Boosting intermediate-term into long-term memory
Department of Physiology and Biophysics, Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1
* Author for correspondence should be addressed (e-mail: lukowiak{at}ucalgary.ca)
Accepted 15 February 2005
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Summary |
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Key words: Lymnaea stagnalis, learning, operant conditioning, snail, behaviour, memory trace
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Introduction |
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Lymnaea are bimodal breathers, therefore it is possible to
modulate one of its respiratory behaviours (i.e. aerial respiration) while
leaving the other (cutaneous) unaffected. We use a non-declarative, operant
conditioning paradigm to decrease aerial respiratory behaviour
(Lukowiak et al., 1996) and
since the snails can still breathe cutaneously our procedure is not harmful. A
three-neuron central pattern generator (CPG), whose sufficiency and necessity
have been demonstrated, drives aerial respiratory behaviour (Syed et al.,
1990
,
1992
). Since non-declarative
memories are stored within the same network that mediates the behaviour
(Dudai, 2002a
), the changes
induced by operant conditioning are stored within the respiratory CPG in
Lymnaea (Spencer et al.,
1999
,
2002
). In fact the molecular
processes necessary for consolidation, reconsolidation (i.e. restabilization
of the memory after it has been made active) and extinction of LTM occur
within RPeD1 (Scheibenstock et al.,
2002
; Sangha et al.,
2003b
,c
).
Previously we showed (Smyth et al.,
2002) that although the behavioural phenotype of ITM was not
apparent 5 h aftertraining, there was enhancement of LTM persistence with
subsequent LTM-training. We now extend these findings and show that ITM leaves
behind a residual molecular memory trace, on which a second bout of
ITM-training builds to cause the formation of LTM. We call this phenomenon
`memory boosting'. This `boosting' of ITM to LTM occurs even if the
behavioural manifestation of the memory is not apparent. However, this `memory
boosting' is (1) impeded by blocking new protein synthesis, (2) interfered
with by behavioural extinction training, and (3) requires the presence of
RPeD1's somata. These findings are all consistent with (1) the hypothesis that
ITM and LTM formation occur in series
(Ghirardi et al., 1995
;
Riedel, 1999
;
Zhao et al., 1995
), and (2) an
emerging view that memory exists in either a labile or a stable state
(Nader, 2003
). Thus, when a
memory is retrieved (i.e. activated) it re-enters the labile state and must go
through a `reconsolidation' phase in order for it to become stable and persist
in the brain.
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Materials and methods |
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Operant conditioning procedure
Individually labeled snails were placed in a 1 l beaker containing 500 ml
of room temperature (1920°C) hypoxic (<0.1 ml O2
l1) water. The water was made hypoxic by bubbling
N2 through it 20 min prior to and during training and testing.
Hypoxia dramatically increases aerial respiratory behaviour
(Lukowiak et al., 1996;
Rosenegger et al., 2004
).
Animals were first given a 10 min acclimatization period, during which they
could freely perform aerial respiration. The onset of operant conditioning
training was initiated by gently pushing the snails beneath the water surface.
During the operant conditioning training session, every time a snail opened
its pneumostome to perform aerial respiration, a sharpened wooden applicator
(0.25 mm diameter) was used to `poke' the pneumostome area to cause the animal
to close the pneumostome. Withdrawal of the snail into its shell typically did
not occur and most snails remained at the surface of the water. The gentle
poke did not cause rotation of the snail and thus a statocyst-dependent reflex
was probably not elicited. The time when every animal attempted to open its
pneumostome was recorded. In between sessions, animals were kept in eumoxic
pondwater and freely performed aerial respiration ad libitum. During
the administration of a memory test (MT), animals were subjected to the
application of tactile stimuli, as in operant conditioning training
sessions.
We also utilized a `change of context' testing procedure. To create the
`different context', N2 was first bubbled through a 750 ml
Erlenmeyer flask containing chopped carrots and water before being bubbled
into the training beaker (Haney and
Lukowiak, 2001). When sensing the presence of carrot odor, the
animals perceive this as a different context and respond as if they have not
received training; i.e. there is an increase in the number of pneumostome
openings. The term `change of context test' means that snails were tested in
the context that they were not trained in. The term `standard
context' refers to the training procedure when nitrogen is bubbled directly
into the beaker containing the snails; the term `carrot-context' refers to the
training procedure where nitrogen is first bubbled through the chopped carrots
before it reaches the beaker containing the snails.
ITM and LTM training procedure
The ITM-training protocol consisted of two 30 min operant conditioning
training sessions in hypoxic pondwater (TS) separated by a 30 min rest
interval in eumoxic pondwater (Lukowiak et
al., 2000). The LTM training procedure, on the other hand,
consisted of two 30-minute operant conditioning training sessions separated by
a 1 h rest interval (Lukowiak et al.,
2000
. A MT was presented to the snails 3 or 24 h after the last
training session. In addition, the appropriate control procedures (e.g. yoked
control experiments; see below) were also previously performed to show that
our training schedules produce associative learning (Lukowiak et al.,
2000
,
2003b
).
In experiments designed to test our `residual memory trace' hypothesis (Figs 2, 3, 4, 5, 6, 7, 8), snails were given the ITM-training protocol on Day 1 (i.e. two 30 min training sessions with a 30 min interval between sessions). On the following day a second similar bout of ITM-training was given. The presence of LTM was tested 24 h later on Day 3. All experiments on memory retention were performed blind. The randomization of a cohort was performed by blindly separating the trained animals into two cohorts (i.e. testing one sub-cohort at 3 h and another at 24 h, etc.).
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Yoked control procedure
These snails received a tactile stimulus to their pneumostome area that was
not contingent upon opening their own pneumostome; rather they received the
tactile stimulus when the snail to which they were `yoked' to opened its
pneumostome in the operant conditioning procedure (Lukowiak et al.,
1996,
2003a
). A similar intensity
tactile stimulus was used as in the operant conditioning group. Since snails
were most often underwater the `yoked-poke' did not cause the pneumostome to
close, as it was not open. We assayed the yoked control snails for memory 24 h
after the last yoked control session. In the memory-test session these snails
now received the tactile stimulus to the pneumostome when they attempted to
open their pneumostome.
Cooling procedure
A 1 l beaker filled with 500 ml of eumoxic water was prechilled and
maintained at 4°C and served as the cooling apparatus. We have previously
shown that the cooling procedure does not adversely affect the snails
(Sangha et al., 2003d).
Cooling snails immediately (within 30 s) following operant conditioning
training, reactivation of memory or extinction training, blocks the
consolidation and reconsolidation processes (Sangha et al.,
2003b
,c
,d
).
Therefore we test whether cooling snails immediately after the first bout of
ITM-training can prevent the establishment of the residual molecular memory
trace.
Cooling has also been used to extend the persistence of memory (Sangha et al., 2002d). Thus, if snails are permitted to undergo consolidation for ITM and then cooled, the duration of the residual molecular memory trace should, if our hypothesis is correct, be enhanced. In these experiments snails were placed in the cooling apparatus 2 h after the last training session (i.e. after the consolidation process has been completed).
To control for any possible `side-effects' of cooling a cohort of snails trained using the ITM-training protocol were kept at room temperature for 2 h. Following this 2 h period, snails were transferred into cold (4°C) eumoxic water for 46 h. Then snails were again trained using the ITM procedure at room temperature (2023°C) and assayed for memory 24 h later (see Fig. 4).
ITM-training in a different context on day 2
In experiments designed to examine if the memory trace is context-specific,
snails were given an ITM-training protocol in the standard context on Day 1.
The following day, they were trained in a different context (i.e. the carrot
context). One day later the snails were tested for memory (i.e. LTM) in the
standard context. Our working hypothesis is that if the first bout of
ITM-training is performed in one context, the residual memory trace will form
for only that specific context. Thus, when the second bout of ITM-training is
performed on the following day in a different context, there will be no LTM
formed 24 h later when tested in the first context.
Extinction training protocol
Extinction training (for full details, see
McComb et al., 2002;
Sangha et al., 2003c
) was
performed by placing ITM-trained snails in a beaker of hypoxic water for two
30 min sessions separated by a 30 min rest interval in eumoxic water. During
the 30 min extinction sessions the reinforcing stimulus (tactile stimulus to
the pneumostome area) was not applied in response to a pneumostome opening.
The two extinction sessions were given to the snails on Day 2. 3 h after the
second extinction session, the second series of ITM-training was performed and
memory was assayed 24 h later. In control experiments
(Fig. 6B), different context
(i.e. carrot) 30 min extinction sessions were given to snails. A bar labeled
E1 or E2 denotes each extinction training session in the figures.
Somata ablation procedure
We have previously shown that the somata of RPeD1 is required for LTM
formation, reconsolidation and extinction
(Scheibenstock et al., 2002;
Sangha et al.,
2003b
,c
,e
).
The ablation procedure used here was performed as in our previous studies.
Briefly, we first anesthetized the animals with 13 ml of 50 mmol
l1 MgCl2 injected through the foot. This
paralyzed the snail, allowing a dorsal midline incision to be made to expose
the snail's brain. Using a fine glass hand-held microelectrode, the RPeD1
somata was ablated by gently `poking' it. In control experiments, the somata
of LPeD1, which is similar in size to RPeD1 but does not play a role in aerial
respiratory behaviour, was ablated. The incision was small enough to allow the
animal to heal without suturing. Animals began to wake from the effects of the
anesthetic within several hours of the surgery. In the experiments reported
here the experimenter performing the behavioural training was unaware of which
neuron had been ablated. The code was only broken after the savings-test.
The ablation of RPeD1's somata, which leaves behind an intact functional
primary neurite where the necessary synaptic interactions occur, does not
adversely affect the snails' ability to perform aerial respiratory behaviour
or to learn associatively (Scheibenstock
et al., 2002; Sangha et al.,
2003b
,c
).
Total breathing time and the number of pneumostome openings were monitored
before and after RPeD1 somata ablation. There were no significant differences
between pre- and post-ablation in either measurement.
Criteria for learning and memory
We have operationally defined both associative learning and memory as
previously (Lukowiak et al.,
1996,
2003b
; Sangha et al.,
2003b
,c
).
In the present study, learning on the particular day was considered present if
the number of attempted pneumostome openings in the last training session
(e.g. TS2) was significantly less than the number of attempted openings in the
first training session (e.g. TS1). In order to be defined as memory, two
criteria had to be met: (1) the number of pneumostome openings in MT was
significantly lower than that of TS1, and (2) the number of pneumostome
openings in the MT was not significantly higher than that of the last training
session (e.g. TS2).
Statistical analysis
To determine whether the experimental manipulation had an effect when
compared to a control group (see below) and whether the number of attempted
pneumostome openings was significantly altered as a result of operant
conditioning or other procedures (yoked control, cooling, extinction, etc.),
we performed repeated-measures one-way ANOVAs, testing both a between-group
factor (i.e. control vs experimental) and a within-group factor (i.e.
training sessions vs savings-test;
Zar, 1999). If the ANOVA was
significant (P<0.05), a post hoc Fisher's LSD protected
t-test was performed to show which groups (i.e. between group) and
sessions (i.e. within group) were significantly different
(Glass and Hopkins, 1996
).
Differences were considered to be significant if P<0.05.
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Results |
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To conclude, however, that the change in behaviour was a true example of associative learning and memory formation, we performed yoked control experiments. In yoked control (N=28) snails (yoked to the ITM-training schedule) a memory test (Yoked MT) was performed 3 h after TS2. We found that the number of attempted openings in Yoked MT was not significantly less than the number of attempted openings in TS1 of the ITM-trained snails (P>0.05) and was statistically greater than the number of attempted openings in TS2 (P<0.01). We also made a between-group comparison of the response in MT in the yoked control and ITM operantly conditioned snails. We found that the number of attempted pneumostome openings in Yoked MT was significantly greater that the number of attempted openings in the 3 h MT (P<0.01) of operantly trained snails. We therefore concluded that two 30 min training sessions separated by an interval of 30 min results in ITM but does not in LTM.
By contrast, when a group of snails (N=155) was subjected to the LTM training procedure (two 30 min training sessions separated by a 1 h interval), ITM and LTM were observed (Fig. 1B), thus learning occurred (ANOVA(154,3)=103.0, P<0.0001). The number of attempted openings in TS2 was significantly less than TS1 (P<0.01). When a randomly picked cohort of the snails was tested 3 h later (3 h MT; N=27) memory was exhibited. That is, there was no significant difference in the number of attempted openings between 3 h MT and TS2 (P>0.05) while the number of attempted openings in 3 h MT was significantly less than in TS1 (P<0.01). When the remaining snails (24 h MT; N=128), were tested for LTM 24 h after TS2, memory was also shown to be present. Thus there w s no significant difference observed between 24 h MT and TS2 (P>0.05) and the number of attempted openings in 24 h MT was significantly less than the number in TS1 (P<0.01). Thus, the LTM-training procedure results in memory that persists for at least 24 h and can also be observed at 3 h (Fig. 1B).
Yoked control snails (to the LTM training procedure) received a memory test either 3 h or 24 h after TS2 (3 h Yoked MT; N=27; 24 h Yoked MT; N=28, respectively). Memory was not demonstrated in either session. We found that the number of attempted pneumostome openings in both MT sessions were not statistically different from TS1 (P>0.05), but were significantly greater than TS2 (P<0.01). Thus, two training sessions of non-contingent tactile stimuli to the pneumostome area with a 1 h interval between sessions did not result in LTM. We also made between-group comparisons of the response in MT in the yoked control and LTM operantly conditioned snails. We found that the number of attempted pneumostome openings of yoked control snails in Yoked 3 h MT and Yoked 24 h MT were significantly greater than (P<0.01) either the 3 h MT and 24 h MT sessions, respectively, of the LTM operantly trained snails.
We thus conclude that: (1) ITM and LTM can be differentially produced by altering the interval between training sessions; and (2) Lymnaea have the capacity of being operantly conditioned (i.e. associative learning) and forming LTM.
A second bout of ITM-training 24 h later causes LTM
We hypothesized that when we tested snails subjected to the ITM-training
procedure for memory 24 h after TS2 (unsuccessfully,
Fig. 1A) that we nonetheless
caused the activation of a `molecular memory trace' in neurons necessary for
memory formation (e.g. RPeD1). We further hypothesized that this residual
molecular memory trace could serve as a foundation upon which a second bout of
ITM-training could produce LTM (i.e. there would be memory in a MT 24 h after
the second bout of ITM-training).
We therefore subjected a naïve cohort of snails (N=37; Fig. 2A) to the ITM-training schedule on 2 consecutive days. Learning occurred on both days (ANOVA(36,5)=20.0, P<0.0001); that is the number of attempted pneumostome openings in TS2 and TS4 were significantly less than the number in TS1 and TS3, respectively (P<0.01 in both cases). Two findings emerged from this experiment. The first finding, as expected, was that there was no memory on TS3. That is, the number of attempted pneumostome openings in TS3 was significantly greater than the number in TS2 (P<0.01), indicating that LTM was not formed. The second finding was that when we tested these snails for memory 24 h after a second bout of ITM-training (Day 3 MT), LTM was evident. That is, when memory was tested 24 h after TS4 (Day 3 MT), the number of attempted pneumostome openings was not statistically different from the number in TS4 (P>0.05), but was significantly different from the number in both TS1 and TS3, respectively (P<0.01 in both comparisons). Thus, the criteria for LTM were met.
Before we could conclude that there was a residual molecular memory trace present in neurons that could serve as a foundation on which to build a LTM memory with further ITM-training (i.e. TS3 and TS4), we had to perform a number of control experiments. The first was a yoked control experiment and a second was to increase the interval between the two ITM-training bouts from 24 h to 48 h.
To show that the LTM observed on Day 3 was not just the result of 2 days of receiving tactile stimuli, a yoked control procedure was used. When we subjected these yoked control snails (Day 3 Yoked; N=37) to a MT Session 24 h after TS4 we found that the number of attempted openings was not statistically different from either TS1 or TS3 of operantly conditioned snails (P>0.05), but was significantly different from TS4 (P<0.01). Most importantly, the number of attempted openings of yoked control snails in Yoked MT was significantly greater than the number of attempted openings in Day 3 MT (P<0.01) of operantly trained snails given the ITM-training procedure on two consecutive days. Thus, 2 consecutive days of non-contingent tactile stimuli (i.e. the yoked control procedure) to the pneumostome did not result in a change in aerial respiratory behaviour (i.e. memory was not observed).
We next imposed a 48 h interval between the two ITM-training bouts (Fig. 2B). Learning occurred on both days (ANOVA(50,4)=27.2, P<0.0001). However, when we tested snails (N=77) for memory 24 h after TS4 we found that the criteria for memory were not met. That is, the number of attempted openings in MT was significantly greater than in TS4 (P<0.01) and was not significantly different from the number in either TS1 or TS3 (P>0.05 for both comparisons). We interpret these data in the following manner. The imposition of a 48 h interval between the two training bouts was sufficient to ensure that there was no vestige of a `residual' memory trace on which to build an LTM memory. Thus we conclude that contingent presentation of a tactile stimulus to the pneumostome utilizing the ITM-training procedure is sufficient to result in a memory that lasts at least 24 h (i.e. LTM) if the second ITM-training sequence occurs within 24 h of the first ITM-training bout.
Cooling during the consolidation period blocks the residual memory trace
We have previously demonstrated that cooling snails immediately (i.e.
within 30 s) after the last training session is sufficient to block the
formation of either ITM or LTM (Sangha et
al., 2003a). We therefore hypothesized that if we cooled snails
immediately after TS2 on Day 1 there would be no vestige of the residual
memory trace for the second bout of ITM-training given on Day 2 to build on,
which would result in LTM. In Fig.
3A, a cohort of snails (N=24) received the ITM-training
procedure on Day 1. Immediately after TS2 (i.e. within 30 s), snails were
placed in water at 4°C for 2 h and then transferred to eumoxic room
temperature water for 22 h. On Day 2, they again received the ITM-training
procedure. Learning occurred on both days (ANOVA(23,4)=7.76,
P<0.0001); that is the number of attempted pneumostome openings in
TS2 and TS4 were significantly less than the number in TS1 and TS3,
respectively (P<0.01 in both cases). When memory (MT) was tested
24 h after TS4 on Day 3, it was not observed. That is, the number of attempted
pneumostome openings in MT was significantly greater than the number in TS4
(P<0.01). Additionally, there was no significant difference
between the number of attempted openings in MT and TS1 or TS3
(P>0.05). Thus LTM was not observed when snails were immediately
cooled after TS2.
To control for the possible adverse effects of cooling on LTM memory formation, we performed the same experiment (Fig. 3B) as in Fig. 3A except the cooling was administered after the consolidation process had occurred (i.e. snails were cooled 2 h after TS2). The snails (N=22) were treated as in A, except that cooling was delayed for 2 h after TS2. Following their 2 h exposure to 4°C water they were returned to room temperature eumoxic pondwater for 20 h. Learning occurred on both days (ANOVA(21,4)=10.9, P<0.0001). When we tested these snails for LTM 24 h after TS4 we found that LTM was present. That is, there was no significant difference between the number of attempted pneumostome openings in MT and TS4 (P>0.05), while there was a significant difference in the number between MT vs TS1 and TS3, respectively (P<0.01). We conclude that it is possible to interfere with the ITM consolidation process by cooling snails such that there is no memory trace to build an LTM memory on with subsequent ITM-training. Thus, LTM is not just the result of two consecutive days of ITM-training. Moreover the data are consistent with the hypothesis that a second bout of ITM-training is sufficient to produce LTM if there is a residual memory trace present in neurons that are necessary for LTM formation.
Cooling extends the memory trace and prolongs the interval between training
While immediate cooling following TS2 is able to block the consolidation
process and thus prevent memory formation, cooling applied after consolidation
paradoxically extends memory persistence
(Sangha et al., 2003d). We
therefore hypothesized that if we cooled snails for a period of 48 h after
ITM-consolidation, a residual memory trace would still be present so that a
second bout of ITM-training would result in LTM. Snails
(Fig. 4) first received
ITM-training on Day 1, and 2 h after TS2 they were placed in 4°C pondwater
for 48 h. They then received a second bout of ITM-training (TS3, TS4).
Learning occurred on both days (ANOVA(37,4)=20.4;
P<0.0001); that is, the number of attempted pneumostome openings
in TS2 and TS4 were significantly less than the number in TS1 and TS3,
respectively (P<0.01). When memory was tested 24 h after TS4 (MT),
there was no significant difference between the number of attempted openings
in MT and TS4 (P>0.05) while the number of attempted openings in
MT was significantly less than both TS1 and TS3 (P<0.01). Thus
memory was present. Thus cooling can preserve the residual memory trace so
that a second bout of ITM-training 48 h after TS2 leads to LTM.
ITM-training followed by another ITM-training in a different context
Context-specific learning, memory formation and extinction have all been
demonstrated in Lymnaea (Haney and
Lukowiak, 2001; McComb et al.,
2002
). We therefore hypothesized that a second bout of
ITM-training would not result in the establishment of LTM, if the second bout
of ITM-training was performed in a different context. Our reasoning was that
the `two different context ITM-training procedures' would result in two
different associations (i.e. one ITM-memory for the standard context and
another, different ITM-memory for the carrot context). Therefore, when we
trained snails using the `different context ITM-training' on Day 2 we would
not be building on the residual memory trace that had been encoded in neurons
for the standard context memory. Since no `foundation' would be present, LTM
for the standard context would not be observed 24 h later. As shown in
Fig. 5A this is exactly what we
found. A cohort of naïve snails (N=26) received ITM-training in
the standard context. On Day 2, they again received ITM-training but this time
in a different context (i.e. carrot; black shading indicates a carrot context
was used and no shading indicates the standard context was used). Learning
occurred on both days (ANOVA(25,4)=22.0, P<0.0001).
However, when we tested for LTM in the standard context 24 h after TS4 (Day 3
MT) LTM was not present. That is, the number of attempted openings in MT was
significantly greater than TS2 (P<0.01) and the number of
attempted openings in MT was not significantly different from TS1
(P>0.05). Although there was learning in both contexts, there was
no LTM.
We also tested for the possibility that training in the standard context on Day 1 and then training in the carrot context on Day 2 produces a LTM on Day 3 for the carrot context. As shown in Fig. 5B, a cohort of naïve snails (N=27) received ITM-training in the standard context on Day 1. On Day 2, they received ITM-training in the carrot context. Learning occurred on both days (ANOVA(26,4)=14.5, P<0.0001). When we tested for memory (MT) in the carrot context 24 h after TS4, LTM was not present. That is, the number of attempted openings in MT was significantly greater than in TS4 (P<0.01) while there was no significant difference between MT and TS3 (P>0.05).
Similar results were found if we reversed the presentation of the contexts used for ITM-training (Fig. 5C; i.e. carrot first, standard second). While learning occurred on both days (ANOVA(27,4)=16.9, P<0.0001) LTM was not shown. That is the number of attempted openings in MT was significantly greater than in TS2 (P<0.01) and there was no significant difference between MT and TS1 (P>0.05). All these data demonstrate that it was not simply the `extra' application of tactile stimuli, even contingent stimuli that lead to LTM formation. Rather, the `extra' training must be within the same context in order to build upon a residual memory trace.
ITM-training followed by extinction training and further ITM-training
We have demonstrated in Lymnaea that extinction training results
in a new memory that co-exists with but occludes the old memory
(Sangha et al., 2003c). We
therefore hypothesized that the imposition of extinction training between the
two ITM-training bouts would prevent the subsequent formation of LTM. We
reasoned that extinction training would occlude or make inaccessible the
memory trace produced by the first bout of ITM-training and thus the second
bout of ITM-training (following the extinction training) would not have a
foundation (i.e. residual memory trace) on which to build to produce LTM. That
is, LTM would not be observable in MT 24 h after Session 4.
A cohort of naïve snails (Fig. 6; N=32) on Day 1 received ITM-training. On Day 2, these snails first received a bout of extinction training (i.e. two 0.5 h sessions separated by a 0.5 h interval, in which they were placed in the hypoxic environment but did not receive the reinforcing stimulus when they opened their pneumostome; E1 and E2). 3 h later they received a second bout of ITM-training. Learning occurred in both ITM-training sessions (ANOVA(31,4)=10.6, P<0.0001). However, when we tested for memory 24 h after TS4, LTM was not present. That is, the number of attempted openings in MT was significantly greater than in TS4 (P<0.01) and there was no significant difference between MT and TS1 or TS3 (P>0.05). Thus, the interposition of extinction training prevented the second bout of ITM-training to produce LTM.
We have previously shown that extinction is also context-dependent
(McComb et al., 2002). We
reasoned that subjecting snails to a `different context extinction training
procedure' (i.e. `carrot-extinction') would not occlude the memory trace for
the standard context. This experiment (Fig.
6B) would also serve to control for the possible time effects of
the interposed extinction sessions on the formation of LTM. The experiment
(N=26) followed the same protocol as in A except that now the
extinction training was performed in the `carrot-context' (black shaded E1 and
E2 bars). Learning occurred on both days (ANOVA(25,4)=23.3,
P<0.0001). However, when we tested for memory 24 h after TS4, LTM
was present. That is, the number of attempted openings in MT was not
significantly greater than in TS4 (P>0.05) but was significantly
less than either TS1 or TS3 (P<0.01). Thus, the interposition of
extinction training in a different context did not prevent the formation of
LTM.
We repeated the experiments shown in Fig. 6 this time using the carrot context for operant conditioning training and either the `carrot' or `standard context' for the extinction training (Fig. 7), and obtained similar results. If a different context extinction training procedure was used (i.e. the standard context) LTM was formed (Fig. 7A), while the interposition of extinction training in the same context (i.e. carrot) prevented LTM formation (Fig. 7B).
A cohort of naïve snails (N=26) received the ITM-training procedure in the carrot-context on Day 1. On Day 2, they received extinction training (E1 and E2, white bars) in the standard context, followed 3 h later by the ITM-training procedure in the carrot context (black bars). Learning occurred on both days (ANOVA(25,4)=17.2, P<0.0001). LTM was present when the interposed extinction training was performed in the standard context [Fig. 7A; i.e. there was no significant difference between the number of attempted openings in MT and TS4 (P>0.05) while MT was significantly less than both TS1 and TS3, respectively (P<0.01)]. By contrast, LTM was not present if the interposed extinction was performed in the carrot context [Fig. 7B; i.e. the number of attempted openings in MT was significantly different from TS4 (P<0.01) and was not significantly different from either TS1 or TS3 (P>0.05)].
Together the results in Figs 6 and 7 show that it is not simply a second bout of ITM-training 24 h after the first ITM-training sessions that results in LTM formation. The residual memory trace, which can be occluded by extinction training in the same but not a different context, has to be present in order for LTM to be produced.
The residual memory trace can lead to LTM formation only if somata of RPeD1 are present
Previously we have shown that the somatata of RPeD1 must be present for LTM
formation, extinction and memory reconsolidation
(Scheibenstock et al., 2002;
Sangha et al.,
2003b
,c
).
Thus, we hypothesized that ablation of RPeD1's somata before ITM operant
conditioning training would prevent a residual memory trace from forming a
foundation for LTM with subsequent ITM-training. That is, because LTM requires
gene transcription and since somata ablation removes the nucleus and thus the
genes, LTM formation should not occur. These data are shown in
Fig. 8. A cohort of naïve
snails had the somata of either RPeD1 (N=19) or LPeD1 (N=9)
ablated 2 days before ITM-training. All snails received two bouts of
ITM-training 24 h apart. Learning occurred on both days
(ANOVA(36,5)=17.7, P<0.0001). When memory was tested
(MT) 24 h after TS4, we found that snails with RPeD1 somata ablated, behaved
differently than those with LPeD1 somata ablated. In the LPeD1 cohort (L-MT)
there was LTM. That is, the number of attempted pneumostome openings in L-MT
was not significantly different than the number in TS4 (P>0.05)
but was significantly less than in TS1 and TS3 (P<0.01). On the
other hand, LTM was not present in the RPeD1 somata-ablated cohort (R-MT).
That is, the number of attempted pneumostome openings in R-MT was
significantly greater than the number in TS4 (P<0.01) and was not
significantly different than the number in either TS1 or TS3
(P>0.05). Thus, we conclude that the somata of RPeD1 must be
present in order for the second series of ITM-training to produce LTM.
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Discussion |
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We show here that following an ITM-training procedure the attempt to retrieve the memory 24 h later (futile at the behavioural level) causes a `residual molecular memory trace' in a neuron, RPeD1, necessary for LTM formation. Activation of this trace is sufficient to enable a second bout of ITM-training to produce LTM. We call this phenomenon `memory boosting'. However, memory boosting only occurs if: (1) RPeD1's somata are present; (2) the second bout of ITM-training occurs within 24 h of the first series; (3) the initial learning undergoes consolidation into ITM; and (4) the second bout of ITM-training occurs in the same context as the first. Finally, extinction training in the same context interposed between the two bouts of ITM-training is capable of occluding or preventing the residual memory trace from being activated and thus prevents LTM formation. Alternatively, perhaps during extinction the residual memory trace is being activated and altered to favor a CS-no US association (non-reinforced association).
These data are all consistent with the hypotheses that: (1) memory exists in either an active (labile) or an inactive (stabile) state and that following its activation it returns to the stabile state in a transcription- and translation-dependent manner; (2) retrieval of ITM causes activation of a molecular memory trace which can in some circumstances form a foundation in which a second ITM-training bout causes LTM to be formed; and (3) the molecular processes underlying LTM build on the molecular processes that cause ITM.
The primary question asked here was `Does the attempted retrieval of an ITM
memory, even if behaviourally memory is not apparent, result in the formation
of a molecular memory trace?' We earlier showed that previous ITM-training
potentiates the duration of LTM produced by subsequent LTM-training,
hypothesizing that potentiation was due to a facilitating priming effect of
the residual ITM trace (Smyth et al.,
2002). We found in the present work that activating the ITM 24 h,
but not 48 h, after the last ITM-training session was sufficient to allow a
second bout of ITM-training to produce LTM
(Fig. 2). Neither yoked
controls nor snails that received ITM-training in two different contexts 24 h
apart subsequently exhibited LTM. Moreover, if the ITM consolidation process
(i.e. the molecular memory trace) was blocked by immediate cooling, a second
bout of ITM-training failed to produce LTM. Thus, LTM formation following a
second bout of ITM-training was not just the result of another session of
ITM-training. To be successful in producing LTM, the second bout of
ITM-training had to build on the presence of a residual memory trace created
by the initial ITM-training bout. The absence of LTM found in the yoked
control snails further shows that it is only contingent reinforcement using an
ITM-specific training procedure that leads to LTM formation.
However, a second bout of contingent ITM-training was not by itself
sufficient to cause LTM formation; the second bout had to be in the same
context as the first ITM-training. Snails show context-dependent memory
(Haney and Lukowiak, 2001),
thus when a different context training regimen is used in the second bout of
ITM-training, LTM is not produced because there is no residual memory trace
for the new context to build upon. The hypothesized residual memory trace
could also be `interfered with' in a number of ways. (1) Increasing the time
interval to 48 h between ITM-training bouts, (2) extinction training in the
same context between training bouts, and (3) cooling to 4°C immediately
after Session 2 to prevent ITM formation and thus a molecular memory
trace.
We have previously shown that the immediate cooling of snails to 4°C
following training blocks the ITM consolidation process
(Sangha et al., 2003d). Thus
we reasoned that the immediate cooling of the snails would block the formation
of the `ITM molecular memory trace' and thus prevent the second bout of
ITM-training from producing LTM. Cooling, however, was only effective in
blocking the production of LTM by the second bout of ITM-training if given
immediately after TS2. If applied 2 h after TS2, cooling had no interfering
effect. In fact, if cooling is applied after consolidation it prolongs the
persistence of the ITM molecular memory trace
(Sangha et al., 2003d
) and
thus extends the effective time interval between the two ITM-training bouts
that lead to the formation of LTM. We have not yet determined how long we can
extend the persistence of the residual memory trace by cooling. We are also
uncertain what cellular processes cause the ITM molecular memory trace to
become ineffective as a foundation for a second bout of ITM-training to build
upon to produce LTM, if the interval between training bouts is increased from
24 h to 48 h. One hypothesis is that interfering behavioural events (e.g.
spontaneous aerial respiration) that occur without reinforcement result in a
`spontaneous extinction' memory that occludes the memory trace. We are
currently attempting to test this hypothesis directly.
We also asked whether the LTM generated by the second series of
ITM-training is dependent on altered gene activity (i.e. transcription). In
Lymnaea, as in other organisms, LTM is dependent on transcription and
translation (Dudai, 2002b;
Scheibenstock et al., 2002
;
Sangha et al., 2003a
). A major
advantage of the Lymnaea model system is that it is possible to
surgically remove the somata of RPeD1 in an otherwise intact naïve snail,
and show that while learning and ITM occur, LTM formation does not because
there is no nucleus (i.e. no genes;
Scheibenstock et al., 2002
).
We found in RPeD1 somata-less snails that a second series of ITM-training did
not result in LTM. The translation of proteins necessary for ITM in these
preparations occurs extra-somally, but the second bout of ITM-training cannot
result in LTM because the nucleus is absent and the transcription of mRNAs
necessary for LTM cannot transpire. Thus, we conclude that the LTM we observe
following the second bout of ITM-training is dependent on transcription in
RPeD1 in addition to translation of new proteins.
Our working hypothesis is that LTM formation is at least a two-step serial
process. The first step parallels ITM formation and only requires new protein
synthesis, which may occur extra-somally
(Scheibenstock et al., 2002;
Spencer et al., 2002
;
van Minnen et al., 1997
).
These new proteins may serve to mark the site for subsequent events necessary
for LTM formation, as suggested in Aplysia
(Martin et al., 1997
). The
second phase of LTM formation requires the transcription of genes, but may not
involve translation of those mRNA transcripts within the somata. Ultimately
these new proteins arrive at the ITM site of encodement (e.g. a presynaptic
terminal) to create LTM. Previously we found that LTM was only observed if we
employed a training regimen that consisted of a single 1 h training session or
had a 1 h interval between training sessions
(Lukowiak et al., 2000
; Sangha
et al.,
2003a
,d
).
We hypothesize that the shorter interval (0.5 h vs 1.0 h) between
training sessions does not promote the downregulation of the suppressive cAMP
response element binding protein (CREB) isoform, and therefore the altered
gene activity (i.e. transcription) necessary for LTM is not initiated. The
ratio of CREB activator to repressor isoform has been considered as a
`molecular switch' to initiate the processes that cause LTM formation
(Sutton et al., 2002
). Thus,
intervals or events that alter the ratio in favor of the activator isoform
would lead to LTM formation. However, a recent report suggests that it is the
downregulation of the suppressor isoform of CREB that is the most important
factor initiating the molecular cascade leading to LTM formation
(Perazzona et al., 2004
). Here
we suggest that the first bout of ITM-training results in the establishment of
a memory trace in RPeD1 (and in other neurons necessary for LTM formation),
permitting a subsequent ITM-training bout to induce the necessary
transcription factors for LTM even if the behavioural manifestation of memory
was not apparent. Hence, our use of the terms `residual memory-trace' and
`memory boost' for the phenomena described here. Our findings are similar in
this respect to the reports that the prior induction of molecular factors
necessary for LTM allowed training procedures, which characteristically do not
produce LTM, to cause LTM (e.g.
Müller, 2000
;
Yin et al., 1995
; but see
Perazzona et al., 2004
).
A two-step LTM formation process, with the first step matching the
processes that underlie ITM formation, is consistent with other reports. For
example, in the crab Chasmagnathus, two phases of cAMP-dependent
protein kinase (PKA) activation are needed for learning to be consolidated
into LTM. The first phase occurs during training and another phase occurs 48 h
after training. This suggests that PKA may play a role in establishing a
memory trace and its activation leads to LTM
(Locatelli et al., 2002).
Using contextual fear conditioning with weak training in mice, researchers
have been able to show that LTM consolidation also has two phases that are
sensitive to PKA and protein synthesis inhibitors
(Bourtchouladze et al., 1998
).
There has been debate about the interrelated nature of the different forms of
memory, as to whether they occur in parallel
(Crow et al., 2003
;
DeZazzo and Tully, 1995
;
Emptage and Carew, 1993
;
Hegde et al., 1997
;
Izquierdo et al., 2002
;
Mauelshagen et al., 1996
;
Tully et al., 1994
) or in
series (Ghirardi et al., 1995
;
Riedel, 1999
;
Sutton et al., 2001
;
Zhao et al., 1995
). Conjoint
serial and parallel processing of memories are also possible
(Sutton et al., 2002
). Our
data clearly show that the processes that cause ITM formation have the
capacity to permit LTM formation following a second series of ITM-training
even if the behavioural phenotype of memory is absent. Nonetheless, the
processes underlying LTM formation are clearly different from those underlying
ITM, as the somata of RPeD1 are necessary for our training regimen to produce
LTM but not ITM. However, the establishment of a memory trace by an
ITM-training protocol can lay a foundation for subsequent training to produce
LTM. This suggests to us that a molecular memory trace is laid down as a
consequence of ITM activation, which serves as a permissive substrate,
sufficient to allow the necessary transcription and translation that is causal
for LTM formation.
![]() |
Acknowledgments |
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