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First published online June 16, 2004
Journal of Experimental Biology 207, 2621-2630 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01073

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Learning and memory in Lymnaea are negatively altered by acute low-level concentrations of hydrogen sulphide

David Rosenegger, Sheldon Roth and Ken Lukowiak^*

Calgary Brain Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1

View larger version (12K): [in a new window]   Fig. 1. Breathing observation experiments. (A) Three 45 min observations were performed, first in standard hypoxia, then in hypoxic H2S (100 µmol l–1), and lastly in standard hypoxic conditions again with 22 naïve snails. In between observation periods the snails were returned to eumoxic pondwater for 1 h. A significant increase in the second session vs the first session was seen (ANOVA F(21,1)=98.5381, P<0.05), and there was no significant difference between sessions 1 and 3 (P>0.05). Session 2 was also significantly different when compared to session 3 (ANOVA F(21,1)=65.8242, P<0.05). (B) A 45 min observation in standard hypoxia followed by 1 h in eumoxia, and then 45 min submerged in hypoxia (i.e. the `more intense hypoxic challenge') and another 45 min observation period in standard hypoxia. The `more intense hypoxic challenge' significantly increases the total breathing time (ANOVA F(28,1)=31.6277, P<0.01). A separate group t-test between the breathing observation for the H2S group (N=22) and the `more intense hypoxic challenge' group (N=29) reveals that there is no statistical difference between the two (t=1.4211, P=0.1714 where d.f.=45.9649).

View larger version (12K): [in a new window]   Fig. 2. Long-term memory (LTM), operant conditioning in `standard' and H2S environments. (A) 18 naïve snails received operant conditioning training under standard conditions (i.e. two 45 min sessions separated by 1 h) with a memory test (MT) performed 24 h later. This cohort exhibited both learning and memory. Learning was shown as the second training session was significantly lower than the first, and memory was shown as the memory test (MT) was significantly lower than the first session but not significantly greater than the second training session (ANOVA F(17,2)=20.9304, P<0.01; sessions 2 and 3 are significantly different from session 1, P<0.01) (*signifies that a session is significantly different from session 1 but not from session 2). (B) A cohort of 23 snails that received the `more intense hypoxic challenge' also demonstrated learning and memory. That is, since the number of openings in session 2 was significantly less than in session 1 (ANOVA F(22,2)=40.6394, P<0.01) learning was demonstrated. Additionally, since the memory test session (MT) was significantly different from session 1 but not different from session 2 memory was shown (ANOVA F(22,2)=66.8919, P<0.01 and F(22,2)=2.5811, P=0.1224, respectively). (C) A separate cohort (N=23) of snails underwent the training and testing protocol in hypoxia + H2S (100 µmol l–1). These snails showed neither learning nor memory. That is, the data in session 2 were not significantly different than that in session 1 (i.e. no learning) and the memory test session (MT) was not significantly different from session 1 (i.e. criteria for memory not met; ANOVA F(22,2)=2.3095, P=0.1112; no significant difference between sessions).

View larger version (11K): [in a new window]   Fig. 3. Learning and memory of snails trained in 50 and 75 µmol l–1 of H2S. (A) A cohort of naïve snails (N=24) were trained using the same LTM training procedure as in Fig. 2 while being exposed to H2S at a concentration of 50 µmol l–1. This cohort of snails showed both learning and memory (ANOVA F(23,2)=23.9962 P<0.01; sessions 2 and 3 significantly different from session 1, P<0.01). (B) Another cohort of naïve snails (N=11) was subjected to the same LTM training procedure as in A except that these snails were exposed to an H2S concentration of 75 µmol l–1. Again both learning and memory occurred (ANOVA F(10,2)=6.6139, P<0.01; sessions 2 and 3 significantly different from session 1, P<0.05 and P<0.01, respectively).

View larger version (13K): [in a new window]   Fig. 4. Yoked control snails do not exhibit LTM. Snails were randomly matched to snails in the operantly conditioned group. These yoked control snails received the same number of stimuli (but in a non-contingent fashion) in sessions 1 and 2. In the third session (MT) 24 h later, snails in the yoked group now received a reinforcing stimulus each time they attempted to open their pneumostome. (A) Yoked controls under standard hypoxic conditions. The yoked group (N=11) had significantly more attempted openings in the memory test (MT) session than did the trained group (N=11) (ANOVA F(21,5)=50.1381, P<0.01). (B) Yoked controls challenged with 75 µmol l–1 H2S hypoxic conditions. Yoked snails (N=11) had significantly more attempted openings in the MT session as compared to the trained group (N=11) (ANOVA F(21,5)=15.4653, P<0.01).

View larger version (32K): [in a new window]   Fig. 5. Snail learning `grade distributions'. Snails were given grades based on their individual performance. Grades were calculated as follows: a 50% reduction or greater is an A, a B is a reduction of 35–49.99%, a C is a 20–34.99% reduction, and an F is a reduction of less than 20%. (A) Grade distributions observed to occur under standard hypoxic conditions (N=2301). (B) Distribution of grades for snails presented with the `more intense hypoxic challenge' (N=23). These snails showed a statistically greater number of A grades and fewer F grades than controls (P=0.0007). (C) Grade distributions seen for snails trained in the hypoxia + 50 µmol l–1 H2S condition (N=24). (D) Distributions for snails trained in the hypoxia + 75 µmol l–1 H2S (N=11). (E) Distributions for training in hypoxia + 100 µmol l–1 H2S condition (N=23).

View larger version (13K): [in a new window]   Fig. 6. The effect of H2S on memory consolidation. Exposure to 100 µmol l–1 H2S for 1 h immediately following the second training session had no effect on the ability of the animals to show memory 24 h later (N=24). Learning was shown since the number of attempted openings in session 2 was significantly less than session 1 (ANOVA F(23,1)=8.6197, P<0.01). Memory was demonstrated since MT was significantly less than session 1 (ANOVA F(23,1)=21.1944, P<0.01), but not significantly more than session 2 (ANOVA F(23,1)=6.5714, P=0.0174).