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Sussex Centre for Neuroscience, School of Life Sciences, University of Sussex, Falmer, Brighton, BN1 9QG, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT ResultsDiscussionMaterials and MethodsREFERENCES |
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). One of
these signaling cascades is the mitogen-activated protein kinase (MAPK)
signaling pathway, which has been implicated in the consolidation of LTM in a
variety of learning paradigms in vertebrate and invertebrate systems (see
Martin et al. 1997;
Atkins et al. 1998;
Berman et al. 1998;
Crow et al. 1998;
Selcher et al. 1999;
Cammarota et al. 2000;
Schafe et al. 2000;
Kelly et al. 2003;
Sharma et al. 2003). Although
MAPK appears to play a major role in memory formation in several types of
aversive classical conditioning paradigms, a role for MAPK in LTM
consolidation after appetitive classical conditioning has yet to be
established.
The well-described chemical conditioning of the feeding behavior of the
pond snail Lymnaea stagnalis enabled us to study the role of MAPK in
reward conditioning. Lymnaea can be reliably conditioned by a single
pairing of a neutral chemical, amyl acetate (the conditioned stimulus [CS])
with a strong feeding stimulant, sucrose (the unconditioned stimulus [US])
(Alexander Jr. et al. 1984). In
this paradigm, a single training trial induces the consolidation of a protein
synthesis-dependent form of memory that can last for up to 21 d
(Alexander Jr. et al. 1984;
Fulton et al. 2005). The
ability to study LTM formation by using a one trial learning paradigm
simplifies the analysis of the temporal cascade of molecular events induced by
conditioning. In addition, key molecular mechanisms of memory consolidation
are conserved in the snail. For example, the transcription factors CREB and
C/EBP that play an important role in transcription-dependent memory
consolidation in other systems have been implicated in associative
conditioning in Lymnaea (Ribeiro
et al. 2003; Hatakeyama et al.
2004; Sadamoto et al.
2004). Another example is the nitric oxide-cGMP signaling pathway.
This pathway has been implicated in learning and memory in a variety of
learning paradigms in other species
(Susswein et al. 2004) and
also plays an essential role in reward learning in Lymnaea
(Kemenes et al. 2002).
We show in this report that MAPK proteins can be detected in the
Lymnaea central nervous system (CNS), and that MAPK activation by
phosphorylation is necessary for food-reward classical conditioning. However,
activation of the MAPK pathway was not restricted to snails exposed to a CS
and US pairing but also occurred when the CS or the US was applied alone. In
all three groups, MAPK activation was found in central ganglia containing the
main interneuronal and motor circuitry for feeding
(Benjamin et al. 2000) and in
lip tissue containing primary chemosensory neurons
(Straub et al. 2004). These
results show that sensory stimulation, as well as reward classical
conditioning, can cause changes in levels of phosphorylated MAPK and that
learning may involve both central and peripheral activation of the MAPK
signaling pathway.
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Results |
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TOPABSTRACTResultsDiscussionMaterials and MethodsREFERENCES |
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We used commercially available anti-pMAPK and anti-MAPK antibodies raised
against mammalian p44 and p42 MAPK proteins for detection of MAPK-like
proteins in the Lymnaea nervous system. The anti-pMAPK antibody
recognizes only dually phosphorylated (activated) MAPK (pMAPK), while the
anti-MAPK antibody recognizes MAPK proteins independent of their
phosphorylation state (total MAPK). These antibodies have been used
successfully to detect MAPK-like proteins in hemocyte protein extracts from
Lymnaea (Plows et al.
2004). Consistent with the results from hemocyte extracts,
although both antibodies detected two bands running very close together at
43 kDa in brain protein extracts, in most experiments the two bands could
not be separated and migrated as a single band
(Fig. 1A). We are confident
that these antibodies are recognizing the Lymnaea homologs of MAPK
because an antibody raised against Aplysia MAPK
(Michael et al. 1998) also
recognized the same band in Western blots of Lymnaea extracts
(Fig. 1A).
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). Importantly, the cerebral giant cells, two symmetrical
neurons that play an essential role in feeding modulation (Yeoman et al.
1994a,b)
and whose gene expression is altered following food-reward conditioning
(Korneev et al. 2005), showed
pMAPK immunoreactivity (Fig.
1D).
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To confirm that Lymnaea is capable of associative memory consolidation after single-trial food-reward conditioning, we trained two groups of snails: a CS+US group presented with a paired presentation of amyl acetate (CS) and sucrose (US), and an unpaired group presented with the two stimuli separated by a time interval of 1 h (Fig. 2A). The feeding response of the snails (number of feeding cycles in 2 min) induced by amyl acetate was measured during training before exposure to sucrose and 1 d after training to test for LTM formation. During training, the responses of both groups were low and not significantly different (P = 0.2), whereas 1 d after training the response of the CS+US group was significantly higher than was the response of the unpaired group (P < 0.001), indicating that associative learning had occurred (Fig. 2B).
To investigate if MAPK phosphorylation is necessary for associative memory
formation in Lymnaea, we used U0126, a specific inhibitor of MAPK
kinase (Favata et al. 1998),
to examine whether it could block the formation of the behavioral memory
trace. Either vehicle or U0126 was injected into the body cavities of two
groups of snails, 30 min before conditioning
(Fig. 3). When tested 1 d after
conditioning, the group injected with U0126 responded to amyl acetate with a
significantly lower number of feeding cycles than did the vehicle-injected
group (P < 0.001) (Fig.
3). The response to amyl acetate of the vehicle-injected group
after conditioning was also significantly higher than was its response before
conditioning (P < 0.001), indicating that learning took place. In
contrast, the response to amyl acetate of the U0126-injected group after
conditioning was not significantly different from its response before
conditioning (P = 0.1), indicating that LTM formation was totally
blocked by U0126 injection. This blocking of the memory formation does not
appear to be due to the impairment of sensory or motor capabilities of the
animals because the locomotor behavior of the snails after injection appeared
normal and the feeding responses of vehicle- and UO126-injected groups during
training were not significantly different at any point during the three-stage
training procedure (water, P = 0.2; amyl acetate, P = 0.3;
amyl acetate + sucrose, P = 0.2)
(Fig. 3). However, this result
is ambiguous for amyl acetate because during training the feeding behavior of
both groups in the presence of amyl acetate was very low and not significantly
different from the behavior following water presentation (vehicle, P
= 0.2; U0126, P = 0.1). To rule out the possibility that amyl acetate
sensory responses were blocked during the CS+US pairing and/or during the
testing of the conditioned response, two further experiments were carried out.
In both experiments UO126 was injected 1 d after conditioning. In the first
experiment, the snails were tested 30 min after injection
(Fig. 4A). During testing, both
groups responded to amyl acetate with normal levels of conditioned response
(Fig. 4A). No significant
difference was found among the groups, showing that the sensory perception of
amyl acetate was not impaired 30 min after U0126 injection. In the second
experiment, the snails were tested for amyl acetate 1 d after injection, 2 d
after training. Again, no significant differences were found between the
groups (Fig. 4B), indicating
that 1 d after U0126 injection the snails are able to perceive amyl acetate
and respond to it normally with an increase in feeding rate. Taken together,
these results strongly suggest that U0126 specifically impairs memory
formation in Lymnaea.
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Injecting UO126 1 d after training has no effect on memory formation (Fig. 4B) unlike the injection 30 min before training (Fig. 3). This indicates that MAPK activation is required for LTM only in the early stages of memory formation either for acquisition or consolidation of the memory trace. A role in consolidation was supported by the experiment shown in Figure 5, where preventing MAPK phosphorylation immediately after CS+US pairing blocked memory formation. Two groups of snails were injected with either vehicle or U0126 immediately after conditioning. When tested 1 d after conditioning, the U0126-injected group showed a significantly lower response to amyl acetate than did the vehicle-injected group (P < 0.05) (Fig. 5).
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It was not possible to test the effect of inhibition of MAPK phosphorylation on short-term memory formation using this conditioning paradigm due to nonspecific arousal effects caused by sucrose presentation that occur early after conditioning (M.J. Ribeiro, unpubl.).
Conditioning induces an increase in levels of MAPK phosphorylation in brain and lip tissue
MAPK kinase inhibition impairs memory formation in Lymnaea,
suggesting that conditioning induces MAPK phosphorylation. More direct
evidence for this hypothesis was obtained by showing an increase in pMAPK
levels 30 min after conditioning. We chose to study changes in levels of pMAPK
at this early time point because we are interested in the early biochemical
events that induce the formation of LTM as measured 24 h after conditioning.
We used the anti-pMAPK antibody and the anti-MAPK antibody, described earlier,
in Western blot analysis. We analyzed protein extracts from combined cerebral
and buccal ganglia (cerebral-buccal; the feeding ganglia) and from the other
ganglia of the brain combined (rest of the brain). We also analyzed protein
extracts from lip tissue. The cerebral and buccal ganglia are known to be
involved in the generation and modulation of the feeding behavior and are thus
good candidates for brain regions involved in conditioning of feeding.
Recently, conditioning-induced phosphorylation of CREB was shown to be
specific for the buccal and cerebral ganglia
(Ribeiro et al. 2003), and
this was another important reason why we targeted these ganglia in our
experiments. We chose to also analyze the lip tissue because backfilling of
the lip nerves showed the existence of putative sensory neuron cell bodies in
the lips (Straub et al. 2004).
Analysis of this tissue is likely to give an insight into the role of the
peripheral nervous system in memory formation. In this experiment, we used two
groups of snails, a naive group and a conditioned CS+US group. We chose to use
a naive nonstimulated control group because we wanted to compare the levels of
MAPK and pMAPK of the conditioned group against an absolute base-line, and
therefore we used a control group that we were sure did not learn anything.
After conditioning, the two groups of snails were divided into two subgroups
each: one used for Western blot analysis and the other used to test for LTM
formation 24 h later. The behavioral response to the CS of the CS+US group 24
h after the single training trial was significantly higher than was the
response of the naive group (CS+US: 16.8 ± 1.6 rasps/2 min; naive: 5.4
± 1.8 rasps/2 min; P < 0.001), confirming that learning
took place. The Western blot data indicated that the CS+US group had higher
levels of pMAPK than did the naive group in both cerebral-buccal extracts and
lip extracts, and this was confirmed statistically by densitometry
(cerebral-buccal, P < 0.05; lip, P < 0.01)
(Fig. 6A,B, respectively).
However, the levels of pMAPK detected in extracts from the rest of the brain
of the CS+US group were not significantly different from levels of the naive
group (P = 0.6; data not shown). The total amount of MAPK did not
differ significantly between the CS+US and naive groups (cerebral-buccal,
P = 0.9; lip, P = 0.8;
Fig. 6A and 6B, respectively;
rest, P = 0.5; data not shown).
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U0126 injection blocks conditioning-induced phosphorylation of MAPK
To confirm that injection of U0126 into the body cavity of the snails blocked the increase in levels of MAPK phosphorylation induced by the training procedure, we injected two groups of snails, one with U0126 and the other with vehicle, 30 min before conditioning. The snails were frozen in liquid nitrogen 30 min after conditioning. A third group of naive snails was frozen at the same time. The protein extracts from the three groups were analyzed by Western blotting using the anti-pMAPK and anti-MAPK antibodies. Snails injected with U0126 had undetectable levels of pMAPK in both cerebral-buccal extracts (Fig. 7A) and lip extracts (Fig. 7B). One-way ANOVAs of pMAPK levels revealed a source of significant difference between the three groups (cerebral-buccal: F(2,9) = 28.9, P < 0.001; lip: F(2,6) = 53.8, P < 0.001). For both types of tissue, Student-Newman-Keuls post hoc statistical analysis showed that conditioned snails injected with vehicle had significantly higher levels of pMAPK compared with that of either conditioned U0126-injected snails (P < 0.05) or naive snails (P < 0.05). These findings confirm that U0126 blocks conditioning-induced MAPK phosphorylation in Lymnaea. One-way ANOVA showed no significant differences in total MAPK levels among the naive, drug-injected, and vehicle-injected groups in cerebral-buccal extracts (F(2,9) = 2.7, P = 0.1) and lip tissue extracts (F(2,6) = 0.95, P = 0.4).
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In the previous experiments, we showed that food-reward conditioning
induces activation of MAPK. The next experiment was designed to establish if
activation of MAPK occurred specifically after CS+US pairing or if it was due
to other aspects of the training procedure such as exposure to novel stimuli
(e.g., the CS) or feeding stimulation (by the US). Levels of pMAPK and total
MAPK were monitored in four groups of snails: naive, CS+US, CS alone, and US
alone. The CS+US group was exposed to a single paired presentation of amyl
acetate and sucrose; the CS alone group was exposed to amyl acetate paired
with water, and the US alone group was exposed to water paired with sucrose
(Fig. 8A). Thirty minutes
later, the snails were frozen in liquid nitrogen. The group of naive snails
was frozen at the same time. A proportion of the snails from each group were
kept alive so that the feeding responses to amyl acetate could be measured 1 d
later. The behavioral results are plotted in
Figure 8B. A one-way ANOVA
showed a significant main effect of group (F(3,79) = 17.8;
P < 0.001). Subsequent Student-Newman-Keuls post hoc comparisons
revealed that the feeding response of the CS+US group was significantly higher
than were the responses of the other groups (P < 0.05). The
feeding responses from naive, CS alone and US alone groups did not differ
significantly. These observations further emphasize the associative nature of
this type of learning and are in agreement with the results obtained by
Kemenes et al. (2002).
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Discussion |
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TOPABSTRACTResultsDiscussionMaterials and MethodsREFERENCES |
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The cerebral and buccal ganglia contain the central pattern generator and
modulatory neurons that control the feeding behavior
(Benjamin et al. 2000) and are
therefore good candidates for sites of plasticity underlying conditioning of
the feeding response. Furthermore, food-reward conditioning has been shown to
selectively increase CREB phosphorylation in the same ganglia
(Ribeiro et al. 2003). In
mammalian systems, phosphorylation of CREB in neurons can occur downstream of
MAPK activation (Thomas and Huganir
2004) and as these pathways seem to be highly conserved, this
might also occur in Lymnaea. The lip tissue around the mouth of the
snail is also a probable site of plasticity because it contains primary
sensory neurons likely to be food receptors
(Straub et al. 2004).
Plasticity in sensory neurons plays an important role in both nonassociative
and associative types of learning in Aplysia
(Castellucci and Kandel 1976;
Hawkins et al. 1983). The fact
that in Lymnaea conditioning increases the levels of activated MAPK
in the cerebral/buccal ganglia and lip tissue suggests that both the CNS and
the peripheral nervous system are involved in memory formation.
Recently, by using extracellular recordings of Lymnaea lip and
tentacle nerves, Straub et al.
(2004) showed that the firing
patterns of these sensory nerves are unaffected by food-reward conditioning.
Their findings suggest that plasticity occurs at the level of synapses located
in central ganglia. However, our results suggest that sensory neurons with
somata in the lips undergo MAPK-dependent plasticity. These peripheral neurons
project through the sensory nerves and synapse with interneurons in the
cerebral ganglia (Straub et al.
2004). Therefore, activation of MAPK in the sensory neurons in the
lips could lead to plasticity in the synapses between the sensory neurons and
interneurons located in the cerebral ganglia. If this is the case, the firing
patterns of these neurons elicited by chemical stimulation of the lips will be
unaffected but the responses of their postsynaptic partners will change.
However, activation of MAPK in the lips of Lymnaea occurred not
only after classical conditioning but also after amyl acetate CS or sucrose US
presentation (Fig. 8D).
Activation of MAPK in sensory neurons following sensory stimulation has been
described before in Caenorhabditis elegans
(Hirotsu et al. 2000) and in
mice (Watt and Storm 2001). In
both organisms, exposure to olfactory stimuli induced MAPK activation in
olfactory sensory neurons. Nevertheless, in mice, the activation of MAPK
induced by olfactory stimulation was unnecessary for the normal olfactory
response observed in cultured sensory neurons
(Watt and Storm 2001),
suggesting a role in plasticity rather than olfactory perception per se. The
fact that, in our study, inhibition of MAPK activation did not impair sensory
perception of amyl acetate and sucrose suggests that this is also the case in
Lymnaea.
Activation of MAPK in brain tissue after exposure to CS or US alone has
been described before in experiments on classical conditioning. For example,
control mice exposed to CS alone or US alone in conditioned taste aversion
experiments (Berman et al.
1998; Swank 2000)
and in contextual fear conditioning experiments
(Sananbenesi et al. 2002)
showed increased levels of activated MAPK compared with that of nonstimulated
controls. In conditioned taste aversion, exposure to CS paired with US
activated MAPK in different regions of the brain compared with exposure to CS
or US alone (Swank 2000).
However, in Lymnaea, our findings suggest that the main ganglia where
MAPK activation is induced by CS alone or US alone are the same as where MAPK
is activated due to CS+US pairing. Nevertheless, it is possible that within
these ganglia, there are neurons where MAPK is activated only by CS+US pairing
and not by CS or US alone. These cells would be important sites of CS/US
association. Unfortunately, immunohistochemistry is not sensitive enough to
detect small changes at the single cell level, and therefore, the existence of
these cells could not be verified. Nevertheless, there is an alternative
mechanism. It is possible that sensory stimulation alone induces MAPK
activation in the same group of cells that undergo the plasticity necessary
for the association between the two stimuli. Activation of MAPK by sensory
stimulation might then be necessary for the animal to learn about the stimulus
it just perceived. For example, animals exposed to CS alone or US alone might
associate the stimulus with the visual environment where it was presented, and
therefore, an associative memory might form. Activation of MAPK induced by
sensory stimulation alone might thus be essential for the formation of these
and other associations. In fact, in conditioned-taste aversion, MAPK
activation elicited by CS presentation in the rat insular cortex is essential
for the formation of LTM of the CS/US association
(Berman et al. 1998). This is a
well-studied example showing that molecular mechanisms triggered by the
presentation of the CS alone can play a very important role in consolidation
of associative memory.
As any type of sensory stimulation might be sufficient for MAPK activation, it is possible that handling alone induces MAPK activation. However, this is unlikely because activation of MAPK was only detected in the cerebral and buccal ganglia, the feeding ganglia, and not in the rest of the brain. Handling stimulation would more likely induce activation of MAPK in all parts of the brain. The selective activation of MAPK in the feeding ganglia is more likely associated with presentation of feeding stimuli, such as sucrose and amyl acetate.
MAPK can be activated by a number of different molecular pathways, and it
has been suggested that in neurons MAPK might be a point of convergence
integrating a wide variety of signals
(Sweatt 2004). Two distinct
molecular pathways that have been shown to act upstream MAPK activation in
mammalian long-term potentiation, the nitric oxide-cGMP pathway and the
cAMP/PKA pathway (Lu et al.
1999; Roberson et al.
1999), have also been shown to play an important role in reward
conditioning in Lymnaea (Kemenes
et al. 2002; G. Kemenes, unpubl.). Both these signaling pathways
might act upstream of the MAPK cascade to induce long-term neuronal
plasticity. Further analysis will be needed to elucidate which of these or
other molecular cascades is activated specifically by the pairing of the CS
and US and how these cascades interact with MAPK in the process of LTM
formation.
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Materials and Methods |
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TOPABSTRACTResultsDiscussionMaterials and MethodsREFERENCES |
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L. stagnalis were bred in our laboratory or obtained from the Free University (Amsterdam, The Netherlands). The snails were kept in groups in large holding tanks containing copper-free water at 18°C-20°C on a 12-h light/12-h dark cycle. The animals were fed lettuce three times and a vegetable-based fish food (Tetra-Phyll, TETRA Werke) twice a week.
Behavioral procedures
Reward classical conditioning was performed on individual snails by using
the previously described one-trial chemical conditioning protocol
(Alexander Jr. et al. 1984). A
0.004% solution of amyl acetate (CS) was paired with a 0.67% solution of
sucrose (US). After a single training trial, the conditioned response is
measured as the number of feeding cycles elicited by a 2-min CS
presentation.
Before training, each snail was placed in an individual Petri dish
containing 85 mL of water and left for 15 min to acclimatize to the new
environment. Five mL of water were pipetted into the dish and the number of
feeding cycles counted for 2 min to establish the level of feeding activity
generated by the general disturbance caused by delivering solutions to the
dish. Five milliliters of the CS were pipetted into the dish, and 2 min after,
5 mL of the US were added for 2 min (CS+US pairing), after which the snails
were transferred to clean water in a holding tank. The feeding behavior of the
snails was monitored throughout this 6-min training period. One day after
training, prior to testing of the conditioned response with the CS, the snails
were returned to individual Petri dishes containing 90 mL of clean water.
Snails were allowed to acclimatize again and were then retested for a
disturbance response by the addition of water, as described earlier. Five
milliliters of the CS were then added to the Petri dish and the number of
feeding cycles counted during the following 2 min. During both training and
testing, the number of feeding cycles measured after water presentation was
not significantly changed, confirming the data of Kemenes et al.
(2002). This finding suggests
that any differences observed in the feeding behavior after conditioning are
not due to a difference in the spontaneous feeding behavior or in the behavior
induced by the mechanical disturbance caused by delivering solutions to the
dish.
In an initial experiment, we re-examined the ability of our snails to be trained by the one-trial chemical conditioning procedure. An unpaired group was used as the control to confirm that learning depends on the close temporal presentation of the CS and US. On the day of training, the unpaired group was exposed to amyl acetate paired with water and, 1 h after, exposed to water paired with sucrose. The CS+US (conditioned) group was exposed to amyl acetate paired with sucrose. To equalize handling between the two groups, the CS+US group was exposed to water paired with water 1 h before exposure to amyl acetate paired with sucrose (procedure summarized in Fig. 2A). The feeding response of the snails induced by amyl acetate was measured during training before exposure to sucrose and 1 d after training to test for LTM formation. During training the feeding behavior of the unpaired group was measured during amyl acetate presentation in the first part of the training, while the feeding behavior of the CS+US group was measured during amyl acetate presentation in the second part of the training. The responses of both groups during training were not significantly different from each other, suggesting that although the responses were measured in different parts of the training this did not affect the validity of the measurements. In another experiment (Fig. 8A), naive, CS alone, and US alone groups were used as controls. Here the CS+US group was exposed to amyl acetate paired with sucrose, the CS alone group was exposed to amyl acetate paired with water, and the US alone group was exposed to water paired with sucrose. The naive group was not handled or exposed to any stimulus on the day of training. Testing for amyl acetate was performed 1 d after training by using a blind protocol. In all the experiments described in this article, the snails were starved for 4 d before training and throughout the training and testing procedures.
All the behavioral data on feeding rates is expressed as mean ±
standard error. The statistical analyses were performed by using parametric
statistics as in previous experiments of this type
(Kemenes et al. 2002;
Fulton et al. 2005). When only
two different groups were compared, pairwise between-group comparisons were
made by using unpaired t-tests, and within-group comparisons were
made by using paired t-tests. Differences between more than two
groups were determined by using one-way ANOVA and subsequent
Student-Newman-Keuls post hoc tests to establish differences between pairs of
groups.
Application of MAPK kinase inhibitor
U0126 is a MAPK kinase inhibitor that specifically inhibits MAPK
phosphorylation (Favata et al.
1998). In the experiments examining the effect of MAPK kinase
inhibition, U0126 (Promega) was first dissolved in 100% methanol and, just
before use, diluted in snail physiological saline (50 mM NaCl, 1.6 mM KCl, 2.0
mM MgCl2, 3.5 mM CaCl2, 10 mM Hepes at pH 7.9)
(Benjamin and Winlow 1981) to a
final concentration of 500 µM in 20% methanol. The solutions were kept at
43°C at all times. Injections of U0126 and vehicle (20% methanol in snail
physiological saline) were performed as described previously
(Kemenes et al. 2002). Two
hundred microliters of U0126 or vehicle were injected into the body cavity of
each snail. As the estimated volume of the hemolymph is 1 mL, the
estimated final concentrations of U0126 and vehicle are one-sixth of the
injected concentration (80 µM U0126 and 3% methanol,
respectively).
Western blotting
For Western blotting studies, we used an antibody raised against
Aplysia MAPK (a gift from Dr. E.R. Kandel's laboratory)
(Michael et al. 1998) and two
commercially available antibodies raised against mammalian p44 and p42 MAPK
proteins: anti-pMAPK and anti-MAPK antibody (Cell Signaling, New England
Biolabs). The anti-pMAPK antibody recognizes only dually phosphorylated
(activated) MAPK (pMAPK), while the anti-MAPK antibody recognizes the MAPK
proteins independent of their phosphorylation state (total MAPK).
For comparison of levels of pMAPK or total MAPK between different groups of snails, the animals were starved for 4 d before the day of training and frozen in liquid nitrogen on the fifth day with no training (naive group), 30 min after conditioning (CS+US group), 30 min after exposure to amyl acetate paired with water (CS alone group), or 30 min after exposure to water paired with sucrose (US alone group). The training procedures were as described in the previous section. Frozen snails were stored at -20°C until dissection. Each frozen snail was thawed at room temperature for 5-10 min before dissection and dissected in ice-cold snail physiological saline. The brain was carefully extracted from the snail and the cerebral and buccal ganglia immediately transferred into 50 µL of ice-cold homogenization buffer (50 mM Tris at pH 7.8, 10 mM KCl, 0.2 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 0.2 mM DTT, 2 µg/mL aprotinin, 1 µg/mL pepstatin A, 100 µg/mL PMSF, and 1 mM Benzamidine). The lips of the snail were dissected by cutting a triangle with vertices between the eyes of the snail and just below the lateral extremities of the mouth aperture. Once dissected, the lip tissue was immediately transferred into 400 µL of ice-cold homogenization buffer (same as above). For each sample, the cerebral and buccal ganglia and lip tissues from seven animals were dissected and transferred into 50 µL or 400 µL, respectively, of ice-cold homogenization buffer and homogenized at 4°C. The homogenized tissue was centrifuged at 10,000g for 10 min at 4°C. The supernatants were then frozen in liquid nitrogen and stored at -70°C. For Western blot analysis, the samples were separated on 10% SDS-polyacrylamide gels and blotted to nitrocellulose membrane. Membrane blocking and antibody incubations were as recommended by the antibody manufacturer. Enhanced chemiluminescence (Pierce) was used for signal detection, and the exposure times were adjusted so that the signals were detected in the linear range. The membranes were first probed with the anti-pMAPK antibody (1:250), stripped, and then reprobed with the anti-MAPK antibody (1:2000). The densitometry was conducted using the ImageMaster Software (Pharmacia Biotech). All the dissections, sample preparation, Western blotting, and densitometry were performed by using a blind protocol.
Statistical analyses of the optical density data were performed by using parametric methods. When only two groups were compared, the differences were determined by using unpaired t-tests. In the experiments when more than two groups were compared, differences were established by using one-way ANOVA, and where appropriate, subsequent Student-Newman-Keuls post hoc tests were performed to establish differences between pairs of groups. All the data was expressed as mean ± standard error.
Immunohistochemistry
The whole dissected CNS was fixed in 1% paraformaldehyde and 1% acetic acid in phosphate buffer (0.1 M sodium dihydrogen orthophosphate, 0.1 M disodium hydrogen orthophosphate) overnight at room temperature. The CNS was subsequently dehydrated and embedded in paraffin wax. Seven-micrometer sections were cut and mounted on microscope slides. Before immunostaining, the sections were dewaxed and rehydrated. The nonspecific binding sites were blocked with 3% BSA, 0.1% Triton X-100 in Tris-buffered saline (TBS; 0.1 M Tris-HCl at pH 7.4, 150 mM NaCl) for 1 h at room temperature. The sections were then incubated with anti-pMAPK antibody (1:50) diluted in 1% BSA and 0.1% Triton X-100 in TBS overnight at 4°C, washed three times in TBS with 0.1% Triton X-100 (10 min per wash), incubated for 1 h at room temperature in goat anti-rabbit alkaline-phosphatase-conjugated (Sigma), diluted 1:100 in 1% BSA and 0.1% Triton X-100 in TBS, and washed again three times in TBS with 0.1% Triton X-100 (10 min per wash). The color reaction was developed for 10-20 min in darkness in 50 mg/mL 5-bromo-4-chloro-3-indolyl-phosphate, 100 mg/mL nitroblue-tetrazolium chloride (both from Roche), 10 mM MgCl2 diluted in 0.2 M Tris (pH 9.5), and 0.4% Triton-X. No immunoreactivity was detected when the primary antibody was omitted (data not shown).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 E-mail m.j.b.ribeiro{at}sussex.ac.uk; fax 44-1273-678535.
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