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Volume 17, Number 21,
Issue of November 1, 1997
pp. 8443-8450
Copyright ©1997 Society for Neuroscience
cAMP Response Element-Binding Protein in the Amygdala Is Required
for Long- but not Short-Term Conditioned Taste Aversion Memory
Raphael Lamprecht,
Shoshi Hazvi, and
Yadin Dudai
Department of Neurobiology, The Weizmann Institute of Science,
Rehovot 76100, Israel
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
In conditioned taste aversion (CTA) organisms learn to avoid
a taste if the first encounter with that taste is followed by transient
poisoning. The neural mechanisms that subserve this robust and
long-lasting association of taste and malaise have not yet been
elucidated, but several brain areas have been implicated in the
process, including the amygdala. In this study we investigated the role
of amygdala in general, and the cAMP response element-binding protein
(CREB) in the amygdala in particular, in CTA learning and memory.
Toward that end, we combined antisense technology in
vivo with behavioral, molecular, and histochemical
analysis. Local microinjection of phosphorothioate-modified
oligodeoxynucleotides (ODNs) antisense to CREB into the rat amygdala
several hours before CTA training transiently reduced the level of CREB
protein during training and impaired CTA memory when tested 3-5 d
later. In comparison, sense ODNs had no effect on memory. The effect of
antisense was not attributable to differential tissue damage and was
site-specific. CREB antisense in the amygdala had no effect on
retrieval of CTA memory once it had been formed, and did not affect
short-term CTA memory. We propose that the amygdala, specifically the
central nucleus, is required for the establishment of long-term CTA
memory in the behaving rat; that the process involves long-term
changes, subserved by CRE-regulated gene expression, in amygdala
neurons; and that the amygdala may retain some CTA-relevant information over time rather than merely modulating the gustatory trace during acquisition of CTA.
Key words:
conditioned taste aversion;
amygdala;
CREB;
learning;
short-term memory;
long-term memory
INTRODUCTION
In conditioned taste aversion (CTA),
organisms learn to avoid a taste if the first encounter with that taste
is followed by malaise (Garcia et al., 1955 ; Bures et al., 1988). CTA
presents to memory research remarkable opportunities but also a unique challenge. The acquisition of CTA is fast (a single trial), and its
memory is robust (high signal-to-noise ratio in the behavioral response) and long-lasting (up to a lifetime). These properties facilitate correlation of molecular and cellular events in brain with
phases of learning, consolidation, retention, and retrieval in a
natural behavioral situation. Moreover, conditioning in CTA tolerates a
delay of hours between the taste [conditioned stimulus (CS)] and the
malaise [unconditioned stimulus (UCS)]. This permits separation in
time of the mechanisms of acquisition of information about the taste
from the association of that information with a reinforcer. Such
separation provides a useful system for investigating incidental
learning, i.e., learning in the absence of an exogenous reinforcer
(Hebb, 1949), a very common but rather neglected type of learning.
However, the uniquely long CS-UCS interval in CTA, so different from
classical instances of conditioning that demand tight temporal
association (less than seconds), also raises the issue of how it is
that the memory of a taste remains specifically associable with malaise
for many hours. Not surprisingly, CTA, although well known to farmers,
encountered difficulties in being accepted as a paradigm of
conditioning in experimental psychology (Garcia, 1981).
Whereas the behavioral characteristics of CTA have been
extensively investigated (Domjan, 1980; Bures et al., 1988), much less
is known about brain systems that subserve it and the molecular and
cellular mechanisms that embody CTA in these areas. It is generally
accepted that the gustatory cortex plays a role in the processing and
memory of the taste stimulus, the parabrachial nucleus in the
association between the taste and malaise, and the amygdala in the
integration and expression of CTA behavior (for review, see Yamamoto et
al., 1994). The role of amygdala is especially interesting, because
this brain structure has been implicated in other types of learning,
especially aversive and emotional conditioning (Davis, 1992; LeDoux,
1993; McGaugh et al., 1993). We recently reported that transient
inhibition of protein synthesis in the central amygdala during CTA
training blocks CTA memory, and that local microinjection into the
amygdala of antisense oligodeoxynucleotides (ODNs) to the
immediate-early gene (IEG) c-fos has a similar effect
(Lamprecht and Dudai, 1996). We have now combined the antisense
technology in vivo (for review, see Cirelli et al., 1995)
with behavioral, molecular, and histochemical analysis to show that in
the amygdala, the cAMP response element-binding protein (CREB),
implicated in multiple types of neuronal plasticity (Frank and
Greenberg, 1994; Carew, 1996), is essential for long-term CTA memory,
but not for short-term memory or for the retrieval of long-term memory
once it has been formed. Our findings suggest that CTA shares
mechanisms with other, more "conventional" forms of learning, and
that long-term changes involving CRE-regulated gene expression take
place in the amygdala in encoding CTA memory.
MATERIALS AND METHODS
Animals
Wistar rats (2-month-old males, 200-250 gm) were caged
individually at 22°C under 12 hr light/dark cycles. Water and food were available ad libitum unless otherwise indicated.
Reagents
The phosphorothioate-modified ODNs were synthesized at the
Biological Services of the Weizmann Institute of Science. The sequence for CREB antisense was 5 -TGGTCATCTAGTCACCGGTG-3, and that for CREB
sense was 5-CACCGGTGACTAGATGACCA-3. Fluorescein
isothiocyanate-labeled CREB antisense (FITC-CREB) was from Biosource
(Camarillo, CA). ODNs were microinjected in physiological saline (0.9%
NaCl). Anti-CREB antibody (06-504) was from Upstate Biotechnology (Lake
Placid, NY). Anti-activation transcription factor 2 (ATF-2) antibody
(SC-242) and anti-protein kinase C (PKC)
antibody (SC-211) were from Santa Cruz Biotechnology (Santa Cruz, CA).
Biotinylated goat anti-rabbit antibody (BA-1000) and avidin
DH-biotinylated horseradish peroxidase (HRP) H reagents (Vectastain,
PK-6100) were from Vector Laboratories (Burlingame, CA). HRP-protein A
(NA-9120) and an enhanced chemoluminiscence (ECL) kit (RPN 2106) were
from Amersham (Buckinghamshire, UK). Anti- -tubulin antibody
(T-9026), HRP-goat anti-mouse antibody (A-9917), cytochrome
c (C-7752), 3,3-diaminobenzidine tetrahydrochloride (DAB;
Sigma Fast tablets, D-4293), and catalase (C-40) were from Sigma (St.
Louis, MO). All other chemicals were of analytical grade or the highest
grade available.
Behavioral procedures
In CTA, saccharin (0.1% w/v, sodium salt) was used as an
unfamiliar taste unless otherwise indicated, and intraperitoneal LiCl
(0.15 M, 2% body weight) was used as a malaise-inducing
agent. The CTA protocol was essentially as described by Rosenblum et al. (1993). In brief, rats were trained over 4 d to get their daily water ration within 10 min/d from 2 pipettes, each containing 10 ml. On day 5 (conditioning day), the rats were presented with saccharin
instead of water. Forty minutes later, they were injected with LiCl
intraperitoneally. On days 6-7 or 6-8 (rest days), the rats were
presented daily for 10 min with two pipettes containing 10 ml of water
each. In the test, performed in an extinction mode on three successive
days (days 8-10 or 9-11), the rats were presented daily with an array
of six pipettes, three containing 4 ml of saccharin and three
containing 4 ml water, and their liquid consumption was recorded. In
the experiments designed to test both short- and long-term memory, all
rats were trained as above, except that on day 5 (conditioning day)
they were presented with 5 ml of saccharin, and 2 or 4 hr after the
injection of LiCl intraperitoneally they were presented with an array
of six pipettes, three containing 3 ml of saccharin and three
containing 3 ml of water for 10 min, and their liquid consumption was
recorded. The same rats were again presented 72 hr after the injection
of LiCl with an array of six pipettes, three containing 4 ml of
saccharin and three containing 4 ml water for 15 min, and their liquid
consumption was recorded again. The aversion index (Rosenblum et al.,
1993) was defined as {[milliliters of water/(milliliters of water + milliliters of saccharin)] × 100} consumed in the test; that is, 50 is chance level, and the higher the aversive index, the more the rats
prefer water to saccharin.
To follow the time course of malaise induced by LiCl injection in CTA
training, rats were injected with LiCl intraperitoneally as above or
with saline as control, placed individually in transparent cages, and
observed. Two behavioral responses were monitored at intervals of 30 min, up to 330 min after the injection: lying on the belly (LOB),
characterized by little or no movement and a prone, flaccid posture
with the chin on the floor of the cage (Meachum and Bernstein, 1990);
and rearing, expressed as lifting the forepaws simultaneously off the
floor in the absence of grooming (Parker, 1982).
Surgical procedures
Microinjection into the amygdala was performed via chronically
implanted cannulas. For surgery, rats were anesthetized with Equithesin
(5.6 ml/kg), restrained in a stereotaxic apparatus (Kopf), and
implanted bilaterally with guide cannulas (stainless steel, 23 gauge)
aimed at the central amygdaloid nucleus [CeA; coordinates in reference
to bregma: anteroposterior (AP),  2.3; lateral (L) ±4; and
dorsoventral (DV), 7.5] (Paxinos and Watson, 1986). In a set of
control experiments, the guide cannulas were implanted bilaterally in
the basal ganglia (coordinates in reference to bregma: AP, 2.3; L,
±4; and DV, 5.5). The cannulae were fixed in place with acrylic
dental cement and secured by two skull screws. A stylus was placed in
the guide cannula to prevent clogging. Animals were allowed 1 week to
recuperate before being subjected to experimental manipulations.
Microinjection
Microinjection of ODNs (4 nmol in 2 µl/hemisphere) was
performed at the times indicated in Results. The stylus was removed from the guide cannula, and a 28 gauge injection cannula was carefully inserted and lowered 1 mm below the tip of the guide cannula. We chose
these injection coordinates, which are just at the bottom of the CeA,
because in our experience this on the one hand minimized the injection
lesion in the CeA and on the other maximized the diffusion of the
injected solution into the CeA, since the solution tended to diffuse
dorsolaterally to the tip of the injection cannula (see below). The
injection cannula was connected via PE20 tubing to a Hamilton
microsyringe driven by a microinjection pump (Carnegie Medicine CMA
100) at a rate of 0.5 µl/min. After microinjection, the injection
cannula was left for an additional 1 min before withdrawal to reduce
efflux of injection liquid along the injection tract. No tremors or
seizures were detected during or after the injection.
In a preliminary set of experiments, using india ink, we estimated the
sphere of diffusion of the solution microinjected into the amygdala as
1.5 ± 0.4 mm3 (n = 4). The
solution diffused in and around the CeA. Occasionally, it also extended
into one or more of the following structures: the ventral horn of the
caudate putamen, the nucleus basalis of Meynert, the dorsal portion of
the internal capsule, the intercalated amygdaloid nuclei, and the
basolateral amygdala. The only common feature of the microinjections,
however, was the bilateral involvement of substantial portions of the
CeA (also see Lamprecht and Dudai, 1996). Microinjection of FITC-CREB
antisense ODNs unveiled a more focused high-concentration sphere of
0.72 ± 0.04 mm3 (n = 3),
spreading mainly dorsolaterally to the tip of the cannula and
uncovering mostly the CeA (both medial and lateral subnuclei), but also
penetrating the intercalated nuclei and limited portions of the
basomedial and basolateral amygdala (Fig.
1A).
Fig. 1.
Sphere of diffusion of microinjected CREB
antisense ODNs into the amygdala (A) and
reduction in CREB-positive nuclei by CREB antisense
(B-D). A, FITC-labeled CREB
antisense was microinjected, and the fluorescence image recorded as
detailed in Materials and Methods. CeM, Central
amygdaloid nucleus, medial division; CeL, central
amygdaloid nucleus, lateral division; BMA, basomedial amygdaloid nucleus, anterior part; BLA, basolateral
amygdaloid nucleus, anterior part; Pir, piriform cortex.
The microinjection cannula track and the lesion induced by the tip of
the cannula are seen within the sphere of FITC diffusion. The schematic
anatomical map is adapted from that of Paxinos and Watson (1986).
B, The number of CREB-positive nuclei in the amygdala of
CREB antisense (AS) versus sense
(S) microinjected rats. In both groups CREB antisense was microinjected into the amygdala 14 hr before CTA training; n = 4 in each group. The
asterisk indicates significance for pair comparison in
which a Scheffe contrast test was used with an of 0.05. C,
D, Immunohistochemistry of CREB using CREB antibodies in CREB
sense (C) and antisense (D)
microinjected rats. The selected fields were taken from the region of
the CeL, indicated in A; dot, edge of the
microinjection cannula-induced lesion.
[View Larger Version of this Image (103K GIF file)]
Histochemistry and image analysis
Images of FITC-CREB antisense were recorded with a Zeiss
Axioskop microscope coupled to a cooled CCD camera (Photometrics, Tuscon, AZ). The computerized microscope system was as described by Kam
et al. (1993). The FITC filters used were from Omega (Brattleboro, VT).
Digital images were acquired and stored using the Priism software
(Applied Precision, Issaqua, WA) (Kam et al., 1992). Composite images
were taken over overlapping regions and fitted using Photoshop 3.0.4 (Adobe, Mountain View, CA).
For immunohistochemical localization of CREB, rats were given
Equithesin (5.6 ml/kg, i.p.) 14 hr after the microinjection of the ODNs
and perfused intracardially by gravity drip infusion with PBS followed
by cold (4°C), fresh 2.5% paraformaldehyde and 5% sucrose in PBS,
pH 7.4. The brain was post-fixed for 48 hr in 1% paraformaldehyde and
30% sucrose in PBS at 4°C. Post-fixed brains were sectioned
coronally on a freezing microtome at a thickness of 50 µm. Sections
were washed three times (5 min each) with PBS and immersed for 30 min
in 49.5% methanol, 49.5% PBS, and 0.9% H2O2, followed by washing five times (5 min each) with PBS and immersion for 20 min in 0.15 M
glycine in PBS, pH 7.4. The sections were then blocked with 20% normal
goat serum (NGS) in PBS and 0.5% Triton X-100 for 1-3 hr at 37°C,
followed by 16 hr incubation at room temperature with the anti-CREB
polyclonal antibody (1:500) and 2% NGS in PBS. The sections were
washed (three times, 5 min each) with PBS and incubated with
biotinylated goat anti-rabbit antibody (1:200) and 2% NGS in PBS for
1.5 hr at room temperature, followed by three additional PBS washings
(5 min each). Finally, an avidin DH-biotinylated horseradish peroxidase
H complex (1:50) was added for 1.5 hr. The sections were then washed
once with PBS and twice with 50 mM Tris-Cl buffer, pH 7.5. Peroxidase activity was determined by reaction with a mixture
containing 0.7 mg/ml DAB and 2 mg/ml urea-H202
in 60 mM Tris-Cl. The sections were washed three times with
Tris buffer, mounted on slides, dehydrated by successive rinses of 70, 95, and 100 ethanol (two times) and 100% xylene (two times), and
covered with Permount.
For cytochrome oxidase staining, rats were treated with
Equithesin as above 4 d after the microinjection of ODNs into the amygdala and perfused intracardially by gravity drip infusion with PBS
followed by cold 2.5% glutaraldehyde, 0.5% paraformaldehyde, and 5%
sucrose in PBS. Brains were post-fixed for 12 hr in 2.5% glutaraldehyde, 0.5% paraformaldehyde, and 15% sucrose in PBS at
4°C. Post-fixed brains were sectioned coronally on a freezing microtome at 50 µm thickness, washed four times, 5 min each, in 0.1 M phosphate buffer, pH 7.6, and incubated for 3-24 hr at
40°C in an oxygenated reaction mixture containing 0.7 mg/ml DAB, 75 µg/ml cytochrome c, and 2000 U/ml catalase (in 0.1%
thymol) in 0.1 M phosphate buffer, pH 7.6. The sections
were then washed four times in 0.1 M phosphate buffer, pH
7.6, mounted on slides, dehydrated by successive rinses in 50, 70, 95, and 100% ethanol (two times) and 100% xylene (three times), and
covered with Permount.
For Nissl staining, brains were processed as above. Sections were
mounted on slides and dehydrated by successive rinses in 95 (three
times) and 100% (three times) ethanol, 2 min each, followed by 30 min
in chloroform. The slides were then rehydrated by successive rinses in
100 (three times), 95 (three times), and 70% ethanol (2 min each). The
slides were stained with 0.25% cresyl violet in 25% ethanol, 231 mM acetic acid, and 18.7 mM sodium acetate for
2 min, washed in water, rinsed 1 min in 25% ethanol, bleached with
50% ethanol and 0.5% acetic acid, and then rinsed in 70, 95 (once),
and 100% (three times) ethanol for 2 min and 100% xylene (three
times) for 15 min, and covered with Permount. A Nikon Labophot microscope and Image 1.41 software (National Institutes of Health, Bethesda, MD) were used for histochemical analysis.
Immunoblotting
CREB antisense ODNs were microinjected as above into the
amygdala of one hemisphere, and CREB sense ODNs were injected into the
amygdala of the contralateral hemisphere. At 14 or 24 hr after injection, the rats were given Equithesin (5.6 ml/kg, i.p.) and perfused intracardially with PBS for 3 min. The brain was rapidly removed, frozen on dry ice, and sectioned in a cryostat until the tip
of the microinjection cannula was visualized. Tissue from the tip of
the cannula at each hemisphere was punched out with a 1-mm-length
15-gauge stainless steel syringe needle blunted at the tip (inner
diameter, 1.3 mm). The tissue thus obtained was homogenized in a
glass-Teflon homogenizer in SDS sample buffer containing 10%
glycerol, 5%  -mercaptoethanol, and 2.3% SDS in 62.5 mM
Tris-Cl, pH 6.8. Aliquots containing equal amounts of protein (25-50
µg) were subjected to SDS polyacrylamide gel electrophoresis in 12%
polyacrylamide (Laemmli, 1970) and Western blotting (Burnette, 1981).
After protein transfer, the nitrocellulose was treated as described
below either in Protocol 1 (anti-CREB or anti--tubulin antibodies)
or in Protocol 2 (anti-ATF-2 or anti-PKC
antibodies).
Protocol 1. The blot was blocked with 1% BSA in washing
buffer (0.9% NaCl, 0.05% Tween 20, and 10 mM Tris, pH
7.6) for 1 hr at room temperature and reacted overnight at 4°C or for
2 hr at room temperature with anti-CREB antibody (1:1000) or
anti--tubulin antibody (1:5000). Blots were washed with washing
buffer three times, 5 min each, followed by a 1 hr incubation at room
temperature with HRP-linked protein A (1:15,000) or HRP-linked goat
anti-mouse (1:5000) for the anti-CREB antibody or the anti--tubulin
antibody, respectively. Blots were washed with washing buffer once for
15 min and then thrice for 5 min before being subjected to ECL
analysis. The anti-CREB antibody recognized the expected ~43KDa band
(Ginty et al., 1993).
Protocol 2. The blot was blocked with 5% milk powder and
0.05% Tween 20 in TBS (150 mM NaCl and 10 mM
Tris-HCl, pH 8.0) for 30 min at room temperature, followed by a 45 min
incubation with anti-ATF-2 (1:50) or anti-PKC (1:800) in
blocking solution at room temperature. The blots were washed twice for
7 min with 0.05% Tween 20 in TBS and incubated with HRP-linked protein
A (PKC blot) or HRP-linked goat anti-mouse (ATF-2 blot)
for 30 min at room temperature, followed by three 5 min washes with 0.05% Tween 20 in TBS and then once with TBS. Here, too, the blots were subjected to ECL analysis.
In both protocols, quantification was performed in a Molecular
Dynamics (Sunnyvale, CA) 300A densitometer. Values were calculated as
percentages of the CREB, ATF-2, or PKC proteins in the antisense-microinjected hemisphere, taking in each individual rat the
value in the contralateral sense-microinjected hemisphere as 100%. In
each rat, the values obtained for CREB, ATF-2, or PKC
were normalized to the level of -tubulin, taken as a stable protein
reference ( 1/2~days; Dustin, 1984).
Statistics
Differences among groups were evaluated using one-way
ANOVA and for repeated measure test two-way ANOVA. For paired
comparisons Scheffe contrast tests were used with an level of
0.05.
RESULTS
CREB antisense reduced the level of CREB protein in
the amygdala
We have determined the effect of CREB antisense ODNs in the
amygdala on the level of the CREB protein by two methods,
immunohistochemistry and immunoblotting. Immunohistochemical analysis
of the central amygdala 14 hr after the injection of antisense or sense
ODNs showed that the antisense significantly reduced the level of CREB protein in the vicinity of the tip of the injection cannulas (Fig. 1B-D). The average reduction over an area of 0.5 mm2 was 37 ± 9% (Fig. 1B;
p < 0.02). Immunoblotting of tissue excised from the
vicinity of the tip of the injection cannula unveiled a similar
magnitude of reduction at 14 hr, which was specific to the CREB
protein; the transcription factor ATF-2 and the enzyme PKC, assayed as controls, were not affected (Table
1). The level of CREB protein returned to
normal within 24 hr (Table 1).
CREB antisense in the amygdala impaired CTA memory
When microinjected into the amygdala 14 hr before CTA training,
CREB antisense markedly reduced CTA memory tested 3-5 d after training
(Fig. 2). In contrast, CREB sense had no
effect. Differences among groups were significant for all
postconditioning days (p < 0.001 for days 1 and
2; p < 0.002 for day 3 of testing). The aversion index
of the CREB antisense group was significantly lower than that of normal
and CREB sense groups in all test days (Scheffe contrast tests). A
group-by-days ANOVA demonstrated a significant difference among groups
(p < 0.001) and a significant decrease in the
aversion index over days (p < 0.001) with no
interaction effect.
Fig. 2.
Impairment of CTA by ODNs antisense to CREB in the
amygdala. Aversion indices are plotted versus the test day. In each
test day, the left (shaded)
bar depicts the aversion index of normal CTA control
animals (n = 23); the center
(solid) bar depicts the aversion index of
animals locally microinjected into the amygdala with antisense ODNs
(n = 16); and the right
(open) bar shows the index of animals
receiving sense ODNs (n = 11). All microinjections into the amygdala were performed 14 hr before CTA training, as detailed
in Materials and Methods. Inset, Spatial and temporal specificity of the antisense effect. The shaded bar
depicts the result for normal controls; the hatched bars
show the results for rats microinjected with antisense.
A, Normal CTA animals; B, antisense
microinjected 14 hr before training 2 mm above the stereotaxic
coordinates used for injection into the amygdala; C,
antisense microinjected into the amygdala 14 hr before the first memory
test, i.e., 36 hr after the completion of training; D,
antisense microinjected into the amygdala 7 d before CTA training; n = 6 animals in each group.
Asterisks indicate significance for pair comparison in
which a Scheffe contrast test was used with an of 0.05.
[View Larger Version of this Image (35K GIF file)]
The effect of CREB antisense was not due to differential
tissue damage
Because ODNs may have toxic effects including tissue damage
in vivo (Chiasson et al. 1994), we wanted to verify that the
differential effect of CREB antisense on CTA was not attributable to
differential tissue toxicity in the amygdala. Toward that end, we
compared cytochrome oxidase- and Nissl-stained sections from animals
microinjected into the amygdala with saline or CREB sense or CREB
antisense ODNs 14 hr before CTA training and killed 4 d later. The
number of Nissl-stained cells and the intensity of cytochrome oxidase activity were compared in selected frames near the cannula tip and
found not to differ between saline (S), CREB sense (Se), and CREB
antisense (A): Nissl staining, 544 ± 27 (S), 516 ± 31 (Se), and 525 ± 69 (A) cells in 0.12 mm2; and
cytochrome oxidase, 128 ± 9 (S), 123 ± 4 (Se), and 126 ± 6 (A) arbitrary pixel units [n = 3 (S);
n = 4 (Se and A)].
CREB antisense did not prevent conditioning several days later
In contrast to its effect on CTA memory when microinjected 14 hr
before training, CREB antisense had no effect on CTA memory when
microinjected into the amygdala several days before training. It should
be noted that ODNs are expected to degrade in vivo within 1-2 d (Chiasson et al., 1994; Konradi et al., 1994), and that in our
hands the level of CREB in the CREB antisense-microinjected amygdala
indeed returned to normal within 1 d (Table 1).
That the time window of the effect of a single microinjection of CREB
antisense on CTA training is limited became evident in two types of
experiments. In experiment 1, rats were microinjected into the amygdala
with CREB antisense 72 hr before CTA training, using saccharin as the
unfamiliar taste; there was no effect on CTA memory tested 3 d
after training (aversion index of 92 ± 5 vs 94 ± 1 in CREB
antisense-microinjected and control animals, respectively). In
experiment 2, we wanted to reinforce the conclusions by using rats in
which the blocking effect of CREB antisense on CTA had already been
demonstrated once, and to generalize the findings to other tastes. We
thus microinjected rats into the amygdala with CREB antisense 14 hr
before CTA training, using saccharin as the unfamiliar taste; in
agreement with the data presented above, we found an impairment in CTA
memory tested 3 d after training (aversion index of 77 ± 6 vs 95 ± 1 in CREB antisense and control animals, respectively;
p < 0.01). However, 1 week later we reconditioned the
same rats, this time using NaCl (0.1 M) as the unfamiliar
taste. The aversion tested 3 d later was normal (97 ± 1). In
a complementary experiment, using other, naive rats, we found that CREB
antisense, microinjected into the amygdala 14 hr before CTA training in
which 0.1 M NaCl was used as the novel taste solution, did
impair the aversion index by (26 ± 4)% when tested 3 d
after training, similarly to the effect on CTA to saccharin. We thus
concluded that the microinjection of ODNs into the amygdala caused no
residual functional damage to this structure with regard to CTA
learning.
The effect displayed anatomical site specificity
Microinjection of the CREB antisense into the basal ganglia (2 mm
above the coordinates used by us for microinjection into the amygdala),
14 hr before CTA training, had no effect on CTA memory (Fig. 2,
inset), indicating site specificity of the effect rather
than general toxicity.
CREB antisense affected long- but not short-term CTA memory
Routinely, CTA memory is tested several days after training (e.g.,
Bermudez-Rattoni et al., 1986; Gallo et al., 1992; Rosenblum et al.,
1993). A problem that might arise in testing CTA memory immediately
after training is that the malaise-producing agent, e.g., LiCl, exerts
a lingering effect; therefore the negative reinforcer might still be
acting while short-term memory is tested. To be able to test short-term
CTA memory, we first determined the time window of the behavioral
effects of intraperitoneal LiCl injection under the conditions used by
us in CTA training. The most prominent behavioral index of malaise in
CTA training is LOB (see Materials and Methods) (Meachum and Bernstein,
1990). LOB was observed during a time window of <2 hr after the
injection of LiCl intraperitoneally (Fig.
3). Rearing (Parker, 1982), which was
taken as an indicator of residual unrest, was observed for a longer
period (Fig. 3). We concluded that the malaise induced by the negative
reinforcer dissipated within <2 hr, and that behavior completely
returned to normal within another 2-3 hr. We then proceeded to test
the effect of CREB antisense in the amygdala on short-term CTA memory.
The antisense was microinjected into the amygdala as above, 14 hr
before the beginning of CTA training, and the animals were tested at 2 or 4 hr (short-term memory) and again at 72 hr (long-term memory) after
the completion of training. As can be seen in Figure 3, CREB antisense
had a significant effect only on long-term memory
(p < 0.001). The data of 4 hr, a point at which
LOB is long undetected, and even rearing is negligible, clearly
demonstrate that the aversion observed was not attributable to the
lingering effects of the UCS but, rather, was attributable to memory.
There was no difference in the amount of liquid consumed by the rats at
2 and 4 hr (5.6 ± 0.6 and 5.5 ± 0.2 ml, respectively), and
combined with the liquid consumed in training (5 ml), it was only
slightly below the total amount drank at 72 hr (11.5 ± 0.3 ml).
Interestingly, no extinction was seen when rats tested for short-term
memory were retested for long-term memory, whereas such extinction is
usually observed in repetitive long-term memory tests (e.g., Fig.
2).
Fig. 3.
CREB antisense in the amygdala selectively impairs
long- but not short-term memory. Aversion indices are plotted versus
the time of test after CTA training. Closed circles,
Rats microinjected with CREB antisense ODNs 14 hr before CTA training;
open circles, control animals injected with saline
instead of CREB antisense. The figure also depicts the time windows of
LOB behavior (dark gray) and rearing (light
gray), i.e., measures of the on-line effect of the malaise
inducing-agent as a function of the time after LiCl injection
intraperitoneally. For further details see Results. The
asterisk indicates significance for pair comparison in
which a Scheffe contrast test was used with an of 0.05.
[View Larger Version of this Image (28K GIF file)]
CREB antisense had no effect on retrieval
We microinjected CREB antisense ODNs into the amygdala 36 hr after
training, i.e., 14 hr before the first memory test. Under these
conditions, no effect on CTA memory was detected (Fig. 2, inset). Hence, CREB antisense in the amygdala had no effect
on detection of saccharin, on its conditioned hedonic valence, or on
other sensory or motor faculties involved in the acquired rejection of
the saccharin solution.
DISCUSSION
Although ample data indicate that the amygdala subserves CTA (for
review, see Yamamoto et al., 1994), its role is still debated, and
questions remain regarding the timing of its contribution (i.e.,
acquisition, retention, or retrieval), its role in the circuits (either
modulatory and transient or long-lasting), and the identity of the
subnuclei involved. Most of the data are based on lesions and suggest
that the effect on CTA depends on the type of lesion (transient or
permanent, affecting fibers of passage or sparing them), the timing of
the lesion relative to training and testing, and the location of the
damage. Whereas some studies unveiled no effect of amygdala lesions on
CTA (Kemble et al., 1979; Hatfield et al., 1992; Galaverna et al.,
1993), others showed that such lesions, especially of the basolateral
amygdala (BLA) (Fitzgerald and Burton, 1983; Simbayi et al., 1986;
Yamamoto and Fujimoto, 1991) and also the CeA (Lasiter and Glanzman,
1982, 1985; Roldan and Bures, 1994; Schafe and Bernstein, 1996),
disrupted CTA. Transient inhibition of protein synthesis in the central amygdala throughout training also disrupted CTA (Lamprecht and Dudai,
1996). Not all of the data are based on inference of function from
dysfunction; some correlative data are also available. The latter
include single-unit recording from the BLA and CeA (Yasoshima et al.,
1995) and in situ hybridization analysis that revealed increased expression of the IEGs mRNA in the CeA after administration of LiCl intraperitoneally, the UCS conventionally used in CTA (Lamprecht and Dudai, 1995).
All in all, the majority of available information does implicate the
amygdala, including CeA, in some aspects of CTA. To determine further
whether CeA is obligatory for CTA and to elucidate the molecular
mechanisms involved, we decided to inhibit the expression of IEGs
transiently in the amygdala in CTA training and testing. We recently
reported that microinjection of ODNs antisense to c-fos into
the central amygdala before CTA training impaired CTA memory tested a
few days later (Lamprecht and Dudai, 1996). c-fos is a
CRE-regulated IEG. We now argue that CREB in the amygdala is indeed
required for normal CTA memory, but only for the long-term form of it;
short-term CTA memory, as well as retrieval of memory once it has been
formed, is unaffected. Although the sphere of drug diffusion in the
brain exceeds the CeA, the only salient common feature of all of the
sections analyzed by us after microinjection of either india ink or
FITC-CREB was the bilateral involvement of substantial portions of the
CeA (see Materials and Methods). Furthermore, although part of the
microinjected solution diffused dorsolaterally along the cannula track,
microinjection 2 mm above the CeA had no effect on CTA (see Results).
We therefore consider the CeA a prime candidate for the site of action
of the antisense in this study.
Before proceeding to discuss potential implications of our results on
the understanding of CTA mechanisms, a few remarks on antisense
technology are pertinent. Whereas ODNs are rapidly degraded in
vivo, phosphorothioate-modified ODNs display markedly increased stability (Campbell et al., 1990). However, the chemical modification may also increase toxicity (Chiasson et al., 1994). In addition to
inhibiting the interaction of the target mRNA with the ribosome and
translation factors, phosphorothioate ODNs form an ODNs-mRNA hybrid
that is a substrate for RNase H, which recognizes DNA-RNA hybrids and
cleaves the RNA (Gao et al., 1991). Other effects include inhibition
rather than activation of RNase H and inhibition of DNA polymerases
(Gao et al., 1991; Helene, 1991), and interaction with other proteins
(Perez et al., 1994). Some of these effects are non-sequence-specific
and increase with the length of the ODNs, the dose, and the duration of
application (Gao et al., 1991; Helene, 1991; Chiasson et al., 1994). In
that respect, the ability to use a single local ODNs application,
because of the single-trial learning situation in the CTA paradigm, is
an advantage. We selected injection of the ODNs into the amygdala once,
14 hr before the beginning of training, because this timing was
previously shown to be effective in inhibition of CREB translation by
CREB antisense in the rat brain in vivo (Konradi et al.,
1994). The reduction of expression that we obtained was partial,
similar to that previously reported in the nucleus accumbens (Widnell
et al., 1996). Apparently, the dependence of brain function on the
level of constitutively expressed CREB is rather critical and sensitive
to a decrease of even ~40%.
Several observations support the assumption that the effects were
specific to the CREB antisense ODNs: (1) there was a clear differential
effect of the sense and antisense ODNs on CTA behavior and on
expression of CREB protein in the injected amygdaloid region; (2) CREB
antisense did not alter the level of ATF-2 and PKC; (3)
the effect on expression of the CREB protein was transient, with a time
course compatible with the published life span of the ODNs in
vivo; (4) the antisense caused no differential anatomical damage
(although it should be noted that the resolution of our anatomical
analysis was limited) (see Coggeshell, 1992); (5) the treatment had no
effect on retraining; (6) the antisense did not affect retrieval under
conditions that affected conditioning; and (7) the effect was confined
to long- but not short-term memory. Taken together, the aforementioned
observations demonstrate that under the conditions used in this study,
the antisense had no short-term toxicity relevant to the expression of
CTA behavior and no long-term toxic effects either. We cannot rule out
the possibility that in addition to its specific suppression of CREB expression, CREB antisense modulates another cellular mechanism, which
is unaffected by CREB sense; this type of reservation applies generally
to studies using inhibitors to infer function from dysfunction.
The amygdala has been repeatedly implicated in learning, especially
aversive and emotional learning (McGaugh et al., 1993; Davis et al.,
1994; Gallagher and Holland, 1994; Yamamoto et al., 1994; Maren and
Fanselow, 1996; Rogan and LeDoux, 1996). In general, two major roles
for amygdala involvement in learning and memory were proposed (without
being mutually exclusive). One is that the amygdala plays a temporary
role in modulating other brain regions in the process of acquisition
and consolidation of aversive memories (McGaugh et al., 1993). The
other is that experience-dependent changes take place in amygdaloid
circuits, which subserve long-term storage of the modified internal
representation of the aversive response. The latter hypothesis
underlies circuit models of fear conditioning (Davis et al., 1994;
Maren and Fanselow, 1996; Rogan and LeDoux, 1996). These models
distinguish in the amygdala two subsystems that subserve fear
conditioning: the basolateral complex, in which sensory information
converges from cortical and subcortical areas, possibly to form the
CS-UCS association in fear conditioning; and the CeA, which receives
projections from the basolateral complex and projects to other brain
regions involved in the fear response. The CeA also receives direct
projections from the parabrachial nucleus and the insular cortex, which
might be particularly significant in CTA.
Our data favor two notions: (1) the role of the central amygdala in CTA
is not limited to fibers of passage, as might be inferred from some
excitotoxic lesion data (Dunn and Everitt, 1988); and (2) the role of
the central amygdala in CTA is not confined to transient modulation of
other circuits during training, and involves lasting modifications in
circuits that include amygdala neurons. The functional nature of such
proposed modifications is not addressed in the present study. They may
bear on specific CTA representations or more general representations of
emotion and fear, and may last for only days or much longer. A role of
amygdala in storage of experience-dependent representations is in
consonance with the types of models suggested for fear conditioning
(Davis et al., 1994; Maren and Fanselow, 1996; Rogan and LeDoux, 1996).
Furthermore, it is tempting to speculate that some amygdala circuit
modules that subserve fear conditioning are also shared with CTA.
The molecular mechanisms implicated in the aforementioned
postulated modifications of amygdala in CTA involve CREB. This is similar to the picture obtained in a number of other experimental systems, which implicate regulation of CREB and CRE-mediated gene expression, via cAMP- and Ca2+-regulated signal
transduction cascades, in long-term circuit alterations subserving
neural development or consolidation of long-term memory (Bourtchuladze
et al., 1994; Bartsch et al., 1995; Yin et al., 1995; Impey et al.,
1996; Liu and Graybiel, 1996; Guzowski and McGaugh, 1997). An increase
in c-Fos, a CRE-regulated IEG product, has been reported to take place
in the nucleus of the solitary tract (NTS) in CTA-trained rats in
response to the CS (Swank and Bernstein, 1994; Houpt et al., 1995). The
latter observation raises the possibility that either in the NTS c-Fos elevation is correlated with, but not obligatory for, CTA expression, or that whenever a trained individual is reexposed to the CS in testing, relearning takes place, which involves IEG modulation in the
NTS. Interestingly, this IEG modulation was not detected in the NTS
ipsilateral to a unilaterally lesioned amygdala (Schafe and Bernstein,
1996). Infusion of c-fos antisense into the fourth ventricle
reduced c-Fos-positive nuclei in the NTS and impaired acquisition and
extinction of CTA in mice (Swank et al., 1996), but such infusion lacks
the site specificity required to make a firm conclusion about the
obligatory role of c-fos in NTS in CTA.
If we now return to the questions posed at the beginning of this
discussion, concerning the role and timing of amygdala contribution to
CTA, the following answers may be offered: the amygdala is required for
proper encoding of long-term CTA memory; CeA is involved; and it
undergoes cellular alterations that suggest some long-term change. We
also propose that whatever circuit mechanisms endow taste aversion
conditioning with its unique tolerance to a very long CS-UCS interval,
they are based on molecular mechanisms that also subserve many other
types of learning. Our data further reinforce the notion that CREB is a
component of such a cross-task, cross-species, and cross-phyla
molecular universal.
FOOTNOTES
Received March 25, 1997; revised Aug. 12, 1997; accepted Aug. 20, 1997.
We thank Z. Kam for advice on the use of the digital microscope system,
The Carl Dominic Center for Brain Research, Rehovot, and the US-Israel
Binational Science Foundation (BSF), Jerusalem, for support, and J. LeDoux and M. Segal for comments on this manuscript.
Correspondence should be addressed to Dr. Yadin Dudai at the above
address.
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J. J. Brightwell, C. A. Smith, R. A. Countryman, R. L. Neve, and P. J. Colombo
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S. Sangha, A. Scheibenstock, and K. Lukowiak
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L. Zhao and R. D. Brinton
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P. J. Colombo, J. J. Brightwell, and R. A. Countryman
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K. P. Kinzig, D. A. D'Alessio, and R. J. Seeley
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S. Faverjon, D. C. Silveira, D. D. Fu, B. H. Cha, C. Akman, Y. Hu, and G. L. Holmes
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Y. Levkovitz and J. M. Baraban
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S. A. Josselyn, C. Shi, W. A. Carlezon Jr, R. L. Neve, E. J. Nestler, and M. Davis
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G. E. Schafe, C. M. Atkins, M. W. Swank, E. P. Bauer, J. D. Sweatt, and J. E. LeDoux
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Y.-Y. Huang, K. C. Martin, and E. R. Kandel
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A. Fiala, U. Muller, and R. Menzel
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H. Gutierrez, R. Gutierrez, L. Ramirez-Trejo, R. Silva-Gandarias, C. E. Ormsby, M. I. Miranda, and F. Bermudez-Rattoni
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T. A. Houpt and R. Berlin
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Compound via MeSH
