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Center for Neural Science, New York University, New York, New York 10003-6621, USA
ABSTRACT
Fear extinction refers to the ability to adapt as situations change by learning to suppress a previously learned fear. This process involves a gradual reduction in the capacity of a fear-conditioned stimulus to elicit fear by presenting the conditioned stimulus repeatedly on its own. Fear extinction is context-dependent and is generally considered to involve the establishment of inhibitory control of the prefrontal cortex over amygdala-based fear processes. In this paper, we review research progress on the neural basis of fear extinction with a focus on the role of the amygdala and the prefrontal cortex. We evaluate two competing hypotheses for how the medial prefrontal cortex inhibits amygdala output. In addition, we present new findings showing that lesions of the basal amygdala do not affect fear extinction. Based on this result, we propose an updated model for integrating hippocampal-based contextual information with prefrontal-amygdala circuitry.
Pavlovian fear conditioning is a behavioral procedure that can be used to
study the brain mechanisms underlying the acquisition and storage of
information about danger, as well as the mechanisms underlying adaptive
responses as situations change (e.g.,
Fanselow 1998
;
LeDoux 2000;
Davis and Whalen 2001;
Maren 2001). In this
procedure, an emotionally neutral stimulus, such as a tone, is paired with an
aversive event, the unconditioned stimulus (US), typically an electric shock.
The pairing of the tone and US results in the transformation of the previously
neutral stimulus into an emotionally potent conditioned stimulus (CS) capable
of eliciting a complex pattern of physiological adjustments that constitute a
fear or defense response, including defensive behavior (freezing), and
supporting changes in autonomic and endocrine activity. If the CS is
subsequently presented repeatedly in the absence of the US, it gradually loses
the ability to elicit these fear-related responses. This weakening of the
ability to elicit a conditioned response is referred to as extinction.
Research over the past few decades has identified neural systems that
underlie the acquisition and storage of the CS-US association and the
expression of fear responses to the CS (for reviews, see
LeDoux 2000;
Davis and Whalen 2001;
Maren 2001). Acquisition and
storage involve circuits that transmit the CS and US to the lateral amygdala
(LA). Expression of conditioned fear involves CS transmission to the LA,
connections from the LA to the central amygdala (CE), either directly or by
way of intraamygdala connections (see below), and then output connections from
the CE to various regions that control specific conditioned responses
(behavioral, autonomic, endocrine).
Progress has also been made in understanding the extinction of conditioned
fear when the CS is repeatedly presented in the absence of the US. Contrary to
common wisdom, extinction is not equivalent to forgetting but instead
represents new learning—learning that the CS no longer reliably predicts
the US (Berman and Dudai 2001;
Bouton 2002;
Myers and Davis 2002). The
neural basis of fear extinction is believed to involve connections between the
medial prefrontal cortex (mPFC) and the amygdala
(Morgan et al. 1993;
Morgan and LeDoux 1995;
LeDoux 1996,
2000;
Garcia et al. 1999;
Quirk et al. 2000;
LeDoux and Gorman 2001;
Garcia 2002;
Grace and Rosenkranz 2002;
Herry and Garcia 2002;
Milad and Quirk 2002;
Myers and Davis 2002;
Paré et al. 2004).
However, the nature of involvement of the mPFC and the amygdala and their
interactions in extinction of fear are still poorly understood.
Current interest in the role of the mPFC in extinction of fear began when
Morgan et al. (1993) reported
that rats with lesions of the mPFC had an increased resistance to extinction.
Damage to the prefrontal cortex had long been known to produce changes in
emotionality as well as perseverative responses in certain cognitive tasks
(Nauta 1971; Luria
1973a,b;
Goldman-Rakic 1987).
Integrating these findings with their observations on fear extinction, Morgan
et al. (1993) proposed that
the resistance to extinction following mPFC damage represented perseverative
tendencies in the emotional domain. They further proposed that connections
between the mPFC and amygdala normally allow the organism to adjust its
emotional behavior when environmental circumstances change, and that some
alteration in this circuitry, causing a loss of prefrontal control of the
amygdala, might underlie the inability of persons with anxiety disorders to
regulate their emotions.
Over the past decade, considerable research has been conducted on the role of the mPFC and the amygdala, and their interactions in fear extinction. This special issue of Learning & Memory features many of the key findings. In this article, we review the original impetus for pursing the role of the mPFC, and survey some of the findings that have come since. We also review parallel research on the role of the amygdala in extinction, and present some new findings regarding amygdala-mPFC interactions.
Prefrontal Cortex Contributions to Fear Extinction
To assess the prefrontal cortex contributions to fear extinction, we first briefly review its general anatomical organization in mammals. Next, we return to the original findings that motivated interest in the role of the mPFC in extinction of conditioned fear, and then we consider the evidence that has subsequently accumulated, which has both confirmed and refined our understanding of mPFC involvement in the regulation of fear.
Anatomical Organization of the Prefrontal Cortex in Mammals
The prefrontal cortex consists of several functionally distinct subregions
(Posner 1992;
Seamans et al. 1995;
Robbins 1996;
Bechara et al. 1998;
Botvinick et al. 1999;
Carter et al. 1999;
Owen et al. 1999;
Smith and Jonides 1999;
Muller et al. 2002;
Heidbreder and Groenewegen
2003). In humans and other primates, these include the lateral
prefrontal cortex, orbital frontal cortex, and mPFC. The lateral prefrontal
cortex, especially the dorsolateral region, is involved in working memory and
executive control functions (Goldman-Rakic
1995; Owen et al.
1999; Smith and Jonides
1999; D'Esposito et al.
2000; Levy and Goldman-Rakic
2000; Miller and Cohen
2001; Curtis and D'Esposito
2003). The orbital frontal cortex is involved in reward,
motivation, and emotional decision making
(Damasio 1990;
Robbins 1996; Rolls
1996,
2000;
Rogers et al. 1999;
Bechara et al. 2000;
Berns et al. 2001). The mPFC
itself has several divisions, including the anterior cingulate cortex and
several more ventral areas that include the infralimbic (IL) and prelimbic
(PL) cortices, and the medial frontal gyrus. The anterior cingulate is divided
into two parts, a dorsal part involved in attention and cognitive control and
a more ventral part involved in emotional regulation
(Bush et al. 2000;
Cohen et al. 2000). The
functional contribution of the ventral mPFC (vmPFC: regions below the ventral
anterior cingulate, and involving the IL and PL, among other regions) in
humans is less clear.
In non-primate species, the prefrontal cortex is poorly developed. Indeed,
it is generally accepted that the lateral prefrontal cortex is a unique
primate specialization (Povinelli and
Preuss 1995; Preuss
1995). However, the orbital frontal cortex and various other mPFC
regions (anterior cingulate, IL, PL) are present in rodents and other
non-primate mammals (Uylings et al.
2003). The role of the mPFC areas in extinction of fear has been
examined, and is reviewed below.
Evidence Implicating the mPFC in Fear Extinction
Research on the neural basis of fear extinction was stimulated by two
studies in the late 1980s showing that damage to sensory processing regions of
the cortex (auditory or visual) leads to increased resistance to extinction
(LeDoux et al. 1989;
Teich et al. 1989). In their
study, LeDoux et al. (1989)
proposed that the sensory cortex was not the locus of extinction, but instead
was a necessary way-station to other regions, possibly the hippocampus or
mPFC. This hypothesis was based on a variety of disparate findings that
implicated the latter two regions in the extinction of one or more behavioral
tasks, although at the time little work had been done on the neural basis of
extinction using fear-conditioning tasks.
Morgan et al. (1993) then
examined the effects of damage to the mPFC on fear extinction of rats, and
found that many more days of exposure to the CS in the absence of the US were
required in order for CS-elicited fear responses to be reduced to pretraining
levels. Follow-up studies systematically examined the effects of damage to the
dorsal mPFC (anterior cingulate cortex) and vmPFC
(Morgan and LeDoux 1995).
Lesions circumscribed to the vmPFC had no effect on the amount of fear
behavior (freezing) expressed on a given trial but greatly increased the
number of CS-alone trials required to extinguish the behavior. Damage to the
dorsal mPFC led to an increased resistance to extinction but also resulted in
an increased amount of freezing behavior expressed on each trial
(Morgan and LeDoux 1995).
With the dorsal mPFC lesions, it was unclear if the effect on extinction was
really due to increased resistance to extinction, because the effect could
also be explained by the overall increase in the expression of conditioned
fear. In contrast, damage to the vmPFC selectively impaired extinction over
days without increasing the expression of fear within trials, and therefore
most subsequent research on the role of the mPFC in extinction has focused on
the vmPFC.
A later study addressing the role of the vmPFC in extinction failed to
replicate Morgan's findings (Gewirtz et
al. 1997). However, this study used fear-potentiated startle, a
training and testing paradigm that differs significantly from the task
described so far, which we will refer to as simple fear conditioning.
Differences between the training task (e.g., partial vs. continuous
reinforcement), testing task, or other procedural differences, may account, at
least in part, for the discrepancy between the findings. Although Gewirtz et
al. (1997) made a strong
effort to make training and testing conditions similar to Morgan et al.
(1993), several
methodological differences still remained. Indeed, studies from several other
research groups using similar simple fear-conditioning tasks, rather than
potentiated startle, have implicated the mPFC in fear extinction. For example,
Morrow et al. (1999) showed
that lesions to the dopaminergic inputs to the mPFC caused a deficit in fear
extinction. Quirk and colleagues (Lebron
et al. 2003) directly replicated the effects of vmPFC lesions on
the extinction of freezing over days reported by Morgan et al.
(1993). Furthermore, Quirk
and collaborators (Quirk et al.
2000) showed that lesions of the IL subregion of the vmPFC impair
the retrieval of extinction learning (from a previous day), but not its
expression or short-term processing (gradual extinction that occurs within a
single session of CS-alone presentations), suggesting a specific role for the
IL in the retrieval of extinction. Particularly dramatic is the finding that
pairing the tone CS with brief IL stimulation reduces freezing in rats,
suggesting that IL stimulation is sufficient to simulate extinction learning
(Milad and Quirk 2002;
Milad et al. 2004).
Results from electrophysiological and imaging studies in rats and humans
provide further support for the view that functional changes in mPFC are
associated with the retrieval of extinction training. Milad and Quirk
(2002) found that IL neurons
respond to CS exposure on the day following extinction training, suggesting
that these cells do not respond to the CS during fear conditioning or during
extinction training, but instead specifically respond when the memory of
extinction training is retrieved. Similarly, Garcia and colleagues observed
that induction of synaptic plasticity by giving LTP-inducing high frequency
stimulation of mediodorsal thalamic inputs to mPFC is associated with the
maintenance of extinction, but not its acquisition
(Herry et al. 1999; Herry and
Garcia 2002,
2003; for review, see
Garcia 2002). Thus, findings
from both the Quirk and Garcia groups suggest that the mPFC is involved in the
retrieval (or consolidation) of extinction as opposed to the initial learning
of the extinction. In a different set of studies, Barrett et al.
(2003) mapped metabolic neural
activity after extinction using a radio-labeled glucose analog and confirmed
that elevated mPFC activity is evident after the retrieval of prior extinction
learning. Interestingly, another related experiment waits to be performed: No
one has yet examined brain metabolism or immediate early gene expression in
animals during extinction acquisition. This would give a better understanding
of the dynamic interaction between specific loci of the different brain areas
thought to be involved in fear extinction. Finally, consonant with the
non-human animal research, PET and fMRI imaging studies have shown that areas
of the mPFC in humans are implicated in fear extinction
(Hugdahl et al. 1995;
Phelps et al. 2004). Results
from human studies of fear extinction are discussed further below, with an
emphasis on clinical implications of mPFC-amygdala interactions.
Overall, throughout this literature the predominant view has been that extinction involves a process by which neural activity in the mPFC comes to regulate the amygdala-mediated expression of conditioned fear responses. However, the amygdala itself has also been implicated in fear extinction, as discussed next.
Amygdala Contributions to Fear Extinction
The fact that the mPFC plays a key role in extinction does not mean that it
alone is involved in this process. An important question concerns whether the
amygdala contributes to fear extinction. Because damage to the amygdala
prevents the acquisition and expression of fear conditioning
(LeDoux 2000;
Davis and Whalen 2001;
Maren 2001), the use of
lesions in evaluating amygdala contributions for fear extinction has been
problematic. We return to this issue below, but for now, because amygdala
regions have unique cytoarchitectonic and connectional characteristics
(Pitkänen et al. 2000a,
2003), we first review the
organization of some of the relevant amygdala areas, including LA, basal
nucleus (B), CE, and the intercalated cell masses (ITC). Then we review
functional evidence that implicates the amygdala in fear extinction.
Anatomical Organization of the Amygdala
At the beginning of this manuscript, we noted that the two main regions of
the amygdala involved in fear conditioning are the LA (the sensory input
region that receives the CS and US) and the CE (the output region that
controls the expression of fear responses). There are direct connections
between the LA and CE, but in addition, the LA communicates with the CE by way
of intermediate connections within the amygdala (Paré et al.
1995,
2003;
Pitkänen et al. 1997;
Savander et al. 1997).
Specifically, the LA projects to the B, accessory basal, and the ITC, and each
of these projects to the CE. In addition, the B projects to the ITC, providing
another link to the CE.
The LA and B are composed of two major types of neurons:
glutamate-containing spiny multipolar cells (pyramidal-like cells) and aspiny
-aminobutyric acid (GABA) immunoreactive cells
(Pitkänen 2001;
Rainnie 2003). The GABAergic
cells are interneurons and constitute the main source of local inhibition,
whereas the excitatory pyramidal-like cells give rise to connections with
other amygdala regions, such as the CE, in addition to initiating collateral
fibers that terminate locally on excitatory and inhibitory cells
(McDonald 1985;
McDonald and Augustine 1993;
Smith and Paré 1994;
Paré et al. 1995;
McDonald and Betette 2001;
Li et al. 2002). ITC cells, in
contrast, are all small, interconnected, densely packed spiny inhibitory
GABAergic neurons that receive connections from the LA and B and project to
the CE (McDonald and Augustine
1993; Paré and Smith
1993,
1998;
Ghashghaei and Barbas 2002;
Royer and Paré 2002).
Thus, the connections from the LA and B to the CE, and from the LA and B to
the ITC are mostly excitatory, and the connections from the ITC to the CE are
mostly inhibitory.
It is important to note that the connections from the LA, B, and ITC to the
CE terminate on different groups within the CE. The LA sends fibers to the
lateral capsular portion of the CE, whereas the ITC sends projections to the
medial part of the CE (Quirk et al.
2003; Paré et al.
2004). The B sends projections to both CE subdivisions
(Krettek and Price 1978;
Paré et al. 1995,
2003;
Collins and Paré 1999;
Royer et al. 1999). This is
significant because the medial part of the CE constitutes the main output
pathway for amygdala projections that control behavioral and autonomic
responses, suggesting that the B may gate the efficacy of LA inputs to
brainstem-projecting CE cells
(Paré et al. 2003).
Interestingly, anatomical (van Groen and
Wyss 1990; McDonald and
Mascagni 1997; McDonald
1998; Pitkänen et al.
2000b) and electrophysiological (Colino and Fernandez de Molina
1986a,b;
Maren and Fanselow 1995;
Ishikawa and Nakamura 2003)
studies suggest that the B is a main locus for hippocampal afferents to the
amygdala. However, damage to the B does not prevent fear conditioning
(Amorapanth et al. 2000; but
see Goosens and Maren 2001;
Nader et al. 2001),
suggesting that the direct pathway from the LA to the CE, or the connection
from the LA to the ITC to the CE is sufficient. It is possible that although
the B is not necessary for fear conditioning, it normally contributes when
still intact.
Evidence Implicating the Amygdala in Fear Extinction
Tone-elicited neural activity in LA neurons increases following pairing of
the tone CS with a US (Quirk et al.
1995; Rogan et al.
1997; Collins and Paré
2000; Repa et al.
2001; Blair et al.
2003; Goosens et al.
2003). During extinction, the responses of many of these cells
return to pretraining levels (Quirk et
al. 1995; Repa et al.
2001; Hobin et al.
2003). However, recently Repa et al.
(2001) also found a
population of cells in which CS-elicited activity remained elevated throughout
extinction training. Such activity is consistent with the idea that extinction
does not erase the original CS-US association
(Rescorla 2001;
Bouton 2002) and that
extinguished fears can be recovered by various manipulations by tapping into
this extinction-resistant activity (Repa
et al. 2001). In addition, fMRI studies in humans have confirmed
that amygdala activation occurs not only during the acquisition, but also
during the extinction of conditioned fear
(Phelps et al. 2004).
It is widely accepted that NMDA receptors contribute to the synaptic
plasticity that underlies learning and memory in a variety of brain systems
(see Morris 1989;
Bliss and Collingridge 1993;
Martin et al. 2000), including
the amygdala systems underlying fear conditioning (e.g.,
LeDoux 2000;
Blair et al. 2001;
Maren 2001;
Walker and Davis 2002). Given
that extinction is believed to be a form of learning, one in which the learned
ability of the CS to elicit fear becomes inhibited, Davis and colleagues
infused the NMDA receptor antagonist D,L-2-amino-5-phosphonopentanoid acid
(AP5) into the LA and B prior to extinction training and found that the
extinction of conditioned fear was disrupted
(Falls et al. 1992).
Furthermore, inhibition of mitogen-activated protein kinase (MAPK), a protein
that contributes to long-term stabilization and storage of fear memories in
the amygdala and other systems (Atkins et
al. 1998; Schafe and LeDoux
2000; Schafe et al.
2000), and that can putatively be activated by calcium entry into
the postsynaptic cells during the opening of NMDA receptors, also blocked
extinction (Lu et al. 2001).
Finally, intra-amygdala infusion of an NMDA agonist, D-cycloserine,
facilitates extinction of fear (Walker et
al. 2002; Ledgerwood et al.
2003). Taken together, these findings indicate that neural
activity in the amygdala that leads to NMDA-dependent plasticity plays an
important role in extinction (Davis
2002; Davis and Myers
2002; Walker and Davis
2002; Davis et al.
2003).
Nevertheless, the precise role of NMDA-dependent plasticity within the
amygdala in extinction remains to be clarified. The studies described above
used the fear-potentiated startle paradigm, which, as noted earlier, differs
in some respects from simple fear conditioning. For example, infusion of AP5
into the LA and B prior to training disrupts learning, but infusion prior to
testing has no effect on the expression of fear responses in the
fear-potentiated startle paradigm
(Miserendino et al. 1990;
Walker and Davis 2000). With
simple fear conditioning, however, infusion of AP5 into the LA and B prior to
training disrupts learning, and this treatment also affects the expression of
fear responses when infused before testing
(Maren et al. 1996;
Lee and Kim 1998;
Rodrigues et al. 2001). This
result is consistent with the finding that AP5 has a significant effect on
synaptic transmission in the LA (Li et al.
1995,
1996;
LeDoux 1996;
Weisskopf et al. 1999), and
thus it is difficult to conclude that the effects of AP5 on simple fear
conditioning are caused by a disruption of plasticity as opposed to
transmission. However, use of a different antagonist, ifenprodil, which
selectively blocks NR2B subunit-containing NMDA receptors, disrupts learning
without affecting expression (Rodrigues
et al. 2001). This suggests that NMDA receptors in the amygdala
are involved in simple fear conditioning, which is consistent with the
fear-potentiated startle results described above. It remains to be determined,
though, whether blockade of NR2B-containing NMDA receptors in the amygdala
affects the extinction of simple fear conditioning. Although preliminary
findings indicate that systemic ifenprodil disrupts extinction of simple fear
conditioning (Sotres-Bayon et al.
2004), this systemic effect could be caused by an effect in the
mPFC or amygdala, or some other region.
Overall, results from human and animal studies strongly support the idea that the amygdala is an important site for the neural plasticity that underlies fear conditioning, and also possibly fear extinction. However, some questions remain unresolved about the neural signaling and neurophysiological response properties of amygdala neurons in extinction. More studies are needed to develop understanding about amygdala involvement during different aspects of extinction (acquisition, consolidation, retrieval); which amygdala nuclei, if any, are critically involved in each aspect; and how these interact with or depend on other structures, such as the mPFC.
Role of mPFC-Amygdala Interactions in Extinction
The presence of strong reciprocal connections between the amygdala and mPFC
suggests that these regions are functionally coupled (McDonald
1991,
1998;
Ghashghaei and Barbas 2002;
Vertes 2004). To evaluate the
possible role of interactions between the mPFC and amygdala in fear
extinction, we first review their interconnections and then consider evidence
for physiological interactions.
Anatomical Connectivity Between the mPFC and Amygdala
The amygdala is robustly connected with the mPFC, with different amygdala
nuclei receiving projections from distinct mPFC regions. For example, using
anterograde anatomical tracing, several studies have investigated the
distribution of mPFC projections to distinct nuclei within the amygdala in
rats (Sesack et al. 1989;
Hurley et al. 1991;
McDonald 1991;
Berendse et al. 1992;
McDonald et al. 1996;
Vertes 2004), and in
non-human primates (Room et al.
1985; Chiba et al.
2001; Ghashghaei and Barbas
2002). In general, these studies indicate that the IL sends a
strong excitatory input to the LA and ITC, whereas the PL sends a similar
input preferentially to the B (Berendse et
al. 1992; McDonald et al.
1996). Interestingly, the B also sends projections back to the PL
(Shinonaga et al. 1994;
Sah et al. 2003), which in
turn sends a heavy projection to the IL
(Pitkänen 2001). The IL
and PL also project to portions of the CE
(McDonald et al. 1996),
suggesting another way for mPFC to influence the amygdala; however, these
projections are relatively sparse and thus are not focused on here.
Thus, the IL and PL are anatomically connected to the amygdala via projections to the LA, B, CE, and ITC, and are accordingly in a position to regulate amygdala function. However, the exact manner in which this regulation occurs is still being debated.
Physiological Interactions Between the mPFC and Amygdala
It has been repeatedly proposed that the reduction of conditioned fear that
occurs during extinction is mediated by the connections from the mPFC to the
amygdala (Morgan et al. 1993;
Milad and Quirk 2002;
Royer and Paré 2002;
Rosenkranz et al. 2003), but
the mechanisms for mPFC-induced suppression of amygdala function are not yet
known.
Consistent with the anatomy, electrophysiological studies in anaesthetized
rats indicate that IL stimulation evokes activity mainly in LA neurons,
whereas PL stimulation evokes activity mainly in B neurons (Rosenkranz and
Grace 2001,
2002). Importantly, the
results reported by Rosenkranz and Grace further suggest that IL and PL
stimulation directly excites inhibitory GABAergic interneurons within the LA
and B, respectively, with a consequent inhibition of LA and B outputs. Indeed,
Rosenkranz et al. (2003) have
recently shown that mPFC stimulation can suppress LA pyramidal cell responses
to a fear-arousing CS.
Given that most mPFC projections to the amygdala are excitatory
(Smith et al. 2000;
Rosenkranz and Grace 2001),
it has been proposed that mPFC inhibition of amygdala output involves the
activation of inhibitory neurons within the amygdala (Rosenkranz and Grace
2001,
2002;
Quirk and Gehlert 2003;
Rosenkranz et al. 2003). Two
models have been proposed. One argues that mPFC projections stimulate
inhibitory GABA-containing interneurons in the LA and B nuclei
(Grace and Rosenkranz 2002),
and that these interneurons, in turn, decrease the responsiveness of
excitatory LA and B projections to CE output cells (see
Fig. 1A). The second model
argues that mPFC projections excite inhibitory projection neurons in ITC,
which, in turn, inhibit CE output neuron activity (see
Fig. 1B), and thereby compete
with excitatory projections from the LA and/or B to the CE
(Paré 2003;
Quirk et al. 2003;
Paré et al. 2004).
According to these competing models, extinction learning could involve mPFC
activation of inhibitory interneurons within the LA/B
(Grace and Rosenkranz 2002),
or alternatively, mPFC activation of inhibitory projections from the ITC to
the CE (Quirk et al. 2003).
In each case, mPFC projections to the amygdala are considered crucial.
Consistent with both models, Quirk et al.
(2003) report that mPFC
stimulation inhibits, and only inhibits, CE output neurons—that is,
mPFC-induced excitation of CE output neurons was never observed. Thus far
there are no published findings that discriminate between these two
models.
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Hypothesis Test: Basal Amygdala (B) Mediates Extinction of Fear
Three lines of evidence suggest that the B might be involved in fear extinction. The first two have already been discussed and will only be briefly mentioned.
First, many studies have explored the role of the LA and B (together as the
basolateral complex) in fear extinction
(Falls et al. 1992;
Lee and Kim 1998;
Lu et al. 2001;
Marsicano et al. 2002;
Walker et al. 2002;
Ledgerwood et al. 2003; Lin et
al.
2003a,b).
Evidence from these findings strongly suggests that the B (as part of the
basolateral complex) should play a role in fear extinction.
Second, the two competing physiological hypotheses described above both imply that the B is involved in fear extinction (there are other possibilities, as described below, but the most direct implication of these two hypotheses, as they are usually described, is that the B should be involved). One suggests that excitatory inputs to B inhibitory cells leads to inhibition of CE neurons, whereas the other suggests that excitatory inputs to B projection cells leads to excitation of inhibitory cells in the ITC, and these inhibit CE. Thus, through either of these circuits, damage to the B should alter fear extinction.
Third, a role for the B in fear extinction is suggested by the role of the
B in contextual fear conditioning (Everitt
and Robbins 1992; Kim and
Fanselow 1992; Phillips and
LeDoux 1992; Maren and
Fanselow 1996; Anagnostaras et
al. 1999) and the fact that extinction is context-dependent
(Bouton and King 1983; Bouton
1988,
1993,
2002). When early studies
found that damage to sensory-processing regions of the cortex led to an
increased resistance to extinction (LeDoux
et al. 1989; Teich et al.
1989), it was proposed that sensory cortex was a necessary
way-station to the mPFC and/or hippocampus. Although the role for the mPFC in
extinction has been largely confirmed, the potential role of the hippocampus
has remained relatively unexplored. Anatomical
(van Groen and Wyss 1990;
McDonald and Mascagni 1997;
McDonald 1998;
Pitkänen et al. 2000b)
and electrophysiological (Colino and
Fernandez de Molina 1986b;
Maren and Fanselow 1995;
Ishikawa and Nakamura 2003)
studies indicate that there are strong reciprocal projections between the B
and the hippocampus, as well as between the B and the mPFC
(Garcia et al. 1999;
Rosenkranz and Grace 1999,
2001,
2002;
Pitkänen 2001;
Ishikawa and Nakamura 2003).
The B has been proposed as the site of contextual inputs to the amygdala in
contextual conditioning (Selden et al.
1991; Everitt and Robbins
1992; Kim and Fanselow
1992; Phillips and LeDoux
1992; Maren and Fanselow
1995; Anagnostaras et al.
1999). Together these data suggest that the B may be a possible
candidate for integrating information from the LA, the hippocampus, and the
mPFC. Indeed, recently, Corcoran and Maren
(2001) found that the
hippocampus is necessary for the context-specific expression of fear
extinction, although others have reported contradictory findings
(Wilson et al. 1995;
Frohardt et al. 2000).
To test whether the B is involved in extinction, we performed bilateral
electrolytic lesions of the B prior to fear conditioning, and evaluated the
effects of these lesions on fear conditioning and fear extinction
(Fig. 2, B lesion protocol).
Consistent with other studies (Amorapanth
et al. 2000; Nader et al.
2001), we found that extensive damage to the B (most lesions
included anterior and posterior damage to the nuclei;
Fig. 3) did not prevent
acquisition of tone-conditioned fear (Fig.
4). Furthermore, lesions of the B did not interfere with the
extinction of tone-elicited fear responses
(Fig. 4). This lack of effect
of B lesions, which was also found in a recent experiment
(Anglada-Figueroa et al. 2003),
confirms that the B is not necessary for cued simple fear conditioning, but
also indicates that the B is not necessary for the extinction of fear
conditioning.
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The dissociation of the basolateral complex into LA and B regions (each of
which has further subregions) has been generally overlooked in fear extinction
research. As noted above, previous studies have shown that basolateral complex
manipulations alter fear extinction, which suggests that the LA and/or B are
involved. Thus, our results showing that B lesions have no effect on fear
extinction implicate the LA, rather than the B, as the part of the basolateral
complex that is involved in fear extinction, similar to its role in fear
conditioning. Consistent with the idea that the LA plays a critical role in
fear extinction is the finding that mPFC stimulation suppresses LA plasticity
(Rosenkranz et al. 2003).
Nonetheless, it is also possible that extinction additionally involves the CE,
given recent studies implicating the CE in fear acquisition
(Paré et al. 2004;
A.E. Wilensky, G.E. Schafe, M.P. Kristensen, and J.E. LeDoux, in prep.).
It should be noted that the fact that fear extinction occurs independently of the B does not necessarily discredit either of the two competing physiological hypotheses about fear extinction described above. That is, the mPFC could suppress amygdala-mediated fear via direct projections to the LA or ITC, rather than to the B. We return to this point below.
An important issue that is emphasized by our finding is that researchers
interested in extinction or other functions mediated by the amygdala should,
when possible, use the known detailed anatomical partitioning of the amygdala
into nuclei and subnuclei (Amaral et al.
1992; Pitkänen et al.
1997, Pitkänen
2001), rather than rely on less precise concepts, such as the
basolateral complex.
Assessment of the Contribution of mPFC and Amygdala Interactions
Implications for Models of mPFC-Amygdala Interactions
The importance of mPFC regulation over amygdala function for the extinction
of conditioned fear is strongly supported by research that has progressed
throughout the past decade. The original findings of Morgan et al.
(1993) showed that ventral
mPFC lesions disrupt fear extinction, and recent results indicate a specific
role for the mPFC, particularly the IL, in the retrieval and possibly the
storage of prior extinction learning
(Quirk et al. 2000;
Garcia 2002;
Morgan et al. 2003). Thus, we
now have a strong basis for the initial proposal that disruption of mPFC
control over amygdala function can result in emotional perseveration
(Morgan et al. 1993;
Morgan and LeDoux 1995).
Nonetheless, much work remains to be done. We still do not know the crucial
neurophysiological mechanisms that underlie mPFC control over
amygdala-mediated fear responses. Our current evaluation of the role for the
B, by performing selective B lesions that spared the LA, indicates that the B
is not a crucial locus for extinction. The possibility remains that mPFC
projections to inhibitory neurons within the LA are critically involved
(Rosenkranz and Grace 2001,
2002;
Grace and Rosenkranz 2002), or
alternatively that mPFC projections to the ITC regulate LA inputs to the CE
(Quirk et al. 2003). Indeed,
as discussed above, Rosenkranz et al.
(2003) have shown that mPFC
stimulation can suppress LA plasticity during conditioning, and can also
suppress LA responses to cues that have been previously fear-conditioned.
These results emphasize the possibility that mPFC-mediated alterations of
plasticity within the LA may be important for extinction learning, and also
that mPFC inputs to inhibitory cells within the LA may be activated during the
retrieval of prior extinction learning.
Model for Hippocampal-Based Contextual Constraints on Extinction
It has long been recognized that extinction depends on the learning of a
new inhibitory (i.e., a CS-NoUS) association that competes with the original
excitatory (i.e., CS-US) association
(Bouton 2002;
Davis and Myers 2002;
Myers and Davis 2002). After
extinction, the original conditioned response can be "renewed" if
the CS is presented in a context that is distinct from the one in which
extinction learning occurred (Bouton
2002). This renewal not only demonstrates that the original
excitatory association remains intact, but also suggests that the extinction
of cued fear is largely dependent on the context. In essence, the retrieval of
extinction learning appears to be contextually constrained.
Corcoran and Maren (2001)
have shown that reversible inactivation of the dorsal hippocampus with
muscimol disrupts the context-specific expression of prior extinction
learning. Specifically, intrahippocampal muscimol infusions appeared to remove
contextual constraints on the retrieval or expression of extinction to a tone
CS, thereby abolishing the capacity of a context change to renew the original
conditioned fear response. Intrahippocampal muscimol had no effect on
tone-elicited fear when rats had not been previously extinguished
(Corcoran and Maren 2001),
indicating that the reduced freezing was not attributable to a general
reduction of fear.
The possibility that hippocampal afferents to the B might be important for
contextual contributions to cued fear extinction was one factor that motivated
the current experiment (see Fig.
5A). However, our results suggest that convergence of mPFC and
hippocampal inputs within the B is not important for the learning or
expression of extinction. Thus, an alternative model is needed, one that
incorporates the current result with the evidence for both mPFC
(Quirk et al. 2000;
Grace and Rosenkranz 2002;
Barrett et al. 2003) and
hippocampal (Corcoran and Maren
2001; Barrett et al.
2003) contributions to extinction of cued fear (see
Fig. 5B).
|
Strong hippocampal projections from the CA1/subiculum to the mPFC have been
identified (Jay and Witter
1991; Conde et al.
1995), and form mainly excitatory synapses
(Carr and Sesack 1996). Recent
electrophysiological findings indicate that stimulation of hippocampal cells
induces monosynaptic, AMPA-receptor-dependent activation of mPFC neurons,
localized primarily within the IL and ventral PL regions
(Degenetais et al. 2003;
Ishikawa and Nakamura 2003).
Moreover, these sites correspond with mPFC stimulation sites that evoke
activity of LA cells (Rosenkranz and Grace
1999,
2001,
2002;
Ishikawa and Nakamura 2003),
and perhaps the ITC as well (Quirk et al.
2003). Overall, these anatomical/electrophysiological findings,
together with the behavioral findings
(Quirk et al. 2000;
Corcoran and Maren 2001;
Barrett et al. 2003;
Rosenkranz et al. 2003),
suggest that hippocampal inputs to mPFC cells may subserve contextual
constraints on the retrieval of cued fear extinction.
Furthermore, Hobin et al.
(2003) found that after
extinction training, CS-elicited firing in LA neurons was suppressed only in
the extinction context. This finding led them to propose a model whereby
context-specific hippocampal-mPFC interactions modulate conditioned fear
responses by inhibiting projections from the LA to the B to the CE. However,
because electrolytic B lesions have no effect on the expression or extinction
of conditioned fear, we propose that Figure
5B more accurately depicts the circuitry underlying contextual
constraints on extinction.
Role of the LA in Extinction Learning
As noted above, intra-amygdala infusions of an NMDA antagonist or MAPK
inhibitor disrupted extinction learning
(Falls et al. 1992;
Lu et al. 2001), and similar
infusions of an NMDA agonist enhanced extinction learning
(Walker et al. 2002;
Ledgerwood et al. 2003). More
recently, endocannabinoids in the amygdala have been implicated, because
cannabinoid receptor 1 receptor knockout mice show disrupted extinction of
fear, and endocannabinoids are elevated in the LA and B during extinction
training (Marsicano et al.
2002). Finally, gastrin-releasing peptide (Grp) is highly
expressed in the LA, and receptors for this peptide are localized on LA
GABAergic interneurons. Mice lacking these Grp receptors showed decreased
inhibition of LA pyramidal neurons, greater conditioned fear, and resistance
to extinction, suggesting that Grp may endogenously regulate LA outputs to the
CE by stimulating inhibitory interneurons
(Shumyatsky et al. 2002).
Collectively, these findings suggest that molecular events within the amygdala
may be crucially activated during extinction learning, and because we have
shown that the B is not needed for the extinction of simple cued fear
conditioning, we emphasize that the LA is the probable candidate for the
extinction-related functional consequences of these molecular signals.
Considerable data have been amassed throughout the past decade to suggest
that both the mPFC and the LA are part of a circuit whereby the
amygdala-processed underlying conditioned fear responses are suppressed by
descending mPFC control. Moreover, this mPFC control over the LA is
specifically needed for the retrieval of prior extinction learning, rather
than for the acquisition of fear extinction per se, and this mPFC-mediated
retrieval of extinction appears to be contextually constrained. Accordingly,
consistent with Hobin et al.
(2003), we emphasize that
hippocampal inputs to the mPFC may set the circumstances for mPFC-mediated
control over amygdala fear circuitry. However, the B does not appear to be
crucially involved. Since molecular events within the LA itself are implicated
in fear learning (e.g. LeDoux
2000; Blair et al.
2001; Maren 2001;
Schafe et al. 2001)
extinction learning may require additional events within the LA that suppress
the fear memory. Thus, synaptic plasticity within the LA may be crucial for
establishing specific mFPC-LA connections that are activated later, at the
time of extinction retrieval.
Clinical Implications of mPFC-Amygdala Interactions
We have emphasized the role of animal studies in suggesting the importance of interactions between the mPFC and amygdala in fear regulation. However, studies in humans also implicate interactions between these two brain structures in several affective disorders.
In fear extinction, the appropriate regulatory interaction between the mPFC
and the amygdala is critical for the organism to adapt as situations change.
Failure in this naturally occurring mechanism can lead to inadequate and
maladaptive affect-related behaviors, referred to as emotional preservation.
Similar emotional preservation occurs in several mental disorders, and may
well involve the dysfunction of the mPFC-amygdala interaction. In fact, it has
been argued that when the mPFC-amygdala interaction is compromised, certain
types of mental disorders can develop, such as depression (Drevets
1998,
1999,
2001,
2003;
Davidson 2002b;
Siegle et al. 2003), anxiety
(Davidson 2002a), and fear
disorders, including posttraumatic stress disorder
(Quirk and Gehlert 2003;
Rothbaum and Davis 2003). For
instance, functional neuroimaging studies have identified that the amygdala is
hyperactive (Siegle et al.
2002) and the mPFC is hypoactive (Drevets et al.
1992,
1998;
Drevets 2003) in depressed
patients. Furthermore, several studies with humans have shown that functional
activity in the mPFC is inversely related to amygdala activity
(Abercrombie et al. 1998;
Davidson et al. 1999,
2000; Davidson
2002a,b;
Anand and Shekhar 2003), and
post-traumatic stress disorder patients show an unusually high activation in
the amygdala and lower than normal activity in the mPFC when exposed to
threatening stimuli (Bremner et al.
1999; Fernandez et al.
2001; Shin et al.
2001).
Conclusions
Animal models suggest that under normal circumstances the mPFC and amygdala orchestrate the control of affective states by regulating each other at particular moments in an emotional experience. Clinical evidence suggests these interactions may be compromised in certain emotional disorders. Advances that are made using animal models of fear extinction are crucial to developing our knowledge, which it is hoped will lead to treatments for these disorders.
MATERIALS AND METHODS
Subjects were adult male Sprague-Dawley rats housed individually in Plexiglas cages. Rats weighed between 325 and 350 g upon arrival at the laboratory and were kept on a 12-12-h light-dark cycle (lights on at 0700). Rats had ad libitum access to food and water throughout the duration of the experiment.
Bilateral electrolytic lesion of the B was made according to previously
published procedures (Amorapanth et al.
2000; Nader et al.
2001). For surgery, rats were anaesthetized with pentobarbital (50
mg/kg, i.p.) and placed into the stereotaxic frame. The skull was exposed and
four holes per side were drilled over the B using a dental drill. Electrolytic
lesions were made by passing positive current (0.5 mA) at each site through a
monopolar electrode insulated with epoxy to within 200 µm of the tip.
Coordinates (in millimeters relative to the skull surface at bregma) were:
anterior/posterior = 2.1, 2.8, 3.3, and 4.2; medial/lateral = 4.9, 4.9, 5.3,
and 5.3; and dorsal/ventral = 9.1, 9.3, 9.2, and 9.3
(Paxinos and Watson 1998).
Respectively, current durations (in seconds) were 12, 15, 15, and 15. Only
rats with lesions localized to the B sparing other amygdala regions were
included in the analysis (n = 7; see
Fig. 3), and 12 rats were
excluded, either because there was insufficient damage to the B bilaterally or
there was damage to other amygdala nuclei. Sham rats (n = 8) received
the same treatment except that electrodes were placed 1-2 mm above the B and
current was not passed through the electrode. After surgery, rats were allowed
to recover in their home cages for 1 wk. A third group (naive: n = 8;
data not shown) did not receive any surgery, but was subjected to the same
behavioral procedures.
Behavioral procedures took place in two different contexts (Context A and Context B) to avoid possible confound influences of the hippocampal-dependent context conditioning. Chambers were contained within a sound isolation cubicle (Model H10-24A; Coulbourn Instruments), and equipped with an overhead infrared motion detector that continuously monitored all movement in the chamber. Recorded activity was acquired at a temporal resolution of 20 msec through a computer running Graphics Notation software (Coulbourn Instruments) that also controlled stimulus presentation. Behavior was also monitored with an infrared camera and recorded on videotape for visual confirmation of the automated recording. Context A consisted of a conditioning chamber with a steel rod floor. A single house light illuminated the chamber. Context B was a modification of Context A in which the metal grid floor was covered with a sheet of transparent plastic previously wiped with peppermint-scented soap and the house light was off. Fear conditioning occurred in Context A, and extinction training took place in Context B. For all rats, CS-US or CS-noUS trials were preceded by a 4-min period in which they were allowed to explore the chamber. On Day 0, rats were habituated to each context for 10 min, with a 2-h interval between each context habituation. On Day 1, fear conditioning occurred. The rats were allowed to explore for 4 min and then received five tone-footshock pairings (CS-US; CS tone = 20 sec, 80 dB, 10 kHz; US footshock = 1 sec, 0.6 mA; intertrial interval = average 2 min [range 1-3 min]). The next day, Day 2, rats were exposed to their first extinction session in Context B. The rats were allowed to explore for 4 min before they were exposed to 20 tone-alone (CS-noUS) presentations. Finally on Day 3, rats were exposed to 15 trials of tone-alone presentations (CS-noUS) in Context B.
Following completion of the behavioral testing, rats were given an overdose of chloral hydrate (25%, 1 cc/100 g) and perfused with physiological saline followed by 10% buffered formalin. The brains were stored in a 30% sucrose, 10% formalin solution for at least 4 d. Brains were then frozen and cut into 40-µm sections using a cryostat, with every other section through the lesion site mounted on a subbed slide and stained with cresyl violet. Sections were examined and images were digitally captured under light microscope.
Freezing was used as the measure of conditioned emotional response. Freezing was defined as immobility with the exception of respiratory-related movement and non-awake or rest body posture (i.e., curled up). Freezing was assessed initially using an automated system, and then confirmed by observing the rat's behavior stored on videotape.
ACKNOWLEDGMENTS
This work was supported by R01 MH46516, R37 MH38774, P50 MH58911, and K05 MH067048 to J.E.L.
FOOTNOTES
Article and publication are at http://www.learnmem.org/cgi/doi/10.1101/lm.79504.
1 E-MAIL fsotres{at}cns.nyu.edu; FAX (212) 995-4704.
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