Department of Neurobiology and Anatomy, University of Texas Medical School, Houston, Texas 77225
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ABSTRACT |
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Crow, Terry,
Juan-Juan Xue-Bian, and
Vilma Siddiqi.
Protein Synthesis-Dependent and mRNA Synthesis-Independent
Intermediate Phase of Memory in Hermissenda.
J. Neurophysiol. 82: 495-500, 1999.
The conditioned stimulus pathway in Hermissenda has been
used to examine the time-dependent mechanisms of memory consolidation following one-trial conditioning. Here we report an intermediate phase
of memory consolidation following one-trial conditioning that requires
protein synthesis, but not mRNA synthesis. In conditioned animals,
enhanced excitability normally expressed during an intermediate phase
of memory was reversed by the protein synthesis inhibitor anisomycin,
but not by the mRNA synthesis inhibitor
5,6-dichloro-1--D-ribobenzimidazole (DRB). Associated
with the intermediate phase of memory is an increase in the
phosphorylation of a 24-kDa protein. Anisomycin present during the
intermediate phase blocked the increased phosphorylation of the 24-kDa
phosphoprotein, but did not block the increased phosphorylation of
other proteins associated with conditioning or significantly change
their baseline phosphorylation. DRB did not reverse enhanced
excitability or decrease protein phosphorylation expressed during the
intermediate phase of memory formation, but it did reverse enhanced
excitability 3.5 h after conditioning. Phosphorylation of the
24-kDa protein may support enhanced excitability during the
intermediate phase, in the transition period between short- and
long-term memory.
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INTRODUCTION |
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Studies of the time-dependent development of
cellular and synaptic plasticity have identified multiple phases in the
formation of memory (DeZazzo and Tully 1995;
Freeman et al. 1995
; McGaugh 1966
, 1968
; Ng and Gibbs
1991
; Rosenzweig 1993
). The components of memory
consolidation can be differentiated based on the contribution of signal
transduction pathways, protein synthesis, and gene induction (Kane et al. 1997
; Nguyen et al. 1994
;
Otani et al. 1989
). Examples of plasticity have been
reported that exhibited an immediate (Kang and Schuman
1996
) or intermediate requirement for protein synthesis (Ghirardi et al. 1995
); however, specific proteins have
not been identified. Short- and long-term memory in the conditioned
stimulus (CS) pathway of conditioned Hermissenda can be
dissociated based on the contribution of mRNA synthesis, protein
synthesis, and protein kinases (Crow and Forrester 1990
,
1993
; Crow et al. 1997
, 1998
; Farley and Schuman 1991
;
Matzel et al. 1990
; Ramirez et al. 1998
).
One-trial conditioning of Hermissenda (Crow and
Forrester 1986
) results in the biphasic development of enhanced
cellular excitability detected in the lateral type-B photoreceptors.
Enhanced excitability reaches asymptotic levels 3 h
postconditioning, followed by a decrease in excitability at 5-6 h
postconditioning, which leads to a second phase of enhanced
excitability at 16-24 h (Crow and Siddiqi 1997
). The
short-term phase of enhancement (
1 h) has been shown to be
independent of protein and mRNA synthesis, whereas the long-term phase
is blocked by both mRNA and protein synthesis inhibitors (Crow
and Forrester 1990
; Crow et al. 1997
). In the
present study, a requirement for protein synthesis, but not mRNA
synthesis during the intermediate phase of memory was observed between
1.5 and 2.5 h postconditioning.
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METHODS |
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Experimental procedures
Three types of preparations were used; an exposed, but otherwise
intact nervous system, an isolated intact nervous system, and isolated
components of the CS pathway consisting of the eye and proximal optic
nerve. Conditioning was conducted with exposed nervous systems followed
by electrophysiological studies of isolated nervous systems at
different times postconditioning. Adult Hermissenda crassicornis were maintained in artificial sea water (ASW) aquaria at 14 ± 1°C on a 12-h light/dark cycle. Before
conditioning, animals were anesthetized with a 0.25-ml injection of
isotonic MgCl2, and a small dorsolateral incision
was made to expose the circumesophageal nervous system. Surgically
prepared animals were transferred to a chamber containing 50 ml of
normal ASW or 50 ml of ASW containing 104 M
5,6-dichloro-1-
-D-ribobenzimidazole (DRB),
10
7 M DRB, or 10
5 M
anisomycin. The one-trial conditioning procedure consisted of a 5-min
presentation of light, the CS (10
4
W/cm2) paired with the application of serotonin
(5-HT) to the region of the cerebropleural ganglion where previous
immunocytochemistry revealed 5-HT reactive processes near the optic
nerve and photoreceptor terminals in the neuropil (Land and Crow
1985
). The final concentration of 5-HT in the ASW was 0.1 mM.
Animals were placed in a chamber with normal ASW or ASW containing the
different inhibitors 15 min before presenting the conditioning trial.
The conditioning trial was presented in the presence of DRB,
anisomycin, or normal ASW. Unpaired control groups received the CS and
5-HT separated by 5 min. For the unpaired control group, the 5-HT was
applied in the dark (infrared illumination) and washed out after the
5-min exposure. Following the conditioning trial, animals were
maintained in ASW containing the different inhibitors before assessing excitability.
Electrophysiology
Intracellular recordings were collected from lateral type B
photoreceptors at times between 0.5 and 3.5 h after the
conditioning trial. A total of 175 animals from the experimental and
control groups were prepared for intracellular recording and
stimulation with extrinsic current using previously published standard
procedures (Crow and Forrester 1990,
1993
; Crow et al. 1997
). Experiments with
the isolated circumesophageal nervous system were conducted in ASW
maintained at 15 ± 0.5°C and had the following composition (in
mM): 460 NaCl, 10 KCl, 10 CaCl2, and 55 MgCl2, buffered with 10 mM HEPES and brought to
pH 7.6 with NaOH. Excitability was assessed with 30-s, 20-mV
depolarizing current steps and in a second group of animals at 1.5 and
3 h postconditioning using 2-s depolarizing pulses at three
current levels: 0.1, 0.15, and 0.2 nA. Averages of spike frequency were
determined by subtracting spontaneous baseline activity from the total
number of action potentials elicited by the 30-s extrinsic current and
dividing by the duration of the current pulse. Membrane potential was
not controlled in experiments examining excitability with 20-mV
depolarizing steps. In experiments using 2-s pulses, spikes were
elicited by the three current pulses from a membrane potential of
60 mV.
Protein phosphorylation and two-dimensional gel electrophoresis
Protein phosphorylation in normal controls and conditioned
groups was examined in components of the CS pathway of preparations exposed to anisomycin or DRB during a 2-h incubation in 0.125 mCi of
32PO4. To minimize
potential animal-to-animal variability in
32PO4 uptake, the eyes and
proximal optic nerves from three animals were used for each treatment
and control procedure in each experimental replication. After the 2-h
incubation the samples were rinsed in an isotonic ice-cold wash
solution (in mM: 460 NaCl, 10 KCl, 5 EDTA, and 100 Tris-HCl, pH 7.8)
and 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), 100 mM NaF, 1 mM sodium
orthovandate, 0.1 mM okadiac acid and stored frozen at
80°C.
Samples were analyzed by two-dimensional gel electrophoresis 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. Gels were exposed to storage phosphor
screens for a period of 24 h. Phosphor screens were computer
scanned and analyzed using ImageQuant software (Molecular Dynamics,
Sunnyvale, CA) for quantitative analysis.
Statistical analysis
A two-way analysis of variance (ANOVA) was used to assess the main effects of the treatments on excitability and membrane potential, and a one-way ANOVA assessed the effects of inhibitors on protein phosphorylation. After the determination of overall significant effects, paired comparisons consisted of Tukey tests. Comparisons of the differences in the ratios between experimental treatments and control gels involved t-tests for correlated means.
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RESULTS |
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Exposure before and after one-trial conditioning to the
translational inhibitor anisomycin (105 M) or
the transcriptional inhibitor DRB (10
4 M and
10
7 M) produced time-dependent differential
effects on enhanced excitability as shown in Fig.
1A. Significant overall
differences between the treatment groups and the controls were detected
by the results of the ANOVA (F4,96 = 63.6; P < 0.001). The main effect of time after
conditioning was significant (F4,96 = 6.5; P < 0.001), and the interaction between
treatments and time after conditioning was also significant
(F16,96 = 1.9; P < 0.05). At 0.5 and 1 h postconditioning, none of the treatment
groups were significantly different from each other
(F3,26 = 0.3; NS; Fig. 1A).
However, the groups conditioned and maintained postconditioning in
anisomycin showed a significant reduction in excitability relative to
the groups conditioned and maintained in normal ASW at 1.5 h
(q = 4.9), 2 h (q = 4.2), 2.5 h (q = 7.9), 3 h (q = 6.7), and
3.5 h (q = 7.4) postconditioning
(P < 0.05 for each comparison). The anisomycin groups
were significantly different from the DRB groups (10
4) at 1.5 h (q = 5.6), 2 h (q = 5.7), and 2.5 h
(q = 4.1; P < 0.05). The anisomycin
groups were not significantly different from the unpaired controls at
1.5 h (q = 3.3), 2 h (q = 2.8), 2.5 h (q = 1.3), 3 h (q = 2.2), and 3.5 h (q = 0.12). In contrast,
excitability measures from the groups conditioned and maintained in DRB
(10
4 M) were not significantly reduced relative
to the groups conditioned in normal ASW or the groups conditioned in
DRB (10
7 M) when examined at 1.5 and 2 h
postconditioning (q = 0.02, 0.6; q = 1.1, 0.5). However, inhibition of mRNA synthesis with DRB (10
4 M) reduced enhanced excitability measured
2.5, 3, and 3.5 h postconditioning as compared with the
conditioned group (q = 4.03; q = 7.9;
q = 5.2; P < 0.05 for all
comparisons). The groups conditioned in 10
7 M
DRB, which does not significantly inhibit mRNA synthesis (Crow et al. 1997
), were not significantly different from the groups conditioned in normal ASW at any time period (Fig. 1A). The
unpaired control group was not significantly different from the DRB
(10
4 M) group at 3.5 h (q = 3.05). The analysis of dark-adapted membrane potential for the
experimental and control groups did not reveal significant overall
differences (F4,96 = 1.55; NS). We
further examined enhanced excitability 1.5 h postconditioning at
levels of depolarization <20 mV for the different experimental groups and the unpaired control group by measuring spike activity elicited by
three levels of depolarizing extrinsic current (0.1, 0.15, 0.2 nA). As
shown in Fig. 1Bb, at 1.5 h postconditioning, fewer spikes were elicited by the 0.2-nA current pulse for the anisomycin preparation as compared with the example from a conditioned group (Fig.
1Ba) or one from the group conditioned in
10
4 M DRB (Fig. 1Bc). The example
from the unpaired control group (Fig. 1Bd) is similar to the
anisomycin group and different from the conditioned group and
conditioned DRB group. However, at 3 h postconditioning, the
example from the group conditioned in DRB (10
4
M) showed fewer elicited spikes (Fig. 1Be) as compared with
excitability assessed at 1.5 h for the DRB
(10
4 M) preparation. The group data for
excitability changes elicited by the three different current levels and
four different treatments is shown in Fig. 1C. The results
of the ANOVA revealed a significant overall effect of treatments
(F3,16= 31.9; P < 0.01) and current levels (F2,32 = 44.9; P > 0.01). The group conditioned and maintained in anisomycin (n = 5) and the unpaired control group
(n = 5) showed a reduction in excitability at all three
current levels as compared with the conditioned ASW (n = 5) and DRB groups (n = 5) (q = 7.8; P < 0.05). The unpaired control group was not
significantly different from the anisomycin group. Overall, these
results show that at an intermediate time in memory consolidation
(1.5-2.5 h) protein synthesis, but not mRNA synthesis is necessary to
support excitability changes produced by one-trial conditioning.
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We have previously shown that one-trial conditioning results in the
phosphorylation of proteins that can be detected several hours after
one-trial conditioning (Crow et al. 1996). To determine whether posttranslational modifications of proteins may be associated with the intermediate phase of memory, we examined changes in protein
phosphorylation postconditioning. Phosphate incorporation into proteins
from conditioned and unpaired controls was analyzed after separation by
2-D PAGE. We found an increase in phosphorylation of a 24-kDa
phosphoprotein detected 2 h after one-trial conditioning that was
not previously reported in our earlier study (Crow et al.
1996
). Phosphate incorporation in the 24-kDa protein from a
conditioned preparation (Fig.
2A) was increased as compared with an unpaired control (Fig. 2B). The analysis of the
group data showed that phosphorylation was significantly greater for the conditioned group (n = 12) as compared with the
unpaired control group (n = 12;
t11 = 2.0; P < 0.05;
Fig. 2C. To determine whether the increased
32PO4 labeling may reflect
an increase in protein synthesis, we conducted control experiments
using 35S-methionine labeling for 2-h
postconditioning. The 35S-methionine labeling of
the 24-kDa protein for the conditioned and unpaired controls was
similar (t5 = 0.57; NS,
n = 6). These results indicate that the increased
32PO4 labeling of the
24-kDa protein for the conditioned group does not reflect an increase
in protein synthesis. In addition, there was a significant difference
between conditioned and unpaired controls in the phosphorylation of the
55 kDa (t11 = 3.92; P < 0.01), 46 kDa (t8 = 4.35;
P < 0.01), and 29 kDa
(t11 = 4.42; P < 0.01) phosphoproteins that had been shown previously to increase with
conditioning and 5-HT application (Crow et al. 1996
).
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We next examined the effect of anisomycin on protein phosphorylation
during the intermediate phase of enhanced excitability following
conditioning. Incubation of components of the isolated CS pathway in
anisomycin (2 h) resulted in significant overall differences in
32PO4 incorporation into
different phosphoproteins (F3,20 = 3.2; P < 0.05). Anisomycin produced a significant
reduction in 32PO4
incorporation into the 24-kDa protein (n = 6;
t5 = 3.9; P < 0.01;
Fig. 3, B and C),
whereas phosphorylation of three other proteins (55, 46, and 29 kDa)
shown previously to exhibit increased 32PO4 incorporation after
one-trial conditioning or 5-HT application were not significantly
affected (Fig. 3C). Moreover, the same anisomycin treatment
(2 h) did not produce a significant decrease in the baseline
phosphorylation of the 55-, 46-, 29-, or 24-kDa proteins in normal
untrained preparations (F3,8 = 0.28;
NS). These results show that the effect of anisomycin during the
intermediate phase of memory is specific to the 24-kDa phosphoprotein,
and on the enhanced phosphorylation of the 24-kDa observed after
one-trial conditioning. As a further control, we examined the effect of a 2-h exposure to DRB on protein phosphorylation because this period of
postconditioning incubation did not reduce enhanced excitability when
tested 2 h after one-trial conditioning (Fig. 1). The incubation
of preparations for 2 h in DRB (104 M) did
not change baseline phosphorylation of the 55-, 46-, 29-, or 24-kDa
phosphoproteins in a normal control group
(F3,8 = 0.13; NS), or significantly
effect 32PO4 incorporation
into the 55-, 46-, 29-, or 24-kDa phosphoproteins of conditioned
animals (F3,12 = 1.1; NS).
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DISCUSSION |
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We have shown that anisomycin and DRB effect enhanced
excitability at different times following one-trial conditioning. The results showing that anisomycin, but not DRB, significantly reduces phosphorylation of a 24-kDa protein 2 h postconditioning provides additional evidence that protein synthesis is required in the intermediate phase of memory consolidation. Our finding that the intermediate phase of memory consolidation does not require mRNA synthesis suggests that activation of translation may occur without the
activation of transcription. The requirement for protein synthesis in
the intermediate phase of memory consolidation may depend on the
learning paradigm, species, and the neural systems responsible for the
maintenance and expression of memory. Increased excitability of
Aplysia sensory neurons produced by protein kinase C
activation measured 3 h after treatment was reported to be
independent of protein synthesis (Manseau et al. 1998).
In addition, there are examples from other species where an
intermediate phase of memory may not be dependent on protein synthesis
(DeZazzo and Tully 1995
). However, an intermediate phase
of synaptic facilitation in Aplysia sensory neurons produced
by exposure to 5-HT has been shown to be protein synthesis dependent
and mRNA synthesis independent (Ghirardi et al. 1995
).
Moreover, there are examples of synaptic plasticity that have an
immediate requirement for protein synthesis (Kang and Schuman
1996
).
The translational dependent intermediate phase does not appear
only to involve an increase in the synthesis of the 24-kDa protein
based on the results of the 35S-methionine
labeling study. Therefore the requirement for protein synthesis may be
necessary but not sufficient, or may reflect an indirect involvement in
phosphorylation such that the change in the phosphorylation of the
24-kDa protein produced by conditioning could be due to changes in the
synthesis of protein kinases in the signal transduction pathways or
phosphatase inhibitors. One protein that has been previously identified
in Hermissenda is calexcitin/cp20, a low molecular weight
GTP- and Ca2+-binding protein (Ascoli et
al. 1997) that is phosphorylated in conditioned
Hermissenda (Neary et al. 1981
). However, the
evidence indicates that the 24-kDa phosphoprotein identified in this
study is not calexcitin. The partial sequence of the 24-kDa protein produced peptides that were similar to the
-thymosin family of actin-binding protein and did not show a sequence homology to calexcitin/cp20 based on a search of the National Center for
Biotechnology Information protein databases that included
calexcitin (unpublished observations). All known vertebrate
and invertebrate
-thymosins bind actin monomers (Nachmias
1993
). Our results would thus support a potential role for
-thymosin in cellular plasticity by modifications in actin
assembly/diassembly in neurons of the CS pathway of conditioned animals.
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ACKNOWLEDGMENTS |
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We thank P. Dash for discussions and D. Parker for typing the manuscript.
This work was supported by National Institute of Mental Health Grants MH-40860 and MH-01363 to T. Crow.
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FOOTNOTES |
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Address for reprint requests: T. Crow, Dept. of Neurobiology and Anatomy, University of Texas Medical School, P.O. Box 20708, Houston, TX 77225.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 20 November 1998; accepted in final form 5 March 1999.
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REFERENCES |
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