Intermediate and long-term memories of associative learning are differentially affected by transcription versus translation blockers inLymnaea
Department of Medical Physiology and Biophysics, Neuroscience Research Group, 3330 Hospital Drive NW, University of Calgary, Calgary, Alberta T2N 4N1, Canada
* Author for correspondence (e-mail: lukowiak{at}ucalgary.ca)
Accepted 18 February 2003
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Summary |
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Key words: Lymnaea stagnalis, intermediate memory, long-term memory, protein synthesis, associative learning
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Introduction |
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The variation in the length of perseverance between ITM and LTM is most
likely due to important molecular dissimilarities that underlie their
encodement. Chief among these differences is the necessity for the
transcription process. While both ITM and LTM require new protein synthesis,
only LTM requires the transcription process. That is, LTM requires both
altered gene activity and protein synthesis, while ITM requires only the
translation process (Davis and Squire,
1984; Rosenzweig et al.,
1993
; McGaugh,
2000
). Data obtained so far strongly support the idea that there
are evolutionarily conserved mechanisms underlying LTM formation. They appear
to involve both a cAMP-dependent MAP kinase signal transduction cascade
culminating in the activation of CREB transcription factors
(Tully, 1998
;
Mayford and Kandel, 1999
;
Silva et al., 1998
) and
formation of the CCAAT enhancer binding protein (C/EBP;
Alberini et al., 1994
;
Taubenfeld et al., 2001
).
Far less is known about the molecular basis underlying ITM. Prior to the
discovery of a memory component of intermediate duration dependent upon
different classes of protein kinase activities than those required for LTM
(Rosenzweig, 1993), it was widely believed that ITM was indistinguishable from
LTM. These shorter-lasting forms of memory have since been distinguished at a
behavioural level through classical conditioning of Aplysia feeding
behaviour (Botzer et al., 1998)
and sensitization of the siphon withdrawal response (a form of non-associative
learning; Sutton et al.,
2001
), as well as through operant conditioning of aerial
respiration in Lymnaea (Lukowiak
et al., 2000
). At the neuronal level, analogues of ITM have been
demonstrated at both Aplysia and Hermissenda CNS synapses
where synaptic transmission is facilitated
(Ghirardi et al., 1995
;
Crow et al., 1999
;
Sutton et al., 2001
). This
form of synaptic facilitation requires protein synthesis but, unlike neuronal
analogues of LTM, does not require transcription, suggesting that the proteins
necessary for ITM formation are translated from pre-existing mRNAs.
A major advantage of conditioning aerial respiratory behaviour in
Lymnaea is that the neural circuitry controlling this behaviour is
well established. A three-neuron central pattern generator (CPG), which is
both necessary and sufficient, mediates aerial respiration (Syed et al.,
1990,
1992
). In the operant
conditioning procedure, snails associatively learn not to perform aerial
respiration as a result of the contingent presentation of a tactile stimulus
to their respiratory orifice, the pneumostome, each time they attempt to open
it. Since Lymnaea are bimodal breathers, satisfying their respiratory
needs via cutaneous and/or aerial respiration, we are able to perform
experiments in which aerial respiratory behaviour is prevented or compromised
without harming them (Lukowiak et al.,
1996
; Taylor and Lukowiak,
2000
). Neural correlates of this operant conditioning have been
demonstrated in the CPG neurons in both isolated ganglia and semi-intact
preparations (Spencer et al.,
1999
,
2002
). A second advantage is
that by modifying the interval between training sessions, the training session
duration, or the number of training sessions per day, ITM versus LTM
can be differentially produced (Lukowiak
et al., 2000
). Given these advantages we have begun to ask how
memory of the associative learning is encoded within this prescribed neuronal
network. One necessary step on this path is to determine whether the different
forms of memory (ITM and LTM) in Lymnaea are differentially affected
by translation versus transcription protein synthesis blockers, as in
other systems studied to date.
To accomplish this task we examined whether a translation blocker,
Anisomycin, and a transcription blocker, Actinomycin D, differentially affect
ITM and LTM. Anisomycin and Actinomycin D have previously been used in studies
to examine their effects on memory consolidation in molluscs as well as other
animals, including mammals (Castellucci et
al., 1988; Nguyen et al.,
1993
; Milner et al.,
1998
; Ramirez et al.,
1998
; Crow et al.,
1999
). We have also used both of these blockers previously in
Lymnaea to demonstrate the necessity for altered gene activity and
new protein synthesis in synaptogenesis as well as to show that isolated
primary neurites (i.e. without the soma) have the capacity to translate mRNA
into functional membrane proteins (Feng et
al., 1997
; Woodin et al.,
1999
; van Minnen et al.,
1997
; Spencer et al.,
2000
).
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Materials and methods |
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Training procedures
Individually labeled snails were placed in a 1 l beaker containing 500 ml
of hypoxic water. The water was made hypoxic by bubbling N2 through
it for 20 min prior to and during training. We refer to this as the `standard'
hypoxic training procedure. We also utilized a `different context' training
procedure, which we will refer to as the `carrot context'. To create the
carrot context, N2 was first bubbled through a 750 ml Erlenmeyer
flask containing chopped carrots and water before being bubbled into the
training beaker (for complete details, see
Haney and Lukowiak, 2001).
`Change of context test' refers to the context in which the snails were
not trained. This test was used as a control to show that, following a
given procedure, e.g. training in the presence of a protein synthesis blocker,
snails were still as responsive as they had been in the initial training
session.
In all experiments, the snails were first given a 10 min acclimatization period, where they could perform aerial respiration freely. The onset of operant conditioning training was initiated by gently pushing the snails beneath the water surface. In between the training and memory test sessions, snails were placed in eumoxic aquaria where they were also allowed to perform aerial respiration freely.
In all of the training, memory test, and change of context test sessions, a gentle tactile stimulus (a sharpened wooden applicator) was applied to the pneumostome area (the respiratory orifice) every time the snail began to open its pneumostome to perform aerial respiration. This tactile stimulus only evoked pneumostome closure; it did not cause the animal to withdraw its foot and mantle area (i.e. the whole-animal withdrawal response), nor did pneumostome stimulation cause the snails to sink to the bottom of the beaker. The time of each attempted opening was recorded and tabulated.
Intermediate and long term memory training procedures
Snails can be differentially trained to produce ITM or LTM
(Lukowiak et al., 2000). We
used similar training procedures here. For ITM, snails were subjected a single
30 min training session, and memory was tested 3 or 4 h later in a 30 min test
session.
For LTM, snails received a single 1 h training session, and memory was tested 6 or 24 h later in a 1 h session.
Following each respective training session we summed the number of attempted pneumostome openings for each animal and calculated the mean and the standard error of the mean (S.E.M.) of the total number of attempted pneumostome openings for each cohort.
A memory or `savings' test session was presented to the snails at the indicated times. This test session was the same as a training session and was performed at varying times after the training session in order to determine how long the memory persists.
The `carrot-context' procedure was used in some experiments. Snails
demonstrate context-specific learning and memory
(Haney and Lukowiak, 2001;
McComb et al., 2002
), so it
was possible to determine if memory was present, or if the observed
behavioural phenotype was the result of an unresponsive animal due to
drug-induced side effects, by altering the context in which we tested the
snails. If the animal was unresponsive due to sickness, altering the context
of the memory test session would not result in an increase in the number of
attempted pneumostome openings. If the observed behaviour was the result of
memory, however, altering the context would result in an increase in
responsiveness.
Injections
The protein synthesis blocker (dissolved in saline) or saline control was
injected into the hemocoel through the foot of the animal. The person
injecting the drug or saline did not perform the training or the memory tests
and the experimenter performing the training/memory test did not know what
each snail had been injected with. Thus all experiments are performed `blind'.
The concentrations used were 12.5 µg Anisomycin ml1 snail
volume and 1 µg Actinomycin D ml1 snail volume, which
were the same as those used effectively in our laboratory to block both the
transcription and translation processes, respectively
(Feng et al., 1997;
van Minnen et al., 1997
;
Hamakawa et al., 1999
). We
also directly demonstrated elsewhere that each of these blockers inhibits
protein synthesis (van Minnen et al.,
1997
; Feng et al.,
1997
).
Results from pilot studies showed that these concentrations of protein synthesis blockers were effective when injected into the whole animal. We recalculated the concentrations so that an amount of 0.1 ml could be injected into snails of 3 ml total volume.
Operational definitions of learning and memory
We have operationally defined memory in previous publications (e.g.
Lukowiak et al., 1996,
1998
,
2000
; Spencer et al.,
1999
,
2002
). Memory was present if
the number of attempted pneumostome openings in the memory test session was
significantly less than the number of attempted openings in the training
session. ITM was tested 3 and 4 h after the single 30 min training session,
whilst LTM was tested 6 h after the last training session. Previously it was
shown that ITM did not persist for longer than 3 h
(Lukowiak et al., 2000
). We
had to test for LTM at 6 h post-training because control snails injected with
Actinomycin D showed drug-induced side effects after this time (see
below).
Statistical analysis
To determine whether there were any detrimental side-effects on aerial
respiratory behaviour, data from snails injected with the protein synthesis
blockers were subjected to a one-way analysis of variance (ANOVA) followed by
a post hoc Fisher's LSD protected t-test to compare each
session. A paired t-test (between groups) was used to determine
whether memory was present (see above). A paired two-sample t-test
for independent groups was used to compare the number of pneumostome openings
in the first session of the different ITM and LTM training cohorts (i.e.
control versus treatment with either of the two blockers).
Significance was at the P<0.05 level.
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Results |
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Snails were observed at 2.5, 4.5, 8 and 24 h following injection. Snails were divided into four groups. One (N=20) served as non-injected controls, while the other groups were injected with saline (N=20), Anisomycin (N=25) or Actinomycin D (N=22), respectively (Fig. 1). The injections and observations were performed by different people, therefore assessment of the snails' breathing behaviour in this and all other experiments reported here was performed `blind'.
We examined whether the various injections significantly affected either the number of pneumostome openings or the duration of each opening by comparison with those of the non-injected snails. We thus calculated and tabulated the mean of the total breathing time (Fig. 1A) and the mean number of openings (Fig. 1B) per 30 min observation session per group.
In the non-injected controls, the total breathing time (ANOVA, F3,19=0.5407, P=0.6564) and the number of pneumostome openings (ANOVA, F3,19=0.5816, P=0.6295) did not change significantly over the four sessions. Therefore, repeated sequential exposure to hypoxic conditions did not alter the breathing behaviour of Lymnaea. Similarly, no significant differences in total breathing time (ANOVA, F3,19=0.1093, P=0.9539) or the number of pneumostome openings (ANOVA, F3,19=0.5247, P=0.6671) were detected in saline-injected snails over the four hypoxic water test sessions. Vehicle injection did not significantly change either the total breathing time or the number of breaths when we compared the saline and non-injection groups (paired t-test: P>0.05).
Injection of the translation blocker Anisomycin did not significantly affect either the total breathing time or the number of pneumostome openings (ANOVA, F3,24=0.6114, P=0.6112 and F3,24=0.259, P=0.847, respectively) at any of the time points. Comparison of the Anisomycin-injected group with the non-injected group showed that there was not a significant difference in total breathing time or in the number of pneumostome openings (paired t-test, P>0.05). We concluded therefore that when studying the effects of injection of Anisomycin on ITM or LTM any time window adequate for the training and testing of ITM and LTM could be used. An injection time of 2.5 h prior to the operant conditioning training session was thus set, allowing enough time for diffusion of the drug within the snail.
By contrast, we found that injection of the transcription blocker Actinomycin D significantly altered aerial respiratory behaviour of Lymnaea beyond 8 h post-injection. There was no significant effect of Actinomycin D on the measured parameters of aerial respiratory behaviour (total breathing time and number of breaths) at 2.5 and 4.5 h post injection (ANOVA, F2,21=2.0797; P=0.1376 and F2,21=1.5849; P=0.217, respectively), nor were any significant differences in the measured respiratory parameters between the non-injected and Actinomycin D groups in the 2.5 h session found (two-sample t-test, P>0.05). There was, however, a significant decrease in both total breathing time and the number of openings in the Actinomycin D group between the first (2.5 h post injection) session and the fourth session (24 h post-injection) (Fisher's LSD protected t-test, P<0.05 and P<0.01; respectively). When we compared the total breathing time in session 3 (the 8 h session) with that of session 1, we obtained a P value of 0.053 (i.e. close to being significant) in a Fisher's LSD protected t-test. Since it was possible that the drug was interfering with the snails' breathing behaviour at periods longer than 8 h post-injection we limited our experiments to 8 h post injection.
Intermediate term memory
A single 30 min training session is sufficient to establish a memory that
persists for 3 but not 4 h (Fig.
2). Two naïve cohorts of 20 snails each received an injection
of saline 2.5 h before training as described in Materials and methods. The
first cohort was tested for memory 3 h after the training session and the
second 4 h after the training session. Memory was present for 3 h
(Fig. 2A). That is, the number
of attempted pneumostome openings in the memory test session was significantly
fewer than in the training session (P<0.01), meeting the criterion
for memory retention. However, when the second cohort was tested for memory 4
h after the training session, memory was not present
(Fig. 2B). That is, the number
of attempted pneumostome openings in the 30 min memory test session was not
significantly different from the number in the training session
(P>0.05). The criterion for memory retention was thus not met. We
therefore conclude that a single 30 min training session is sufficient for
memory retention of 3 h but not 4 h.
|
Knowing that we were able to produce an ITM that persisted for 3 h, we could test the effects of Actinomycin D and Anisomycin on other naïve cohorts of snails. We first tested the effect of the transcription blocker Actinomycin D. 20 snails were pre-injected with Actinomycin D 2.5 h before the training session (Fig. 3A). These injected snails performed similarly to the control snails. That is, the number of attempted pneumostome openings in the 30 min training session was similar (i.e. not statistically different; P>0.05) to the number we observed in Fig. 2. When we tested for memory in these Actinomycin D-injected snails we found that memory was present. That is, the number of attempted pneumostome openings in the memory test session was significantly less (P<0.01) than the number of attempted openings in the training session. To show that the statistically lower number of attempted openings in the memory test session was due to memory retention and not a side effect of the drug, we challenged these snails with the carrot-odour context 1 h later. As can be seen (Fig. 3A) the number of attempted openings in the carrot context session was significantly different from the number in the memory test session (P<0.01) but was not different from the number in the initial training session (P>0.05). We thus conclude that Actinomycin D does not prevent the formation of ITM.
|
We next performed a similar experiment using the translation blocker Anisomycin. Again a naïve cohort of snails (N=20) was pre-injected with Anisomycin 2.5 h before the initiation of training (Fig. 3B). The number of attempted pneumostome openings in the training session was not different from the number observed in the control snails (Fig. 2) or in the Actinomycin D group (Fig. 3A; P>0.05 in both cases). When we tested for memory retention 3 h later, however, we found that memory was not present. That is, the number of attempted pneumostome openings was not significantly different from the number in the single training session (P>0.05). Since these snails performed as they did in the initial training session we did not challenge them with the carrot-odour context. We conclude from this experiment that the translation blocker Anisomycin prevents the formation of ITM and, while protein synthesis is necessary for ITM, altered gene activity is not.
Long term memory
A single 1 h training session is sufficient to establish a memory that
persists for 24 h (Fig. 4). A
naïve cohort of 20 snails was pre-injected with saline 2.5 h before
training commenced and 24 h later was tested for memory retention in a 1 h
test. As can be seen, memory was present for at least 24 h. That is, the
number of attempted pneumostome openings in the memory test session was
significantly fewer than in the training session (P<0.01), meeting
the criterion for memory retention. These data confirm our earlier reported
results (Lukowiak et al.,
2000) and show that we can study the effects of protein synthesis
inhibitors on the establishment of LTM using a single 1 h training
session.
|
We first tested the effect of the transcription blocker Actinomycin D on the LTM consolidation process by injecting a naïve cohort of snails (N=20; Fig. 5A) with Actinomycin D 2.5 h before the training session. Following the training session we tested whether the learning was consolidated into memory. As can be seen, memory was not observed as the number of attempted pneumostome openings in the 1 h memory test session was not significantly different from the number in the training session. Since these snails performed as they did in the initial training session we did not challenge them with the carrot-odour context. Notice also that the number of attempted pneumostome openings in the training session was not statistically different (P>0.05) from the number of attempted openings in the training session in the control snails in Fig. 4. Thus Actinomycin D, while not affecting the ability of the snails to perform aerial respiration, did block the establishment of memory.
|
In a similar manner we tested the ability of the translation blocker, Anisomycin, to prevent memory formation. Thus another cohort of naïve snails (N=20; Fig. 5B) was pre-injected with the blocker 2.5 h before training commenced. As can be seen the learning was also not consolidated into memory. Thus, the number of attempted openings in the 1 h memory test session was not significantly different from the operant training session (P>0.05). Again the number of attempted openings in the operant training session in these Anisomycin pre-injected snails was not significantly different from the number in the LTM control (Fig. 4) or Actinomycin D groups (Fig. 5A; P>0.05). This demonstrates that Anisomycin did not alter the responsiveness of the snails but did alter their ability to consolidate the learning into LTM. Since in both the Actinomycin D and Anisomycin experiments the number of attempted openings in the memory test session was statistically as large as in the initial training session and in the LTM control group (Fig. 4), we did not have to alter the context to show that the snails were still responsive. We therefore conclude that LTM is dependent on both altered gene activity and new protein synthesis.
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Discussion |
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Injection of the translation blocker Anisomycin into the hemocoel did not
alter the responsiveness of the snails in either the ITM- or LTM-training
procedure, but did prevent the consolidation of associative learning into both
ITM and LTM. In our model system new protein synthesis is therefore a
necessary step for encoding of both ITM and LTM. The transcription blocker
Actinomycin D, in the time period used, also had no effect on the snails'
responsiveness during training and, importantly, had no effect on the ITM
formation, indicating that the transcription process is not a necessary step
in ITM formation. The transcription process is necessary for encoding LTM,
however, as Actinomycin D prevented its establishment. These findings are
consistent with the hypotheses that the consolidation of learning into LTM is
dependent on both altered gene activity and protein synthesis
(Davis and Squire, 1984),
whilst the transformation of learning into ITM is only dependent on protein
synthesis (Rosenzweig et al.,
1993
; McGaugh,
2000
; Sutton et al.,
2001
). A recent abstract, utilizing a one-trial associative
learning procedure in Lymnaea, similarly concludes that LTM can be
blocked by both translation and transcription blockers
(Fulton et al., 2002
).
The hypothesis that there are at least two stages in longlasting memory
formation, each dependent on different molecular mechanisms and subsequent
cellular events, is supported by our data. With respect to ITM, our results
are consistent with the hypothesis that the molecular message necessary for
the induction and maintenance of ITM is already transcribed and only need be
translated for ITM encodement. A requirement for protein synthesis but not
mRNA synthesis in the intermediate phase of memory consolidation has
previously been found in Hermissenda
(Crow et al., 1999).
Furthermore, the Aplysia sensory-motor neuron synapse undergoes a
5HT-induced facilitation termed ITF (intermediate term facilitation, lasting a
few hours) that requires only translation
(Ghirardi et al., 1995
;
Martin et al., 1997
;
Sutton et al., 2001
). In
addition, complementary data from three other studies are consistent with the
hypothesis that the new proteins necessary for ITM are produced from
pre-existing mRNA transcripts (Mauelshagen, 1998;
Martin et al., 1997
;
Manseau et al., 1998
). The
synaptic ITF seen at the Aplysia sensory-motor neuron synapse may in
part form the basis of a behavioural memory (ITM) that persists for a similar
time course, and is similarly disrupted by translational but not
transcriptional blockers, following tail-shock induced siphon-withdrawal
sensitization in reduced preparations
(Sutton et al., 2001
).
Interestingly, evidence for protein translation outside of the soma has
been emerging in both vertebrate and invertebrates (for a review, see
Giuditta et al., 2002). For
example, it has been demonstrated that the isolated primary neurite (i.e. with
the soma removed) of a Lymnaea neuron has the capacity to synthesize
de novo proteins from injected mRNA
(van Minnen et al., 1997
). New
proteins translated from the injection of `foreign' mRNA into the primary
neurite of Lymnaea were functionally inserted into the membrane
(Spencer et al., 2000
).
Similarly, in culture following the removal of the soma of a mechano-sensory
neuron in Aplysia, the remaining primary neurite was capable of local
translation of mRNAs (Martin et al.,
1997
). Since ITM is dependent on protein synthesis from a
pre-existing message, it is possible that the protein synthesis responsible
for ITM could occur in extrasomal regions at the site of plasticity (see
below).
Experiments where the soma of RPeD1 in Lymnaea in the freely
moving animal was ablated but the primary neurite left functionally intact
before operant conditioning training, showed that the snails are still capable
of performing aerial respiratory behaviour and are still able to learn and
form ITM (Scheibenstock et al.,
2002); however, they could not form LTM. We hypothesize that LTM
formation did not occur because RPeD1, one of the CPG neurons, is a site of
LTM formation. Since the RPeD1 nucleus was not present, the necessary
transcription process for LTM could not occur. These snails were capable of
forming ITM, so we further hypothesize that the necessary protein synthesis
for ITM occurs extra-somally, possibly at the actual sites of synaptic
plasticity, i.e. the pre- or post-synaptic specializations. Such locally
synthesized proteins in snails where the soma is intact may serve to mark the
site of plasticity so that new proteins being synthesized in the soma are
delivered to specific sites (Martin et
al., 1997
; Manseau et al.,
1998
).
Why are there two different forms of longer-lasting memory, each with a
different time course and susceptibility to interruption by different classes
of protein synthesis blockers? Rosenzweig et al.
(1993) put forward a scheme to
explain the existence of ITM, with the central hypothesis that the underlying
biochemical mechanisms (i.e. an assortment of classes of protein kinase
activities and the difference in the requirement of transcription) responsible
for each form of memory are not arranged in a serial fashion but rather occur
in parallel. ITM forms sooner and persists for a shorter period of time than
LTM, thus ITM may serve a function somewhat analogous to a memory cache of the
CPU of a personal computer. According to this scheme, ITM allows a memory to
be maintained until such time as LTM (which depends on events in the nucleus)
can be induced, the newly synthesized proteins transported and becoming
functional. The proteins subserving ITM could additionally serve as
`signposts' or `markers' that ultimately enable the proteins encoded by the
transcription process to arrive at the proper site of plasticity so that
memory will become encoded only at specific sites and not globally within the
neuron.
At the behavioural level it has been shown that prior ITM can augment the
ensuing persistence of LTM (Smyth et al.,
2002). In those experiments a training procedure that only
produced a memory persisting for 3 h was shown to significantly augment the
persistence of memory produced by an LTM-training procedure. This augmentation
occurred up to 5 h after the last ITM training session, even though there was
no behavioural evidence that ITM was present. Thus while the biochemical and
molecular processes at the neuronal level that encode ITM and LTM may occur in
a parallel, non-sequential, fashion it appears that there is an augmenting
effect of the `ITM-process' on the `LTM-process'. It has been suggested that
LTM formation is in itself a two-step process, whereby the first step
parallels ITM formation and involves only protein synthesis, while the second
step requires transcription to produce new products capable of mediating the
physiological and morphological synaptic changes characterized by LTM
(Feudenthal-Ramiro, 2000).
It is not clear why the processes that encode ITM only persist for a few
hours. One hypothesis is that the new proteins initiated by the ITM-training,
which are responsible for the encodement of the memory, `fall below' some
threshold level and evidence of memory can no longer be demonstrated
behaviourally. Support for this comes from the ITM sensitization seen in
Aplysia reduced preparations, which is dependent on continued
cAMP-dependent protein kinase (PKA) activation
(Sutton et al., 2001). As PKA
activation decreases with time the translation of mRNAs ceases and the changes
induced by local protein synthesis fall below some lower limit, resulting in a
synapse that is no longer facilitated. In the case of the tail-shock induced
siphon-withdrawal response sensitization seen in Aplysia
(Sutton et al., 2001
) the fall
in PKA-induced local protein synthesis appears to happen quite quickly, within
90 min, whilst in Lymnaea the fall in local protein synthesis
persists for at least 3 h. In the tail-shock induced sensitization in
Aplysia and in other preparations (including honeybee, rodents and
humans; Kamin, 1957
;
Menzel, 1983
;
Sutton et al., 2001
) there is
often a U-shaped graph of memory retention. That is, memory is strong
initially, fades after some period, and then regains its strength. In the case
of tail-shock induced sensitization of the siphon-withdrawal response in
Aplysia, this means that if a memory test is administered after ITM
is over (90 min or so), but before LTM becomes apparent (some 15+ h after the
last tail shock) there is no evidence of memory. We have not yet seen such a
phenomenon in our Lymnaea operant conditioning studies. However, we
do know (Smyth et al., 2002
)
that even though ITM may not be apparent behaviourally after 3 h, its
underlying neuronal substrates are able to exert an augmenting influence of
subsequent LTM formation.
The data presented here are the first to our knowledge in Lymnaea
where the dependence of ITM and LTM on protein synthesis has been
demonstrated. These new findings further strengthen the use of
Lymnaea as model system (Benjamin
et al., 2000) in studies to investigate the causal neuronal
mechanisms underlying associative learning and its various forms of
memory.
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Acknowledgments |
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