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
Pascaline De Caigny and
Ken Lukowiak*
Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Drive NW,
Calgary, Alberta, Canada T2N 4N1
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Fig. 1. Crowding reversibly alters total breathing time (TBT). A naïve cohort
of snails (N=23) received a 30-min hypoxic challenge (10–12
snails/500 ml; Pre-obs). The mean TBT (± s.e.m.) is plotted on the
y-axis. These snails were then placed into crowded conditions (20
snails/100 ml) for 24h and were then given a second hypoxic challenge
(Post-obs 1). Snails were then placed into an uncrowded aquarium (two
snails/100 ml) for 24h. A Kruskal–Wallis (non-parametric ANOVA) was
performed on these data (KW=19.326; P<0.01) followed by a Dunn's
Multiple Comparison test. TBT was significantly less in Post-obs 1 compared
with Pre-obs (*P<0.01). TBT in Post-obs 2 was
significantly greater than in Post-obs 1 (P<0.01). There was no
statistical difference between Pre-obs and Post-obs 2
(P>0.05).
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Fig. 2. The traditional training procedure and crowding. (A) The traditional
training procedure results in LTM, i.e. MT is significantly less than TS1
(*P<0.01) but not significantly greater than TS2
(P>0.05). Yoked control snails also do not demonstrate LTM as
Yoked is not significantly different from TS1 (P>0.05) but is
significantly greater than TS2 (P<0.01). (B) A cohort of
naïve snails (N=19) were placed in crowded conditions for 24h
prior to TS1. In TS2 (separated by an hour, in which the snails were placed in
their normal aquarium; N), the snails received a tactile stimulation each time
they began to open their pneumostome. Twenty-four hours after TS2, the memory
test (MT) was performed. As can be seen, LTM is not formed; that is, when the
data were analyzed (one-way ANOVA) we found that while TS2 is significantly
smaller from TS1 (*P<0.01; demonstrating learning and a
1h memory), MT is significantly greater than TS2 (P<0.01) but not
significantly different from TS1 (P>0.05). (C) A cohort of
naïve snails was placed in crowded conditions for 24h immediately after
TS2. A similar analysis as in A was done on these snails (N=22). TS2
is significantly less than TS1 (*P<0.01) and also the
MT (P<0.01). Values are means ± s.e.m.
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Fig. 3. Long-term (LTM) and intermediate-term (ITM) memory formation following the
memory augmentation procedure. (A) A group of naïve snails
(N=20) received the KCl bath immediately before a 30-min TS in
hypoxic pond water. We tested for memory 24h later (MT). Each time the snail
attempted to open its pneumostome it received a tactile stimulus to the
pneumostome. These snails were then challenged (2h later) to a
change-of-context (carrot context) session (CT). The data were subjected to a
repeated-measures ANOVA (F19,2=22.183; P<0.01)
followed by a Tukey–Kramer comparison test. The number of attempted
openings in MT is significantly less than in TS
(*P<0.01). The number of attempted openings in CT is
not significantly different from in TS (P>0.05) but is
significantly greater than MT (P<0.01). (B) As in A, except memory
was tested 2h after TS. MT is significantly less than TS, showing that ITM had
formed (*P<0.05). Furthermore, there was no significant
difference between the response when the context was changed (CT) and TS
(P>0.05). The analysis in B was obtained using a Friedman's Test
followed by a Dunn's Multiple Comparison test. Values are means ±
s.e.m.
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Fig. 4. Crowding, the memory augmentation procedure and LTM formation. Snails
(N=52; 26 operantly conditioned, 26 yoked controls) were described in
Fig. 3A. (A) Snails that
received the operant conditioning procedure were randomly divided into two
groups immediately following TS. One group (N=12) was subjected to
crowded conditions for 24h, while the other group (N=14) was
maintained for 24h in the control, uncrowded conditions. All snails were then
tested for memory (MT). The data were subjected to a one-way ANOVA
(F51,4=5.107 P<0.01) followed by a
Tukey–Kramer comparison test. As can be seen (B) LTM was not present.
That is, the number of attempted openings in MT was not statistically
different from TS (P>0.05). (C) By contrast, snails not subjected
to crowding exhibited LTM. The number of attempted openings in MT of this
group was statistically different from TS (*P<0.01).
Yoked control snails subjected to either crowding or uncrowded conditions
showed no statistical difference from TS (P>0.05 for each
comparison) nor were they statistically different from each other
(P>0.05). Values are means ± s.e.m.
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Fig. 5. Delay of crowding and LTM formation. (A) A cohort of naïve snails
(N=23 operantly conditioned and N=23 yoked controls) was
trained using the memory augmentation procedure and placed in an uncrowded
aquarium for 1h immediately after training. Following this period, all snails
were subjected to crowded conditions for 23h. All snails were then tested for
memory (MT). Data were subjected to a repeated-measures ANOVA
(F45,3=10.982; P<0.01) followed by a
Tukey–Kramer comparison test. Snails that were subjected to the operant
conditioning procedure exhibited LTM (i.e. MT was significantly less than TS;
*P<0.01). When these same snails were subjected to a
change of context challenge (CT, carrot context) 2h later they did not exhibit
LTM. That is, CT is not significantly different from TS (P>0.05).
Snails subjected to the yoked control procedure also did not exhibit LTM (i.e.
yoked is not significantly different from TS, P>0.05). In
addition, the response of the yoked control snails was not significantly
different from the response to CT. (B) Another cohort of naïve snails
(N=40; 20 operantly conditioned and 20 yoked control snails) was
subjected to operant conditioning and the yoked control procedure,
respectively. Immediately after their respective training procedures they were
placed into a crowded aquarium for 1h. Following this period, all snails were
placed into an uncrowded aquarium for 23h. All snails were then tested for
LTM. Data were subjected to a repeated-measures ANOVA
(F39,2=0.5398; P>0.05) followed by a
Tukey–Kramer comparison test. The operantly trained snails do not
exhibit LTM (MT is not significantly different from TS, P>0.05).
In addition, the yoked control snails subjected to the same crowded and
uncrowded conditions do not exhibit LTM (Yoked is not significantly different
from TS). Values are means ± s.e.m.
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Fig. 6. Crowding before training blocks LTM formation. (A) A naïve cohort of
snails (N=56; 28 operantly conditioned and 28 yoked controls) was
subjected to 24h of crowding immediately before the memory augmentation
procedure. Following training, all snails were placed in an uncrowded aquarium
for 24h and then tested for LTM (MT). Data were subjected to a
repeated-measures ANOVA (F55,2=2.378; P>0.05)
followed by a Tukey–Kramer comparison test. Neither the operantly
conditioned snails nor the yoked control snails exhibited LTM. That is, MT is
not significantly different from TS (P>0.05) nor is Yoked
different from TS (P>0.05). Finally, Yoked is not significantly
different from MT (P>0.05). (B) Crowding for 1h immediately before
the memory augmentation procedure is sufficient to block LTM formation.
Another naïve cohort of snails (N=19; 10 operantly conditioned
and nine yoked controls) was subjected to 1h of crowding immediately before
training. Following training, all snails were placed in an uncrowded aquarium
for 24h and then tested for LTM (MT). Data were subjected to a
repeated-measures ANOVA followed by a Tukey–Kramer comparison test.
Neither the operantly conditioned snails nor the yoked control snails exhibit
LTM (F18,2=0.8510; P=0.4364). That is, MT is not
significantly different from TS (P>0.05) nor is Yoked different
from TS (P>0.05) Finally, Yoked is not significantly different
from MT (P>0.05). Values are means ± s.e.m.
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Fig. 7. Crowding does not block intermediate-term memory (ITM). (A) A naïve
cohort of snails (N=46; 23 operantly conditioned and 23 yoked control
snails) was subjected to 1h of crowding immediately after the memory
augmentation procedure and then placed into an uncrowded aquarium for an
additional 1h period. The snails were then tested for memory (MT). That is,
memory was tested 2h after training. A Kruskal–Wallis (non-parametric
ANOVA) was performed on these data (KW=27.726; P<0.01) followed by
a Dunn's Multiple Comparison test. In the cohort that received operant
conditioning training, ITM is present as MT is significantly less than TS
(*P<0.01). These snails were then challenged with a
change-of-context session (CT) 2h later. Memory is not present as CT is not
significantly different from TS (P>0.05). The snails that received
the yoked control procedure (Yoked) do not exhibit ITM (Yoked is not
significantly different from TS; P>0.05). (B) Another cohort of
snails (N=27 operantly conditioned and 27 yoked controls) was crowded
for 24h before the memory augmentation procedure. Following training they were
all placed into an uncrowded aquarium for 2h before the memory test (MT or
Yoked). Data were subjected to a repeated-measures ANOVA
(F53,3=31.651; P<0.01) followed by a
Tukey–Kramer comparison test. Snails that had been operantly conditioned
exhibit ITM when tested (MT; *P<0.01). The yoked
control snails do not exhibit ITM (Yoked not significantly different from TS;
P>0.05). Snails that had received operant conditioning training
were also challenged 2h after MT with a change-of-context test (CT). In CT,
the number of attempted openings was not statistically different than TS
(P>0.05), indicating that snails were not sick. (C) As in B except
snails were only crowded for 1h before the operant conditioning training or
the yoked control procedure. A Kruskal–Wallis (non-parametric ANOVA) was
performed on these data (KW=43.536; P<0.01) followed by a Dunn's
Multiple Comparison test. As in B, ITM is present in the operantly conditioned
snails (MT is significantly different from TS; *P<0.01)
but is not present in the yoked control (Yoked is not significantly different
from TS; P>0.05). Also as in B, when the context was changed,
snails behaved as though they were naïve (i.e. CT is not significantly
different from TS; P>0.05). Values are means ± s.e.m.
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Fig. 8. Aerial respiratory behaviour and LTM formation in CPW. (A) Total breathing
time (TBT) was unaffected by placing snails in crowded pond water (CPW) for
24h. TBT was first calculated for a cohort of naïve snails (Pre-obs;
N=20). The snails were then placed in CPW for 24h after which TBT was
then calculated (Post-obs). A paired t-test was performed on these
data. There is no difference in TBT between the Pre-obs and the Post-obs
sessions (t=0.4442; P>0.05). (B) A 1h exposure to CPW (OC
in figure) immediately before the memory augmentation procedure does not block
LTM formation. The data were subjected to a repeated-measures ANOVA
(F18,3=7.299; P<0.01) followed by a
Tukey–Kramer comparison test. The operantly conditioned snails
(N=10) exhibit LTM (MT is significantly less than TS;
*P<0.01) while yoked control snails (N=10) do
not exhibit LTM (Yoked is not significantly different from TS;
P>0.05). Additionally, the operantly conditioned snails behaved as
naïve snails to the change-of-context challenge (CT is not significantly
different from TS; P>0.05). (C) A 1h exposure to CPW immediately
after operant conditioning training (memory augmentation procedure) does not
block LTM formation. A naïve cohort of 20 snails (N=20; 10
operantly conditioned and 10 yoked controls) received their respective
training procedures and was then immediately placed into CPW for 1h. Following
the 1h exposure to CPW, snails were moved to an uncrowded aquarium for 23h.
The data were subjected to a repeated-measures ANOVA
(F19,3=5.384; P<0.01) followed by a
Tukey–Kramer comparison test. When tested for LTM (MT), snails that
received operant conditioning training exhibited LTM. That is, MT is
significantly less than TS (*P<0.05). Yoked control
snails do not exhibit LTM (i.e. Yoked is not significantly different from TS;
P>0.05). The operantly conditioned snails were also challenged
with a change-of-context test (CT) 2h after TS. In CT, snails behaved as
naïve snails. That is, CT is not significantly different from TS
(P>0.05). Values are means ± s.e.m.
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Fig. 9. The overcrowding effect is not caused by the physical lack of space. (A)
When snails (N=18) were exposed to crowding with clean shells for 24h
prior to the memory augmentation procedure, LTM forms
(*P<0.05). This was determined with the use of a
Wilcoxon matched-pairs signed ranks test, which showed that the number of
pneumostome openings significantly decreases in the MT compared with the TS.
(B) As in A, except that in addition to clean shells we added crowded pond
water (CPW) as in Fig. 8. The
data were subjected to a paired t-test (t=2.391;
*P<0.02) and show that LTM is formed despite the snails
being exposed to CPW and clean snail shells.
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