Induction of LTM following an Insulin Injection

The CTA training procedures and time lines of food deprivation. A, All snails were first given a pretest 10 min before the training. In this observation period (1 min), the feeding responses (i.e., number of bites) were counted following a 15-s application of 100 mm sucrose (the CS) to the lip of the snail. For taste aversion training, the 100 mm sucrose CS was paired with the 200 mm KCl US. The interstimulus interval was 15 s between the onset of the CS and US. A 90-s ITI was interposed between each pairing of the CS-US. Snails received 10 paired CS-US trials on a single day. Controls included a backward-conditioned (US-CS) group and a naive group to validate associative learning. For the naive control group, only water was applied to the lips instead of the CS and US. In the post-test sessions, snails were again challenged with the CS, and the number of bites was recorded in the 1-min interval after a 15-s application of the CS. B, Food-deprivation status was defined in the following manner: (1) the day when snails began food deprivation is called day 0; (2) day −1 snails had ad libitum access to food (AF), that is, they were not food deprived (FD); (3) day 1 and day 5 snails were food deprived for 1 and 5 d, respectively, after food deprivation, had ad libitum access to food (AF); (4) day 12 snails had ad libitum access to food for 7 d following training on day 5; and (5) day 13 snails were food deprived for an additional 1 d following 7 d of ad libitum access to food. C, Snails were first placed in the test chamber for 10 min to acclimate to the training apparatus. Following the acclimatization, a pretest session was performed, and 10 min after the pretest, CTA training was performed. After CTA training, several post-test sessions were performed to verify the memory expression.

Memory formation in day 1, day 5, and day 13 cohorts of snails. The experimental schedule for each cohort of snails is shown at the top of figures. A, The number of bites elicited by the CS (a 100 mm sucrose solution) in 1 min in the pretest session and the post-test sessions for day 1 snails are shown. The feeding response to the CS in the forward conditioned group was significantly reduced at the three memory test sessions (10 min, 1 d, and 7 d), compared with those observed for the backward-conditioned and naive control snails (two-way ANOVA, n = 10 each, F_(6,81) = 8.79, p < 0.0001 for interaction, F_(2,81) = 29.5, p < 0.0001 for conditioning, F_(3,81) = 11.6, p < 0.0001 for test, Holm post hoc test **p < 0.01). B, As in A except the training and testing were performed on the day 5 snails (5 d of food deprivation before CTA training). Learning and memory are not expressed on the three post-training tests (10 min, 1 d, and 7 d; two-way ANOVA, n = at least 6, F_(6,57) = 0.50, p = 0.80 for interaction, F_(2,57) = 1.18, p = 0.33 for conditioning, F_(3,57) = 1.47, p = 0.23 for test). C, As in A, B except that after the 10-min memory test snails were given ad libitum access to food for 7 d. Snails were thus tested for memory following this 7-d period. No LTM was observed. Finally, snails were given a single day of food deprivation, and memory was tested the following day. LTM was now observed (i.e., day 13 snails; two-way ANOVA, n = 10 each, F_(6,81) = 2.27, p = 0.045 for interaction, F_(2,81) = 4.29, p = 0.024 for conditioning, F_(3,81) = 2.41, p = 0.073 for test, Holm post hoc test *p = 0.031 for forward vs backward; *p = 0.024 for forward vs naive). The error bars indicate SEM.

Real-time PCR of MIP II, MIP I, and MIPR mRNA expression in the CNS of day 12 and day 13 good and poor snails. The data were normalized by the mRNA amounts of HSP40 and GPx. A, MIP II mRNA level was significantly increased in day 13 good learner CNS (one-way ANOVA, n = 5 each, F_(2,12) = 7.95, p = 0.0063, Holm post hoc test *p = 0.013 for day 13 good vs day 12; p = 0.0133 for day 13 good vs day 13 poor). B, C, There was no significant difference in the MIP I and MIPR mRNA levels among day 12, day 13 poor learners, and day 13 good learners (one-way ANOVA, n = 5 each, MIP I: F_(2,12) = 3.11, p = 0.081, MIPR: F_(2,12) = 0.56, p = 0.58). The error bars indicate SEM. n.s., not significant.

Insulin injection into day 12 snails and LTM. The experimental schedules are shown at the top of the figures. A, Number of bites elicited by the CS in 1 min in the pretest session and the post-test sessions in day 5 snails following 7 d of ad libitum access to food and 1 h after drug injections in the day 12 snails. LTM was only expressed in day 12 snails that were injected with bovine insulin. Importantly, note that the simultaneous injection of insulin plus the antibody to the insulin receptor did not allow memory to be recalled (two-way ANOVA, n = 40 for insulin injected group and n = 20 for vehicle-injected control and simultaneous injection group, F_(6,231) = 3.51, p = 0.0024 for interaction, F_(2,231) = 2.97, p = 0.056 for conditioning, F_(3,231) = 8.70, p < 0.0001 for test, Holm post hoc test **p = 0.0031 for insulin vs vehicle; p = 0.0049 for insulin vs simultaneous injection). B, The insulin injection did not alter feeding behavior in day 12 naive snails (one-way ANOVA, n = 10 each, F_(2,27) = 0.09, p = 0.91). The error bars indicate SEM.