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 Previous Article
The Journal of Neuroscience, May 1, 1998, 18(9):3480-3487
Phosphorylation of Mitogen-Activated Protein Kinase by One-Trial
and Multi-Trial Classical Conditioning
Terry
Crow1,
Juan-Juan
Xue-Bian1,
Vilma
Siddiqi1,
Yuan
Kang2, and
Joseph T.
Neary2, 3
1 Department of Neurobiology and Anatomy, University of
Texas Medical School at Houston, Houston, Texas 77225, and
2 Research Service, Veterans Affairs Medical Center and
3 Departments of Pathology, and Biochemistry and Molecular
Biology, University of Miami School of Medicine, Miami, Florida 33125
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ABSTRACT |
The pathway supporting the conditioned stimulus (CS) is one site of
plasticity that has been studied extensively in conditioned Hermissenda. Several signal transduction pathways have
been implicated in classical conditioning of this preparation, although
the major emphasis has been on protein kinase C. Here we provide
evidence for the activation and phosphorylation of a mitogen-activated protein kinase (MAPK) pathway by one-trial and multi-trial
conditioning. A one-trial in vitro conditioning
procedure consisting of light (CS) paired with the application of 5-HT
results in the increased incorporation of 32PO4
into proteins detected with two-dimensional gel electrophoresis. Two of
the phosphoproteins have molecular weights of 44 and 42 kDa, consistent
with extracellular signal-regulated protein kinases (ERK1 and ERK2).
Phosphorylation of the 44 and 42 kDa proteins by one-trial conditioning
was inhibited by pretreatment with PD098059, A MEK1 (ERK-Activating
kinase) inhibitor. Assays of ERK activity with brain myelin basic
protein as a substrate revealed greater ERK activity for the group that
received one-trial conditioning compared with an unpaired control
group. Western blot analysis of phosphorylated ERK using antibodies
recognizing the dually phosphorylated forms of ERK1 and ERK2 showed an
increase in phosphorylation after one-trial conditioning compared with
unpaired controls. The increased phosphorylation of ERK after one-trial
conditioning was blocked by pretreatment with PD098059.
Hermissenda that received 10 or 15 conditioning trials
showed significant behavioral suppression compared with pseudo-random
controls. After conditioning and behavioral testing, the conditioned
animals showed significantly greater phosphorylation of ERK compared
with the pseudo-random controls. These results show that the ERK-MAPK
signaling pathway is activated in Pavlovian conditioning of
Hermissenda.
Key words:
Pavlovian conditioning; MAPK pathway; signal
transduction; cellular excitability; ERK cascade; Hermissenda
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INTRODUCTION |
Several cellular signaling pathways
have been implicated in studies of neuronal plasticity underlying
learning and memory. The activation of cAMP-dependent protein kinase is
a requirement for long-term facilitation in Aplysia and
long-term retention of conditioning in Drosophila (for
review, see DeZazzo and Tully, 1995 ; Bailey et al., 1996). In
Hermissenda the protein kinase C (PKC) pathway is involved
in the induction of short-term plasticity (Matzel et al., 1990; Crow et
al., 1991; Schuman and Clark, 1994) and changes in excitability
associated with multi-trial Pavlovian conditioning (Farley and
Auerbach, 1986; Neary et al., 1986; Farley and Schuman, 1991; Frysztak
and Crow, 1997). One-trial conditioning in Hermissenda
produces long-term behavioral suppression (Crow and Forrester, 1986)
and short- and long-term enhancement (STE and LTE, respectively) in
sensory neurons of the conditioned stimulus (CS) pathway (Crow and
Forrester, 1991, 1993). Short- and long-term memory after conditioning
can be dissociated based on the role of mRNA synthesis, protein
synthesis, and the contribution of PKC (Crow and Forrester, 1990, 1991,
1993; Crow et al., 1997). Recent support for the independence of short-
and long-term memory stems from studies showing that downregulation of
PKC or pretreatment with kinase inhibitors blocked the induction of
STE; however, LTE was normally expressed (Crow and Forrester, 1993).
These results indicate that other signaling pathways contribute to
plasticity associated with short- and long-term memory in
Hermissenda. A mitogen-activated protein kinase (MAPK)
pathway involving extracellular signal-regulated protein kinase (ERK)
has been implicated in cellular growth and plasticity (for review, see
Neary, 1997; Robinson and Cobb, 1997). MAPK activation and
phosphorylation have been previously shown for LTP induction (English
and Sweatt, 1996) and long-term facilitation of cultured
Aplysia neurons (Martin et al., 1997); however, MAPK
phosphorylation has not been previously reported in Pavlovian
conditioning. In this report, we provide evidence for the contribution
of ERK in classical conditioning of Hermissenda. We show
that in vitro one-trial conditioning and multi-trial
Pavlovian conditioning result in the activation and phosphorylation of
ERK1 and ERK2.
Portions of this research have appeared previously in abstracts
(Xue-Bian et al., 1997a,b)
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MATERIALS AND METHODS |
Experimental paradigm. Adult Hermissenda
crassicornis obtained from Sea-Life Supply (Sand City, CA) were
used in the experiments. Animal maintenance was as described previously
(Crow and Forrester, 1991). For phosphorylation studies and kinase
assays, the circumesophageal nervous systems were removed, and the
eyes, including proximal optic nerve, were isolated. As shown
previously, the eye is a discrete structure containing five
photoreceptors (Alkon and Fuortes, 1972). To minimize potential
animal-to-animal variability in 32PO4 uptake,
eyes from three to four animals were used for each experimental
treatment and for control procedure in each experimental replication.
The isolated eyes were placed into vials containing artificial seawater
(ASW) at 15°C having the following composition (in mM):
460 NaC1, 10 KC1, 10 CaCl2, and 55 MgCl2, buffered with 10 HEPES, and adjusted to pH
7.6 with dilute NaOH. The isolated eyes were randomly distributed to
the different treatment groups. The vials containing the isolated eyes
were maintained at 15°C in a dark room to provide a 12 min period of
dark adaptation before applying the different treatments.
Conditioning procedure and baseline test of phototactic
behavior. The details of the conditioning procedures and methods
for testing phototactic behavior have been described previously in detail (Crow and Alkon, 1978; Crow and Offenbach, 1983; Crow, 1985) and
will be described only briefly in this report. Animals were tested
before conditioning to determine baseline latencies to initiate
locomotion in response to a test light. Animals that did not respond
within a 15 min criterion period during the pretraining measurements
were not used in the conditioning experiments. Animals were placed into
228-mm-long glass tubes filled with ASW. A foam plug inserted through
an opening confined the animal to one end of the tube. The tubes were
attached by spring clips to a modified turntable enclosed in an
incubator maintained at 15°C. Animals were dark-adapted for 12 min
before testing phototactic behavior. A light spot
(10 4 W/cm2, white light) was
projected onto the center of the turntable, illuminating a circular
area 15-16 cm in diameter, and the elapsed times to initiate
locomotion in the presence of the test light were recorded. Previous
research has shown that the increase in the time taken by the animals
to locomote into a test light can be accounted for by an increase in
the latency to initiate locomotion (Crow and Offenbach, 1983). After
baseline measurements, animals were randomly assigned to conditioned
and pseudo-random control groups. The conditioning phase consisted of
10 or 15 trials of the 10 sec CS (light) and the unconditioned stimulus
(US, high-speed rotation) with an average intertrial interval of 3-4
min. The intensity of the CS was the same as that for the test light
used to establish baseline responding of phototactic behavior during the pretest condition. The pseudo-random control group received 10 or
15 trials of the CS and US programmed on explicitly unpaired schedules.
For each conditioning session, both conditioned and pseudo-random
control animals were selected from the same animal shipment. All
animals received behavioral testing identical to the pretraining
(baseline) test measurement for phototaxis 10 min after the last
conditioning trial. Animals that did not initiate locomotion in the
presence of the CS within 15 min during the post-test received a
maximum latency score. Assessment of conditioning was determined by
computing suppression ratios that compared post-training phototactic
behavior with pretraining test scores. The ratio is expressed as
A/(A + B), where A
represents pretraining scores, and B represents
post-training scores.
The in vitro one-trial conditioning procedure consisted of
the presentation of a 5 or 10 min period of light
(~104 W/cm2; CS) paired with
104 M 5-HT applied to the isolated
eye-nerve preparations. After the conditioning trial preparations were
washed, lysed, and frozen at  80°C. Control groups received one 5 min trial of light or one unpaired presentation of light (10 min) and
the application of 5-HT (104 M) using
procedures described previously (Crow and Forrester, 1986). The
one-trial conditioning procedure when applied in vivo has
been shown to result in long-term (24 hr) behavioral suppression (Crow
and Forrester, 1986).
Protein phosphorylation. For studies of protein
phosphorylation after one-trial conditioning, the treatment and control
groups were incubated for 2 hr in 32PO4.
Previous work has shown that the incorporation of phosphate into
protein is linear with respect to time for 1-4 hr (Neary et al.,
1981). The in vitro conditioning procedure consisted of a 5 min trial of light paired with 104 M
5-HT and was followed by a wash with ASW. The treatment group received
the 2 hr incubation in 32PO4 followed by
one-trial in vitro conditioning. The control groups received
the 2 hr incubation in the 32PO4 followed by 5 min of light. The incubation solution consisted of 200 µl of
oxygenated ASW containing 11 mM glucose and 0.125 mCi of
32PO4 (carrier-free) (New England Nuclear).
After the 2 hr incubation the samples were quickly rinsed twice in an
isotonic ice-cold wash solution (in mM: 460 NaCl, 10 KCl, 5 EDTA, and 100 Tris-HCl, pH 7.8). After the wash, samples were lysed in
a modified lysis solution containing 9.2 M urea, 2%
Nonidet P-40, 5%  -mercaptoethanol, and 2% carrier ampholytes
(1.6% pH 5-8, 0.4% pH 3.5-10) and stored frozen at 80°C.
Two-dimensional gel electrophoresis. Aliquots of the samples
from the treatment and control conditions were analyzed by a horizontal
system for two-dimensional gel electrophoresis (Pharmacia, Piscataway,
NJ) using a first-dimension isoelectric focusing (IEF) gel with an
immobilized pH gradient (4-7) and a precast SDS polyacrylamide (8-18% linear gradient) second-dimension gel (Görg et al.,
1988; Görg, 1991). For the first dimension, the IEF phase was
conducted for a total of 58.5 kVhr. The gels were then equilibrated for 20 min in a solution containing 2% SDS, 50 mM Tris, 6 M urea, 9% glycerol, and 65 mM DTT and placed
on precast SDS polyacrylamide (gradient) gels for the second dimension.
Electrophoresis in the second dimension was conducted for 1.65 kVhr.
Gels were stained and fixed in 0.1% Coomassie brilliant blue in 50%
trichloroacetic acid, destained in 7% acetic acid, and dried. Gel
staining verified that approximately equivalent amounts of protein were
applied to each gel. Autoradiographic exposures of the gels were
performed using storage phosphor screens for 24 hr. Phosphor screens
were computer scanned and analyzed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA) for quantitative analysis. All samples were
coded so that two-dimensional gel electrophoresis, autoradiographic exposures, phosphor screen scanning, and analysis were performed by
individuals not knowing the previous experimental treatments of the
samples, i.e., totally blind experimental procedure. Phosphoprotein molecular weights were estimated by comparison with prestained protein
standards (Bio-Rad, Hercules, CA).
ERK phosphorylation. ERK phosphorylation was measured in
preparations consisting of the eyes and proximal optic nerves after one-trial in vitro conditioning or in groups that received
10 or 15 conditioning trials. Control groups received an unpaired trial
of light (10 min) and 5-HT (in vitro procedure) or 10 or 15 pseudo-random presentations of the CS and US. The activation of ERK was
determined by immunoblotting using polyclonal antibodies raised against
the dually phosphorylated and active epitope in ERK1/p44 and ERK2/p42
(antiactive MAPK; Promega, Madison, WI). To verify that approximately
equal amounts of ERK were applied to each lane, identically prepared
membranes were probed for total ERK1 and ERK2 (Santa Cruz
Biotechnology, Santa Cruz, CA). The lysates from the different
treatments were resolved in SDS polyacrylamide gels and transferred to
polyvinylidene difluoride membranes. Membranes were blocked with 0.5%
gelatin in Tris-buffered saline and Tween (TBST), pH 7.5, 10 mM Tris buffer, 100 mM NaCl, and 0.1% Tween 20 or 5% dry milk in TBST for 1 hr at room temperature. The membranes were incubated in the primary antibody for 2 hr at room temperature, washed, and incubated in the secondary antibody (anti-rabbit IgG connected with HRP) for 1 hr. The inmunocomplexes were detected with
enhanced chemiluminescence reagent (Amersham, Arlington Heights, IL)
following the manufacturer's procedures. The relative intensity of the
immunoblotting on autoradiograms was quantified by using imaging
densitometry on the output of a digital video camera attached to a
microscope. Relative enzyme activation was determined by normalization
of the density of images from phosphorylated ERK with the total ERK
from parallel experiments from the same sample.
ERK assays. ERK activity was measured as described
previously (Neary and Zhu, 1994). In brief, after the one-trial
in vitro conditioning or unpaired control procedures,
preparations were lysed in a buffer containing 20 mM Tris,
pH 7.0, 0.27 M sucrose, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1 mM
dithiothreitol, 1 mM sodium orthovanadate, 10 mM glycerophosphate, 5 mM sodium
pyrophosphate, and 1% Triton X-100. The lysates were centrifuged in a
microfuge for 5 min at 4°C. Aliquots of the supernatants (20 µl)
were assayed at 30°C for 20 min in a final reaction solution
containing 10 µM ATP (0.2 µCi
[ 32P]ATP; New England Nuclear), 10 mM
MgCl2, 1 µM okadaic acid (LC Laboratories), and 0.33 mg/ml bovine brain myelin basic protein (Sigma,
St. Louis, MO) in a final volume of 40 µl. Under these conditions,
the reaction is linear with respect to time and enzyme concentration.
Reactions were stopped by pipetting 20 µl aliquots onto 1 × 2 cm strips of phosphocellulose paper and immediately placing the strips
in 75 mM phosphoric acid. Strips were washed for a minimum
of 2 hr and rinsed three times for 5 min each in 75 mM
phosphoric acid and once in ethanol. Strips were dried and transferred
to scintillation vials, and radioactivity was assessed by liquid
scintillation counting. ERK activity was expressed as picomoles of
32P transferred per minute per milligram of protein.
Protein concentrations were determined by the modified Lowry procedure
described by Peterson (1983) with bovine serum albumin as standard.
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RESULTS |
Protein phosphorylation
We examined changes in protein phosphorylation in the
photoreceptors after the in vitro one-trial conditioning
procedure. Previous studies have shown that conditioning results in the
increased phosphorylation of several phosphoproteins associated with
short- and long-term enhancement of excitability in lateral type B
photoreceptors (Crow et al., 1996). In the present study we examined
two additional phosphoproteins after one-trial conditioning with a
molecular mass of 44 and 42 kDa. An example of
32PO4 labeling of the 44 and 42 kDa
phosphoproteins after two-dimensional electrophoretic separation is
shown in Figure 1. The animals that received one trial of light paired with 5-HT (104
M) showed a greater level of 32P incorporation
into the two phosphoproteins compared with the controls that received
one trial of light (Fig. 1). The group data [mean ± SEM
experimental/control (E/C) ratio] shown in Figure 2 represents the quantitative analysis of
the 32P content of the 44 and 42 kDa phosphoproteins from
the conditioned group (n = 7) and light-only control
group (n = 7), as determined by densitometry. A
significant increase in protein phosphorylation relative to the
light-only control groups was detected for the 44 (mean = 2.4 ± 0.3; t(6) = 2.7; p < 0.03)
and 42 (mean = 1.8 ± 0.4; t(6) = 2.4;
p < 0.05) kDa phosphoproteins. These results show that
two phosphoproteins with molecular masses consistent with ERK1 and ERK2
show an increased phosphorylation for the conditioned groups relative
to the controls. In addition, the immunoblots of two-dimensional gels
probed with a phosphotyrosine antibody (PY 20; Transduction
Laboratories, Lexington, KY) revealed two spots that co-migrated with
the 44 and 42 kDa phosphoproteins as would be expected if these
proteins are ERK1 and ERK2 (data not shown).
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Figure 1.
Phosphate incorporation in 44 and 42 kDa proteins
from paired and control groups as analyzed by two-dimensional gel
electrophoresis. Laser prints of a storage phosphor screen scan showing
32PO4 labeling of phosphoproteins after
two-dimensional electrophoretic separation. The example shown on the
top is from a group that received one 5 min conditioning
trial of light paired with 5-HT (104
M). The control group shown on the bottom
received one 5 min trial of light. An increase in
32PO4 incorporation was detected for the
paired group compared with the controls.
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Figure 2.
Group data (n = 7) showing
mean ± SEM E/C ratios of densitometric measurements for the 44 and 42 kDa phosphoproteins after the one-trial conditioning procedure
(paired light and 5-HT, 5 min) and the light-only control (5 min).
One-trial conditioning resulted in a significant increase in
32PO4 incorporation for the 44 (mean = 2.4 ± 0.3; p < 0.03) and 42 (mean = 1.8 ± 0.4; p < 0.05) kDa phosphoproteins
compared with the control groups.
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The phosphorylation studies suggest that the ERK signaling pathway may
be involved in the cellular plasticity observed after one-trial
conditioning. To further investigate the potential role of ERK in
conditioning, we treated preparations with PD098059, A MEK-1
(ERK-activating kinase) inhibitor (Dudley et al., 1995). The
phosphorylation of four phosphoproteins from conditioned and control
animals was examined after 30 min pretreatment with PD098059 (50 µM). The group data (mean ± SEM E/C ratio) shown in
Figure 3 represents the densitometric
analysis of the four phosphoproteins after conditioning that were
pretreated with PD098059. Two of the phosphoproteins, 55 and 29 kDa,
have been shown to exhibit an increase in 32P incorporation
after the one-trial conditioning procedure (Crow et al., 1996). As
shown in Figure 3, pretreatment with PD098059 resulted in a significant
decrease in 32P incorporation for the conditioned groups
(n = 6) relative to the light-only control groups
(n = 6) for the 44 (mean = 0.59 ± 0.17;
t(5) = 2.4; p < 0.05) and 42 (mean = 0.34 ± 0.04; t(5) = 15.6;
p < 0.001) kDa phosphoproteins. In contrast to the
results for the 44 and 42 kDa phosphoproteins, the 55 and 29 kDa
phosphoproteins were not significantly affected by the 30 min
pretreatment with PD098059. However, both the 55 and 29 kDa
phosphoproteins were shown to exhibit increased 32P
incorporation with one-trial conditioning. These results show that an
inhibitor of the ERK activator blocks increased protein phosphorylation
of two phosphoproteins hypothesized to be ERK1 and ERK2. In addition,
these findings support the specificity of the actions of the inhibitor
because most phosphoproteins, including the 55 and 29 kDa
phosphoproteins, were not significantly affected by PD098059.
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Figure 3.
Group data (n = 6) showing
mean ± SEM E/C ratios of densitometric measurements for four
phosphoproteins examined after one-trial conditioning (paired light and
5-HT, 5 min) and one-trial conditioning for a group pretreated with the
MEK1 inhibitor PD098059 (50 µM). Treatment with PD098059
before one-trial conditioning produced a significant reduction in
32PO4 incorporation into the 44 (mean = 0.59 ± 0.17; p < 0.05) and 42 (mean = 0.34 ± 0.04; p < 0.001) kDa phosphoproteins
relative to the conditioned group not treated with PD098059. In
contrast, two other phosphoproteins (55 and 29 kDa) in which
phosphorylation has been shown to increase with one-trial conditioning
were not affected by pretreatment with PD098059.
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Assay of ERK activity in conditioned and unpaired controls
The results of the experiments that examined the effects of the
MEK1 inhibitor on protein phosphorylation suggest that the 44 and 42 kDa phosphoproteins may be ERK1 and ERK2, respectively. To provide
evidence for the activation of ERK after one-trial conditioning, we
assayed ERK activity. We also examined the issue of pairing specificity
of light and 5-HT by examining ERK activity in unpaired control groups.
The group data (mean ± SEM) shown in Figure
4 represent the ERK activity for the
group that received one trial of paired light and 5-HT
(n = 6) and a control group that received one unpaired
trial and light and 5-HT (n = 3). As shown in Figure 4,
the conditioning trial resulted in a significant increase in ERK
activity (mean = 130.7 ± 3) compared with the unpaired
control group (mean = 102.7 ± 6.7;
t(7) = 3.8; p < 0.001). The
results provide further evidence for the involvement of ERK in
one-trial conditioning.
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Figure 4.
Group data showing mean ± SEM ERK activity
measured as phosphorylation of an ERK substrate, brain myelin basic
protein. ERK activity for the paired group (mean = 130.7 ± 3; n = 6) that received one trial of light and 5-HT
was significantly greater than the unpaired group (mean = 102.7 ± 6.7; n = 3) that received one
unpaired trial of light and 5-HT. *p < 0.001.
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Western blot analysis of ERK
To provide additional evidence for the role of active ERK in
one-trial conditioning, we examined phosphorylation of ERK using antibodies recognizing the dually phosphorylated forms of ERK1 and ERK2
compared with antibodies recognizing total nonphosphorylated and
phosphorylated ERK1 and ERK2. Immunoblot analysis with the antibody
recognizing total ERK revealed a single band consistent with the
molecular weight of ERK. Moreover, a pretreatment control with an
ERK-blocking peptide (Santa Cruz Biotechnology) prevented antibody
staining on Western blots. This result supports the specificity of the
ERK signal observed on the immunoblots. As shown in Figure 5A, one conditioning trial of
light paired with 5-HT (104 M)
resulted in an increased amount of phosphorylated ERK compared with
controls that received one unpaired trial of light and 5-HT, although
the total ERK was equivalent in these two conditions (Fig.
5A). As shown in Figure 5B, samples pretreated
with PD098059 (50 µM) 30 min before the presentation of
the conditioning trial did not show the typical increased
phosphorylation of ERK as exhibited by the group that received one
trial of light and 5-HT (see Fig. 5B). The group data from
three independent experiments were consistent with the example shown in
Figure 5B. Pretreatment before conditioning with PD098059
resulted in a significant decrease in phospho-ERK relative to the
groups that received one conditioning trial (mean = 55;
t(2) = 3.7; p < 0.05). The
group data (mean ± SEM) shown in Figure 5C represents
the increased phosphorylation of ERK in the conditioned group relative
to the unpaired controls detected with the antibody that recognized the
dually phosphorylated forms of ERK1 and ERK2 (one-trial conditioning,
mean = 6.5 ± 2.6; unpaired controls, mean = 2.2 ± 0.5). The difference in phospho-ERK between the conditioned groups
(n = 4) and unpaired controls (n = 4)
was statistically significant (t(3) = 3.5;
p < 0.05). These findings are consistent with the
previous studies that showed that conditioning increases the
phosphorylation of the 44 and 42 kDa phosphoproteins (ERK1 and ERK2)
and that the increased phosphorylation is dependent on pairing light
with 5-HT.
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Figure 5.
Western blot analysis of phosphorylation and
activation of ERK. The preparations were processed for immunoblotting
using the chemiluminescence detection method with antibodies
recognizing nonphosphorylated and phosphorylated ERK1 and ERK2 or
antibodies recognizing the dually phosphorylated forms of ERK1 and ERK2
(Promega). The antibody used for the immunoblotting studies stained a
single band on immunoblots, consistent with the molecular weight of
ERK. A, After one 10 min trial of light paired with 5-HT
(104 M), an increased amount of
phosphorylated ERK was detected compared with unpaired controls that
received 10 min of unpaired light and 5-HT (see Materials and Methods).
Neither paired light and 5-HT nor unpaired light and 5-HT changed the
total amount of ERK detected with the antibody that recognized both
phosphorylated and nonphosphorylated ERK. B,
Pretreatment with PD098059 30 min before one-trial conditioning blocks
the phosphorylation of ERK detected after conditioning.
C, Group data showing mean ± SEM ratios of
phospho-ERK/ERK for the groups that received one 10 min trial of light
paired with 5-HT (n = 4) and the controls that
received one 10 min trial of unpaired light and 5-HT
(n = 4). One-trial conditioning resulted in a
significant increase in ERK phosphorylation. *p < 0.05.
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The analysis of one-trial in vitro conditioning indicates
that ERK is phosphorylated and activated in groups receiving the conditioning trial of light and 5-HT compared with unpaired controls. However, the potential relationship between ERK activity and behavior has not been established with the in vitro procedure. To
address this issue we conditioned animals and established that
conditioning produced behavioral phototactic suppression relative to
pseudo-random controls that received the same number of explicitly
unpaired CS and US presentations. The mean suppression ratios for the
conditioned group (n = 17) and pseudo-random controls
(n = 15) tested 10 min after 10 or 15 conditioning
trials is shown in Figure 6. The
behavioral analysis revealed that there were no significant differences
between the groups receiving 10 trials or 15 trials, so the two groups were combined for the final analysis. As shown in Figure 6,
conditioning produced a significant suppression of phototactic behavior
(mean = 0.18 ± 0.04) compared with the pseudo-random
controls (mean = 0.36 ± 0.06; t(30) = 2.5; p < 0.01). After conditioning and behavioral
testing, preparations were prepared for analysis of phosphorylated ERK.
Examples of the Western blot analysis of ERK in the eyes and proximal
optic nerve preparations from the conditioned and pseudo-random
controls is shown in Figure
7A. As shown in the Western
blots of Figure 7A, phospho-ERK was greater for the conditioned group compared with the pseudo-random controls. However, neither procedure changed the total amount of ERK detected with the
antibody that recognized both phosphorylated and nonphosphorylated ERK
(Fig. 7A). The statistical analysis of the group data shown in Figure 7B, consisting of ratios of phospho-ERK/ERK from
conditioned preparations (mean = 1.6 ± 0.15;
n = 5) and pseudo-random controls (mean = 1.3 ± 0.10; n = 5), revealed that conditioning resulted in
a significant increase in phosphorylated ERK
(t(4) = 3.4; p < 0.02) (Fig.
7B). These results provide evidence that behavioral conditioning results in a significant increase in the phosphorylation of ERK that can be detected shortly after behavioral testing.
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Figure 6.
Group data depicting the mean ± SEM
suppression ratios for the conditioned group (n = 17) and the pseudo-random controls (n = 15).
Conditioning produced a significant behavioral suppression in response
to the CS after 10 or 15 conditioning trials. *p < 0.01.
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Figure 7.
Western blot analysis of phosphorylation of ERK
after 10 or 15 conditioning trials compared with pseudo-random
controls. A, After 10 conditioning trials the amount of
phosphorylated ERK was greater than the pseudo-random control group.
Neither conditioning nor the control procedure changed the total amount
of ERK detected with the antibody that recognized both phosphorylated
and nonphosphorylated ERK. B, Group data showing the
mean ± SEM ratio of phospho-ERK/ERK for the conditioned group
(n = 5) and the pseudo-random controls
(n = 5). The conditioned group (mean = 1.6 ± 0.15) showed a significant increase in phosphorylated ERK
compared with the controls (mean = 1.3 ± 0.10).
*p < 0.02.
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DISCUSSION |
The major findings from our studies are that a one-trial in
vitro conditioning procedure results in the increased
phosphorylation of ERK, as detected by 32PO4
labeling and antibodies recognizing the dually phosphorylated forms of
ERK1 and ERK2 and an increase in ERK activity. In addition, inhibition
of the ERK activator MEK1 blocks increased phosphorylation of ERK1 and
ERK2, as detected by 32PO4 labeling and
antibodies that recognize phospho-ERK1 and -ERK2. Finally, we have
provided evidence that classical conditioning that produces significant
behavioral suppression results in a significant increase in ERK
phosphorylation compared with pseudo-random controls.
Signal transduction in the CS pathway
Studies of the nervous system in general and specifically the CS
pathway in conditioned Hermissenda have documented the role of PKC in enhancement of synaptic efficacy, cellular excitability, and
classical conditioning (Farley and Auerbach, 1986; Neary et al., 1986;
Alkon et al., 1988; Matzel et al., 1990; Crow et al., 1991; Crow and
Forrester, 1993; Schuman and Clark, 1994; Frysztak and Crow, 1997). In
addition, PKC plays a role in several examples of cellular and synaptic
plasticity in both vertebrates and invertebrates (Akers et al., 1986;
Malinow et al., 1989; Braha et al., 1990; Sacktor and Schwartz, 1990;
Sugita et al., 1992). In Hermissenda, PKC inhibitors have
been shown to block the induction of short-term plasticity (Matzel et
al., 1990; Crow et al., 1991; Crow and Forrester, 1993) and reverse the
expression of established plasticity in conditioned animals (Farley and
Schuman, 1991; Frysztak and Crow, 1997). Moreover, downregulation of
PKC has also been shown to be effective in blocking the induction of
short-term plasticity (Crow et al., 1991; Crow and Forrester, 1993).
However, long-term enhancement produced by one-trial conditioning does
not depend on the activation of PKC (see next section).
Independence of short- and long-term plasticity
Recent studies of Hermissenda have shown that short-
and long-term enhancement may be independent parallel processes,
because they can be dissociated based on the role of mRNA synthesis,
protein synthesis, and the contribution of protein kinase C (Crow and Forrester, 1990, 1991, 1993; Crow et al., 1997). Downregulation of PKC
and preconditioning treatment with kinase inhibitors block the
induction of short-term enhancement, although long-term enhancement can
still be expressed (Crow and Forrester, 1993). This finding suggests
that additional signaling pathways are engaged to support the
consolidation of long-term memory in Hermissenda. ERK, a
member of the mitogen-activated protein kinase family, is an attractive candidate for contributing to long-term enhancement. The ERK cascades are highly conserved, being found in all eukaryotic organisms (Robinson
and Cobb, 1997). MAPK cascades are a series of cytosolic enzymes that
can transmit extracellular signals to the nucleus (for review, see
Neary, 1997). These cascades consist of at least three protein kinases
that are activated sequentially: a MAPK kinase kinase such as Raf-1
activates a MAPK kinase such as MEK1, which in turn activates a MAPK
such as ERK. The activated ERK can translocate to the nucleus (Chen et
al., 1992; Lenormand et al., 1993), where it can phosphorylate or
induce transcription factors leading to the activation of genes and the
expression of proteins needed for differentiation or proliferation. In
addition to nuclear targets, ERK can phosphorylate membrane,
cytoskeletal and cytoplasmic proteins and thus may be involved in other
cellular functions (Minshull et al., 1994), including the regulation of ion channels (Adams et al., 1997). Growth factors activate the ERK
cascade by means of a well established sequence of receptor autophosphorylation and protein-protein interactions, leading to
stimulation of Raf-1. G-protein-coupled receptors (GPCRs), such as
serotonin receptors (Hershenson et al., 1995; Launay et al., 1996), can
also activate the ERK cascade. Soluble protein kinases such as Src
(Chen et al., 1994; Luttrell et al., 1996), PKC (Wang et al., 1992; van
Biesen et al., 1996), and Pyk2 (Lev et al., 1995) often link GPCRs to
the ERK cascade. PKC (Nishizuka, 1995) and Pyk2 (Lev et al., 1995) can
be activated by increases in intracellular calcium, thereby linking
calcium-dependent signaling pathways to the ERK cascade. In this
manner, activation of ERK by these protein kinases may play a role in
short- and/or long-term calcium-dependent functions in the CNS.
Interestingly, ERK has recently been shown to be involved in LTP
(English and Sweatt, 1996) and long-term facilitation of cultural
Aplysia neurons (Martin et al., 1997). It was recently
reported that procedures that produce long-term facilitation result in
the translocation of MAPK in the presynaptic, but not postsynaptic
neurons in Aplysia sensorimotor co-cultures. In addition,
MAPK is required for long-term but not short-term facilitation (Martin
et al., 1997). In hippocampal slices the p42 MAPK isoform, but not the
p44 MAPK isoform, is activated in CA1 after LTP induction. The ERK
signaling cascade is attractive in Hermissenda plasticity
because upstream components may involve the activation of PKC and a MEK
activator such as Raf. This would provide for the convergence point
within the signaling pathway to support input from the CS and US
pathways. Alternatively, the PKC and ERK cascade may represent two
independent parallel pathways that support short- and long-term memory
for Pavlovian conditioning. Our findings indicate that ERK is
phosphorylated and active soon after one-trial and multi-trial
conditioning. However, it is not known in Hermissenda, or in
other species, that ERK phosphorylation is uniquely responsible for
long-term memory.
Multi-trial Pavlovian conditioning
The one-trial conditioning procedure was shown to result in
long-term (24 hr) behavioral suppression, although tests of behavioral changes immediately after the conditioning trial could not be performed
(Crow and Forrester, 1986). It was shown previously that 10 conditioning trials did not result in pairing specific behavioral
suppression when animals were tested 1 or 30 min after conditioning
(Crow, 1983). In the present study, the CS duration and
inter-trial-interval were modified from the procedures used in the
earlier study to produce the most efficacious conditions for the
expression of Pavlovian conditioning after a few conditioning trials.
Excitability changes and Pavlovian conditioning
Studies of cellular correlates of Pavlovian conditioning in
Hermissenda have identified both changes in synaptic
efficacy (Frysztak and Crow, 1994, 1997) and changes in excitability of cells in the CS pathway (Crow and Alkon, 1980; Crow, 1985; Frysztak and
Crow, 1993, 1997; Farley and Han, 1997). Indeed, conditioned enhancement of excitability appears to be a general characteristic of
both vertebrate and invertebrate Pavlovian conditioning. Excitability changes in hippocampal pyramidal neurons have been detected in vitro after Pavlovian conditioning (Moyer et al., 1996; Thompson et al., 1996). The cellular correlates of short- and long-term enhancement after one-trial conditioning of Hermissenda
involve changes in excitability. It is interesting to note that the
Kv4.2 channel, a voltage-activated potassium channel localized to
dendrites in the hippocampus, is a substrate for MAPK phosphorylation
(Adams et al., 1997). The reduction in the conductance of an A-type
K+ channel in Hermissenda has been
proposed as a mechanism of Pavlovian conditioning (Alkon et al., 1982).
It is therefore intriguing that activation of the ERK cascade could
potentially be regulating diverse K+ conductances
that have profound effects on cellular excitability.
| |
FOOTNOTES |
Received Dec. 31, 1997; revised Feb. 20, 1998; accepted Feb. 23, 1998.
This work was supported by National Institute of Mental Health Grants
MH40860 and MH01363 to T.C.
Correspondence should be addressed to Terry Crow, Department of
Neurobiology and Anatomy, University of Texas Medical School, P.O. Box
20708, Houston, TX 77225.
| |
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Top
Abstract
Introduction
