Response to operant conditioning and yoked controls at 24 h.

<p>Number of average (± SEM) pneumostome openings in 30 min during training (TR) and the test for LTM 24 h later (test @ 24 h) in the CM (Chilton Moor) and TC1 (Trans Canada 1) populations following contingent (white bars) or yoked (grey bars) training. ** = significantly different from TR (P<0.01).</p

Duration of memory retention following operant conditioning.

<p>Number of average (± SEM) pneumostome openings in 30 min during training (TR: average response across four training groups is displayed) and the test for LTM (test @ 24 h, 3 d, 5 d or 8 d) in the CM (Chilton Moor) and TC1 (Trans Canada 1) populations following training in control conditions. ** = significantly different from TR (P<0.01).</p

Effect of low calcium on the duration of memory retention.

<p>Number of average (± SEM) pneumostome openings in 30 min during training (TR: average response across two training groups is displayed) and the test for LTM (test @ 24 h or 3 d) in the CM (Chilton Moor) and TC1 (Trans Canada 1) populations following exposure to low environmental calcium (pale grey: 20 mg/l) for 1 week before and during training/testing. ** = significantly different from TR (P<0.01).</p

RPeD1 burst activity in naïve versus trained <i>Lymnaea</i>.

<p>Mean (±SEM) burst activity in RPeD1 in naïve <i>Lymnaea</i>, 3 h following training (i.e. representing intermediate-term memory test) and 24 h following training (i.e. representing long-term memory test) following exposure to: A) control conditions (white bars: naïve: N = 11; ITM: N = 9; LTM: N = 9); B) low calcium availability (pale grey bars: naïve: N = 10; ITM: N = 7; LTM: N = 6); C) crowding (dark grey bars: naïve: N = 9; ITM: N = 10; LTM: N = 10) or D) a combination of low calcium availability and crowded conditions (black bars: naïve: N = 9; ITM: N = 13; LTM: N = 10). *  =  significant difference between naïve and trained bursting behaviour (P<0.05: SNK test).</p

Burst activity in RPeD1 and breathing behaviour 3 h post-stress exposure.

<p>RPeD1 burst activity in naïve <i>Lymnaea</i>. A) Mean (±SEM) burst activity in untrained <i>Lymnaea</i> following exposure to control conditions (white bars: N = 11), low calcium availability (pale grey bars: N = 10), crowding (dark grey bars: N = 9) or a combination of low calcium availability and crowded conditions (black bars: N = 9). Representative traces show burst activity in RPeD1 over 10 min in naïve <i>Lymnaea</i> exposed to B) control conditions, which did not differ significantly from those exposed to low calcium only; and C) combined stressors, which did not differ significantly from those exposed to crowding only. Breathing activity in untrained <i>Lymnaea</i> 1 week prior to exposure (pre-obs) and 3 h following exposure (post-obs) to D) control conditions (N = 12) or E) combined stressors (N = 12). Control animals did not alter their breathing rate, whereas combined stressors significantly depressed aerial breathing behaviour.</p

Behavioural assessment of intermediate-term and long-term memory.

<p>Mean (± SEM) number of pneumostome opening attempts during training (TR1 and TR2) and the test for intermediate-term memory 3 h following training (A to D: test @ 3 h) or long-term memory 24 h following training (E to H: test @ 24 h). <i>Lymnaea</i> were exposed to:control conditions (white bars: A: N = 18; E: N = 13); low calcium availability (pale grey bars: B: N = 18; F: N = 12); crowding (dark grey bars: C: N = 18; G: N = 12) or a combination of low calcium availability and crowded conditions (black bars: D: N = 18; H: N = 12). **  =  significant difference between the first training session (TR1) and second training session (TR2) or the memory test (P<0.01, paired t-test).</p

Cooling blocks memory formation.

<p>A) Snails (n=9) received 20 paired presentations of the CS-US on one day. These snails exhibited STM when tested 10 min after the last pairing (Post 1D, white bar), but did not exhibit LTM 24h later (checked bar). B) A second group of snails (n = 8) was trained as in A except the cold block was applied immediately after the last CS-US pairing. Both STM and LTM were blocked. C) A naive group (n = 8) received 20 CS-US pairings but the pairings were spread out over 2 days (i.e. 10/day). STM was observed but not LTM. D) As in C except that the cold block was immediately applied following the 10th CS-US pairing each day (n=8). Neither STM nor LTM formed. Each graph was composed of the scores of pre-test, 10 min post-test and 24-h post-test.</p

Effects of cold-block on the feeding response to the CS at various time points, (A) 0-min delay (n=8), (B) 4-min delay (n=8), (C) 7-min delay (n=8), (D) 10-min delay (n=8), (E) 15-min delay (n=8), and (F) 180-min delay (n=8).

<p>Conditioning was performed with 5 paired CS-US presentations for 4 days. Each scattered graphs were composed of 8 snails’ individual data, the mean of feeding scores was connected along with daily change. Within a day the scores of pre-test were shown first then immediate post-test scores were followed. The immediate post-test in 15 min and 180 min delay was applied to snails twice at 10 min and 27 min after the final CS-US pairing. In the cases snails received 15 min delay cold-block, they were examine to CS application twice before and after the cold-block, while 180 min delayed snails were tested before the cold-block twice. The scores of second immediate post-test in E and F were not included.</p

Dynamic changes in the feeding response of group 3 (4D×5 trials) each day (n=9) (A).No significant differences were detected after (B) backward conditioning, 5US+CS pairings for 4 days (n=7) or (C) after applying the cold-block immediately after conditioning each day (n=8). Values were compared to the pre-test value (1D pre) in Figure 3A if there was a significant main effect evaluated by repeated measures of ANOVA.

<div><p>Data are presented as mean ± standard error.</p> <p>*<i>p</i> <0.05, ** <i>p</i><0.01, +<i>p</i><0.0001.</p></div

Taste avoidance conditioning was reversible (A) after immediate cold-block, and (B) during the amnesic state when PKC activation resulted in STM on the final day of conditioning.

<div><p><b>A</b>) Snails experienced 0-min delay cold-block after conditioning for 4 days, and were then conditioned without cold-block for the next 4 days (n=7). Note that the learning kinetics to obtain the new memory formation were similar to those shown in Figure 3A.</p> <p>B) Snails did not exhibit memory when the cold block was applied immediately following the CS-US pairing (i.e. days 1-3). However, on day 4 before the training procedure snails were exposed to the PKC activator bryostatin for 45 min, and then received the 5 CS-US pairings in ‘normal’ water. This resulted in STM but not LTM formation (n=5). Note that 5 paired CS-US presentation was insufficient to cause STM in Figure 3A. Bryo represents the time to soak animals in bryostatin containing water on day 4 before the conditioning paradigm in the horizontal axis.</p> <p>* <i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, +<i>p</i><0.0001.</p></div