Associative learning and memory in Lymnaea stagnalis: how well do they remember?
Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada, T2N 4N1
* Author for correspondence (e-mail: lukowiak{at}ucalgary.ca)
Accepted 18 March 2003
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Summary |
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Key words: associative learning, operant conditioning, long-term memory, Lymnaea, invertebrate learning, invertebrate memory
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Molluscan model systems for the study of learning and memory |
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Some of the first studies in the `modern' search for the engram took the
comparative physiological and psychological approach. For example, in the
early 1900s, Piéron
(1911), Dawson
(1911
) and Thompson
(1917
) used snails in attempts
to discover how learning occurred. However, these studies were, by and large,
forgotten, and it really was not until tests that were more natural and
meaningful to the organisms were used that a full appreciation of the learning
capabilities of gastropods became apparent.
The 1960s saw a burst of activity that is still evident today to study how the nervous system is `wired-up' to mediate specific behaviours and how changes in the behaviour brought about by training procedures are reflected or caused (the real goal) by changes in the activity of specific neurons. Thus, preparations such as Aplysia, Hermissenda, Pleurobranchaea and Tritonia gained popularity. Eric Kandel, with his share of the Nobel Prize for Medicine and Physiology in 2000, attained one of the pinnacles of science in part by using Aplysia.
All the species mentioned above are marine creatures, and, with few exceptions (e.g. the land slug Limax and the land snail Helix), conventional wisdom from the 1970s through to the early 1990s was that the freshwater gastropods just did not have the `right stuff' to be used in the quest for the Holy Grail. This review will focus on why that conventional wisdom was incorrect and why using Lymnaea might just be a very useful path to take to grasp the Grail in hand.
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Lymnaea as a model system for neurobiology |
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The Audersirks in the early 1980s
(Alexander et al., 1982) were
probably the first to really appreciate that Lymnaea had the capacity
for associative learning, but, for reasons not understood by us, this path was
not taken up by others until very recently. Now, at least three different
laboratories in the UK and Japan (e.g. Ito
et al., 1999
; Ono et al.,
2002
; Staras et al.,
1999
) have embarked on studies examining associative learning and
its long-term memory in Lymnaea, following the pathway opened up by
the Audersirk's pioneering studies. Again, readers are directed to a recent
review on these studies (Benjamin et al.,
2000
). If we might editorialize here, we think that the feeding
circuitry is just too complicated for investigators to show that changes in
neuronal activity are causal for memory, and that is why we did not go down
that particular pathway in search of the engram. However, we could be
wrong.
Our main reason for moving from Aplysia to Lymnaea to
study learning and memory was the finding that a three-neuron network drove an
important homeostatic function in Lymnaea, aerial respiratory
behaviour. By using a combination of cell culture and in vivo
transplantation techniques, Syed et al.
(1990,
1992
) were able to directly
demonstrate both the sufficiency and necessity of the three-neuron central
pattern generator (CPG) to drive aerial respiratory behaviour. Few, if any,
other neural circuits have been described that meet both the sufficiency and
necessity tests. If this behaviour could undergo associative learning and its
consolidation into long-lasting memory [intermediate term memory (ITM),
lasting 34 h, and long-term memory (LTM), lasting longer than 5 h] then
we would have a preparation where we might be able to study the causal
neuronal mechanisms of learning and memory directly.
Lymnaea are bi-modal breathers, obtaining oxygen either through cutaneous or aerial respiration. Typically, in eumoxic conditions, cutaneous respiration dominates and aerial respiration seldom occurs. To `motivate' Lymnaea, we make the pond water hypoxic by bubbling N2 through the training beaker for 20 min (Fig. 1) and, in these hypoxic conditions, aerial respiration predominates. Briefly, to operantly condition (a form of associative learning) the snails, we apply a relatively weak tactile stimulus (a sharpened wooden applicator is used) to their pneumostome, the respiratory orifice, each time they attempt to open it. This negative reinforcement causes the snail to close its pneumostome but does not cause the animal to withdraw its foot and mantle area (i.e. the whole-animal withdrawal response). Pneumostome stimulation also does not cause the snails to sink to the bottom of the beaker.
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We found that Lymnaea have the ability to be operantly conditioned
(Lukowiak et al., 1996) and
that this learning undergoes consolidation into either ITM or LTM (Lukowiak et
al., 2000). Depending on the training procedure used, LTM persistence could be
as long as one month (Lukowiak et al.,
1998
). All the necessary controls to show that this change of
behaviour is a bona fide example of associative learning have been
performed. We are now at the point where we can begin to determine the causal
neuronal basis of learning and its consolidation into memory.
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Definitions of learning and memory |
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Where is the non-declarative memory of operant conditioning formed and stored? |
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Molluscan neurons are unipolar. That is, they have a single process
emerging from the soma (properly known as the primary neurite, but more often
than not called the axon) and this is where the majority of synaptic
interactions occur (Bullock and Horridge,
1965; Kandel,
1979
; Fig. 2).
Moreover, molluscan neurons possess an ability to function `normally' for long
periods of time without their soma. It is therefore possible to surgically
remove the soma of RPeD1, leaving behind a functional primary neurite
sufficient to mediate normal neuronal activity and aerial respiratory
behaviour (Haque, 1999
;
Scheibenstock et al., 2002
).
The isolated primary neurites (i.e. without the soma) of Lymnaea are
also capable of de novo protein synthesis of injected novel mRNA, and
the newly synthesized protein can be functionally integrated into the membrane
(van Minnen et al., 1997
;
Martin et al., 1997
;
Spencer et al., 2000
). Because
LTM is dependent on both altered gene activity and new protein synthesis
(Kandel, 2001
), our working
hypothesis is that, if RPeD1 is a site for either the formation or storage of
LTM, removal of its soma, and thus its nucleus, before operant
conditioning training should prevent the formation of LTM. We therefore
determined whether selective ablation of the RPeD1 soma (i.e. leaving intact
its primary neurite) would result in an inability to encode or access LTM of
the operantly conditioned aerial respiratory behaviour.
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We found that RPeD1 soma-ablated snails
(Fig. 3) had the ability to
associatively learn and could form ITM (which is dependent on new protein
synthesis but not altered gene activity remember, an isolated neurite
is still capable of de novo protein synthesis) but they could not
consolidate the learning into LTM
(Scheibenstock et al., 2002).
Based on these data, it would appear that RPeD1 is a necessary site for LTM
formation.
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However, it was possible that in RPeD1 soma-ablated snails, LTM was encoded
but either could not be accessed or retrieved without the soma. We therefore
ablated the soma of RPeD1 after learning and memory consolidation had
occurred (N=10) and found that LTM was present
(Fig. 4). As an extra control,
we challenged these snails with a `different-context'
(Haney and Lukowiak, 2001)
test. The snails responded as they did in the initial training session (when
the soma of RPeD1 was present). Thus, RPeD1 soma-ablated snails still had the
ability to access or retrieve a previously encoded memory. Thus, we concluded
that RPeD1 is a site for LTM formation and storage and we think this
is the first instance where a single neuron has been shown to be a necessary
site for LTM formation. Whether it is the only necessary site for this memory
remains to be determined.
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Behavioural learning and memory: the assignment of marks |
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We have shown above that a single neuron is a site of memory formation and storage. But the inquisitive reader might ask: `how robust is the learning, and how robust is the memory in our model system?'
The approach we have taken to answer this question is to combine all of our
data obtained using one specific training procedure and then examine this
population as to how well or how poorly each individual learned and
remembered. Since we teach and give examinations to students in a university,
it seemed appropriate to give `marks' or to `grade' each snail and then view
the performance level of `the class as a whole'. The data set below
(N=1500 snails) uses the following marking system: a mark of `A'
is assigned if the last training session is greater than a 50% reduction
compared with the initial training session; a mark of `B' is assigned if the
last training session is a 3550% reduction of the initial session; a
mark of `C' is assigned is there is a 2035% decrease; and an `F' is
assigned if the decrease is less than 20%.
Fig. 5A shows the overall learning and memory curves for snails trained using the procedure of two 45-min training sessions (each session separated by a 1-h interval) and testing for memory 1 day later. As a group, there was a 46% decrease in the number of attempted pneumostome openings in Session 2 compared with Session 1. An analysis of variance (ANOVA) showed that there was a significant effect (P=0.0001, N=1490) of training on the number of attempted pneumostome openings (i.e. learning occurred). Overall, the class mark assigned would be a B+. Not too bad! As is typical (sad to say) in the overwhelming majority of schools (including universities), following training (i.e. lectures) a test is given (i.e. memory test) and marks assigned based on performance. We tested 490 snails for memory 24 h later (the remaining 1000 snails were used for other experiments and were not tested for 24 h memory, thus they are not included here) and found that the criteria for memory were met. As a class, we would have assigned a mark of A, since, as a group, there was greater than a 50% decrease in the number of attempted pneumostome openings compared with Session 1. Most teachers and school boards we know would be extremely satisfied with this overall class result. When we assigned individual grades based on the memory test, we found the following grade distribution (Fig. 5B): 57% of snails received an A, 20% a B; 11% a C and 12% an F. Thus, the vast majority of our snails (77% received an A or a B) show very good learning and memory. We conclude that learning and memory in these snails is both reproducible and robust.
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What is also interesting to us is why one in 10 snails do not form memory. We do not know what is different about these snails. These `memory-challenged' snails can serve as a positive control when we begin to examine the molecular changes that cause memory to be formed and stored. We may also be able to give `remedial education' to these snails in the form of more training or training using a different procedure in an attempt to get them to a pass level. This may also allow us to determine what is different about their nervous systems compared with those of the other 90% of the snails.
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Where to next? |
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We may now be able to directly test in an identified single neuron which
genes are turned on or off, what proteins constitute the physical basis of
memory storage, and where within the cell these proteins are located.
Transcription factors analogous to those found in Aplysia (CREB1a,
CREB2 and C/EBP; Alberini et al.,
1994: Bartsch et al.,
1995
; Carew and Sutton,
2001
; Kandel,
2001
; Silva et al.,
1998
) have now been cloned in Lymnaea, and preliminary
data have shown them to be present in RPeD1
(Hatakeyama et al., 2002
;
Sadamoto et al., 2002
). Thus,
by taking genomic and proteomic approaches, we may be able to specify the
nature of the changes that encode memory in RPeD1.
Finally, although we have shown that RPeD1's soma is necessary for
LTM we have not shown that changes in it are sufficient for LTM. It
may be possible to directly determine if RPeD1's soma is both necessary and
sufficient for LTM by transplantation experiments (see
Syed et al., 1992). If RPeD1's
soma is both necessary and sufficient for LTM then transplantation of an
`educated' RPeD1 to a naïve snail would result in the recipient snail
exhibiting the behavioural phenotype of a trained snail with LTM.
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Concluding remarks |
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Will the engram be found? Possibly, but as in any quest there will be interesting and important diversions along the way that will grab our attention and make us rethink many of our accepted beliefs. The next few years will certainly bring lots of fun, but also frustration, as we again rediscover the axiom that `the more we really know the less we know about our subject'. It may be that all we will ever get are just more tantalizing glimpses of the Grail, which always appears to be just a little ahead and around the corner.
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