical inputs, it may suggest that plasticity at the two inputs is sensitive
to different patterns of pre- and postsynaptic stimulation. Consistent with this interpretation, in vitro studies have shown that
some stimulation protocols induce LTP more readily at thalamic inputs (Humeau et al., 2005), whereas others induce
LTP selectively at cortical inputs (Humeau et al., 2003). Insofar
as specific patterns of neural activity reflect different events in
T. Sigurdsson et al. / Neuropharmacology 52 (2007) 215e227
223
Fig. 5. Differences in LTP magnitude and longevity at thalamic and cortical inputs to the LA. (A) In this experiment, LTP was induced at thalamic and cortical
inputs to the LA in awake, freely behaving animals. Although the magnitude of LTP was initially greater at cortical compared to thalamic inputs, it decayed back to
baseline within 3 days, whereas LTP was observed at thalamic inputs for at least 6 days (B). Adapted with permission from Doyère et al. (2003).
the external world, the stimulus requirements for LTP at thalamic and cortical inputs may enable plasticity at these two inputs to store different kinds of information. More work is
needed to systematically examine the exact stimulus parameters that give rise to LTP at the two inputs and how they relate
to external events.
What mechanism might underlie these differences in LTP?
One study found that dendritic spines that are postsynaptic to
thalamic inputs are larger than spines that are postsynaptic to
cortical afferents. This might facilitate LTP induction at thalamic inputs when presynaptic stimulation is paired with postsynaptic action potentials (Humeau et al., 2005). The two
inputs have also been found to display different short-term
plasticity profiles in vivo: whereas the thalamic inputs display
primarily depression during paired-pulse stimulation, cortical
inputs show more facilitation (Sigurdsson et al., 2006). This
may determine how well the two inputs respond during
high-frequency stimulation protocols that are used to induce
LTP. Differences in the expression of postsynaptic receptors
between the two inputs are another possibility. There is evidence to suggest that NMDA receptors make a larger contribution to synaptic transmission at thalamic inputs in vivo (Li
et al., 1995, 1996a; Rogan, 1998). However, with one exception (Weisskopf and LeDoux, 1999a) in vitro studies have
not found such differences between the two inputs (Mahanty
and Sah, 1999; Szinyei et al., 2003; Tsvetkov et al., 2004;
Humeau et al., 2005), a discrepancy that may reflect differences between the in vivo and in vitro preparations. Recent
studies also suggest that LTP induction at the two inputs may
involve separate molecular mechanisms (Humeau et al.,
2005). Such pharmacological differences are particularly interesting since they may allow the two inputs to be dissociated
pharmacologically and their individual contributions to fear
conditioning assessed.
Synaptic plasticity at the two inputs may also be differentially involved in the long-term storage of fear memories.
LTP at thalamic inputs has been shown to last for at least
6 days in awake behaving animals, whereas LTP at cortical
inputs, although initially larger in magnitude, decays back
to baseline within 3 days (Doyère et al., 2003; Fig. 5). An
important question for future research will be to examine
whether the synaptic changes observed at the two inputs following fear conditioning also display differences in their persistence. Most studies have examined synaptic changes only
up to 2 days following fear conditioning (McKernan and
Shinnick-Gallagher, 1997; Zinebi et al., 2001; Tsvetkov et al.,
2002; Schroeder and Shinnick-Gallagher, 2004). However,
one study demonstrated synaptic changes at cortical inputs
10 days after learning (Schroeder and Shinnick-Gallagher,
2005). Whether thalamic inputs show similarly or more persistent changes following conditioning has not been examined.
Another possibility for examining the contribution of the
two inputs to fear memory storage might be to compare different temporal components of CS-evoked responses in the LA
during fear conditioning. It is well established that the shortest-latency CS-responses in the LA must be mediated by the
thalamic inputs whereas cortical inputs may contribute to longer response latencies (Quirk et al., 1995, 1997; Rogan et al.,
2005). Interestingly, although plasticity of CS responses is observed at the shortest response latencies during fear conditioning, longer-latency responses show more persistent changes
(Repa et al., 2001). One intriguing possibility is that plasticity
at these longer response latencies reflects, in part, synaptic
plasticity at cortical inputs onto LA neurons. It will be important to determine whether this is the case, for example by functionally inactivating the auditory cortex while recording CS
responses in the LA.
In addition to examining how plasticity differs at the thalamic and cortical inputs, it will also be important to address
the functional consequences of these differences. One approach is to examine the effects of lesions of either the auditory thalamus or auditory cortex on fear learning. One such
study found that when lesions were done after fear conditioning, cortical lesions impaired the retention of conditioned fear
whereas thalamic lesions did not (Campeau and Davis, 1995a).
This suggests that the cortical inputs may be more involved in
the long-term storage of fear memories. However, lesion studies are an imperfect way of addressing this issue since they do
224
T. Sigurdsson et al. / Neuropharmacology 52 (2007) 215e227
not selectively block the expression of plasticity, but cause
a more general disruption of auditory processing. An alternative method would be to selectively impair plasticity at either
input using pharmacological agents that act selectively at one
input or the other, as previously mentioned. Yet another strategy may be to selectively induce depotentiation of either input,
thus reversing the synaptic changes brought about by fear
conditioning (Lin et al., 2003a). Given our incomplete understanding of the relative contributions of the thalamic and
cortical auditory inputs to fear conditioning, these and other
experimental approaches will be important for future studies
on synaptic plasticity in the amygdala.
4. Conclusion
Considerable progress has been made in relating activitydependent changes in synaptic strength to learning and memory since Bliss and Lomo first described LTP in 1973. We have
argued that studies linking fear conditioning to LTP in the
amygdala constitute an important part of this progress. The
findings reviewed here strongly suggest that LTP-like synaptic
changes occur as a result of fear learning, and that such
changes are necessary for fear memories to be formed. Nevertheless, our understanding of the cellular mechanisms mediating fear conditioning is far from complete. Accordingly, we
have highlighted several important questions which we think
will be important for future research: how CSeUS contiguity
and contingency is computed by individual neurons, the role
of inhibitory transmission and plasticity and how synaptic
plasticity at different sensory inputs contributes to fear learning and memory. Although these questions are specific to
fear conditioning and synaptic plasticity in the amygdala,
the answers may unravel more general mechanisms of plasticity that will apply to other memory circuits as well as different
forms of learning and memory.
Acknowledgments
This work was supported by NIH Grants R01 MH46516,
R37 MH38774, K05 MH067048, and P50 MH58911 to
J.E.L., an NIMH NRSA MH077458 to C.K.C. and CNRSPICS to V.D.
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