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Vol. 63, Issue 1, 44-52, January 2003
Department of Pharmacology, College of Medicine, National Cheng-Kung University, Tainan, Taiwan
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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If fear memory is expressed by a long-term potentiation (LTP) of synaptic transmission in the amygdala, then reversal of LTP (depotentiation) in this area of the brain may provide an important mechanism for amelioration of anxiety and post-traumatic stress disorder. Herein, we show that low-frequency stimulation (LFS) of the external capsule elicits a depotentiation in the lateral nucleus of the amygdala. The induction of depotentiation requires activation of N-methyl-D-aspartate receptors and voltage-dependent calcium channels but is independent of adenosine A1 and metabotropic glutamate group II receptors. Extracellular perfusion or loading cells with protein phosphatase (PP) 2B (calcineurin) inhibitors prevents depotentiation. The same stimulating protocol applied to the amygdala in vivo attenuates the expression of fear memory measured with fear-potentiated startle and reduces conditioning-elicited phosphorylation of Akt and mitogen-activated protein kinase (MAPK). This is paralleled by an increase in the activity of calcineurin. In addition, application of calcineurin inhibitor blocks LFS-induced extinction of fear memory and MAPK dephosphorylation. Taken together, this study characterizes the properties of LFS-induced depotentiation in the amygdala and suggests an involvement of calcineurin cascade in synaptic plasticity and memory storage.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The
prevention of fear development or, more importantly, the erasure of
fear memory is a major challenge to neuroscientists and psychiatrists
today. Fear conditioning is the process by which a cue comes to induce
an elevated startle when it is consistently paired with an aversive
stimulus, such as a foot shock (Pavlov, 1927
; Davis, 2000; LeDoux,
2000). It represents not only an animal model of fear and anxiety but
also a model of long-term neural plasticity and memory because, once
conditioned, animals can be left untrained for at least 1 month without
a loss of startle susceptibility (Campeau and Davis, 1995). Previous
studies suggested that LTP of synapses from auditory thalamus and
cortex to the LA underlay the encoding of fear memory (McKernan and
Shinnick-Gallagher, 1997; Rogan et al., 1997). If this is true, then
depotentiation in these synapses may result in an attenuation of fear
memory. In an analogous situation, it has been shown that
administration of LFS to the amygdala in vivo blocked the development
and expression of kindled seizures, a phenomenon termed quenching
(Weiss et al., 1995). A recent report demonstrated that depotentiation
could be induced at the EC-basolateral amygdala synapse
(Aroniadou-Anderjaska et al., 2001); however, the underlying mechanism
was not elucidated.
In the present study, we first show that depotentiation occurs in the amygdala that is capable of reversing tetanus-induced LTP. Depotentiation is dependent on N-methyl-D-aspartate (NMDA) receptor and VDCC activation and requires postsynaptic phosphatase activity. By behavioral assessment, we further demonstrate that administration of LFS to the EC or LA using depotentiation-like parameters attenuates the expression of fear memory. Quenching stimulation, which also increases the activity of calcineurin, reduces fear training-induced Akt and MAPK phosphorylation. Thus, quenching could be attributed to an increased calcineurin activity, which dephosphorylates key proteins in the amygdala and results in the extinction of conditioned fear.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Slice Preparation and Electrophysiological Recordings. Male Sprague-Dawley rats, 4 to 5 weeks old, were decapitated and their brains rapidly removed and placed in cold oxygenated artificial cerebrospinal fluid (ACSF) solution. Subsequently, the brain was hemisected and cut transversely posterior to the first branch and anterior to the last branch of the superior cerebral vein. The resulting section was glued to the chuck of a Vibroslice tissue slicer (Campden Instruments, Silbey, UK). Transverse slices of 450 µm thickness were cut and the appropriate slices placed in a beaker of oxygenated ACSF at room temperature for at least 1 h before recording. ACSF solution had the following composition (in mM): 117 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 25 mM NaHCO3, 1.2 mM NaH2PO4, and 11 mM glucose. The ACSF was bubbled continuously with 95% O2/5% CO2 and had a pH of 7.4.
A single slice was transferred to the recording chamber, in which it was held submerged between two nylon nets and maintained at 32 ± 1°C. The chamber consisted of a circular well of a low volume (1-2 ml) and was perfused constantly at a rate of 2 to 3 ml/min. Extracellular field potentials were recorded by electrical stimulation of the external capsule, which contained fibers from the auditory cortex to the lateral amygdala, with a concentric bipolar stimulating electrode (SNE-100; Kopf Instruments, Bern, Germany). Electrical stimuli (150 µs in duration) were delivered at a frequency of 0.05 Hz. Intracellular recording microelectrodes were pulled from 1.0-mm microfiber capillary tubing on a Brown-Flaming electrode puller (Sutter Instruments, San Rafael, CA). The electrodes were filled with 4 M potassium acetate, with resistance ranging from 70 to 130 M
.
Baseline field potentials were adjusted to ~30 to 40% of the maximal
responses. LTP was elicited by three trains of tetanus (100 Hz, 1 s, at 1.5-min intervals) at the same stimulation intensity used for
baseline. In most experiments, bicuculline (1 µM) was present in the
perfusion solution. Data were expressed as mean ± S.E. The data
were analyzed by analysis of variance and Student's t test,
and p < 0.05 was considered statistically significant.
Surgery and Quenching.
Rats were anesthetized with sodium
pentobarbital (50 mg/kg, i.p.) and mounted on a stereotaxic apparatus.
Two stimulating electrodes (MS303/2; Plastic Products, Roanoke, VA)
were implanted bilaterally into the EC or LA. The coordinates were
anteroposterior,
2.8 mm; mediolateral, ± 5.3 mm; dorsoventral, 7.8
mm, according to the method of Paxinos and Watson (1986). Three jewelry
screws were implanted over the skull, serving as anchors, and the whole assembly was affixed on the skull with dental cement. The rats were monitored and handled daily and were given 5 to 7 days to recover.
The animals were then subjected to training and behavioral tests
performed the next day. Quenching stimulation (5 Hz for 3 min at the
intensity of 0.1 mA) was given at 10 min after training (after
training) or 1 h after testing (after testing).
Fear Conditioning.
Fear conditioning was measured using the
potentiated startle paradigm (Cassella and Davis, 1986). Male
Sprague-Dawley rats (100-150 g) were trained and tested in a startle
chamber (San Diego Instruments) in which cage movement results in the
displacement of an accelerometer. Startle amplitude was defined as peak
accelerometer voltage within 200 ms after startle stimulus onset. The
acoustic startle stimulus was a 50-ms burst of white noise at an
intensity of 95 dB. The visual conditioned stimulus (CS) was a 3.7-s
light flash produced by an 8-W fluorescent bulb attached to the back of
a stabilimeter. The unconditioned stimulus (US), which serves as the
aversive component necessary for fear conditioning, was a 0.6-mA foot
shock with a duration of 0.5 s. On the training day, rats were
placed in the startle chamber and received 10 CS-foot shock pairings.
Unpaired control rats received the same number of CS and US
presentation, but in an unpaired, pseudorandom fashion.
Western Blot Analysis.
Unless noted in the experiments,
groups of sham control and LFS animals were sacrificed by decapitation
at 10 min after training or 1 h after testing. The lateral and
basolateral subregions of the amygdala were sonicated briefly in
ice-cold buffer (50 mM Tris-HCl, pH 7.5, 0.3 M sucrose, 5 mM EDTA, 2 mM
sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 4 µg/ml
aprotinin). After sonication, the samples were centrifuged at 7500 rpm
for 15 min and the supernatant was obtained after pelleting the crude
membrane fraction by centrifugation at 50,000 rpm for 1 h at
4°C. Protein concentration in the soluble fraction was then measured
using a Bradford assay, with bovine serum albumin as the standard.
Equivalent amounts of protein for each sample were resolved in 8.5%
SDS-polyacrylamide gels, blotted electrophoretically to Immobilon-P
transfer membrane (Millipore, Bedford, MA), and blocked
overnight in Tris-buffered saline (50 mM Tris-HCl, pH 7.5, 150 mM NaCl)
containing 3% bovine serum albumin. For detection of the
phosphorylated forms of MAPK, blots were incubated with
anti-phospho-ERK (New England Biolabs, Beverly, MA) antibodies. To
control the content of the specific protein per lane, membranes were
stripped with 100 mM 
-mercaptoethanol and 2% SDS in 62.5 mM
Tris-HCl, pH 6.8, for 30 min at 70°C and reprobed with a mouse
monoclonal anti-pan-ERK (BD Transduction Laboratories, Lexington, KY)
antibody. An enhanced chemiluminescence kit (Amersham Biosciences,
Piscataway, NJ) was used for detection. Western blots were
developed in the linear range used for densitometry. The density of the
immunoblots was determined by an image analysis system installed with a
software BIO-ID (Vilber Lourmat, Marne La Vallée, France). To
assess for changes in the activation of MAPK, total kinase levels were
first normalized to total protein levels. Activated kinase levels in
trained animals were normalized to total kinase levels and then were
expressed as a percentage of those in control animals.
Calcineurin Activity Assay.
Rats trained with light alone
were sacrificed by decapitation immediately after trials. The LA and
BLA were microdissected and frozen on dry ice. Phosphatase assay was
performed according to the instructions of the calcineurin assay kit
(Promega Corp., Madison, WI). Pooled LA and BLA areas were homogenized
in ice-cold buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM EGTA, 5 mM EDTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 4 µg/ml aprotinin) and centrifuged at 100,000 rpm for 1 h to remove particular matter. Supernatants were
added to the reaction buffer from the kit and incubated the reaction at
30°C for 10 min. The reaction buffer contains 50 mM imidazole, pH
7.2, 0.2 mM EGTA, 10 mM MgCl2, 1 mM
NiCl2, 50 µg/ml calmodulin, and 0.02%
-mercaptoethanol. FK-506 (1 µM) in the supernatant completely
blocked Pi release, indicating that the measured
phosphatase activity reflects calcineurin function. The enzyme activity
was expressed in nanomoles of Pi released per minute per milligram of protein from the substrate. The calcineurin substrate sequence is RRA(pT)VA.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Induction of LTP in the LA Neurons.
In amygdala slices of 4- to 6-week-old rats, delivery of three sets of tetanic stimulation (TS;
100 Hz for 1 s) to the EC at an interstimulus interval of 1.5 min
produced a robust enhancement of synaptic responses in the LA neurons
that persisted for more than 2 h. The slopes of fEPSP were
213.4 ± 18.2 and 200.1 ± 15.2% (n = 6) of
pretetanus level at 1 and 2 h after the stimulation, respectively
(Fig. 1). To examine whether LTP was
NMDA-receptor dependent, 50 µM
D-2-amino-5-phosphonovaleate (D-APV)
was added to the perfusion medium 5 min before, during, and 10 min
after TS. Consistent with previous reports, LTP was blocked by
D-APV (Huang and Kandel, 1998). The slopes of
fEPSP were 122.6 ± 4.9 and 91.6 ± 4.5% (p < 0.001, n = 6, unpaired t test) at 1 and
2 h after the stimulation.
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Depotentiation by LFS. We attempted to establish a protocol for investigating depotentiation and found that, when delivered at 10 min after TS, reliable depotentiation could be induced by 15 min of stimulation at 1 Hz, 7.5 min at 2 Hz, or 3 min at 5 Hz. Because it is easier to handle and quench the animals (see Quenching) with a shorter LFS protocol, 5 Hz stimulation for 3 min was used to elicit depotentiation. In six experiments, the slopes of fEPSP returned to 118.0 ± 8.1 and 104.9 ± 11.4% of pretetanus level at 1 and 2 h after TS, respectively (Fig. 1). In another set of experiments, in which LFS was applied at 1 h (instead of 10 min) after TS, this LFS protocol caused similar depotentiation. The fEPSP measured at 2 h after TS was 112.0 ± 10.1% (n = 6) of pretetanus value, which was not significantly different from that LFS applied at 10 min after TS (p > 0.1).
Several neurotransmitter systems have been implicated in the mediation of depotentiation. We first examined whether depotentiation is NMDA receptor-dependent by application of D-APV. Because NMDA receptor antagonists block the induction of LTP, D-APV was applied at 5 min after TS. LFS was then given at 5 min after D-APV perfusion, and the drug was removed at the end of LFS. Figure 1 shows that D-APV (50 µM) blocked depotentiation. The fEPSPs measured at 1 and 2 h after TS were 231.7 ± 16.0 and 181.3 ± 7.1% (n = 6), respectively, of pretetanus value, which were significantly different from that without D-APV application (p < 0.01, unpaired t test). To further ensure NMDA dependence, LFS was delivered in the presence of the noncompetitive antagonist MK-801 (10 µM). Depotentiation was blocked in a similar way by MK-801. The fEPSPs measured at 1 and 2 h after TS were 234.1 ± 23.8 and 197.6 ± 16.1% (n = 6) of pretetanus value, respectively. In hippocampal CA1 neurons, depotentiation has been shown to be blocked by adenosine A1 receptor antagonists, suggesting an involvement of this receptor (Larson et al., 1993; Fujii et al., 1997; but see De Mendonca et al., 1997). We tested whether this is the
case in the amygdala by application of a selective adenosine receptor
antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). DPCPX (500 nM)
was applied 5 min before LFS and removed from the perfusion medium
immediately after LFS. Consistent with previous reports showing that
there were significant amounts of endogenous adenosine present in the
extracellular space (Dunwiddie et al., 1981), DPCPX increased the fEPSP
by 28 ± 6%. The effect was reversible within 20 min of washing.
As summarized in Table 1, DPCPX did not
affect depotentiation. The fEPSPs measured at 1 and 2 h after TS
were 123.1 ± 12.5 and 109.1 ± 12.5% (n = 6) of baseline, respectively, which was not significantly different
from that without DPCPX treatment.
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-ethylglutamic acid (EGLU), suggesting the
involvement of mGluR II (Li et al., 1998). To examine the involvement
of mGluR II, EGLU (20 µM) was applied 5 min before and during LFS.
EGLU did not affect depotentiation (Table 1). The fEPSPs measured at 1 and 2 h after TS were 131.7 ± 21.9 and 97.9 ± 17.2%
(n = 6) of baseline, respectively.
Aside from NMDA receptors, Ca2+ could enter the
cell through VDCCs. Indeed, NMDA-independent forms of synaptic
plasticity in several brain areas, including the amygdala (Weisskopf et
al., 1999), depend on L-type VDCCs (Christie et al., 1997; Kapur et al., 1998; Morgan and Teyler, 1999). We therefore tested whether inhibition of L-type VDCCs affected amygdala depotentiation. Figure 2 shows that blocking L-type VDCCs with
nifedipine (20 µM) partially prevented LFS-induced depotentiation.
The fEPSP slopes were 197.9 ± 16.1 and 154.9 ± 16.8%
(n = 6) of baseline at 1 and 2 h, respectively, after the induction of LTP. We applied another selective L-type VDCC
blocker, nimodipine. Nimodipine (2 µM) blocked depotentiation. The
fEPSPs were 227.9 ± 18.9 and 173.7 ± 14.2% of baseline 1 and 2 h after TS, respectively, which was not statistically
different from control without receiving LFS (p > 0.05, Fig. 2).
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Involvement of Calcineurin in Synaptic Depotentiation in the
Amygdala.
One likely signal pathway that is
Ca2+-dependent and may be involved in the
reversal of LTP is the serine/threonine protein phosphatase calcineurin
(O'Dell and Kandel, 1994; Staubli and Chun, 1996; Zhuo et al., 1999).
We examined whether amygdala depotentiation is associated with a change
in phosphatase activity by application of specific calcineurin
inhibitors. Figure 3 shows that
cyclosporin A, an immunosuppressant that inhibits calcineurin in a
complex with cyclophilin (Liu et al., 1991), blocked depotentiation in a concentration-dependent manner (F2,15 = 15.48, p < 0.001). In 10 µM cyclosporin A, the fEPSP slopes
were 170.4 ± 16.1 and 146.3 ± 12.3% (n = 6) of baseline at 1 and 2 h, respectively, after the induction of
LTP. In 100 µM, the fEPSP slopes were 207.8 ± 21.7 and
196.8 ± 12.4% (n = 6) of baseline at 1 and
2 h, respectively, after the induction of LTP, which was
significantly different from LFS group (p < 0.01) but
was not statistically different from the group not receiving LFS
(p > 0.05). Similarly, another selective calcineurin
inhibitor, FK-506 (100 µM), also blocked depotentiation (fEPSPs were
233.8 ± 10.9 and 190.4 ± 24.0% of baseline 1 and 2 h,
respectively, after TS, n = 6) (Fig. 3). As a control,
we examined whether basal synaptic strength is regulated by
calcineurin. We found that both cyclosporin A and FK-506 had no
significant effect on basal fEPSP (Table 1). The lack of effect of
calcineurin inhibitors on basal synaptic strength may suggest that
calcineurin either is not constitutively active at synapses during low
levels of synaptic activity or is functionally expressed in adult but
not young rats (Wang and Kelly, 1997).
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Quenching.
In the first set of experiments, rats were
implanted bilaterally with stimulating electrodes into the EC or LA and
were given 5 to 7 days to recover. The rats were then given 10 pairs of
light (CS) and foot shock (US) and tested 24 h later (pre-LFS
test). After initial training, animals exhibited fear of the light,
which manifested as an increase in acoustic startle. They were
subsequently divided into three groups: sham-operated control, 0.1-Hz
LFS, and 5-Hz LFS. LFS groups were given low-frequency stimulation (0.1 or 5 Hz for 3 min) bilaterally 1 h after pre-LFS tests and were
retested 23 h later, whereas the sham control group did not receive electrical stimulation. Figure 5A
illustrated the mean startle potentiation in control and LFS groups.
After LFS, the degree of potentiation was significantly reduced in rats
of the 5-Hz post-LFS group compared with pre-LFS test
(t9 = 3.45, p < 0.01). In contrast, startle amplitudes in animals of the sham-operated and 0.1 Hz post-LFS groups were not significantly different from their
pre-LFS tests (t5 = 3.5, p = 0.34). The differential results obtained from 0.1- and 5-Hz stimulation could have be caused by the different number of
stimulations. Therefore, additional control experiments were performed
in which rats received three sessions of stimulation and each session
consisted of three trains of 100-Hz stimulation at an intersession
interval of 1 min. As shown in Fig. 5A, startle amplitude was not
changed between pre- and poststimulation tests in these rats,
indicating that 100-Hz stimulation was unable to reduce conditioned
fear. Figure 5B illustrates the electrode tip locations in these
experiments. Only rats with cannula tips at or within the boundaries of
LA and BLA were included in the data analysis.
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Quenching Induces Dephosphorylation of Akt and MAPK.
We have
recently shown that fear conditioning caused a selective activation of
phosphoinositide-3 kinase in the amygdala (Lin et al., 2001). It is of
interest to determine whether conditioning-elicited Akt phosphorylation
is affected by quenching stimulation. Rats were implanted bilaterally
with stimulating electrodes and trained with startle reflex paradigm.
After training, the rats were divided into three groups; after-test,
after-training, and sham-operated control groups. The group of
after-training was given LFS 10 min after fear conditioning, whereas
the group of after-test received LFS 1 h after pre-LFS test (Fig.
6A). The sham-operated rats were similarly implanted with electrodes without receiving LFS. Figure 6B
shows that LFS after training significantly reduced the degree of Akt
phosphorylation (101.6 ± 4.3%, n = 6, p < 0.01 versus conditioned). Similarly, Akt
phosphorylation was inhibited by LFS after pre-LFS test (100.9 ± 6.2%, n = 6; p < 0.01 versus
conditioned). By comparison, Akt phosphorylation in sham-operated
control rats was comparable with those of conditioned animals
(p = 0.28, unpaired t test). No change in
the immunoreactivity against Akt was detected, suggesting that the
total amount of Akt was not altered (Fig. 6C).
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;
Schafe et al., 2000; Lin et al., 2001). We next investigated the effect
of quenching stimulation on conditioning-elicited phosphorylation of
MAPK. As shown in Fig. 7, LFS after
training significantly reduced the degree of MAPK phosphorylation (p42, 106.3 ± 12.4%; p44, 113.7 ± 23.2%; n = 6, p < 0.01 versus conditioned). Similarly, MAPK
phosphorylation was inhibited by LFS after pre-LFS test (p42,
122.0 ± 13.3%; p44, 117.4 ± 21.3%; n = 6;
p < 0.01 versus conditioned). In contrast, MAPK
phosphorylation in sham-operated control rats (p = 0.85) or after 0.1-Hz stimulation (p = 0.71) was not
significantly different from those of conditioned animals. These
results suggest that fear training-induced MAPK phosphorylation was
markedly attenuated after LFS. Figure 7 also shows that the immunoreactivity against MAPK was not changed, indicating that the
total amounts of MAPKs were not altered.
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Calcineurin Inhibitor Blocks Both Extinction and
Dephosphorylation.
It has been proposed that synaptic plasticity
and memory storage is determined by the balance between protein
phosphorylation and dephosphorylation mediated by cAMP-dependent
protein kinase and phosphatases. Therefore, a decrease in the
phosphorylated state of Akt and MAPK after quenching stimulation could
result from a recruitment of phosphatase calcineurin. To examine this possibility, animals were intravenously administered cyclosporin A (1, 10, or 20 mg/kg) immediately after the pre-extinction tests. As shown
in Fig. 8A, rats that received vehicle
showed a significant decrease in the startle after quenching
stimulation. In contrast, rats given intravenous infusion of
cyclosporin A before stimulation exhibited less reduction. An analysis
of variance revealed that cyclosporin A produced a dose-dependent
inhibition of LFS-induced decrease in startle responses
(F3,19 = 27.71, p < 0.001). In
addition, a parallel experiment determined that cyclosporin A prevented quenching-induced dephosphorylation of MAPK (Fig. 7A). Thus,
cyclosporin A blocked LFS-induced startle reduction at the same dose
that inhibited MAPK dephosphorylation.
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) before LFS stimulation.
Figure 8A showed that memory retention in verapamil-treated rats
(205.2 ± 46.8%, n = 4) was significantly better
than that of the LFS group.
Calcineurin Activity Is Increased after Quenching Stimulation. We directly determined the involvement of calcineurin in quenching by measuring the released Pi from the phosphopeptide substrate that was insensitive to okadaic acid but could be blocked by FK-506. The rates of Pi released from LA and BLA in unpaired control and conditioned rats were 4.5 ± 0.3 (n = 8) and 4.8 ± 0.3 nmol/min/mg (n = 8), respectively. The value was increased to 6.9 ± 0.4 nmol/min/mg after quenching stimulation (n = 8, p < 0.001 versus unpaired or conditioned). By contrast, calcineurin activity was unaltered in rats given 0.1-Hz stimulation (5.0 ± 0.4, n = 7, p = 0.5 versus conditioned) (Fig. 8B).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The major findings of this study are that 1) LFS is capable of eliciting depotentiation in the in vitro amygdala slices and, in parallel, reduces conditioned fear in whole animals; 2) inhibition of protein phosphatase 2B by calcineurin inhibitors blocks depotentiation in slices and prevents LFS-induced extinction of fear memory in animals; and 3) fear training-induced phosphorylation of Akt and MAPK in the rat amygdala is reduced after quenching stimulation, which is accompanied by an increase in the enzymatic activity of calcineurin. The paralleled effects of LFS (quenching) on depotentiation in the in vitro slices and on phosphorylation of protein kinases and retention of fear memory in vivo provide the direct evidence suggesting that synaptic plasticity and memory storage are regulated by protein phosphorylation and dephosphorylation in the amygdala.
Properties of Amygdala Depotentiation.
In hippocampal CA1
neurons, the reversal of LTP or depotentiation was thought to result
from activation of NMDA or adenosine A1 receptors
during LFS, which modulates Ca2+ entry or
intracellular cAMP levels, leading to a decrease of previously enhanced
responses (Fujii et al., 1991, 1997; Otmakhova and Lisman, 1998). We
investigated the cellular mechanism of synaptic depotentiation at the
EC-LA synapse, which carries axons from secondary auditory and
perirhinal cortices to the amygdala and is important for mediation of
fear conditioning (Davis et al., 1993; Campeau and Davis, 1995).
We found that synaptic depotentiation at the EC-LA synapse was
independent of adenosine A1 or mGluR II receptors
but required activation of NMDA receptors, L-type Ca2+ channels, and calcineurin. In addition,
loading of LA neurons with calcineurin inhibitor blocked
depotentiation. Taken together, these results suggest that during LFS,
Ca2+ enters through NMDA receptors and L-type
Ca2+ channels, resulting in the activation of
calcineurin (PP2B). Many protein kinases including Akt and MAPK are
substrates of PP2A, and PP2A seems to be the major kinase phosphatase
in eukaryotic cells that down-regulates activated protein kinases
(Andjelkovic et al., 1996; Millward et al., 1999). Thus, calcineurin
may dephosphorylate inhibitor-1, resulting in an activation of PP1 and
PP2A. Active PP1 and 2A then inactivated protein kinases, which in
their phosphorylated state were required for synaptic plasticity. This
is consistent with genetic studies showing that depotentiation was
abolished completely in mice lacking the predominant calcineurin
isoform (Zhuo et al., 1999). Alternatively, calcineurin could directly act on ligand-gated ion channels independent of PP1 and PP2A (Yakel, 1997).
). It is possible
that by our stimulating protocol, when nifedipine or nimodipine blocked
VDCCS, depotentiation was not induced because of insufficient calcium
influx through NMDA receptors. Depotentiation occurs only when
additional calcium entered through VDCCs that were evoked on top of
NMDA depolarization.
In the lateral amygdala, LTP was dependent on NMDA receptors but not on
VDCCs when it was induced by TS (Huang and Kandel, 1998; Lin et al.,
2001). Conversely, LTP was dependent on VDCCs but not on NMDA receptors
when induced by pairing presynaptic stimulation with postsynaptic
depolarization (Weisskopf et al., 1999). Interestingly, in behavioral
study, bilateral infusion of NMDA receptor antagonists into the LA
impaired both short- and long-term memory of fear conditioning, whereas
VDCC blockade selectively impaired long-term memory (Bauer et al.,
2002). These results suggest the differential contribution of NMDA
receptor and VDCC in different types of memory formation in the LA. It would be interesting for the future studies to determine the relative contribution of both types of Ca2+ influx in
initiating second-messenger systems and/or gene expression that
underlie memory extinction.
Quenching Effect.
Here we verify the hypothesis that
depotentiation in the amygdala may weaken or extinguish memory
performance by first showing that bilateral administration of LFS to
the amygdala in vivo produced a profound effect on the expression of
fear memory. Secondly, fear training-elicited Akt and MAPK
phosphorylation was attenuated after quenching stimulation. This was
paralleled by an increase in the enzymatic activity of calcineurin,
suggesting that dephosphorylation of key proteins in the amygdala by
serine/threonine phosphatase contributes to memory storage.
Furthermore, converging evidence indicates that the AMPA receptor
itself may be the critical substrate of calcineurin that mediates the
expression of quenching extinction. Phosphorylation on Ser845 of the
GluR1 subunit regulates the open-channel probability of AMPA receptors,
whereas on Ser831, it increases the apparent single-channel conductance
(Derkach et al., 1999; Banke et al., 2000). In this respect, it has
been shown that LTD induction is associated with persistent
dephosphorylation of GluR1 at Ser845 and dephosphorylation occurs at
Ser831 when previously activated synapse is depotentiated (Lee et al.,
2000). In addition, phosphorylation of GluR1 regulates synaptic
trafficking of AMPA receptors and the enhancement of AMPA receptor
endocytosis after LTD induction is blocked by calcineurin inhibitors
(Beattie et al., 2000; Ehlers, 2000). Thus, LFS may initiate an influx
of Ca2+ into cells through NMDA receptors or
L-type Ca2+ channels (Huang and Kandel, 1998;
Weisskopf et al., 1999), leading to the activation of calcineurin.
Calcineurin induces dephosphorylation of GluR1, a process that
decreases the synaptic function of AMPA receptors by altering channel
properties and/or promoting the endocytosis. In conclusion, in a
combination of behavioral, pharmacological, and biochemical
experiments, we show for the first time that LFS elicits depotentiation
in in vitro amygdala slices and reduces conditioned fear in whole
animals; both effects seem to result from an increase in calcineurin activity.
), suggesting that sensitized fear response
is created by the stress of combat trauma. In the present study, we
have characterized the molecular mechanisms underlying depotentiation
in the amygdala and defined the parameters for quenching fear memory in
an attempt to provide better pharmacological strategies for treating
anxiety disorders.
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Acknowledgments |
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We thank Drs. M. D. Lai and B. Mirasty for valuable discussion and comments on the manuscript.
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Footnotes |
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Received April 30, 2002; Accepted September 19, 2002
This study was supported by National Science Council grant NSC89-2320-B006-011 and by Academic Excellence Program of the Ministry of Education grant 89-B-FA08-1-4, Taiwan, Republic of China.
Address correspondence to: Dr. Po-Wu Gean, Department of Pharmacology, College of Medicine, National Cheng-Kung University, Tainan, Taiwan 701. E-mail: powu{at}mail.ncku.edu.tw
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Abbreviations |
|---|
LTP, long-term potentiation;
LFS, low-frequency
stimulation;
PP, protein phosphatase;
EC, external capsule;
NMDA, N-methyl-D-aspartate;
VDCC, voltage-dependent calcium channel;
LA, lateral nucleus of amygdala;
MAPK, mitogen-activated protein kinase;
ACSF, artificial cerebrospinal
fluid;
CS, conditioned stimulus;
US, unconditioned stimulus;
ERK, extracellular signal-regulated kinase;
BLA, basolateral amygdala;
FK-506, tacrolimus;
TS, tetanic stimulation;
fEPSP, field excitatory
postsynaptic potential;
D-APV, D-2-amino-5-phosphonovaleate;
MK-801, dizocilpine maleate;
DPCPX, 8-cyclopentyl-1,3-dipropylxanthine;
EGLU, 2S--ethylglutamic
acid;
mGluR II, metabotropic glutamate group II receptor;
AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid;
GluR1, glutamate receptor 1.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-ethylglutamic acid.
J Neurosci
18:
1662-1670 in synaptic depotentiation in hippocampus.
Proc Natl Acad Sci USA
96:
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), 5-Hz LFS (
), 0.1-Hz LFS
(
), and 100-Hz (
) rats in experiments shown in A.
