Canadian Association of Neurosciences Review: learning at a snail's pace.
ABSTRACT While learning and memory are related, they are distinct processes each with different forms of expression and underlying molecular mechanisms. An invertebrate model system, Lymnaea stagnalis, is used to study memory formation of a non-declarative memory. We have done so because: (1) We have discovered the neural circuit that mediates an interesting and tractable behaviour; (2) This behaviour can be operantly conditioned and intermediate-term and long-term memory can be demonstrated; and (3) It is possible to demonstrate that a single neuron in the model system is a necessary site of memory formation. This article reviews how Lymnaea has been used in the study of behavioural and molecular mechanisms underlying consolidation, reconsolidation, extinction and forgetting.
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ABSTRACT: Retinoic acid, a metabolite of vitamin A, is proposed to play an important role in vertebrate learning and memory, as well as hippocampal-dependent synaptic plasticity. However, it has not yet been determined whether retinoic acid plays a similar role in learning and memory in invertebrates. In this study, we report that retinoid signaling in the mollusc Lymnaea stagnalis, is required for long-term memory formation following operant conditioning of its aerial respiratory behaviour. Animals were exposed to inhibitors of the RALDH enzyme (which synthesizes retinoic acid), or various retinoid receptor antagonists. Following exposure to these inhibitors, neither learning nor intermediate-term memory (lasting 2 hours) was affected, but long-term memory formation (tested at either 24 or 72 hours) was inhibited. We next demonstrated that various retinoid receptor agonists promoted long-term memory formation. Using a training paradigm shown only to produce intermediate-term memory (lasting 2 hours, but not 24 hours) we found that exposure of animals to synthetic retinoids promoted memory formation that lasted up to 30 hours. These findings suggest that the role of retinoids in memory formation is ancient in origin, and that retinoid signaling is also important for the formation of implicit memories, in addition to its previously demonstrated role in hippocampal-dependent memories.Neurobiology of Learning and Memory 06/2014; · 3.33 Impact Factor
- Comparative Biochemistry and Physiology A-molecular & Integrative Physiology - COMP BIOCHEM PHYSIOL PT A. 01/2009; 153(2).
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ABSTRACT: The aerial respiratory behaviour of Lymnaea can be operantly conditioned to form a long-term memory (LTM) that will persist for >24hrs. LTM formation is dependent on altered gene activity and new protein synthesis, with the N-methyl-D-aspartate (NMDA) receptors, mitogen activated protein kinase (MAPK), and protein kinase C (PKC) pathways playing a critical role. LTM can also undergo extinction, whereby the original memory is temporarily masked by a new memory. Here we investigate if the formation of an extinction memory uses similar molecular pathways to those required for LTM formation. We find that the formation of the extinction memory can be blocked by inhibitors of NMDA receptors, PKC, and MAPK suggesting that extinction memory formation uses similar mechanisms to that of 'normal' memory formation.Neurobiology of Learning and Memory 12/2012; · 3.33 Impact Factor
CLASSIFICATIONS OF LEARNING AND MEMORY
The ability to learn and form a memory enables organisms to
adapt to a changing environment so as to be better able to
survive. Learning and memory are behavioural manifestations of
activity within individual neurons and neuronal circuits. While
learning and memory are interrelated, they are separate processes
each with different underlying molecular mechanisms and forms
of expression. Learning can be broadly defined as the acquisition
of a new behaviour, while memory is defined as the ability to
both store and recall the new information.
Learning and memory (or their correlates) have been studied
at behavioural, systems, neuronal, and sub-cellular levels in
organisms ranging from humans to worms. In this short review
we will focus on research in an invertebrate model system that
has provided insight into the underlying mechanisms of memory
formation. While an understanding of the causal mechanisms of
learning and memory formation in snails is of heuristic interest,
our main reason for employing such a molluscan model system
is to gain insight as to how learning and memory occur in us. It
appears that the ‘substrates’ for learning and memory have been
fairly conserved in all organisms throughout evolution.
ABSTRACT: While learning and memory are related, they are distinct processes each with different
forms of expression and underlying molecular mechanisms. An invertebrate model system, Lymnaea
stagnalis, is used to study memory formation of a non-declarative memory. We have done so because:
1) We have discovered the neural circuit that mediates an interesting and tractable behaviour; 2) This
behaviour can be operantly conditioned and intermediate-term and long-term memory can be
demonstrated; and 3) It is possible to demonstrate that a single neuron in the model system is a necessary
site of memory formation. This article reviews how Lymnaea has been used in the study of behavioural
and molecular mechanisms underlying consolidation, reconsolidation, extinction and forgetting.
RÉSUMÉ: L’apprentissage au pas d‘escargot. Bien que l’apprentissage et la mémoire soient deux fonctions
connexes, leurs processus sont distincts et chacun a des formes d’expression différentes et des mécanismes
moléculaires sous-jacents différents. Nous utilisons un système dans un modèle invertébré, la Lymnaea stagnalis,
pour étudier comment se forme une mémoire non déclarative. Nous avons utilisé ce modèle parce que : 1) Nous
avons découvert un circuit neural qui assure la médiation d’un comportement intéressant et observable; 2) Ce
comportement peut être conditionné en cours d’étude et la mémoire à moyen et à long terme peut être démontrée;
3) Il est possible de démontrer dans ce modèle qu’un seul neurone est nécessaire pour la formation de la mémoire.
Cet article revoit comment la Lymnaea a été utilisée pour étudier les mécanismes comportementaux et moléculaires
sous-jacents à la consolidation, à la reconsolidation, à l’extinction et à l’oubli.
Can. J. Neurol. Sci. 2006; 33: 347-356
THE CANADIAN JOURNAL OF NEUROLOGICAL SCIENCES347
CANADIAN ASSOCIATION OF
NEUROSCIENCES REVIEW: Learning at a
Kashif Parvez, David Rosenegger, Michael Orr, Kara Martens, Ken Lukowiak
From the Hotchkiss Brain Institute, University of Calgary, Calgary, AB, Canada.
RECEIVED MAY 11, 2006. ACCEPTED IN FINAL FORM AUGUST 18, 2006.
Reprint requests to: Ken Lukowiak, Hotchkiss Brain Institute, University of Calgary,
3330 Hospital Drive N.W., Calgary, Alberta, T2N 4N1, Canada.
Moreover, there are many experimental advantages (see below)
to using invertebrate model systems over mammalian
preparations. Thus, gaining an understanding of causal
mechanisms of memory formation in a snail may help us better
understand how memory systems function in higher organisms
such as in humans. Since this paper will focus primarily on how
memory is formed following learning we first need to describe
how memory is classified.
Memory encompasses a broad spectrum of sub-types. Studies
on humans have examined both declarative and non-declarative
forms of memory. Declarative (or explicit) memory is the
memory of facts and events, while non-declarative (implicit or
procedural) memory is the memory of ‘how’ to do things (e.g.
motor memory). However, there are a number of complexities
associated with declarative memory that have made it more
difficult to study. For example, declarative memories are initially
formed in different neuronal circuits in the brain than the ones
where they are ultimately stored. The distributed nature of
declarative memory makes it difficult to determine if an
unexpressed memory has been forgotten (i.e. no longer stored) or
is not accessible at a specific time (i.e. a failure in retrieval).
Moreover, whether a memory is no longer stored (i.e. forgotten)
or just not able to be accessed at a specific time is problematic.
These problems may be avoided by studying non-declarative
memory. Non-declarative memories are stored in the circuit that
mediates the behavior.1-3 Thus, we know ‘where’ the memory is
stored if we can determine which neurons are responsible for
generating the behavior. Since the memory is stored within the
circuit that mediates the behaviour, we eliminate inaccessibility
as an explanation for lack of memory expression.
Probably, the most basic and fundamental form of learning is
non-associative learning. Habituation and sensitization are two
well-known examples of non-associative learning. In the case of
habituation the animal learns and remembers not to respond to a
‘benign’ (i.e. harmless) stimulus that has been repeatedly
presented to it. The analysis of how such learning and memory
are encoded in neurons was greatly aided by the development of
a set of parametric characteristics of habituation that all animals
exhibited4and the development of model systems such as
Aplysia. Model organisms showed that habituation was similar in
intact animals and in reduced semi-intact preparations, which
allowed simultaneous study of behaviour and neurophysiology.5-7
Sensitization, on the other hand, can de defined as an increase in
response amplitude following the presentation of another distinct
non-contingent stimulus. Again, our understanding of how
sensitization is mediated at the neuronal level came from the
development of model systems where behaviour and neural
activity could be studied simultaneously.8Finally, with both
habituation and sensitization it was shown that long-lasting
memory was dependent on new protein synthesis and altered
A more complicated and important form of learning is
associative learning. Associative learning can itself be divided
into two types; classical conditioning and operant
conditioning.3,10,11In classical conditioning (i.e. Pavlovian
conditioning) a ‘neutral’ stimulus, known as the conditional
stimulus (CS) is temporally paired with a stimulus, known as the
unconditional stimulus (US), that inevitably elicits a response
(the unconditional response; UR). With repeated CS-US
pairings, but not US-CS pairings (backwards conditioning), the
CS comes to elicit the UR. That is, the animal learns that the CS
predicts the occurrence of the US. The most famous example of
this form of learning is that of Pavlov’s dogs who learned, after
a number of CS-US pairings, that the presentation of a tone,
signaled the presentation of a food-substance. Thus, the dogs
salivated (the UR) on hearing the tone (CS) even if the US (the
food-substance) was not presented.
In contrast to classical conditioning, operant conditioning
involves application of a reinforcement stimulus contingent upon
spontaneous performance of a specific behaviour.12-14With
repeated contingent presentation of the reinforcing stimulus the
occurrence of the behaviour changes.10The reinforcing stimulus
can result in either increasing the occurrence of the behaviour
(positive reinforcement) or decrease its occurrence (negative
reinforcement). In short, operant conditioning results in an
animal learning the consequence of its behaviour.
LYMNAEA STAGNALIS AS A MODEL SYSTEM
A vast variety of model systems ranging from invertebrates
Drosophila,22Hermissenda23) to non-human primates are
available to researchers for the study of memory formation. Each
model system comes with benefits and drawbacks. For example,
one benefit of using a rodent model system is that they have all
the neural structures hypothesized to play key roles in human
declarative memory formation and storage (e.g. the
hippocampus, amygdala and cortex). The equivalent brain
structures in rodents are also necessary for memory formation
and recall. However, in rodents as in humans, declarative
memory is stored ‘somewhere’ (i.e. in multiple areas) in the
cortex. Attempts have been made to directly study how and
where the various components of the memory (e.g. visual,
olfactory, auditory, emotional, etc) are stored and accessed.
There has been progress in localizing auditory fear conditioning
(i.e. classical conditioning) to the lateral amygdala.24,25Another
form of associative learning, conditioned taste aversion (CTA),
has been localized to the insular and prepiriform cortices and the
amygdala.26-28However, as of yet the necessary molecular
changes in neurons that cause memory formation in the
aforementioned structures have not been elucidated.
With the above caveats in mind we have taken to using an
invertebrate model system to study memory formation of a non-
declarative memory. We have done so because: 1) We have
discovered the neural circuit that mediates an interesting,
tractable behaviour; 2) This behaviour can be operantly
conditioned and long-lasting memory can be demonstrated; and
3) It is possible to demonstrate that a single neuron in the model
system is a necessary site of memory formation.
THE CANADIAN JOURNAL OF NEUROLOGICAL SCIENCES
respiration. Adult Lymnaea measure approximately 25 mm in shell
Lymnaea with its pneumostome open, performing aerial
Lymnaea stagnalis, a freshwater pond snail, is an ideal model
system for the study of learning and memory. There are many
advantages to studying learning and memory in this mollusc.
Lymnaea are bimodal breathers, meaning they can exchange O2
and CO2cutaneously (through their skin) or aerially through an
orifice (i.e. pneumostome; Jones, 1961; Lukowiak et al, 1996).
To aerially respire, the snail approaches the water surface, opens
its pneumostome and begins gas exchange. The snail must come
to the air-water interface for aerial respiration and it is easy to
observe this behaviour.29
Another advantage to Lymnaea is that it has a simple central
nervous system consisting of several thousand neurons. Some of
the unusually large neurons have been characterized and are
easily identified. Most importantly, a three neuron central pattern
generator (CPG) has been thoroughly characterized and proven
both necessary and sufficient for controlling aerial respiratory
behaviour in Lymnaea.30,31
CENTRAL PATTERN GENERATOR
The aerial respiratory CPG in Lymnaea consists of three
identified interneurons. Right Pedal Dorsal 1 (RPeD1) is a
dopaminergic interneuron that initiates the respiratory rhythm.
Right Pedal Dorsal 1 receives chemosensory inputs from the
pneumostome area32to modulate its activity. The remaining two
interneurons in the CPG are Visceral Dorsal 4 (VD4) and the
Input 3 Interneuron (IP3I). Visceral Dorsal 4 initiates
pneumostome closure by excitation of VK closer motor neurons.
Input 3 Interneuron initiates pneumostome opening by excitation
of VI/J opener motor neurons. All synaptic connections between
the 3 interneurons are monosynaptic. Right Pedal Dorsal 1
excites IP3I via a biphasic (inhibitory then excitatory) synaptic
connection. Input 3 Interneuron and VD4 have mutually
inhibitory synaptic connections as do RPeD1 and VD4. The
activities of VD4 and IP3I alternate to generate the rhythmic
opening and closing of the pneumostome (see Lukowiak33for a
description of how the emergent network properties cause
rhythmic activity in this circuit). The sufficiency and necessity of
the 3-neuron CPG in producing rhythmogenesis was tested by
reconstructing the CPG in vitro and performing cell killing and
Thus Lymnaea was demonstrated to possess an easily
observable behaviour (aerial respiration) that is driven by a
‘known’ neural network; it only remained to be shown that the
behaviour could exhibit both associative learning and long-term
memory (LTM). This was accomplished by our laboratory in
199615when it was shown that aerial respiratory behaviour could
be operantly conditioned and that LTM resulted following
OPERANT CONDITIONING OF AERIAL RESPIRATORY BEHAVIOUR
Operant conditioning of aerial respiratory behaviour in
Lymnaea is performed by placing snails into a beaker filled with
hypoxic pond-water (PW). The PW is made hypoxic by bubbling
N2gas through it for 20 minutes. Hypoxia is used to increase the
snails’drive to perform aerial respiration. In hypoxic conditions,
snails crawl to the air-water interface to open their pneumostome
and perform gas exchange with the external environment. When
they open their pneumostome we apply a negative reinforcement
in the form of a gentle tactile stimulus. This reinforcing stimulus
is of sufficient strength to cause pneumostome closure but does
not elicit the whole-animal withdrawal response. With repeated
application of the negative reinforcement, snails reduce the
number of attempted pneumostome openings when placed in the
We record the number of attempted pneumostome openings
during training sessions (TS1 and TS2) and a ‘savings-test’
(memory test; MT). We defined learning and memory
operationally.15,34,35Learning is present if the number of
attempted pneumostome openings in the last training session is
significantly less than the number of attempted pneumostome
openings in the first training session. In order for memory to be
present when ‘savings’ is tested (memory test, MT), two criteria
must be met: 1) the number of attempted pneumostome openings
in the MT is not significantly greater than that of the last training
session and 2) the number of attempted pneumostome openings
in the MT is significantly less than that of the first training
While the ‘savings-test’ is a suitable method to test for
memory, we also use a second memory assessment method (a
‘probe-test’). In the ‘probe-test’ method, we compare total
breathing time before and after training. Thus, memory is
assessed in the absence of the presentation of reinforcing stimuli.
There are both advantages and disadvantages to these two
memory assessment methods. The disadvantage of the ‘savings-
test method’is that reinforcing stimuli are applied during the MT.
LE JOURNAL CANADIEN DES SCIENCES NEUROLOGIQUES
Volume 33, No. 4 – November 2006
Figure 2: A schematic diagram illustrating the connectivity of the three
interneuron CPG that controls aerial respiration in Lymnaea. Included
in the diagram is the neuron that regulates the whole-body withdrawal
reflex (i.e. RPeD11) and the neurons responsible for the opening (i.e. VK
motor neurons) and closing (i.e. VI/J motor neurons) of the
Additional learning may occur, creating the possibility that
reduction in aerial respiration is due to this additional
reinforcement and not the memory phenotype. The advantage of
the ‘savings-test’ method is that, following the MT, another
different context-MT (see below) can be given showing that the
change in behaviour is a bona fide reflection of memory. The
advantage of the ‘probe-test’ method is reinforcing stimuli are
not applied during the MT, but there are two disadvantages. The
first is that the pre-training observation session (OBS1) may
trigger latent inhibition and thus affect memory formation. The
second disadvantage is the post-training observation session
(OBS2) is an extinction session. If another observation session in
a new context is given after the post-training observation session,
we cannot be sure if naïve respiratory levels are due to context
specificity or occlusion by extinction. In our model system, both
the ‘savings-test’method and ‘probe-test’method yield the same
conclusions and are valid methods for the assessment of
memory. Shown below is a diagram illustrating different training
procedures and procedures used to assess forgetting. Figure 3A
and 3B show the ‘massed’and ‘spaced’training procedures used
to produce ITM and LTM, respectively, as measured by a
savings-test method. Figure 3C and 3D show the ‘massed’ and
‘spaced’ training procedures used to produce ITM and LTM,
respectively, as measured by the probe-test method. Forgetting
can be assessed by giving snails memory tests at various time
points after training has ceased.
In addition to using two memory assessment methods to
ensure the validity of our experimental findings, we use an
important control, the yoked control procedure. A yoked control
snail is given a non-contingent stimulus (i.e. a stimulus when the
pneumostome is not open) every time a snail from the
experimental cohort receives a contingent stimulus (i.e. a
stimulus when pneumostome opening is initiated). As a result,
the experimental snail associatively learns and forms memory,
but the yoked control snail cannot as there is no association
present.15The yoked control procedure serves to control for
phenomena that may alter behaviour or internal state of the snail
such as: 1) stress associated with stimulus application, 2)
handling and 3) the training environment.
In addition to the yoked control, a ‘change of context’
procedure is used to ensure the behaviour of snails is not altered
by experimental conditions (i.e. stress, drug exposure, handling
or the training environment). When trained snails are placed in a
novel context, they respond as if they were naïve. That is, they
show more respiratory activity in the novel context than in the
trained context. Typically we train snails in hypoxic pond-water
that lacks the presence of an odorant (i.e. the standard context).
To create a ‘novel context’, N2is first bubbled through a flask
containing, for example, a slurry of carrot and water. The
resulting carrot-odorant N2is bubbled into the training beaker
prior to training or testing. For complete details see Haney and
As already mentioned, learning and memory, while related,
are two separate and distinct processes.1Behaviourally learning
is the acquisition of a new or altered behaviour, while memory
refers to the retention of what is learned. There are also
fundamental molecular differences between learning and
memory and these will be discussed. The process that leads to the
formation of memory following learning has come to be known
as consolidation. The concept of consolidation stems from
clinical observations made by Ribot in 1882. He noted that after
traumatic brain injury, recent memories were more likely to be
forgotten than memories of earlier events.37The hypothesis
postulated that after learning, memory existed in a fragile state
THE CANADIAN JOURNAL OF NEUROLOGICAL SCIENCES
Figure 3: A diagram illustrating training procedures used to produce
memory in Lymnaea and to measure the forgetting of memory. (A) To
produce ITM, snails are given a ‘massed’ training procedure that
consists of two 0.5h training sessions separated by a 0.5h rest interval
and then memory is tested for at 3h (3h MT) using the ‘savings-test’
method. If snails are given a MT 24h after training (24h MT), memory is
no longer present and hence forgotten. (B) To produce LTM, snails are
given a ‘spaced’ training procedure that consists of two 0.5h training
sessions separated by a 1h rest interval and then memory is tested for at
24h (24h MT) using the ‘savings-test’ method. The procedure to assess
forgetting is performed by giving snails MTs at 24h, 48h and 72h (24h
MT, 48h MT and 72h MT, respectively). (C) An alternative method to the
‘savings-test’ method is to use the ‘probe-test’ method. A breathing
observation (OBS1) is performed to measure mean total breathing time
of a cohort of naïve snails. Twenty four hours later, the snails are given
‘massed’ training (i.e. ITM training procedure; two 0.5h training
sessions separated by a 0.5h rest interval). Memory is tested at 3h by
performing another breathing observation (3h OBS2) to measure mean
total breathing times. If memory is tested using the ‘probe-test’method at
24h after training (24h OBS2), memory is no longer present and hence
forgotten. (D) Snails are treated as in C except they are given ‘spaced’
training (i.e. LTM training procedure; two 0.5h training sessions
separated by a 1h rest interval) and memory is tested at 24h. The
procedure to assess forgetting is performed by giving snails breathing
observations at 24h, 48h and 72h (24h OBS2, 48h OBS2 and 72h OBS2,
and was vulnerable to interference. With time the memory was
stabilized and became insusceptible to interference by amnesiac
treatments.38It was observed that the shorter the interval
between the training and the amnesiac treatment, the more
prominent the retrograde amnesia. Therefore, if enough time had
passed after training, the amnesiac treatment was no longer able
to disrupt memory. That is, the memory had become fixed and
stable and, hence ‘consolidated’.39,40
We noted above that memories could be classified as
declarative or non-declarative. In addition memories may also be
distinguished by their temporal characteristics as follows: 1)
short-term memory (STM; lasting seconds to minutes), 2)
intermediate-term memory (ITM; lasting up to 3 hours); and 3)
long-term memory (LTM; lasting greater than 6 hours).3,41,42 We
found that we could in Lymnaea differentially produce ITM and
LTM by specific training procedures. We can broadly classify the
two procedures as ‘massed’ (ITM-training) or ‘spaced’ (LTM-
training) training. For example, if we subject snails to two 30-
min training sessions separated by a 0.5h interval only ITM is
formed. However, if we increase the interval between the
training sessions to 1h, LTM (persisting at least 1 day) forms.
Thus, depending on which training procedure we choose to use
we can get ITM or LTM.16
Short-term memory does not require the synthesis of new
protein rather it results from the transient modification of already
existing proteins (e.g. phosphorylation of proteins).
Intermediate-term memory requires de novo protein synthesis
from pre-existing mRNA but ITM can also be protein synthesis
independent as shown in Aplysia.43,44Long-term memory
requires both de novo protein synthesis and altered gene activity
(for review see Milner et al, 1998). We first determined if
memory in our Lymnaea model system conformed to these rules.
To show that ITM and LTM require protein synthesis, snails are
subjected to procedures that inhibit or reduce their rate of protein
synthesis. For example, if snails are injected with the protein
synthesis inhibitor anisomycin, before training neither ITM nor
LTM results. As a control, snails injected with saline at the same
time continue to exhibit memory. If, however, snails are injected
prior to training with the transcription inhibitor, actinomycin D,
ITM forms but LTM formation is blocked. A further attribute of
our model system is that we are able to subject snails, without
harming them, to cooling to 4oC. Cooling has been used to
interfere with memory consolidation by other several labs.45,46
Cooling interferes with both protein synthesis and gene
transcription. Thus, if snails are subjected to 1h of cooling
immediately after training, neither ITM nor LTM result. If
however, the same duration of cooling (1h) is delayed by 1h after
training memory formation is not interfered with.47
One of the reasons we have used the Lymnaea model for
studying memory formation is that we have a very through
knowledge of the underlying neuronal circuitry. Thus, we should
be able to show that molecular events in CPG neurons are
necessary for memory formation. To do this we chose to utilize
an advantage of molluscs, namely the ability to surgically
remove the soma of an identified neuron while leaving behind a
functional primary neurite. Since the synaptic specializations
(pre and post) of invertebrate neurons all reside on the primary
neurite, removal of the soma does not interfere with synaptic
interactions. Moreover, the primary neurite is still capable of de
novo protein synthesis and is capable of surviving for weeks
after surgery.2,48What is missing, after this procedure is
performed, are the genes. Thus, we can directly ask whether
altered gene activity in the soma of a particular neuron is need
for LTM formation. We made a decision to first examine the role
played in memory formation by the RPeD1, the neuron that
initiates CPG rhythmogenesis. Another reason for choosing
RPeD1 is that we had already shown specific changes in neural
activity could be correlated with changes in behaviour following
memory formation.49We therefore removed either the soma of
RPeD1 or, as a control, the soma of a similar sized neuron
(LPeD1) that is not a member of the respiratory CPG from naïve
snails. We found that both groups (i.e. RPeD1 and LPeD1 soma
ablated snails) demonstrated learning and ITM. However, the
RPeD1-soma ablated snails did not exhibit LTM. That is,
removal of only the somata of a single neuron prevented LTM
memory formation without altering the ability of the snail to
learn or form ITM.2LPeD1 soma ablated animals were still
capable of forming and exhibiting LTM.
A question that has arisen is whether LTM formation first
requires ITM formation (i.e. LTM formation occurs serially) or
can it occur without ITM formation (i.e. a parallel process). If
ITM and LTM are the result of parallel molecular processes one
could block the ITM producing process and not interfere with the
LTM molecular process. This strategy has been attempted by a
number of different laboratories.50-56For example in a one-trial
step down inhibitory avoidance task studied in rats, 11 different
treatments blocked what we have termed ITM but not LTM and
18 treatments either blocked or enhanced LTM alone and left
However, if ITM and LTM are the result of parallel processes,
then an ITM training procedure no matter how many times
presented should only produce ITM and never result in the
formation of LTM. Studies in our lab and others have shed doubt
on the notion that ITM and LTM formation occur in parallel by
demonstrating that training procedures that would not normally
result in LTM (i.e. ITM-training) can in fact be made to produce
LTM.57-62If an ITM training protocol is given to snails, the
memory produced persists for 3h but not 24h (i.e. ITM). If the
parallel hypothesis was correct, then a subsequent ITM training
procedure the following day could only result only in ITM since
the molecular pathways underlying ITM and LTM formation
occur in parallel. However, we found that a subsequent bout of
ITM training a day after the first bout of ITM training produces
LTM. This suggests that the first ITM training protocol produced
a residual molecular memory trace that persists longer than the
behaviourally observable memory. Activation of the residual
molecular memory trace with a second bout of ITM training was
able to boost this trace into LTM consistent with the hypothesis
that ITM and LTM are serial processes.58,63
Another demonstration of how the processes that lead to LTM
occur in series was demonstrated in Drosophila. In Drosophila,
behavioural experiments have shown that olfactory conditioning
(i.e. a form of classical conditioning) can produce an associative
LTM. Training is performed by repeated pairings of an odor and
electrical shock and a different odor and no electrical shock. To
test for conditioned odor avoidance responses (i.e. memory) fruit
flies are placed in a T-maze and allowed to distribute themselves
into the arms of the maze containing the two odors. Memory is
LE JOURNAL CANADIEN DES SCIENCES NEUROLOGIQUES
Volume 33, No. 4 – November 2006
observed if fruit flies avoid the odor that was paired to electrical
shock. LTM is produced by multiple ‘spaced’ training sessions
separated by rest intervals, a training paradigm that we also use.
In contrast, ‘massed’training is unable to produce LTM. To show
that they could boost memory, they created transgenic flies that
carried a heat shock inducible activator CREB2 isoform. Three
hours after heat shock, massed training produced LTM in the
transgenic flies. This demonstrated that activating CREB2 in
transgenic flies permitted LTM formation by a procedure that
would not normally produce LTM.64These findings suggest that
activation of the activator isoform of CREB2 is part of a serial
pathway that allows LTM formation to occur.
While the role of the activator isoform of CREB2 was
explored in LTM formation, the repressor isoform of CREB2
was also studied in another invertebrate model system. In
Aplysia, a repressor isoform of CREB was identified and
characterized and termed ApCREB2. The repressor isoform of
ApCREB2 is targeted for phosphorylation by protein kinases
such as PKA, PKC, MAPK and CAMK. Normally, a single pulse
of 5-HT produces short-term facilitation that lasts minutes. But
when anti-ApCREB2 was injected into the nucleus of sensory
neurons to ‘inactivate’ the repressor isoform of ApCREB2, a
single 5-HT pulse produced long-term facilitation.65This long-
term facilitation required protein synthesis and altered gene
activity. These experiments showed that induction of CREB2 de-
repression could permit the formation of long-term facilitation
by a procedure that would normally produce short-term
facilitation. These findings suggest that the de-repression of the
repressor isoform of CREB2 is part of a serial pathway that
allows LTM formation to occur.
While the previous two studies manipulated CREB activity,
another study activated an upstream kinase, PKA, to produce
LTM. In the honeybee, the associative olfactory conditioning
(i.e. classical conditioning) of the proboscis extension response
results in the formation of LTM that was associated with
persistent PKA activation. A single pairing of the odor stimulus
(carnation; CS) followed by presentation of sucrose solution
(US) does not result in LTM formation. However, multiple
pairings produce a LTM that was associated with persistent PKA
activation. If photo-release of caged cAMP in the antennal lobes
is coupled with a single conditioning trial, LTM is formed.66
These findings suggest that activation of the PKA pathway is
also a part of the serial pathway that allows LTM formation to
All the above mentioned results in Lymnaea, Drosophila,
Aplysia and the honeybee are consistent with the hypothesis that
LTM formation involves processes that occur in series.
While the idea of memory consolidation has existed for a long
time, what happens to the memory afterwards has only been
under recent investigation. Upon activation, a stable and
consolidated memory becomes
Reconsolidation, first observed by Misanin et al,67is the process
by which an activated memory undergoes another process of
stabilization to return it to an inactive state. While undergoing
reconsolidation, the memory can be updated and/or changed to
incorporate new information.68,69More recent studies have
examined the mechanisms underlying reconsolidation.46,70-76
active and labile.
There exist some similarities between consolidation and
reconsolidation. CREB activation has been shown to be a
requirement in both consolidation77-82and reconsolidation .
As well, both processes have been shown to require new protein
synthesis.69,71,72,74,83,84Finally, NMDA receptors have been
shown to be involved in consolidation,77,85,86
reconsolidation.72,73,87While it appears that some molecular
cascades are involved in both consolidation and reconsolidation,
some differences have been identified. BDNF has been shown to
be required for consolidation of contextual fear memory but not
its reconsolidation. In the same study, Zif268 was shown to be
involved in the reconsolidation of contextual fear memory but
not its consolidation.88C/EBP (CAAT/enhancer-binding protein)
is required for consolidation.76,89However C/EBP does not seem
to be needed for reconsolidation.76This suggests that
consolidation and reconsolidation may trigger similar initial
molecular cascades, resulting in the activation of CREB.
Downstream from CREB however, the various molecular
cascades and proteins involved may be different.
To demonstrate reconsolidation in Lymnaea, we activate a
previously formed memory by placing the snails in the same
context-training beaker, and then attempt to block newly labile
memory using a variety of experimental methods. Application of
protein synthesis inhibitors, RNA transcription blockers or
ablation of the soma of RPeD1 after activation of the memory
perturbs the reconsolidation process. In all of these scenarios,
long-term memory was no longer present or abolished. However,
if the memory is not activated before the application of these
interventions, LTM is still present.74Thus, perturbations of
protein synthesis and RNA transcription inhibit both
consolidation and reconsolidation suggesting that similar
molecular mechanisms underlie both processes. Interestingly,
when snails were over-trained to produce a well-rehearsed
memory, the memory eventually becomes independent of protein
synthesis and transcription. This suggests that, somehow,
repeated reconsolidation transforms the memory into a very
stable state to the point that it can no longer be modified.90
Extinction typically occurs when reinforcing stimuli are no
longer applied.91,92Extinction research is of great interest as it
potentially can be used as a therapeutic tool to treat substance
addiction and fear disorders. A question that has arisen is
whether extinction is unlearning or is it new learning and
memory formation that occludes the original memory.93-98We
have attempted to directly determine whether extinction is
unlearning. To cause extinction, snails that possess LTM are
placed in the training context and are allowed to perform
pneumostome opening without any reinforcement. That is, they
learn that it is ‘acceptable’ to open their pneumostomes. If
extinction occurs snails will perform as if they are naïve snails.
This is what we have found.96,99However, this still does not
address the question as to whether extinction is unlearning. The
original studies on extinction performed by Pavlov92
demonstrated the phenomenon, which he termed ‘spontaneous
recovery’. That is, following extinction training, the original
memory was not seen. However, with time the original memory
could be evoked again by presentation of the CS, suggesting that
extinction training is not unlearning of the original learning. We
have also seen spontaneous recovery in our studies.99
THE CANADIAN JOURNAL OF NEUROLOGICAL SCIENCES
One theory of extinction postulates that extinction involves
the weakening of pre-existing connections. Support for this
theory of extinction assumed that the original conditioning was
erased.91However, a defining characteristic of extinction is that
spontaneous recovery can be observed.92If extinction training-
sessions are given after LTM has formed, the extinction memory
occludes the original memory. That is, extinction is not
unlearning but a new memory that acts in opposition of the
original LTM. However, the extinction memory itself can be
forgotten causing the original memory to return (i.e. spontaneous
MECHANISMS OF EXTINCTION
There is strong support to demonstrate that extinction is an
active process that involves new learning and, therefore, new
protein synthesis, to produce a memory that occludes the original
conditioning.93-95Extinction is an active process that involves
new learning to produce a memory that occludes the original
conditioning. Extinction has been observed in many model
systems.17,96-100Using a number of the experimental attributes of
our model system outlined above we set out to rigorously test the
hypothesis that extinction is new learning and memory that
occludes the older memory. We were able to show that the
process of extinction requires the somata of RPeD1 to be present
(just as LTM formation requires RPeD1’s somata).99While we
have identified a neuron necessary for extinction (RPeD1), no
one particular brain structure has been identified in mammalian
systems to be responsible for extinction.97Thus highlighting
another advantage to the Lymnaea model system.
Of all the topics that we deal with in our laboratory the one
that we are most often questioned about by non-neuroscientists
is forgetting. The most frequently asked question is, “How can I
stop forgetting things and improve my memory?” We often
answer, much to the chagrin of the questioner, that the biggest
problem with forgetting is ‘not forgetting’but rather the inability
to forget! We first need to define what we mean by forgetting.
The Oxford English Dictionary defines the verb forget as “To
lose remembrance of; to cease to retain in one's memory” or “To
fail to recall to mind; not to recollect.” Because we are dealing
with non-declarative memory that is stored within the neural
circuit that mediates the behaviour (i.e. the CPG
network)2,49,101,102we define forgetting as the obliteration of the
memory and therefore the associated learned behaviour.
While forgetting is correlated with time, it is not caused by
the passage of time.103 There are several theories on forgetting
with the two most prominent being: 1) Failure to retrieve; and 2)
Retroactive interference. The retrieval failure hypothesis states
that the memory cannot be accessed and hence it is dubbed
‘forgotten’. Certainly, we have all experienced this form of
forgetting. While a failure to retrieve a memory might explain
how forgetting of a declarative memory may occur, retrieval
failure cannot explain forgetting of a non-declarative memory.
We study a non-declarative memory that is formed and forgotten
both in the same circuit.2Since we know the circuit, we can test
for retrieval failure. To test this, we can produce two different
context specific memories in the snail (i.e. standard and carrot).
If we use a training procedure (two 45 min training sessions on
two successive days) in the standard context to produce a LTM
that persists for seven days and a training procedure in the carrot
context to produce a LTM that persists for only one day (two 30
min sessions on one day) we can then directly show that memory
is not due to an inability to retrieve the memory. If the memory
in the carrot context was forgotten due to retrieval failure, then
placing the animals in the standard context should not manifest
the original long-lasting memory. If the animal still has the LTM
for the standard context, then we know that the memory is
accessible and forgetting is not due to retrieval failure. We
observe the latter finding that demonstrates forgetting is not due
to retrieval failure.
The interference theory states that related events that occur
after LTM formation cause forgetting. When they are trained,
snails make the association that pneumostome opening results in
the delivery of a tactile stimulus. When snails are not being
trained, they are free to perform aerially respiration ad libitum.
Animals learn anew that pneumostome opening will not result in
the delivery of a tactile stimulus. Interfering events can be
prevented by submerging animals after training to prevent
spontaneous opening of their pneumostome. Thus, animals will
not learn the new association and therefore retain the original
association. We found that preventing interfering events
extended the persistence of LTM (i.e. delayed forgetting).35The
molecular mechanisms of forgetting are still unknown. We have
hypothesized that forgetting is an active process. That is,
forgetting requires altered gene activity and new protein
synthesis in order to obliterate the memory. Thus, cooling snails
after the completion of consolidation can extend the persistence
of memory (i.e. delay forgetting). Cooling reduces the snails’
metabolic rate and subsequently reduce the amount of protein
synthesis that occurs.47,58Most importantly, we can directly
demonstrate that forgetting resembles learning something new
and remembering it since if we remove the soma of RPeD1 after
the LTM consolidation process, the snails are unable to forget
(they are also unable to form new LTM if operantly conditioned
in a new context) indicating that forgetting requires access to the
It has been demonstrated that stress and emotions can be
strong modulators of memory formation; however, experimental
results have been varied and often contradictory.105For example,
rats that were exposed to uncontrollable restraint and tail-shock
stress demonstrated normal learning during water maze training,
but animals showed impaired memory compared to unstressed
controls when tested the next day.106However, memories for
very traumatic or highly emotional events are often extremely
long-lasting.107,108Given the complexity of animal behaviour and
the many diverse ways different stressors could act on learning
and memory formation, the disagreement in the literature is not
surprising. Preliminary evidence has shown that a single, highly
aversive stimulus (submersion in KCl), contingent with
pneumostome opening, is sufficient to alter the breathing
behaviour of Lymnaea.109Yoked controls, change of context
controls, and handling controls indicate that this change is a bona
fide example of single-trial operant conditioning.
Stressors such as predators, inter- and intra-specific
competition for food, habitat and procreational resources can
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Volume 33, No. 4 – November 2006
impart a high strain on the energy allocation of an organism.
Learning about predators is expected to have adaptive payoffs in
any species that can alter their behaviour during times of
predation risk.110Lymnaea respond to crayfish predators by
using predator-avoidance behaviours such as the full body
withdrawal response when under attack.111It is possible that
snails which can detect the presence of a predator, in a
presumably stressful situation in which they respond with
defensive behaviors, will show a change in their ability to learn
and remember when given an operant training paradigm in the
presence of that predator. Preliminary experiments in our
laboratory suggest when Lymnaea are operantly conditioned in
the presence of a crayfish predator memory formation is
enhanced.112How this crayfish exposure affects the neural
substrates responsible for this behavior and to what extent this
occurs is currently under investigation. In future work, we hope
to investigate the alteration of memory formation by stress.
Another avenue of research we would like to pursue is to
explore anatomical studies associated with memory formation.
We have as yet to determine what changes in synapse profile and
cytoarchitecture occur throughout neurons that are involved in
memory formation (i.e. the 3 interneurons of the CPG mediating
aerial respiratory behaviour). However, we have undertaken a
functional proteomics approach to identify changes in protein
expression associated with LTM formation using the entire CNS
of the snail. Preliminary analyses have indicated that LTM
formation results in a reduction in some specific membrane
proteins and an increase in the expression of many specific
cytosolic proteins. Once these proteins have been identified, we
can begin to construct a possible picture of what changes occur
in the Central nervous system of Lymnaea to form LTM.
This work was supported by a grant from CIHR to KLand the
lab. KP is supported by a scholarship from the Alberta Heritage
Foundation for Medical Research (AHFMR) and the
Neuroscience Canada Foundation. KM is supported by a
studentship from NSERC.
Milner B, Squire LR, Kandel ER. Cognitive neuroscience and the
study of memory. Neuron. 1998; 20 (3):445-68.
Scheibenstock A, Krygier D, Haque Z, Syed N, Lukowiak K. The
Soma of RPeD1 must be present for long-term memory
formation of associative learning in Lymnaea. J Neurophysiol.
2002; 88 (4):1584-91.
Dudai Y. Memory from A to Z. Oxford: Oxford University Press;
Thompson RF, Spencer WA. Habituation: a model phenomenon for
the study of neuronal substrates of behavior. Psychol Rev. 1966;
Castellucci V, Carew TJ, Kandel ER. Cellular analysis of long-term
habituation of the gill-withdrawal reflex of Aplysia californica.
Science. 1978; (202):1306-8.
Kupfermann I, Castellucci V, Pinsker H, Kandel ER. Neuronal
correlates of habituation and dishabituation of the gill-withdrawal
reflex in Aplysia. Science. 1970; (176):1740-8.
Pinsker H, Kupfermann I, Castellucci V, Kandel ER. Cellular
analysis of behavioral reflex habituation in Aplysia. Fed Proc.
Castellucci V, Kandel ER. Presynaptic facilitation as a mechanism
for behavioral sensitization in Aplysia. Science. 1976; 194
9.Castellucci VF, Frost WN, Goelet P, Montarolo PG, Schacher S,
Morgan JA, et al. Cell and molecular analysis of long-term
sensitization in Aplysia. J Physiol (Paris). 1986; 81 (4):349-57.
10. Kimble GA. 'Hilgard and Marquis' conditioning and learning. 2nd
ed. New York: Appleton-Century-Croft; 1961.
11. Carew TJ, Sahley CL. Invertebrate learning and memory: from
behavior to molecules. Annu Rev Neurosci. 1986; 9:435-87.
12. Skinner BF. Are theories of learning necessary? Psychol Rev. 1950;
13. Thorndike E. Animal intelligence. New York: The Macmillan Co.;
14. Lipsitt LP. Learning processes in the human newborn. Sensitization,
habituation, and classical conditioning. Ann N YAcad Sci. 1990;
608:113-23; discussion 23-7.
15. Lukowiak K, Ringseis E, Spencer G, Wildering W, Syed N. Operant
conditioning of aerial respiratory behaviour in Lymnaea
stagnalis. J Exp Biol. 1996; 199 (Pt 3):683-91.
16. Lukowiak K, Adatia N, Krygier D, Syed N. Operant conditioning in
Lymnaea: evidence for intermediate- and long-term memory.
Learn Mem. 2000; 7 (3):140-50.
17. Rankin CH. Context conditioning in habituation in the nematode
Caenorhabditis elegans. Behav Neurosci. 2000; 114 (3):496-505.
18. Glanzman DL. The cellular basis of classical conditioning in
Aplysia californica--it's less simple than you think. Trends
Neurosci. 1995; 18 (1):30-6.
19. Hawkins RD, Kandel ER, Bailey CH. Molecular mechanisms of
memory storage in Aplysia. Biol Bull. 2006; 210 (3):174-91.
20. Lukowiak K, Sahley CL. The in vitro classical conditioning of the
gill withdrawal reflex of Aplysia californica. Science. 1981;
21. Menzel R. Searching for the memory trace in a mini-brain, the
honeybee. Learn Mem. 2001; 8 (2):53-62.
22. Skoulakis EM, Grammenoudi S. Dunces and da Vincis: the genetics
of learning and memory in Drosophila. Cell Mol Life Sci. 2006;
23. Crow T, Tian LM. Pavlovian conditioning in Hermissenda: a circuit
analysis. Biol Bull. 2006; 210 (3):289-97.
24. LeDoux JE, Cicchetti P, Xagoraris A, Romanski LM. The lateral
amygdaloid nucleus: sensory interface of the amygdala in fear
conditioning. J Neurosci. 1990; 10 (4):1062-9.
25. Rogan MT, LeDoux JE. LTP is accompanied by commensurate
enhancement of auditory-evoked responses in a fear conditioning
circuit. Neuron. 1995; 15 (1):127-36.
26. Lasiter PS, Deems DA, Garcia J. Involvement of the anterior insular
gustatory neocortex in taste-potentiated odor aversion learning.
Physiol Behav. 1985; 34 (1):71-7.
27. Lasiter PS, Glanzman DL. Cortical substrates of taste aversion
learning: dorsal prepiriform (insular) lesions disrupt taste
aversion learning. J Comp Physiol Psychol. 1982; 96 (3):376-92.
28. Lasiter PS, Glanzman DL. Cortical substrates of taste aversion
learning: involvement of dorsolateral amygdaloid nuclei and
temporal neocortex in taste aversion learning. Behav Neurosci.
1985; 99 (2):257-76.
29. Jones J. Aspects of respiration in Planorbis corneus (L) and
Lymnaea stagnalis (L) (Gastropoda: Pulmonata). Comp.
Biochem. Physiol. 1961; (4):1-29.
30. Syed N, Winlow W. Respiratory behaviour in the pond snail
Lymnaea stagnalis. II. Neural elements of the central pattern
generator (CPG). J Comp Physiol. 1991; 169:557-68.
31. Syed NI, Bulloch AG, Lukowiak K. In vitro reconstruction of the
respiratory central pattern generator of the mollusk Lymnaea.
Science. 1990; 250 (4978):282-5.
32. Inoue T, Takasaki M, Lukowiak K, Syed N. Inhibition of the
respiratory pattern-generating neurons by an identified whole-
body withdrawal interneuron of Lymnaea stagnalis. J Exp Biol.
1996; 199 (Pt 9):1887-98.
33. Lukowiak K. Central pattern generators: some principles learned
from invertebrate model systems. J Physiol (Paris). 1991; 85
34. Lukowiak K, Sangha S, McComb C, Varshney N, Rosenegger D,
Sadamoto H, et al. Associative learning and memory in Lymnaea
stagnalis: how well do they remember? J Exp Biol. 2003; 206 (Pt
THE CANADIAN JOURNAL OF NEUROLOGICAL SCIENCES
35. Sangha S, McComb C, Lukowiak K. Forgetting and the extension of
memory in Lymnaea. J Exp Biol. 2003; 206 (Pt 1):71-7.
36. Haney J, Lukowiak K. Context learning and the effect of context on
memory retrieval in Lymnaea. Learn Mem. 2001; 8 (1):35-43.
37. Ribot T. Diseases of memory. New York: Appleton-Century-Crofts;
38. Muller GE, Pilzecker A. Experimentelle Beiträge zur Lehre vom
Gedächtnis. Z. Psychol. Erganzungsband. 1900; 1:1-300.
39. McGaugh JL. Time-dependent processes in memory storage.
Science. 1966; 153 (742):1351-8.
40. Squire LR, Alvarez P. Retrograde amnesia and memory
consolidation: a neurobiological perspective. Curr Opin
Neurobiol. 1995; 5 (2):169-77.
41. Rosenzweig M. Historical perspectives on the development of the
biology of learning and memory. In: J Martinez, R Kesner,
editors, translator and editor Neurobiology of learning and
Memory. San Diego: Academic Press; 1998; p. 1-54.
42. Rosenzweig MR, Bennett EL, Colombo PJ, Lee DW, Serrano PA.
Short-term, intermediate-term, and long-term memories. Behav
Brain Res. 1993; 57 (2):193-8.
43. Sutton MA, Bagnall MW, Sharma SK, Shobe J, Carew TJ.
Intermediate-term memory for site-specific sensitization in
aplysia is maintained by persistent activation of protein kinase C.
J Neurosci. 2004; 24 (14):3600-9.
44. Sutton MA, Ide J, Masters SE, Carew TJ. Interaction between
amount and pattern of training in the induction of intermediate-
and long-term memory for sensitization in aplysia. Learn Mem.
2002; 9 (1):29-40.
45. Morrison GE, van der Kooy D. Cold shock before associative
conditioning blocks memory retrieval, but cold shock after
conditioning blocks memory retention in Caenorhabditis elegans.
Behav Neurosci. 1997; 111 (3):564-78.
46. Yamada A, Sekiguchi T, Suzuki H, Mizukami A. Behavioral
analysis of internal memory states using cooling-induced
retrograde amnesia in Limax flavus. J Neurosci. 1992; 12
47. Sangha S, Morrow R, Smyth K, Cooke R, Lukowiak K. Cooling
blocks ITM and LTM formation and preserves memory.
Neurobiol Learn Mem. 2003; 80 (2):130-9.
48. Spencer GE, Lukowiak K, Syed NI. Transmitter-receptor
interactions between growth cones of identified Lymnaea
neurons determine target cell selection in vitro. J Neurosci. 2000;
49. Spencer GE, Syed NI, Lukowiak K. Neural changes after operant
conditioning of the aerial respiratory behavior in Lymnaea
stagnalis. J Neurosci. 1999; 19 (5):1836-43.
50. Izquierdo LA, Barros DM, Vianna MR, Coitinho A, deDavid e Silva
T, Choi H, et al. Molecular pharmacological dissection of short-
and long-term memory. Cell Mol Neurobiol. 2002; 22 (3):269-87.
51. Crow T, Redell JB, Tian LM, Xue-Bian J, Dash PK. Inhibition of
conditioned stimulus pathway phosphoprotein 24 expression
blocks the development of intermediate-term memory in
Hermissenda. J Neurosci. 2003; 23 (8):3415-22.
52. DeZazzo J, Tully T. Dissection of memory formation: from
behavioral pharmacology to molecular genetics. Trends
Neurosci. 1995; 18 (5):212-8.
53. Emptage NJ, Carew TJ. Long-term synaptic facilitation in the
absence of short-term facilitation in Aplysia neurons. Science.
1993; 262 (5131):253-6.
54. Hegde AN, Inokuchi K, Pei W, Casadio A, Ghirardi M, Chain DG,
et al. Ubiquitin C-terminal hydrolase is an immediate-early gene
essential for long-term facilitation in Aplysia. Cell. 1997; 89
55. Mauelshagen J, Parker GR, Carew TJ. Dynamics of induction and
expression of long-term synaptic facilitation in Aplysia. J
Neurosci. 1996; 16 (22):7099-108.
56. Tully T, Preat T, Boynton SC, Del Vecchio M. Genetic dissection of
consolidated memory in Drosophila. Cell. 1994; 79 (1):35-47.
57. Ghirardi M, Montarolo PG, Kandel ER. A novel intermediate stage
in the transition between short- and long-term facilitation in the
sensory to motor neuron synapse of aplysia. Neuron. 1995; 14
58. Parvez K, Stewart O, Sangha S, Lukowiak K. Boosting
intermediate-term into long-term memory. J Exp Biol. 2005; 208
59. Smyth K, Sangha S, Lukowiak K. Gone but not forgotten: the
lingering effects of intermediate-term memory on the persistence
of long-term memory. J Exp Biol. 2002; 205 (Pt 1):131-40.
60. Sutton MA, Masters SE, Bagnall MW, Carew TJ. Molecular
mechanisms underlying a unique intermediate phase of memory
in aplysia. Neuron. 2001; 31 (1):143-54.
61. Zhao WQ, Polya GM, Wang BH, Gibbs ME, Sedman GL, Ng KT.
Inhibitors of cAMP-dependent protein kinase impair long-term
memory formation in day-old chicks. Neurobiol Learn Mem.
1995; 64 (2):106-18.
62. Riedel G. If phosphatases go up, memory goes down. Cell Mol Life
Sci. 1999; 55 (4):549-53.
63. Parvez K, Moisseev V, Lukowiak K. A context-specific single
contingent-reinforcing stimulus boosts intermediate-term
memory to long-term memory. Eur J Neuro Sci. 2006; 24
64. Yin JC, Del Vecchio M, Zhou H, Tully T. CREB as a memory
modulator: induced expression of a dCREB2 activator isoform
enhances long-term memory in Drosophila. Cell. 1995; 81
65. Bartsch D, Ghirardi M, Skehel PA, Karl KA, Herder SP, Chen M, et
al. Aplysia CREB2 represses long-term facilitation: relief of
repression converts transient facilitation into long-term
functional and structural change. Cell. 1995; 83 (6):979-92.
66. Muller U. Prolonged activation of cAMP-dependent protein kinase
during conditioning induces long-term memory in honeybees.
Neuron. 2000; 27 (1):159-68.
67. Misanin JR, Miller RR, Lewis DJ. Retrograde amnesia produced by
electroconvulsive shock after reactivation of a consolidated
memory trace. Science. 1968; 160 (827):554-5.
68. Nader K. Memory traces unbound. Trends Neurosci. 2003; 26
69. Milekic MH, Alberini CM. Temporally graded requirement for
protein synthesis following memory reactivation. Neuron. 2002;
70. Anokhin KV, Tiunova AA, Rose SP. Reminder effects -
reconsolidation or retrieval deficit? Pharmacological dissection
with protein synthesis inhibitors following reminder for a
passive-avoidance task in young chicks. Eur J Neurosci. 2002; 15
71. Nader K, Schafe GE, Le Doux JE. Fear memories require protein
synthesis in the amygdala for reconsolidation after retrieval.
Nature. 2000; 406 (6797):722-6.
72. Pedreira ME, Perez-Cuesta LM, Maldonado H. Reactivation and
reconsolidation of long-term
Chasmagnathus: protein synthesis requirement and mediation by
NMDA-type glutamatergic receptors. J Neurosci. 2002; 22
73. Przybyslawski J, Sara SJ. Reconsolidation of memory after its
reactivation. Behav Brain Res. 1997; 84 (1-2):241-6.
74. Sangha S, Scheibenstock A, Lukowiak K. Reconsolidation of a
long-term memory in Lymnaea requires new protein and RNA
synthesis and the soma of right pedal dorsal 1. J Neurosci. 2003;
75. Sekiguchi T, Yamada A, Suzuki H. Reactivation-dependent changes
in memory states in the terrestrial slug Limax flavus. Learn Mem.
1997; 4 (4):356-64.
76. Taubenfeld SM, Milekic MH, Monti B, Alberini CM. The
consolidation of new but not reactivated memory requires
hippocampal C/EBPbeta. Nat Neurosci. 2001; 4 (8):813-8.
77. Bourtchuladze R, Frenguelli B, Blendy J, Cioffi D, Schutz G, Silva
AJ. Deficient long-term memory in mice with a targeted mutation
of the cAMP-responsive element-binding protein. Cell. 1994; 79
78. Dash PK, Hochner B, Kandel ER. Injection of the cAMP-responsive
element into the nucleus of Aplysia sensory neurons blocks long-
term facilitation. Nature. 1990; 345 (6277):718-21.
memory in the crab
LE JOURNAL CANADIEN DES SCIENCES NEUROLOGIQUES
Volume 33, No. 4 – November 2006
79. Guzowski JF, McGaugh JL. Antisense oligodeoxynucleotide-
mediated disruption of hippocampal cAMP response element
binding protein levels impairs consolidation of memory for water
maze training. Proc Natl Acad Sci U S A. 1997; 94 (6):2693-8.
80. Kogan JH, Frankland PW, Blendy JA, Coblentz J, Marowitz Z,
Schutz G, et al. Spaced training induces normal long-term
memory in CREB mutant mice. Curr Biol. 1997; 7 (1):1-11.
81. Lamprecht R, Hazvi S, Dudai Y. cAMP response element-binding
protein in the amygdala is required for long- but not short-term
conditioned taste aversion memory. J Neurosci. 1997; 17
82. Yin JC, Wallach JS, Del Vecchio M, Wilder EL, Zhou H, Quinn
WG, et al. Induction of a dominant negative CREB transgene
specifically blocks long-term memory in Drosophila. Cell. 1994;
83. Kida S, Josselyn SA, de Ortiz SP, Kogan JH, Chevere I, Masushige
S, et al. CREB required for the stability of new and reactivated
fear memories. Nat Neurosci. 2002; 5 (4):348-55.
84. Debiec J, LeDoux JE, Nader K. Cellular and systems
reconsolidation in the hippocampus. Neuron. 2002; 36 (3):527-
85. Abeliovich A, Paylor R, Chen C, Kim JJ, Wehner JM, Tonegawa S.
PKC gamma mutant mice exhibit mild deficits in spatial and
contextual learning. Cell. 1993; 75 (7):1263-71.
86. Morris RG, Anderson E, Lynch GS, Baudry M. Selective
impairment of learning and blockade of long-term potentiation by
an N-methyl-D-aspartate receptor antagonist, AP5. Nature. 1986;
87. Summers MJ, Crowe SF, Ng KT. Administration of DL-2-amino-5-
phosphonovaleric acid (AP5) induces transient inhibition of
reminder-activated memory retrieval in day-old chicks. Brain Res
Cogn Brain Res. 1997; 5 (4):311-21.
88. Lee JL, Everitt BJ, Thomas KL. Independent cellular processes for
hippocampal memory consolidation and reconsolidation.
Science. 2004; 304 (5672):839-43.
89. Alberini CM, Ghirardi M, Metz R, Kandel ER. C/EBP is an
immediate-early gene required for the consolidation of long-term
facilitation in Aplysia. Cell. 1994; 76 (6):1099-114.
90. Sangha S. Memory formation, reconsolidation, extinction and
forgetting in Lymnaea stagnalis: PhD Thesis. Calgary: University
of Calgary; 2004. p. 227.
91. Rescorla R, Wagner A. A theory of Pavlovian conditioning:
variations in the effectiveness of reinforcement and
nonreinforcement. In: A Black; W Prokasy, editors. Classical
Conditioning II: Current Research and Theory. New York:
Appleton-Century-Crofts; 1972. (A Black; W Prokasy editors).
92. Pavlov IP. Conditioned reflexes. London: Oxford UP.; 1927.
93. Berman DE, Dudai Y. Memory extinction, learning anew, and
learning the new: dissociations in the molecular machinery of
learning in cortex. Science. 2001; 291 (5512):2417-9.
94. Flood JF, Jarvik ME, Bennett EL, Orme AE, Rosenzweig MR.
Protein synthesis inhibition and memory for pole jump active
avoidance and extinction. Pharmacol Biochem Behav. 1977; 7
95. Vianna MR, Szapiro G, McGaugh JL, Medina JH, Izquierdo I.
Retrieval of memory for fear-motivated training initiates
extinction requiring protein synthesis in the rat hippocampus.
Proc Natl Acad Sci U S A. 2001; 98 (21):12251-4.
96. McComb C, Sangha S, Qadry S, Yue J, Scheibenstock A, Lukowiak
K. Context extinction and associative learning in Lymnaea.
Neurobiol Learn Mem. 2002; 78 (1):23-34.
97. Myers KM, Davis M. Behavioral and neural analysis of extinction.
Neuron. 2002; 36 (4):567-84.
98. Richards WG, Farley J, Alkon DL. Extinction of associative
learning in Hermissenda: behavior and neural correlates. Behav
Brain Res. 1984; 14 (3):161-70.
99. Sangha S, Scheibenstock A, Morrow R, Lukowiak K. Extinction
requires new RNA and protein synthesis and the soma of the cell
right pedal dorsal 1 in Lymnaea stagnalis. J Neurosci. 2003; 23
100. Schwaerzel M, Heisenberg M, Zars T. Extinction antagonizes
olfactory memory at the subcellular level. Neuron. 2002; 35
101. Lowe MR, Spencer GE. Perturbation of the activity of a single
identified neuron affects long-term memory formation in a
molluscan semi-intact preparation. J Exp Biol. 2006; 209 (Pt
102. McComb C, Varshney N, Lukowiak K. Juvenile Lymnaea ventilate,
learn and remember differently than do adult Lymnaea. J Exp
Biol. 2005; 208 (Pt 8):1459-67.
103. Jenkins J, Dallenbach K. Obliviscence during sleep and waking.
Am J Psychol. 1924; 35:605-12.
104. Sangha S, Scheibenstock A, Martens K, Varshney N, Cooke R,
Lukowiak K. Impairing forgetting by preventing new learning
and memory. Behav Neurosci. 2005; 119 (3):787-96.
105. Shors TJ. Learning during stressful times. Learn Mem. 2004; 11
106. Kim JJ, Koo JW, Lee HJ, Han JS. Amygdalar inactivation blocks
stress-induced impairments in hippocampal long-term
potentiation and spatial memory. J Neurosci. 2005; 25 (6):1532-
107. Cahill L, McGaugh JL. Mechanisms of emotional arousal and
lasting declarative memory. Trends Neurosci. 1998; 21 (7):294-9.
108. Bohannon JN, 3rd. Flashbulb memories for the space shuttle
disaster: a tale of two theories. Cognition. 1988; 29 (2):179-96.
109. Martens K, Lukowiak K. Long-term memory in Lymnaea using
one-trial operant training (abstract). In Society for Neuroscience.
Washington, D.C., USA; 2005.
110. Coolen I, Dangles O, Casas J. Social learning in noncolonial
insects? Curr Biol. 2005; 15 (21):1931-5.
111. Rigby MC, Jokela J. Predator avoidance and immune defence: costs
and trade-offs in snails. Proc Biol Sci. 2000; 267 (1439):171-6.
112. Orr MV, Lukowiak K. Learning in stressful environments: Effect of
predator presence on learning and memory in the pond snail
(abstract). In Society for Neuroscience. Washington, D.C.; 2005.
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