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 Previous Article
The Journal of Neuroscience, June 1, 2001, 21(11):4116-4124
Amygdalar NMDA Receptors Are Critical for the Expression
of Multiple Conditioned Fear Responses
Hongjoo J.
Lee1,
June-Seek
Choi1,
Thomas H.
Brown1, 2, 3, and
Jeansok J.
Kim1, 3
Departments of 1 Psychology and 2 Cellular
and Molecular Physiology, and 3 Interdepartmental
Neuroscience Program, Yale University, New Haven, Connecticut
06520-8205
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ABSTRACT |
There is conflicting evidence regarding the issue of whether NMDA
receptors in the basolateral amygdalar complex (BLA) are critically
involved in the expression of conditioned fear. This matter was
addressed by infusing the rat BLA with
D,L-2-amino-5-phosphonovaleric acid (APV), a competitive
NMDA receptor antagonist. APV infusion into the BLA was reported to
block the expression of conditioned fear when measured by freezing but
not when measured by fear-potentiated startle response to a loud noise.
To examine this issue further, here we used multiple indices of
conditioned fear, including analgesia, 22 kHz ultrasonic vocalization
(USV), defecation, and freezing. Rats with bilateral BLA cannula
implants underwent fear conditioning consisting of 10 tone-footshock
pairings. Before context and tone fear-retention tests, animals
received intra-BLA infusions with APV (2.5 µg/side) or artificial
CSF. Both tone and context tests demonstrated that the
expression of conditioned freezing, USV, defecation, and analgesia were
significantly impaired by intra-amygdalar infusions of APV. In a second
set of experiments, intra-BLA infusions of APV markedly impaired the
normal expression of postshock fear responses during training, as
measured by freezing, USV, and defecation. Immediate postshock fear
expression was predictive of subsequent fear retention to the tone and
context when the animals were not infused. These results are consistent
with the hypothesis that amygdalar NMDA receptors participate in normal
synaptic transmission and therefore the overall functioning of the amygdala.
Key words:
learning; memory; amygdala; LTP; fear conditioning; synaptic plasticity; emotion
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INTRODUCTION |
Long-term synaptic potentiation
(LTP) is commonly suggested as a candidate neurophysiological mechanism
through which acquired fear is encoded in the amygdala (Chapman et al.,
1990 ; Kim et al., 1991; Davis et al., 1993; Lam et al., 1996; Maren and
Fanselow, 1996; LeDoux, 2000). This idea was based in part on the
demonstration of LTP in the amygdala in vitro (Chapman et
al., 1990) and in vivo (Clugnet and LeDoux, 1990), as well
as on extensive synaptic studies of the hippocampus, in which it has
been shown that associative LTP exists (Barrioneuvo and Brown, 1983);
this LTP is governed by a Hebbian (Hebb, 1949) mechanism (Kelso et al.,
1986), and this Hebbian form of associative LTP has some of the
ingredients required to construct a platform for Pavlovian conditioning
(Kelso and Brown, 1986; Brown et al., 1990).
A once tenable belief was that the NMDA subtype of glutamate ionotropic
receptor (NMDAR) was essential for inducing LTP, but that it had
no role in the expression of LTP or normal synaptic transmission
(Collingridge et al., 1983; Teyler and DiScenna, 1987; Morris et al.,
1990). In this case, a suitable NMDAR antagonist might conceivably
block the acquisition of conditioned fear without having any effect on
its expression. The idea that amygdalar LTP might participate in fear
conditioning was supported by numerous studies reporting that
intra-amygdalar infusions of the competitive NMDAR antagonist
D,L-2-amino-5-phosphonovaleric acid (APV) effectively blocked the acquisition of fear conditioning (Miserendino et al., 1990;
Campeau et al., 1992; Fanselow and Kim, 1994; Maren et al., 1996).
The receptor mechanisms responsible for the induction and expression of
amygdalar LTP remain uncertain and may depend on the particular
synapses and input pathway (Chapman et al., 1990; Weisskopf and LeDoux,
1999; LeDoux, 2000), as demonstrated in the hippocampus (Harris and
Cotman, 1986; Brown et al., 1989; Grover and Teyler, 1990; Zalutsky and
Nicoll, 1990; Johnston et al., 1992). One report (Chapman and
Bellavance, 1992) suggested that APV can interfere with LTP induction
in the basolateral amygdalar complex (BLA), but only in such high
concentrations that the drug markedly impaired normal synaptic
transmission (but see Huang and Kandel, 1998). Similarly, single-unit
recordings indicate that normal auditory-evoked responses in the
amygdala are considerably attenuated by APV, suggesting that NMDARs are
involved in normal synaptic transmission of the auditory pathway to the
amygdala that putatively mediates auditory fear conditioning (Li et
al., 1995). These results were foreshadowed and are reinforced by APV
studies on the visual cortex, in which APV was found to substantially
interfere with normal synaptic transmission (Miller et al., 1989; Shatz
1990).
These data notwithstanding, two reports have concluded that APV
infusions into the amygdala have virtually no effect on the expression
of conditioned fear, as measured by fear-potentiated startle responses
to a loud noise (Miserendino et al., 1990; Campeau et al., 1992).
However, two subsequent studies did find a significant impairing effect
of APV on the expression of conditioned fear, as measured by freezing
(Maren et al., 1996; Lee and Kim, 1998). Attempting to reconcile these
disparate findings, Lee and Kim (1998) hypothesized that there may be a
divergence of fear conditioned response (CR) centers within the
amygdala, such that NMDARs are involved in synaptic transmission
mediating the expression of some measures of conditioned fear (such as
freezing) but not others (such as fear-potentiated startle). The
present experiments further explore this hypothesis.
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MATERIALS AND METHODS |
Experiment 1: effects of intra-amygdalar APV on the expression of
multiple conditioned fear responses
Previous studies have found that intra-BLA infusions of APV
impaired the expression of conditioned freezing to tone, light, and
context conditioned stimuli (CSs) in Long-Evans rats (Maren et al.,
1996; Lee and Kim, 1998). The goal of experiment 1 was to determine the
NMDAR dependence of the expression of conditioned fear using four
established fear measures, including analgesia (Helmstetter and
Bellgowan, 1993), the 22 kHz ultrasonic vocalization (USV) (a distress
signal) (Blanchard et al., 1991), defecation (Fanselow and Kim, 1992),
and freezing (Blanchard and Blanchard, 1969). As part of an ongoing
study of fear conditioning in different stocks of rats, Sprague Dawley
rats were used in the experiment assessing conditioned freezing, USV,
and defecation, whereas Long-Evans rats were used in the conditioned
analgesia experiment. These measures were used to assess conditioned
fear to a specific context and a discrete tone.
Subjects. Experimentally naive male Sprague Dawley and
Long-Evans rats (initially weighing 275-325 gm) were obtained from Charles River (Wilmington, MA) and housed individually in a
climate-controlled vivarium on a 12 hr light/dark cycle (lights on at
7:00 A.M.) with ad libitum access to food and water. All
experiments were conducted during the light phase of the cycle and were
in strict compliance with the Yale Animal Resource Center guidelines.
Surgery. All rats were anesthetized via intraperitoneal
injection of a ketamine (30 mg/kg)-xylazine (2.5 mg/kg) solution, with
supplemental injections given as needed. Under aseptic conditions, a
stereotaxic instrument (Stoelting, Wood Dale, IL) was used to implant
26 gauge guide cannulas (Plastics One Inc., Roanoke, VA) bilaterally
into the BLA (from bregma: anteroposterior,  2.3 mm; mediolateral, ±5
mm; dorsoventral, 7.7-8.0 mm). Implanted cannulas were cemented to
three anchoring screws on the skull. During 7 d of postoperative
recovery, the rats were adapted to transportation and handling, and
each dummy cannula was removed and replaced with a clean one.
Drugs and injection. APV (Research Biochemicals, Natick,
MA), dissolved in artificial CSF (ACSF), pH 7.4, was microinfused into
the BLA (bilaterally) by backloading the drug up a 33 gauge internal
cannula into polyethylene (PE 20) tubing connected to 10 µl Hamilton
microsyringes (Hamilton Company, Reno, NV). The internal cannula
extended 1.0 mm beyond the guide cannula. An injection volume of 0.3 µl (per side) was delivered using a Harvard PHD 2000 syringe pump
(Harvard Apparatus, Inc., South Natick, MA) over the course of 3 min
(at a rate of 0.1 µl/min). The internal cannula remained in place for
at least 30 sec after the infusions before being pulled out.
Because the intra-BLA infusion parameter used in the present study is
similar to those used by Davis and colleagues (Miserendino et
al., 1990; Falls et al., 1992), the extent of APV diffusion in the
amygdala should be reasonably comparable between these studies. In
addition, our drug infusion parameter is similar to other studies that
dissociated drug effects in the BLA from the central nucleus
(Fanselow and Kim, 1992; Roozendaal and McGaugh, 1997; Shors and
Mathew, 1998). Hence, any effects exerted by APV on the expression of
conditioned fear likely occur because of the blockade of NMDA
receptors within the BLA. However, the possibility of APV spreading to
and affecting immediate adjacent regions [especially the overlying
lateral amygdalar nucleus (LA)] cannot be ruled out.
Fear conditioning apparatus and procedure. Training and
testing took place in two modular operant test chambers, each equipped with speaker modules (Coulbourn Instruments, Allentown, PA), located in
a controlled acoustic room (Industrial Acoustics Company, Inc., New
York, NY). The two chambers differed in several regards: chamber A was
rectangular (27 cm width × 28 cm length × 30.5 cm height), whereas chamber B was octagonal (26.5 cm diameter × 25 cm
height). Chamber A had front and back walls made of clear Plexiglas and two side walls made of metal plates, whereas chamber B had all eight
walls constructed of clear Plexiglas. Finally, chamber A was placed in
a wooden isolation box (46 cm width × 53 cm length × 49 cm
height) that was painted white, whereas chamber B was placed in a
similar box that was painted black.
The grid floor of chamber A was composed of 16 stainless steel bars
(4.5 mm diameter) spaced 17.5 mm center-to-center and wired to a
Coulbourn precision-regulated animal shocker. The grid floor of chamber
B was composed of 17 stainless steel bars (5 mm diameter) spaced 15 mm
apart and wired to a second, identical Coulbourn shocker. The floor
grid and base pan of each chamber were washed thoroughly with tap water
and dried completely before training and testing. Fecal boli were
counted during training and testing days.
The experiments took place over the course of 5 consecutive days. On
day 1, rats were placed in either chamber A or chamber B
(counter-balanced). Both cages were wiped with 5% ammonium hydroxide solution, and the overhead room light was on. After 1 min, animals were
presented with 10 coterminating tone-footshock pairings (tone, 2.9 kHz, 82 dB, 10 sec; footshock, 1 mA, 1 sec) with 1 min intertrial intervals (ITIs). Animals were removed 1 min after the last shock and
returned to their home cages (see Table
1 for the experimental design). On
the second day, rats were infused with either APV or ACSF and placed in
the same chamber as on day 1 for 8 min of context testing. The
following day, all rats underwent the same context testing as on day 2, but without infusions.
The fourth day entailed a tone-retention test in a context shift.
Animals trained in chamber A were tested in chamber B and vice versa.
The floor of each chamber was replaced with Plexiglas, which was
scattered with sawdust. In addition, the overhead lights were turned
off, and each internal chamber was wiped with a 1% acetic acid
solution. These changes produced a reliable context shift. The animals
that received APV on day 2 were infused with ACSF, whereas the animals
that received ACSF on day 2 were infused with APV. The tone-retention
test consisted of 1 min of baseline followed by 8 min of continuous
tone. On the final day, animals underwent the same tone test as on day
4 in the absence of infusions. To minimize the extent of necrosis
around the cannula tip produced by multiple insertions of the injection
cannula, animals were infused before context test (day 2) and tone test
(day 4), but not before context extinction test (day 3) and tone
extinction test (day 5) (cf. Lee and Kim, 1998).
Freezing and USV data collection and analysis. The stimulus
presentations were controlled and the freezing data were collected by
an IBM personal computer (PC) equipped with the Coulbourn LabLinc Habitest Universal Linc System. Although the collection of the vocalization and freezing data was fully automated, each session was
recorded for video and audio analysis off-line, if necessary, using an
infrared light source and miniature video camera (CB-21; Circuit
Specialists, Inc., Mesa, AZ).
A 24 cell infrared activity monitor that detects the movement of the
emitted infrared (1300 nm) body-heat image from the animals in the
x-, y-, and z-axes was mounted on top
of each chamber and used to assess freezing behavior (cf. Lee and Kim,
1998). In brief, the total time of inactivity exhibited by each animal
was measured using a computer program, and freezing was defined as
continuous inactivity lasting  3 sec. Any behavior that yielded an
inactivity of <3 sec was recorded as general activity.
A heterodyne bat detector (Mini-3; Noldus Information Technology,
Wageninge, The Netherlands) was used to transform high-frequency (22 ± 5 kHz) ultrasonic vocalizations into the audible range. The
output of the bat detector was fed through an audio amplitude filter
(Noldus), which filtered out signals falling below an amplitude range
that was individually adjusted for each animal. The resulting signal
was then sent to an IBM PC equipped with Noldus UltraVox vocalization
analysis software. The software converted the signal into vocalization
onset and offset times according to the following specifications: an
onset was recorded if its duration was 30 msec, and the offset was
recorded if the onset of the ensuing episode was 40 msec apart. If
the interval was <40 msec, the two bouts were counted as a single episode.
A custom-written analysis program was used to generate a raster plot
representing the distribution of freezing and vocalization episodes
over the entire session.
Conditioned analgesia apparatus and procedure. Long-Evans
rats underwent 3 consecutive days of fear conditioning (without infusions). Ten tone-footshock pairings were presented daily in the
same manner as described for day 1 above. Our pilot data indicated that
3 d of training produced reliable conditioned analgesia to the
tone CS. At 2-3 hr after the training sessions on days 1, 2, and 3, each subject was gently restrained under a hand towel on a
Tail-Flick Analgesia Meter (model 0570-001L; Columbus Instruments, Columbus, OH) for ~20 sec of adaptation, during which the tail of the
animal was placed on the heat lamp source (which was turned off on days
1 and 2). This procedure was repeated four times with ~30 sec
interval periods between adaptations.
On day 3 of training, all animals underwent tail-flick experiments
(four measures) with the lamp intensity setting preset to 8, which
produced ~10 sec baseline tail-flick responses as measured using the
automatic tail-flick detection mode. On day 4 of testing, animals were
infused with either APV or ACSF into their BLA before the tail-flick
responses were measured in the manner described on day 3, the only
change being that the tone CS came on 10-15 sec before the third
tail-flick measure and remained on all through the fourth measure. The
speaker module used during training was placed next to the tail-flick
apparatus. The experimenter performing the tail-flick procedure was
unaware of the drug treatment the animal had received.
Histology. At the completion of all behavioral testing, the
subjects were overdosed with ketamine HCl and xylazine and perfused intracardially with 0.9% saline followed by 10% buffered formalin. The brains were removed and stored in 10% formalin for at least 2 weeks before slicing. Transverse sections (80 µm) were taken through
the extent of the cannula placement, mounted on gelatinized slides, and
stained with cresyl violet dye. An observer unaware of the behavioral
data determined the locations of the cannula tips, and subjects with
inaccurate cannula placements were excluded from the statistical analysis.
Experiment 2: effects of APV on expression of postshock fear
responses and acquisition of conditioned fear
Based on the results of experiment 1, it is clear that
intra-amygdalar infusions of APV during retention tests can profoundly impair the expression of conditioned fear, as measured by freezing, USV, defecation, and analgesia. The goal of experiment 2 was to examine
the effects of intra-amygdalar infusions of APV during training and to
relate postshock fear responses to subsequent retention tests in which
none of the animals were infused. The only precedent to this experiment
was a study by Lee and Kim (1998) showing that when APV was
administered to Long-Evans rats just before fear conditioning, it
considerably attenuated postshock freezing during training. The
relationship between postshock fear responses and subsequent
performance on retention tests might potentially be relevant to the
interpretation of the mechanisms of action of APV. Specifically, if
intra-amygdalar APV blocks the acquisition of fear conditioning by
directly interfering with normal synaptic transmission (of CS and/or US
pathways) in the amygdala, then the effect of APV on postshock fear
responses should correlate with the level of conditioned fear during
the retention test.
Subjects, surgeries, and behavioral training. Two groups of
naive Sprague Dawley male rats (275-325 gm) received bilateral cannula
implants and drug infusions in a manner identical with that of
experiment 1.
On day 1, animals were infused with either APV (2.5 µg/0.3 µl per
side) or ACSF (0.3 µl per side) and placed in the chamber. After 1 min, animals were presented with 10 coterminating tone-footshock pairings. The animals were removed 1 min after the last shock and
returned to their home cages. On day 2, the animals were placed back in
the trained context for 8 min of context testing. Animals were not
infused during this testing period. On day 3, animals were given a
tone-retention test, which consisted of 1 min of baseline followed by 8 min of continuous tone. Animals did not receive infusions before the
tone test.
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RESULTS |
Experiment 1
Histology
Figure 1 shows a photomicrograph of
a transverse brain section stained with cresyl violet from a typical
rat with bilateral guide cannulas implanted in or just dorsal to the
BLA, as well as a composite of the injection sites based on a
reconstruction of cannula placements (Paxinos and Watson,
1997).
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Figure 1.
Top, Photomicrograph showing a
transverse brain section stained with cresyl violet from a rat with
bilateral guide cannulas implanted in the BLA.
Arrowheads indicate guide cannula tip positions.
Bottom, Location of injection sites based on a
reconstruction of guide cannula placements in the BLA from experiment 1 animals.
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Conditioned freezing
Figure 2A depicts
the mean percentage of freezing from tone-shock-trained animals in
both group I (open circles, n = 9) and group
II (filled circles, n = 10). As
shown, animals in both groups exhibited robust postshock freezing
during the ITIs on day 1 of infusion-free training.
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Figure 2.
A, Mean percentage of freezing (± SE) during the 1 min baseline and during the intervening 10 tone-shock
pairings (denoted by small bars) of the 1 min ITIs with
no infusions. Group I received ACSF during the context test and
APV during the tone test, whereas group II received APV during the
context test and ACSF during the tone test. B, Mean
percentage of freezing (± SE) during 8 min context-retention testing
after intra-BLA infusions of ACSF or APV (Day 2) and
during 8 min context retesting the next day (Day 3).
C, Mean percentage of freezing (± SE) during an 8 min
tone-retention test in context B after intra-BLA infusions of ACSF or
APV (Day 4) and during a tone retest the next day
(Day 5).
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The context test (Fig. 2B) shows that group II
animals (which received intra-amygdalar infusions of APV before context
testing) showed significantly impaired freezing (on day 2) in
comparison with the group I animals that received ACSF infusion
(F(1,17) = 61.45; p < 0.01). When retested in the same context in the absence of infusions
(on day 3), group II animals (which had received APV infusions on day
2) now froze significantly more than they did on day 2 (F(1,9) = 15.08; p < 0.01). In contrast, group I animals (which had received an ACSF
infusion on day 2) exhibited greater freezing on day 2 than on day 3 (F(1,8) = 6.21; p < 0.05). This pattern is consistent with selectively impaired day 2 extinction in APV-infused animals and is supported by a significant
interaction between the drug treatment and the testing day
(F(1,17) = 20.22; p < 0.01).
The tone test (Fig. 2C) shows that the animals that received
APV infusions before retention testing demonstrated significantly attenuated freezing in comparison with the animals that received ACSF
(F(1,17) = 5.80; p < 0.05). When retested to the tone without infusions (on day 5), the
animals that had received APV on the previous tone test froze
considerably more than the animals that received ACSF on the previous
tone test (F(1,17) = 4.56;
p < 0.05). Again, there was a significant interaction
between the drug treatment and the testing day
(F(1,17) = 19.60; p < 0.01), consistent with the hypothesis that APV blocks extinction during
the tone test on day 4.
To appreciate better the impairing effects of intra-BLA APV on the
expression of conditioned fear across time, Figure
3 displays freezing as event raster plots
that show the distribution of freezing episodes (raw data) from a
typical APV and ACSF animal during context and tone tests. Here and
elsewhere we operationally define the term "typical" to denote that
the animal was selected on the basis that its performance was at the
median of the distribution for its group. Each point on the raster plot
represents an episode of freezing. Note that no subjectivity entered
into these dramatic differences in freezing, which were scored by the
apparatus as indicated earlier, and that these results are genuinely
representative in the operationally defined sense described above.
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Figure 3.
Examples of conditioned freezing raster plots
indicating the distribution of freezing episodes from a typical ACSF
and APV animal during context testing after infusions (Day
2) and context retesting without infusions (Day
3) (A), and during tone-retention testing
after infusions (Day 4) and tone retesting
without infusions (Day 5) (B). The
raster is divided into 1 min blocks in which each dot
represents a time-stamped event of freezing.
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Conditioned USV
Figure 4A depicts
the mean duration of USV from tone-shock-trained animals in both group
I (open circles) and group II (filled circles). As indicated, animals in both groups exhibited robust postshock USV during day 1 of infusion-free training.
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Figure 4.
A, Mean duration of USV (± SE)
during the 1 min baseline and during the intervening 10 tone-shock
pairings (denoted by small bars) of the 1 min ITIs with
no infusions. Group I received ACSF during the context test and
APV during the tone test, whereas group II received APV during the
context test and ACSF during the tone test. B, Mean
duration of USV (± SE) during 8 min of context testing after intra-BLA
infusions of ACSF or APV (Day 2) and 8 min of context
retesting the next day (Day 3). C, Mean
duration of USV (± SE) during the 8 min tone-retention test in context
B after intra-BLA infusions of ACSF or APV (Day
4) and during the tone retest the next day (Day
5).
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The context test (Fig. 4B) shows that group II
animals (which received intra-amygdalar infusions of APV before
testing) showed significantly impaired USV (on day 2) in comparison
with the group I animals (which received ACSF infusion)
(F(1,17) = 26.55; p < 0.01). When retested in the same context in the absence of infusions (on day 3), group II animals (which had received APV on day 2) produced
significantly more vocalizations than they had on day 2 (F(1,9) = 6.22; p < 0.05). In contrast, group I animals (which had received an ACSF
infusion on day 2) generated significantly more USV on day 2 than on
day 3 (F(1,8) = 18.96;
p < 0.01). Once again, this pattern is consistent with
selectively impaired day 2 extinction in the APV-infused animals and is
supported by a significant interaction between drug treatment and
testing day (F(1,17) = 19.54;
p < 0.01).
The tone test (Fig. 4C) shows that the animals that received
APV infusions before retention testing demonstrated significantly attenuated USV in comparison with the animals that received ACSF (F(1,17) = 22.90; p < 0.01). When retested to the tone in the same context without infusions
(day 5), the animals that had received APV on the previous tone tests
generated significantly more USV than the animals that had received
ACSF on the previous tone test (F(1,17) = 6.87; p < 0.05). Again, there was a significant interaction between the drug
treatment and the testing day (F(1,17) = 35.07; p < 0.01), consistent with the hypothesis
that APV blocks extinction during the tone test on day 4.
The impairing effects of intra-BLA infusions of APV on the expression
of conditioned USV and extinction are depicted in Figure 5 as raster plots. Once again, these
results are from typical rats, meaning that the animals were selected
on the basis that their performance was at the median of the
distribution for the group. The raster plot shows a typical ACSF and
APV animal during the context test with infusions (day 2) and without
infusions (day 3) (Fig. 5A) and a typical ACSF and APV
animal during the tone test with infusions (day 4) and without
infusions (day 5) (Fig. 5B). Overall, the USV results
closely parallel freezing results (Fig. 2) and indicate that intra-BLA
infusions of APV block the expression of the conditioned USV response
to the tone and context stimuli and also impede extinction.
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Figure 5.
Examples of the USV raster plots in which each
dot represents a time-stamped episode of vocalization.
USV emissions are shown from a typical ACSF and APV animal during
context testing after infusions (Day 2) and context
retesting without infusions (Day 3)
(A), and during tone-retention testing after
infusions (Day 4) and tone retesting without
infusions (Day 5) (B).
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Conditioned defecation
Figure 6 depicts the mean boli
counts from APV and ACSF animals during 10 pairings of tone-footshock
training on day 1 (no infusions) (Fig. 6, left), during the
context-retention test on day 2 (with infusions) and the
context-retention retest on day 3 (no infusions) (Fig. 6,
middle), and during the tone-retention test on day 4 (with
infusions) and the tone-retention retest on day 5 (no infusions) (Fig.
6, right). In both the context- and tone-retention tests,
intra-BLA infusions of APV significantly impaired conditioned
defecation (F(1,17) = 7.66;
p < 0.05 and F(1,17) = 22.23; p < 0.01, respectively). Furthermore, there
were significant interaction effects between the drug treatment and the
testing day (F(1,17) = 13.17;
p < 0.01 for context;
F(1,17) = 45.61; p < 0.01 for tone). Thus, the defecation results parallel precisely those
from freezing and USV. Again these results indicate that
intra-BLA infusions of APV block the expression of conditioned fear to
the tone and context stimuli and also impede extinction.
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Figure 6.
Mean number of boli (± SE) during 10 tone-shock
pairings (Day 1), an 8 min context-retention test after
intra-BLA infusions of ACSF or APV (Day 2), an 8 min
context retest (Day 3), an 8 min tone-retention test
after intra-BLA infusions of ACSF or APV (Day 4),
and an 8 min tone retest (Day 5). Open
circles and filled circles represent the groups
that received ACSF and APV, respectively, on days 2 and 4. (None of the
groups received infusions on days 1, 3, and 5.)
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Conditioned analgesia
Tail-flick latencies were examined in an additional set of 19 animals. Figure 7 shows the mean
tail-flick latencies from APV and ACSF animals measured before and
during the tone CS presentation. As can be seen, ACSF-treated animals
exhibited significantly increased latencies to tail flick (conditioned
analgesia) during the tone period in comparison with the pretone period
(F(1,7) = 92.05; p < 0.01). In contrast, APV-treated animals did not demonstrate reliably
increased latencies to tail flick during the tone CS period when
compared with the pretone period
(F(1,10) = 3.98; p > 0.05). These conditioned analgesia results are in agreement with
conditioned freezing, USV, and defecation responses and indicate that
intra-BLA infusions of APV block the expression of multiple conditioned
fear responses.
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Figure 7.
Mean tail flick latencies (± SE) of animals
during the baseline measures (minutes 1 and
2) and the tone presentation (indicated by a
horizontal line) (minutes 3 and
4), after receiving intra-BLA infusions of ACSF
or APV.
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Experiment 2
The guide cannula tip locations were similar to those for the
animals in experiment 1 (in or just dorsal to the BLA; data not shown).
Intra-BLA infusions of APV produced a clear and obvious attenuation of
the immediate postshock reactions of the animal to the 10 tone-footshock pairings on day 1. Postshock freezing and USV were
plotted as a function of time, whereas a cumulative measurement was
obtained for defecation (Fig.
8A). Clearly, all three
response measurements were dramatically attenuated during training in
the APV group (n = 10) relative to the ACSF controls (n = 10) (F(1,18) = 45.55; p < 0.01 for freezing;
F(1,18) = 23.78; p < 0.01 for USV; and F(1,18) = 6.75;
p < 0.05 for defecation).
View larger version (25K):
[in this window]
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|
Figure 8.
A, Mean percentage of freezing
(left) and duration of USV
(middle) (± SE) during the 1 min baseline and
during the intervening 10 tone-shock pairings (denoted by small
bars) of the 1 min ITIs after intra-BLA infusions of ACSF or
APV, and mean number of boli (right) (± SE) during
training. B, Mean percentage of freezing and duration of
USV, and mean number of boli (± SE) during context testing on day 2 and tone testing on day 3.
|
|
The context- and tone-retention tests indicated that APV infusions into
the BLA (on training day 1) dramatically blocked the acquisition of
fear conditioning to the context (day 2) and the tone (day 3) on all
three response measures (Fig. 8B): freezing to
context (F(1,18) = 38.13;
p < 0.01), USV to context
(F(1,18) = 12.40; p < 0.01), defecation to context (F(1,18) = 10.91; p < 0.01), freezing to tone
(F(1,18) = 13.99; p < 0.01), USV to tone (F(1,18) = 8.76;
p < 0.01) and defecation to tone
(F(1,18) = 15.68; p < 0.01).
One might expect the sizeable differences in immediate postshock
responding (during training) to be predictive of or reflected in
subsequent retention testing. Indeed, the immediate postshock fear
responses were predictive of subsequent retention to the context and
cue. Comparing training (day 1) and subsequent retention of the context
(day 2) or tone (day 3), the correlations (Pearson's correlation
coefficients) for all response measures were significant: freezing to context (r = 0.82; p < 0.01), vocalization to context (r = 0.71;
p < 0.01), defecation to context (r = 0.51; p < 0.05), freezing to tone (r = 0.52; p < 0.05), vocalization to tone
(r = 0.70; p < 0.01), and defecation
to tone (r = 0.54; p < 0.05).
When examining only the APV-infused animals, the highest correlation
was freezing to context (r = 0.65; p < 0.05). Correlations within the APV group were clearly attenuated by a
floor effect resulting from the fact that overall response levels were
so low during retention, especially to the tone (Fig.
8B). When examining only the ACSF group, the highest
correlations were for USV to context (r = 0.77;
p < 0.01) and tone (r = 0.73;
p < 0.01). Correlations for freezing were low because
of an obvious ceiling effect during acquisition (Fig.
8A). However, it is the combined group correlations that are most relevant to the point of this analysis.
To persue further how much of the variance in retention could be
accounted for by postshock responding during training, we performed a
multiple linear regression (using all three immediate postshock fear
measures). The multiple correlation coefficients were as follows:
freezing to context (r = 0.86; p < 0.01) (accounting for 74% of the variance in retention), vocalization
to context (r = 0.75; p < 0.01)
(accounting for 56% of the variance in retention), defecation to
context (r = 0.75; p < 0.01), freezing
to tone (r = 0.66; p < 0.05),
vocalization to tone (r = 0.70; p < 0.05), and defecation to tone (r = 0.70;
p < 0.05).
| |
DISCUSSION |
APV action during retention testing
The first experiment demonstrated that, on several response
measures, NMDA receptors in the amygdala are critically involved in the
expression of conditioned fear. Specifically, we found that
intra-amygdalar infusions of APV before context- and tone-retention tests significantly interfered with the expression of conditioned fear,
as assessed by USV, defecation, analgesia, and freezing (Figs. 2-7).
When combined with previous findings (Maren et al., 1996; Lee and Kim,
1998), we conclude that the impairing effects of intra-amygdalar APV on
the expression of conditioned fear are wide-ranging across different CS
modalities (tones, lights, contexts) as well as numerous fear responses
(freezing, USV, defecation, analgesia). Not surprisingly, we also found
APV effects on acquisition (experiment 2) and extinction of conditioned
fear (experiment 1).
There are several mechanisms by which intra-amygdalar APV might affect
the acquisition, expression, and extinction of conditioned fear (Table
2), five of which are considered here.
The first possibility is that APV selectively, directly, and
exclusively blocks the NMDAR-mediated synaptic plasticity involved in
learning (acquisition and extinction) without affecting the normal
synaptic transmission necessary to induce or express fear (possibility 1). In this case, APV should block acquisition and extinction but not
the expression of conditioned fear. The present results, combined with
other published findings (Chapman and Bellavance, 1992; Li et al.,
1995; Maren et al., 1996; Lee and Kim, 1998), are sufficient to reject
this possibility, at least in the most general case.
Three other possibilities are that intra-amygdalar APV significantly
affects synaptic transmission in the amygdala along the CS pathway
(possibility 2), the unconditioned stimulus (US) pathway (possibility
3), or both CS and US pathways (possibility 4). Finally, we could
consider the possibility that APV has a purely motoric effect, because
of altered synaptic transmission, which is downstream from all
learning-related mechanisms (possibility 5). Recall that Lee and Kim
(1998) did make reference to APV effects on CR centers, which could be
interpreted in a manner similar to this last possibility.
According to possibility 3, conventional thinking suggests that
intra-BLA APV should block acquisition (because of the blockade of US
synaptic transmission) but not affect the expression or extinction of
fear CRs. However, our results, along with those of others (Falls et
al., 1992; Maren et al., 1996; Lee and Kim, 1998), also demonstrate
that APV impairs both the expression and extinction of conditioned
fear. Importantly, the present results demonstrate that APV impairs the
expression of conditioned fear based on several response measures that
were used to examine retention to both a tone and a context. We can
thus reject the general hypothesis (possibility 3) that intra-BLA
infusions of APV selectively and exclusively affect synaptic
transmission in the US pathway.
Possibility 5 can easily be eliminated because it predicts no APV
effect on acquisition or extinction and only a motor impairment in CR
or unconditioned response (UR) production. The prediction is that APV
only has an effect when present, and that this effect is to attenuate
URs or CRs. In contrast, both possibility 2 and possibility 4 predict
that APV should block the acquisition, expression, and extinction of
conditioned fear, consistent with this study and other findings (Maren
et al., 1996; Lee and Kim, 1998). A reasonable hypothesis is that APV
affects fear conditioning, not only by preventing synaptic plasticity,
but also or instead by impeding synaptic signaling of the CS and
possibility also the US.
Recall that there is no convincing neurophysiological evidence that
NMDA receptors are selectively involved in synaptic plasticity but not
involved in normal synaptic transmission in the amygdala (LeDoux, 2000); however, there is evidence to the contrary
(Chapman and Bellavance, 1992; Weisskopf and LeDoux, 1999), as in the
visual cortex (Miller et al., 1989; Shatz, 1990). In the absence of
additional data, the default assumption cannot be that amygdalar NMDARs
generally participate only in plasticity but not normal synaptic transmission.
Considering these results, a weakened version of the Lee and Kim (1998)
hypothesis might be that the expression of conditioned fear, as
measured by the general paradigm developed by Brown et al. (1951),
which entails conditioned enhancement of "pre-existing stimulus-response tendencies," is functionally unique. However, even
this weakened version of this hypothesis has now been refuted by
results from another laboratory. A recent study by Fendt (2001) showed
that APV infusions into the LA, which is located directly above
the BLA, significantly attenuated the expression of both conditioned
freezing and fear-potentiated startle [the same measure used by Davis
and colleagues (Miserendino et al., 1990; Campeau et al., 1992; Gewirtz
and Davis, 1997)]. It is possible that the discrepant findings
with the expression of potentiated startle might be because of APV
being infused into the LA (study by Fendt) rather than the BLA
[studies by Davis and colleagues (Miserendino et al., 1990; Campeau et
al., 1992; Gewirtz and Davis, 1997)]. It should be noted, however,
that the expression of conditioned freezing is significantly impaired
regardless of whether APV was infused into the BLA (Maren et al., 1996;
Lee and Kim, 1998) (present study) or into the LA (study by Fendt).
Interestingly, another laboratory has independently found that
intra-BLA APV infusions significantly impaired the expression of
conditioned (fear-related) defeat responses in hamsters
(K. L. Huhman, personal communication).
Given the results of Fendt (2001), the Lee and Kim hypothesis cannot
further accommodate the view of intra-amygdalar APV affecting the
expression of conditioned fear, as measured by freezing, USV, defecation, and analgesia, but not startle. In the absence of strong
and independent evidence to the contrary, we conclude that the
fear-potentiated startle paradigm is not unique with respect to
receptor mechanism or circuitry, but that there is something special
about the particular procedures used by Davis and colleagues (Miserendino et al., 1990; Campeau et al., 1992; Gewirtz and Davis, 1997).
APV action during training
The second experiment demonstrated that the expression of
postshock fear responses during training was also dramatically impaired by intra-amygdalar infusions of APV when measured by freezing, USV, or
defecation. The only other study to show this effect was done by Lee
and Kim (1998), who used freezing as a response measure. That study was
performed in an attempt to reconcile the discrepant data of Davis and
colleagues (Miserendino et al., 1990; Campeau et al., 1992). The
reasoning of Lee and Kim (1998) made three assumptions. The
first was that APV might not block the expression of fear when the fear
levels are very high. Second, they assumed the levels to be higher in
the studies by Davis and colleagues (Miserendino et al., 1990; Campeau
et al., 1992; Gewirtz and Davis, 1997). Finally, they reasoned that the
fear levels should be higher during training than during subsequent
retention tests. However, they did observe a strong APV effect on
postshock freezing. We have now demonstrated the robustness and
generality of their findings by switching to Sprague Dawley rats and
including multiple fear measures.
We also examined the relationship between postshock fear responses and
subsequent performance on retention tests, mainly because this
relationship had not been examined previously and it seemed potentially
relevant to the interpretation of the postshock fear responses and
therefore the mechanisms of action of APV. What we found was that the
immediate postshock expression of our three response measures during
training was highly predictive of subsequent retention to both context
and tone (see Results). This was demonstrated mainly through regression
analysis but also by comparing group mean differences (Fig. 8).
Whatever the immediate postshock fear responses reflect, they are
surprisingly predictive of subsequent retention. Clearly more work is
needed to interpret postshock fear responses.
Meaning of discrepant findings
Davis and colleagues (Miserendino et al., 1990; Campeau et al.,
1992; Gewirtz and Davis, 1997) reported that intra-BLA APV blocks the
acquisition but not the expression of a fear-potentiated startle
response to both a light and tone CS (Miserendino et al., 1990; Campeau
et al., 1992). Based on these reports, they concluded that APV affects
synaptic plasticity but does not affect normal synaptic transmission.
Clearly, their results are neither general nor robust, because several
independent laboratories have determined, using a variety of response
measures and varying conditions, that APV does affect the expression of
first-order (CS-US) conditioning (present results) (Maren et al.,
1996; Lee and Kim, 1998; Fendt, 2001) (K. L. Huhman, personal communication).
The question of whether APV affects the expression of first-order
conditioning is critical to the inference that learning-related changes
reside in the amygdala (Gewirtz and Davis, 1997). The latter inference
assumes that APV blocks the acquisition and extinction of fear, but not
normal synaptic transmission. The conclusion that APV does not affect
normal synaptic transmission is based solely on the report that APV
failed to significantly affect the expression of first-order
conditioning (Miserendino et al., 1990; Campeau et al., 1992). As we
have seen, this claim is not valid in any general sense and may be
unique to some very specific aspect of the procedures used by these
investigators. The same reservation applies to the claim that
BLA infusion with APV blocks second-order (CS2-CS1) conditioning at a
concentration that does not affect the expression of first-order
conditioning (Gewirtz and Davis, 1997).
The conclusion that learning-related changes must reside in the
amygdala is derived from the fact that this is the locus of the cannula
and from the belief that APV has no relevant effects on amygdalar
function other than blocking synaptic plasticity. Clearly the latter
conclusion is incorrect in a general sense. Even if one imagines that,
in some constrained parameter space, APV has no effect on the
expression of one measure of first-order conditioning, it does not
follow that the synaptic transmission involved in second-order
conditioning is unaffected. The latter inference cannot be the default
assumption because the general results refute that position.
If fear conditioning occurs in some yet-to-be identified brain
structure or structures that require interactions with the amygdala, as
some have suggested (Cahill et al., 1999), and if intra-amygdalar APV
substantially interferes with the normal synaptic transmission of CS
and possibly US information to the extra-amygdalar structure or
structures (recall discussion of Table 2), then fear conditioning could
be blocked by APV infusion into the amygdala even if the actual
learning occurs outside the amygdala. Nevertheless, it is also
important to recognize that the fact that APV affects CS-US signaling
transmission in the amygdala does not necessarily exclude the
possibility of fear learning occurring within the amygdala, and
possibly involving a specific subtype or subtypes of NMDA receptors
(Rodrigues et al., 2000).
Summary and conclusions
We are forced to concede that the results of the APV experiments
do not strengthen the hypothesis that an NMDAR-dependent form of
associative LTP in BLA mediates fear conditioning (LeDoux, 2000). We
have seen a robust effect of APV in impairing the expression of
conditioned fear to both context and tone CSs, as assessed by freezing,
USV, defecation, and analgesia. Similarly, other studies have found
that intra-amygdalar APV impairs the expression of conditioned
fear-potentiated startle (Fendt, 2001) and defeat responses (K. L. Huhman, personal communication). These data do not rule out a direct
effect of APV on the acquisition or extinction of fear; however, as we
have seen in generalizing the findings of Lee and Kim (1998), the
results do suggest that APV interferes with normal synaptic
transmission in the amygdala in a manner that could indirectly affect
the mechanisms involved in fear conditioning.
| |
FOOTNOTES |
Received Nov. 27, 2000; revised March 2, 2001; accepted March 21, 2001.
This work was supported by the Whitehall Foundation, by Grant NIA
P60AG10469 from the Claude D. Pepper Older Americans Independence Center, and by a Yale Junior Faculty Fellowship (J.J.K.) and National Institute of Mental Health Grant MH58405 (T.H.B.). We
thank Markus Fendt for sharing his data and Kevin B. Baker for his
assistance in the analgesia experiment.
Correspondence should be addressed to Jeansok J. Kim, Department of
Psychology, 2 Hillhouse Avenue, Yale University, New Haven, CT
06520-8205. E-mail: jeansok.kim{at}yale.edu.
| |
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[Abstract]
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J. W. Koo, J.-S. Han, and J. J. Kim
Selective Neurotoxic Lesions of Basolateral and Central Nuclei of the Amygdala Produce Differential Effects on Fear Conditioning
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S. Malkani, K. J. Wallace, M. P. Donley, and J. B. Rosen
An egr-1 (zif268) Antisense Oligodeoxynucleotide Infused Into the Amygdala Disrupts Fear Conditioning
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M. R. Holahan and N. M. White
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T. Lee and J. J. Kim
Differential Effects of Cerebellar, Amygdalar, and Hippocampal Lesions on Classical Eyeblink Conditioning in Rats
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March 31, 2004;
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M. R. Tinsley, J. J. Quinn, and M. S. Fanselow
The Role of Muscarinic and Nicotinic Cholinergic Neurotransmission in Aversive Conditioning: Comparing Pavlovian Fear Conditioning and Inhibitory Avoidance
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F. Zinebi, J. Xie, J. Liu, R. T. Russell, J. P. Gallagher, M. G. McKernan, and P. Shinnick-Gallagher
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[Abstract]
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P. SAH, E. S. L. FABER, M. LOPEZ DE ARMENTIA, and J. POWER
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Physiol Rev,
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A. Savonenko, T. Werka, E. Nikolaev, K. Zielinski, and L. Kaczmarek
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D. R. Catherall
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[Abstract]
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E. Santini, R. U. Muller, and G. J. Quirk
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H. T. Blair, G. E. Schafe, E. P. Bauer, S. M. Rodrigues, and J. E. LeDoux
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S. M. Rodrigues, G. E. Schafe, and J. E. LeDoux
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M. Fendt
Injections of the NMDA Receptor Antagonist Aminophosphonopentanoic Acid into the Lateral Nucleus of the Amygdala Block the Expression of Fear-Potentiated Startle and Freezing
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[Abstract]
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TOP
ABSTRACT
INTRODUCTION
