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First published online August 8, 2008
Journal of Experimental Biology 211, 2678-2688 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.020347
Crowding, an environmental stressor, blocks long-term memory formation in Lymnaea
Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1
* Author for correspondence (e-mail: lukowiak{at}ucalgary.ca)
Accepted 5 June 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: long-term memory, Lymnaea, crowding, stress, block of memory formation
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
McEwen and Sapolsky, 1996;
Kim and Diamond, 2002).
Stressors can take the form of either physical (e.g. heat shock) or
psychological (e.g. public speaking) challenges and have the ability to alter
the processes of memory formation and recall
(Yerkes and Dotson, 1908;
Shors, 2006;
Joels et al., 2006;
Martens et al., 2007a;
Martens et al., 2007b).
Depending on the specific stressor and when and how the stress is perceived,
memory formation and/or its recall may be enhanced or impaired (e.g.
de Quervain et al., 1998;
Bowman et al., 2003; Cahil et
al., 2003; Joels et al., 2006;
Martens et al., 2007a;
Orr and Lukowiak, 2008).
Stress has the ability to modulate memory formation and memory recall as it is
a dynamic brain process (Hebb,
1949; Lewis,
1979). There are various indeterminate factors influencing whether
memory formation and/or memory recall will be augmented or impaired by stress
(Bowman et al., 2003;
Luine, 2002;
Vanitallie, 2002;
Martens et al., 2007a;
Martens et al., 2007b). A
long-term goal of our research is to determine how a specific environmental
stressor, crowding, modifies memory formation using a model system in which it
is possible to study memory formation at the single-cell level. As a first
step in this process, the present study examines whether crowding alters
long-term memory (LTM) formation at the behavioural level.
Crowding is a stressor that alters genomic and behavioural activity in both
vertebrates and invertebrates (Boranic and
Poljak-Blazi, 1983; Roman et
al., 2004; Reber et al.,
2006; Holt, 2006).
As a step towards elucidating the causal neuronal mechanisms underlying how
stress modifies memory, we devised a series of experiments utilizing the
Lymnaea model system to determine whether crowding either affects the
ability to form LTM or hinders its ability to be recalled. Previously,
crowding in Lymnaea was shown to significantly alter genomic
activity, specifically affecting growth rate, embryonic development and
reproduction, and retarding growth (Colton,
1908; Crabb, 1929;
Forbes and Crampton, 1942;
Noland and Carriker, 1946;
Voronezhskaya et al.,
2004).
Lymnaea is a model system used to elucidate the neuronal
mechanisms underlying memory formation
(Lukowiak et al., 2003a;
Lukowiak et al., 2003b;
Birmingham et al., 2004;
Parvez et al., 2006;
Lukowiak et al., 2008). In
particular, aerial respiratory behaviour has proven to be very tractable in
attempts to uncover the causal mechanisms of LTM formation, as this behaviour
is driven by a three-neuron central pattern generator (CPG) whose sufficiency
and necessity has been shown (Syed et al.,
1990; Syed et al.,
1992; Lukowiak et al.,
2003b). In addition, RPeD1, one of the three CPG neurons, has been
shown to be a necessary site for the molecular processes required for LTM
formation, reconsolidation, extinction and forgetting
(Scheibenstock et al., 2002;
Sangha et al., 2003c;
Sangha et al., 2003d;
Sangha et al., 2005;
Lattal et al., 2006).
Importantly, using this model system, we have recently demonstrated that
stress has the ability to modify memory formation
(Martens et al., 2007a;
Martens et al., 2007b;
Orr and Lukowiak, 2008). In
the present study we report that crowding, during a critical 1h period just
prior or immediately after operant conditioning, prevents LTM but does not
block intermediate-term memory (ITM) formation or recall of an already formed
LTM.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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20°C).
Aerial respiratory behaviour and operant conditioning
The traditional training procedure
Snails were removed from their home aquaria and placed into a 1-litre
beaker containing 500 ml of hypoxic pond water (PW; <0.1 ml O2
l–1). PW was made hypoxic by bubbling N2 gas
through the water for 20 min prior to introducing the snails. The animals were
given a 10-min acclimatization period prior to a 30-min training session. By
subjecting the snails to a hypoxic challenge, the animals increased their rate
of aerial respiration (Lukowiak et al.,
1996; Lukowiak et al.,
1998). The animals were operantly conditioned by applying a gentle
tactile stimulus with a wooden applicator to their pneumostome as the
pneumostome began to open. The stimulus was strong enough to cause the snails
to close their pneumostome yet gentle enough that the snails did not perform
the full-body withdrawal response. This pneumostome closer response is a
graded part of the whole-snail escape response
(Orr et al., 2007). Every time
the snail opened its pneumostome and received the stimulus during the training
period, the time was recorded for use in future yoked control experiments. All
behavioural experiments were done `blind' such that the person performing the
training paradigm was unaware of the status of the cohort being tested.
In order to cause LTM, the traditional training procedure utilized in the
present study consisted of two 30-min training sessions (TS1 and TS2) with a
1h interval between the sessions (Lukowiak
et al., 1998), after which time the snails were randomly selected
to be returned to either their home aquaria or to crowded conditions for a
specified time (see below). The snails were then tested for memory (MT; i.e. a
`savings test') using a similar test to that used during training
sessions.
The memory augmentation procedure
A second, faster training procedure that results in LTM formation was also
used in the present study (Martens et al.,
2007a). In this procedure, snails were exposed to a noxious,
aversive 25 mmoll–1 KCl stimulus immediately prior to a
single 30 min TS as described in the traditional training procedure. Snails
were placed in individual Petri dishes (37 mm) containing 4 ml of 25
mmoll–1 KCl for 30–35 s. This volume was sufficient to
cover the foot of the snails but was not enough to submerge them. The KCl bath
caused snails to withdraw into their shells. The snails were then placed in
the hypoxic training beaker for acclimatization for 10 min, followed by the 30
min TS (see above). This procedure results in LTM that persists for up to 36h
(Martens et al., 2007a).
Change of context
In some experiments, a `carrot context' was also employed. This consisted
of bubbling N2 through a 750 ml Erlenmeyer flask containing blended
carrot and then through the water in the training beaker
(Haney and Lukowiak, 2001).
`Change-of-context' testing was employed as an internal control to test for
injury or unresponsiveness, as LTM recall is context-specific.
Crowded conditions
In crowded conditions, snails were maintained for a specified period of
time (1–24h) at a density of 20 snails per 100 ml of PW (normal density
is two snails per 100 ml of PW). Snails can be maintained at these and greater
densities for 2 to 3 months without an increase in mortality
(Crabb, 1929;
Forbes and Crampton, 1942;
Noland and Carriker, 1946)
although growth may be compromised. Maintaining snails at these densities for
24h was not considered to be harmful. We added naïve (i.e. untrained)
snails to the home `crowded aquaria' to create crowding.
Crowded pond water (CPW)
In some experiments, only `crowded pond water' (CPW) was used. In order to
obtain CPW snails were crowded for 24h at a density of 20 snails per 100 ml of
PW. The water was then used during experiments. Thus, the experimental snails
did not directly experience the crowded conditions.
Empty shells
Another control condition used for some experiments was generated by
placing clean shells from deceased snails in the training beaker with
experimental snails at the identical density to that used in the crowding
experiments (20 snails per 100 ml of PW).
Crowded pond water and empty shells
As a final control procedure, we combined CPW with empty shells.
Yoked control procedure
The yoked control procedure is similar to the operant conditioning training
procedure. The difference is that the yoked control snails are `poked' in
their pneumostome area, not when they attempt to open their pneumostome but at
the exact time as the snail to which they are yoked opens its pneumostome and
receives the tactile stimuli. Thus, these yoked control snails receive exactly
the same number of tactile stimuli delivered at the same time as the operant
conditioned snails during the training sessions. However, during the memory
test (MT), yoked control snails receive the tactile stimulus when they attempt
to open their pneumostome. Thus, when the behaviour of yoked control snails is
compared with the behaviour of operantly conditioned snails, we compare the
response of each respective group in MT.
Breathing observation procedure
The total breathing time (TBT) of snails was also observed before and after
crowding to ascertain whether the combination of stresses (crowding and the
hypoxic challenge) results in abnormal aerial respiratory behaviour. The
breathing observations consisted of placing snails in hypoxic water for a
10-min acclimatization period. The snails were then gently pushed below the
surface of the water. We monitored each snail and recorded the time each snail
kept its pneumostome open during the 30-min observation session. These snails
were then either crowded or placed in their eumoxic home aquaria for 24h, at
which point another observation session was performed. TBT was then compared
between the two sessions. Tactile stimuli were not delivered to the
pneumostome during these observation sessions, thus allowing snails to perform
aerial respiration as often as necessary. In both operant conditioning
procedures used here (described above) snails received the tactile stimulus to
their pneumostome as soon as they attempted to open the pneumostome. We are
thus not able to compare the TBT with the number of attempted openings.
Operational definition of memory
In order for memory to be present, the number of attempted pneumostome
openings (i.e. the number of tactile stimuli delivered) during the MT must be
significantly less (P<0.05) than the number of attempted
pneumostome openings administered during the TS. ITM (persisting up to 3h) is
dependent on de novo protein synthesis, whereas LTM (lasting at least
24h) depends on both de novo protein synthesis and gene transcription
(Sangha et al., 2003b;
Sangha et al., 2003c;
Parvez et al., 2006;
Martens et al., 2007a).
Statistical methods
Parametric data were analyzed using a repeated-measures analysis of
variance (ANOVA) followed by a Tukey–Kramer post-hoc test. For
groups that did not pass the normality test, a Friedman's test was conducted,
followed by a Dunn's Multiple Comparison post-hoc test. When groups
were not matched, a Kruskal–Wallis test was performed, followed by a
Dunn's Multiple Comparison post-hoc test. Other data were analyzed
using a one-way ANOVA test and a Tukey–Kramer post-hoc test.
Post-hoc tests were conducted to determine which measurement(s)
differed. When comparing TBT, a paired t-test was used. In all cases,
significance was considered to be at least P<0.05.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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TBT is significantly less in eumoxia than it is in hypoxia
(Lukowiak et al., 1996).
Therefore, in the present study, we determined whether crowding caused TBT to
be suppressed in eumoxia. Experiments were repeated as above except that the
TBT of the snails was observed in eumoxia (N=20). The TBT in eumoxic
conditions 24h before crowding was 74.8±8.2 s. Following 24h of
crowding, TBT was significantly decreased to 51.3±3.1 s
(P<0.01). TBT following 24h in the uncrowded condition was
77.6±8.5 s, which was not significantly different to that of the
Pre-obs session (P>0.05). Thus, crowding causes significant
decreases in TBT in both hypoxia and eumoxia, but this change is not
permanent. Together these data demonstrate that crowding is a stressful
stimulus, as the normal homeostatic response to hypoxia is altered.
Crowding and the traditional training procedure
A cohort of 30 naïve snails was randomly distributed into two groups:
(1) an operant conditioning group and (2) a yoked control group. The operant
conditioning group was subjected to the traditional training procedure (TS1
and TS2) and was tested (MT) for LTM 24h later. The yoked control group
received identical training except that, during each TS, they received the
non-contingent tactile stimulus. Following the traditional training procedure,
the operantly conditioned group demonstrated LTM while the yoked control
snails did not (Fig. 2A). Thus,
as previously reported, this training procedure results in associative
learning and LTM.
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Having shown that, following the traditional training procedure, crowding prior to training blocked LTM formation, we next tested whether crowding after the traditional training procedure would also block LTM formation. Thus, another cohort of naïve snails (N=22) was given the traditional training procedure and placed into crowded conditions for 24h (24h OC) immediately after TS. When MT was tested, LTM was not present. MT was significantly greater than TS2 but not significantly different from TS1. However, TS2 was again significantly less than TS1 (Fig. 2C). Therefore, these data indicate that crowding before or after the traditional operant conditioning training procedure blocks LTM formation in L. stagnalis. Moreover, crowding before training does not appear to interfere with the ability to learn or to remember for 1h, as TS2 was significantly different from TS1.
Crowding and the memory augmentation procedure
Martens et al. demonstrated that snails subjected to the KCl bath prior to
a single 30 min TS, which normally only results in a memory persisting for
2–3h (i.e. ITM), exhibited memory 24h later (i.e. LTM)
(Martens et al., 2007a). We
replicated those results (Fig.
3A). The snails demonstrated LTM, since the number of attempted
pneumostome openings in MT was significantly less than the number of attempted
openings in TS1. We then challenged this cohort of snails with a
change-of-context test (CT). The snails did not demonstrate memory in the new
context, as the number of attempted pneumostome openings in CT was
significantly greater than MT but was not different from TS1. These data
reveal that the decreased number of attempted pneumostome openings in TS was
not the result of sickness or other side effects caused by the KCl bath.
Another cohort of snails was exposed to the memory augmentation procedure to
determine whether memory is observed 2h after TS1. As expected, MT was
significantly less than TS1, suggesting that memory is detected 2h after
training. This 2h memory was also context specific, as changing the context
(CT) resulted in the snails behaving as though they did not possess memory,
i.e. CT was significantly greater than MT but not significantly different from
TS1 (Fig. 3B).
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Crowding immediately after the training procedure prevents the formation of LTM
To determine whether crowding also blocks LTM formation using the memory
augmentation procedure, as it did during the traditional training procedure,
the effect of crowding immediately after the memory augmentation procedure was
also examined (Fig. 4).
Following TS1, the operantly conditioned snails (N=26) were randomly
divided into two groups: 12 snails were placed into a crowded aquarium for
24h, while the remaining 14 were placed into an uncrowded aquarium for 24h.
Snails subjected to crowding (Fig
4B) did not exhibit LTM, as the number of attempted pneumostome
openings in MT was not significantly different from the number of attempted
openings in TS. However, LTM was present in those snails that were housed in
an uncrowded aquarium for 24h (Fig
4C). Moreover, the number of attempted openings in MT of the
snails subjected to crowded conditions was significantly greater than the
number of attempted openings in MT of the snails in uncrowded conditions. To
further demonstrate that crowding blocked LTM formation, yoked control snails
were subjected to similar crowded and uncrowded conditions following TS
(Fig 4). The response in MT of
the yoked control snails was statistically similar between the two groups,
revealing that crowding had no effect on how yoked control snails respond in
MT compared with yoked control snails maintained in uncrowded conditions.
Thus, we conclude that the immediate crowding of snails for 24h following
training prevents LTM formation.
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We next tested whether delaying crowding for 1h following TS also blocked LTM formation using the memory augmentation procedure. Following 1h in uncrowded conditions, the trained snails (N=23) were subjected to crowded conditions for 23h before testing MT for LTM. Results reveal that LTM was present (Fig. 5). To control for possible crowding side effects, the snails were subjected to the carrot context (CT) 2h later. In CT, the snails behaved as naïve snails. Finally, we also subjected another cohort (N=23) of snails to the yoked control procedure and waited 1h before subjecting these snails to crowded conditions. Results reveal that the number of attempted openings in yoked control snails was statistically the same as in TS but was significantly different from the number of attempted openings in MT.
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Immediate crowding for 1h is sufficient to block LTM formation
The consolidation process necessary for LTM following the memory
augmentation procedure occurs within 1h following TS
(Martens et al., 2007a). This
led us to ask if crowding snails for only 1h immediately after training was
sufficient to impair LTM formation. Immediately after training, snails were
subjected to crowded conditions for 1h, after which they were subjected to
uncrowded conditions for 23h. Results reveal that LTM was not present
(Fig. 5B). Yoked control snails
subjected to the same crowding challenge gave similar results.
Crowding before training can also block LTM formation
A naïve cohort of 56 snails was subjected to crowded conditions for
24h prior to TS1 to determine whether crowding before operant training blocked
LTM. The snails were trained using the memory augmentation procedure. Results
reveal that LTM was not present 24h later, as the number of attempted openings
in MT was not significantly different from TS1
(Fig. 6A). Yoked control snails
showed a similar response. Thus, crowding for 24h prior to training altered
the snails' ability to form LTM.
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Crowding and ITM formation
LTM formation in Lymnaea, requires both altered gene activity and
new protein synthesis, while ITM, which persists for 2–3h in
Lymnaea, is dependent only on new protein synthesis
(Sangha et al., 2003a;
Sangha et al., 2003b;
Lattal et al., 2006). We
therefore asked if crowding also compromises ITM formation following the
memory augmentation procedure. Immediately following training, the snails were
placed in crowded conditions for 1h, after which they were returned to normal
conditions for a further 1h before testing for ITM. Results revealed that ITM
was present. To control for the possibility that the decreased number of
attempted openings in MT was not a reflection of memory but rather a side
effect of crowding, the context of the memory test was changed. In this test,
snails behaved as though they were naïve. Finally, the yoked control
snails, which were subjected to the same crowding challenge and tested at the
same time as the operantly conditioned snails, also showed no evidence of
memory.
We then determined whether crowding prior to training had a deleterious effect on ITM formation following the memory augmentation procedure (Fig. 7B). Snails were kept in crowded conditions for 24h before TS. Following TS they were maintained in uncrowded conditions for 2h before MT. We found that ITM was present. When challenged with a change-of-context test 2h later, they behaved as naïve snails. Finally, the yoked control snails showed no evidence of memory. Thus, with 24h of crowding before training, ITM was still formed. In a second experiment (Fig. 7C), a cohort of snails was subjected to crowded conditions for 1h only prior to the memory augmentation procedure. Following TS1, snails were placed into uncrowded conditions for 2h before testing for memory. We found memory to be present. Moreover, when challenged with the change-of-context test they behaved as naïve snails. Finally, the response in MT of the yoked control snails was not significantly different from TS. Thus, we concluded that crowding either before or after TS does not block ITM, suggesting that the effect crowding has on LTM formation is due to an alteration of the transcription process.
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Aerial breathing behaviour and memory formation in CPW
In the next series of experiments, we determined whether snails had to
experience crowding with other live snails (i.e. `rubbing shoulders') in order
for LTM formation to be blocked or whether there was a chemical signal, e.g.
in the water released by snails in crowded conditions, that would be
sufficient to block LTM. A 30-min breathing observation (Pre-obs) was
performed as in Fig. 1. These
snails were then placed in an aquarium containing CPW for 24h but were
maintained in uncrowded conditions (i.e. two snails per 100 ml of PW). A
30-min post-observation was then performed; no significant changes in TBT of
the snails was demonstrated (Fig.
8A). Thus, unlike crowding, CPW did not alter breathing
behaviour.
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Having demonstrated that CPW is not sufficient to block LTM formation, we asked whether crowding with empty clean snail shells would block LTM. We placed a cohort of naïve snails with clean, empty snail shells for 24h before the memory augmentation procedure. We found that this crowding was insufficient to block LTM formation (Fig. 9A) either before or after training (data not shown). Finally, we tested whether we could use a combination of clean shells of deceased snails (same density used as for live snails), to produce crowding, together with CPW, to alter either aerial respiratory behaviour or block LTM formation. We found that the combination of shells and CPW was insufficient to block the formation of LTM (Fig. 9B). Thus, we conclude that exposure to CPW alone or exposure to a combination of shells and CPW is not sufficient to alter aerial respiratory behaviour or LTM formation.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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), we hypothesise that crowding interferes with the necessary
genomic activity to produce LTM in neurons, such as RPeD1, that are necessary
for LTM formation (Scheibenstock et al.,
2002).
The data showing that LTM, but not ITM, is blocked show that the effects of
crowding are specific to memory formation and are not the result of general
malaise. If crowding induced a general depression of behaviour, this would
have been observed in the change-of-context challenge and in the blockage of
ITM. However, in all cases with the change-of-context controls, snails behaved
as they did in the initial TS. We believe that the blockage of LTM formation
by crowding is relatively specific. It remains to be determined whether
crowding alters any other easily observable behaviours (e.g. feeding or
locomotion) or whether crowding blocks LTM formed following either appetitive
or aversive food conditioning (Azami et
al., 2006; Sugai et al.,
2007).
Crowding of snails at densities equal to or greater than those we used in
the present study (Colton,
1908; Crabb, 1924; Forbes and
Crampton, 1942; Noland and
Carriker, 1946) negatively affected the extent and rate of growth,
reproductive success and development of recently hatched Lymnaea.
However, these previous studies employed chronic crowding, whereas acute
crowding (1h to a maximum of 24h) was investigated in the present study.
Whether chronic crowding (i.e. days to weeks) would have any different
effect(s) on aerial respiration and/or memory formation remains to be
determined.
Crowding has been used as a stressor in studies investigating behaviours
such as memory formation, immune system functioning, and longevity in both
vertebrates and invertebrates (Gems et
al., 1998; Vanitallie,
2002; Bowman et al.,
2003; Roman et al.,
2004). For example, Gems et al. showed that crowding in
Caenorhabditis elegans arrests development
(Gems et al., 1998). Mice have
also been observed to become more susceptible to nematode infection when
crowded due to stress-mediated immuno-depression
(Abu-Madi and Lewis, 1997).
However, our present study is the first we know of to use crowding as a
stressor to block LTM formation in a model system where it may be relatively
easy to demonstrate how this stressor acts at the single cell level, as the
molecular processes that cause LTM formation occur within RPeD1
(Scheibenstock et al.,
2002).
TBT significantly decreases following crowding. Intuitively, this appears
to be an inappropriate response. However, it is not. When exposed to an
environmental stressor such as hypoxia or prey detection, Lymnaea
respond first with compensatory and then adjustive changes in metabolism,
respiration and heart rate (Hochachka et
al., 1996; Taylor et al.,
2003; Orr et al.,
2007). Therefore, a paradox exists between compensatory and
adjustive changes. Compensatory responses involve changes in ventilation and
cardiac output (e.g. they increase) and in turn tend to minimize the fall in
blood oxygen. These compensatory responses represent an attempt by the
organism to continue to meet cellular metabolic demands. However, if the
compensatory changes fail to stem the drop in oxygen, Lymnaea switch
to an adjustive strategy, i.e. TBT is depressed
(Taylor et al., 2003). This
adjustive response represents an attempt to minimize oxygen requirements,
thereby avoiding hypoxic impairment
(Hochachka et al., 1996). We
hypothesize that since there is a metabolic cost of LTM formation (i.e.
altered gene activity and new protein synthesis), a consequence of
Lymnaea assuming the adjustive strategy as a result of crowding is
that LTM formation will be suppressed. That is, we suggest that the blocking
of LTM formation is a side effect of crowding on overall genomic activity.
Whether or not crowding at similar densities as we used here with
non-conspecifics (e.g. other species of snails or with non-predatory
vertebrates) would have the same effect on LTM formation remains to be
determined. However, it seems unlikely based on our findings with the clean
shell data.
A different environmental stressor, predator detection, alters both aerial
respiratory behaviour and LTM formation
(Orr et al., 2007;
Orr and Lukowiak, 2008).
However, upon predator detection, aerial respiratory behaviour increases and
LTM is significantly enhanced. To date, in Lymnaea, crowding has been
the only stressor we have found that blocks LTM formation by itself. If we had
used only the memory augmentation procedure to produce associative learning
and the subsequent formation of LTM, we could possibly have interpreted our
findings to indicate that the reason LTM formation was blocked was that there
was too much stress. That is, the memory augmentation procedure uses a
stressor (KCl bath) to enhance memory formation and, coupled with an
additional stress (crowding), LTM would be blocked. As Martens et al.
demonstrated, too much stress blocks LTM formation
(Martens et al., 2007a).
However, we also showed that crowding blocks LTM using the traditional
training procedure, which does not utilize a stressor (e.g. KCl bath) to
enhance memory formation. Thus, we conclude that the effect of crowding either
immediately before or after operant conditioning for a period as short as 1h
is sufficient to block LTM formation.
As previous data demonstrate, stress can modify LTM formation either by
enhancing it or, as in our case, suppressing it (e.g.
Yerkes and Dodson, 1908;
Shors, 2006). For example,
using an acute heat shock as a stressor, Beck and Rankin showed that LTM
formation could be blocked in C. elegans
(Beck and Rankin, 1995).
Likewise, with an acute stress (e.g. inescapable exposure to a cat), spatial
memory in rats was impaired (Sandi,
2004). By contrast, acute stress improved eye blink conditioning
in both animals (Shors, 2006)
and healthy humans (Duncko et al.,
2007). Therefore, many factors, including the type of stress, the
response of the organism to the stress, the nature of the task and the
previous history of the organism, determine whether acute stress will suppress
or enhance memory formation. Chronic stress has also been demonstrated to
impair hippocampal-dependent spatial memory in rats without affecting their
motor skills (Kleen et al.,
2006). Equally, LTM can be enhanced through fearful stressors
(Blank et al., 2002;
Nijholt et al., 2004;
Orr and Lukowiak, 2008). Thus,
conflicting data exist regarding the effect that stress has on LTM formation.
The effect that stress will have on the formation and/or recall of LTM is
dependent on a myriad number of factors, including the age and gender of the
organism, its previous history dealing with stress and whether or not the
stress is in any way controllable by the animal. It is thus problematic to
predict ahead of the actual data what effect a particular stressor will have
on LTM. This is one of the main reasons why it has been so difficult to
understand how stress alters memory formation.
For crowding to block LTM formation it must occur either immediately before
or after the training procedure and it can be as short as 1h. These data are
consistent with the notion that memory modification is time dependent, i.e. it
does not occur instantaneously (McGaugh,
2000; Nielson and Powless,
2007). We have not yet determined either the minimal duration of
crowding or the minimal gap between training and crowding necessary to block
LTM formation. Previously, we have found that to block LTM formation
successfully, cooling to 4°C must occur within 15 min of training and must
be at least 1h duration (Sangha et al.,
2003b). We hypothesize that the ability of crowding to block LTM
formation will have similar time parameters; however, these experiments are
still to be performed. Recent data show that, in humans, arousal after
learning is capable of enhancing memory formation even when the arousal
stimuli are delayed by up to 30 min
(Nielson and Powless, 2007).
Whether delaying crowding by 30 min following training will affect the ability
of crowding to block LTM formation remains to be determined. We do know,
however, that crowding does not block ITM formation, which is dependent on new
protein synthesis. The fact that ITM formation is resistant to blockade by
crowding also reinforces our notion that the effects of crowding are specific
to LTM formation and not the result of some epiphenomenon.
We initially hypothesized that, as for some other stressors
(Diamond et al., 1996;
Kirschbaum et al., 1996;
de Quervain et al., 1998;
Diamond et al., 1999;
Payne et al., 2002;
Roozendaal, 2002;
Woodson et al., 2003),
crowding would impair memory recall. However, this was not the case, because
even if we crowded snails for 23h before giving them a memory test, LTM was
still apparent. As LTM was still present in the memory recall experiments, we
concluded that the ability of crowding to block memory formation was therefore
not the result of a deficit in a general metabolic process that caused snails
to perform aerial respiration to a greater degree and thus mask LTM.
Earlier researchers (Crabb,
1929) hypothesized that the reduced growth of snails in crowded
conditions was due to a `factor' in the water that the snails were maintained
in. Voronezhskaya et al. also found that `chemicals' released into the water
by snails in crowded conditions delayed embryonic development of
Lymnaea (Voronezhskaya et al.,
2004). Our initial working assumption was that water from the
crowded snail aquarium would be sufficient to block LTM formation. However,
our attempts to block LTM formation with just water taken from a crowded
aquarium (CPW), or a combination of CPW and empty snail shells allow us to
conclude that in order to block LTM formation snails must experience crowding
with other live snails. It is possible that substances released by other live
snails in the mucus that snails secrete to move may contain the substance(s)
sensed by snails that causes LTM to be blocked. Further experimentation will
be needed to test this hypothesis.
In conclusion, our data reveal that stress associated with crowding blocks LTM formation and that there is a critical period persisting for 1h following training when crowding blocks LTM formation. However, crowding during this period does not block ITM formation. Thus, we hypothesize that this stressor acts on genomic activity to prevent the molecular processes necessary for LTM from being initiated or brought to completion.
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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