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First published online October 18, 2006
Journal of Experimental Biology 209, 4273-4282 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02520

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Repeated cocaine effects on learning, memory and extinction in the pond snail Lymnaea stagnalis

Kathleen Carter¹, Ken Lukowiak², James O. Schenk^1,3 and Barbara A. Sorg^1,*

¹ Program in Neuroscience, Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA 99164, USA
² Department of Physiology and Biophysics, Neuroscience Research Group, University of Calgary, T2N 4N1, Canada
³ Department of Chemistry, Washington State University, Pullman, WA 99164, USA

View larger version (6K): [in a new window]   Fig. 1. Michaelis-Menten analysis of dopamine uptake in isolated snail brains measured by rotating disk electrode voltammetry. Sequential addition of dopamine to the preparation showed a Km of 0.9 µmol l-1 dopamine and a Vmax of 558 pmol dopamine s-1 g-1 wet mass. A total of 40 animals was used for this experiment (N=8; 5 pooled brains used for each experiment). Because the concentration of outside dopamine ([dopamine]o) varied slightly as a function of the rate of uptake when each sequential addition of exogenous dopamine was made, individual data points are shown rather than averaged data. The data were fitted to the Michalis-Menten equation using non-linear regression. The curve does not significantly deviate from the model (P=0.36 via the runs test).

View larger version (5K): [in a new window]   Fig. 2. Inhibition of dopamine uptake by cocaine in isolated snail brains measured by HPLC. Cocaine doses ranging from 0.03-10.0 µmol l-1 were used, and dopamine uptake is reported as mean ± s.e.m. of dopamine levels remaining outside the tissue (N=6 for controls; N=3 for 0.03 µmol l-1; N=4 for all other doses). The IC50 for cocaine was estimated to be approximately 0.03 µmol l-1.

View larger version (12K): [in a new window]   Fig. 3. Basal respiration in control and cocaine-exposed snails. To determine changes in respiratory behavior due to cocaine exposure, total breathing time was measured in snails placed in hypoxic pond water over a 45 min session before exposure to cocaine and 2 days after the last (fifth) exposure. Values are means ± s.e.m. (A) Animals exposed to 0.1 µmol l-1 cocaine (N=13) showed significantly higher total breathing time after exposure to cocaine than before exposure. No significant change in total breathing time was observed in controls (N=12). (B) Total number of pneumostome openings was also tabulated for the same snails as in A. Animals receiving 0.1 µmol l-1 cocaine showed a non-significant trend toward more pneumostome openings after exposure. *P<0.05, compared within the treatment group to the before exposure values.

View larger version (12K): [in a new window]   Fig. 4. Respiratory behavior in freely behaving, yoked and operantly conditioned snails. Pneumostome activity was measured over three 45 min training sessions for (A) freely behaving animals (N=18), (B) yoked animals (N=17) and (C) operantly conditioned animals (N=31). Values are means ± s.e.m. for number of pneumostome openings. Animals operantly conditioned show a significant decrease in pneumostome openings across sessions. The other two treatment groups show no difference in pneumostome openings over the training time course. 1, Training session 1; 2, Training session 2; MT, Memory Test; NS, not significant. *P<0.05 compared with performance during Training session 1 within each treatment group.

View larger version (8K): [in a new window]   Fig. 5. Learning and memory in control and cocaine-exposed snails. (A) Control (N=31; from Fig. 4C) and (B) 0.1 µmol l-1 cocaine-treated (N=15). Values are mean ± s.e.m. for number of pneumostome openings. Both groups demonstrated learning and memory. 1, Training session 1; 2, Training session 2; MT, Memory Test; *P<0.05 compared with performance during Training session 1 within each treatment group.

View larger version (15K): [in a new window]   Fig. 6. Test for savings of extinction memory during reinstatement in control and cocaine-exposed snails. After operant conditioning, animals were exposed to extinction training but no pneumostome stimulus was administered (shown by ovals E1, 2 and 3; no data indicated). Testing for extinction memory was conducted during the Test for Savings 2 h after the last extinction training session, and this Test for Savings session is shown for the same animals as those used for the data in Fig. 5. (A) Control and (B) 0.1 µmol l-1 cocaine treated. Values are mean ± s.e.m. for number of pneumostome openings. Animals treated with 0.1 µmol l-1 cocaine did not meet the requirements for extinction memory during reinstatement. Snails pretreated with 0.1 µmol l-1 cocaine showed a significant reduction in the number of pneumostome openings on the Test for Savings compared with the Memory Test. Training session 1; 2, Training session 2; MT, Memory Test; TS*P<0.05 compared with MT within the same treatment group.

View larger version (10K): [in a new window]   Fig. 7. Respiratory behavior after pretreatment with 0.1 µmol l-1 cocaine in freely behaving snails over time. Comparison of animals pretreated with 0.1 µmol l-1 cocaine and given operant training (shown in Fig. 6B) with freely behaving animals similarly pretreated but given no pneumostome stimulation (N=15) indicates that respiratory behavior does not change over the time frame used for trained animals. Values are mean ± s.e.m. for number of pneumostome openings. Training session 1; 2, Training session 2; MT, Memory Test; N.S.=not significant; E1-3, extinction training sessions. *P<0.05 compared with Training session 1 within the same treatment group.

View larger version (16K): [in a new window]   Fig. 8. Comparison of normalized performance between control and cocaine-exposed snails. Performance of control animals and animals treated with 0.1 µmol l-1 cocaine. Data are reported as mean ± s.e.m. of pneumostome openings measured on the Test for Savings (TS) as a percent of openings on Training session 1 (A) and the last extinction day (B). Animals treated with 0.1 µmol l-1 cocaine showed a significant reduction in extinction memory during reinstatement compared with control snails. 2, Training session 2; MT, Memory Test. *P<0.05, compared with controls on the same day.