Short-term memory reactivation of a weak CS-US association promotes long-term memory persistence in conditioned odor aversion

Abstract

In conditioned odor aversion (COA), the association of a tasteless odorized solution (the conditioned stimulus [CS]) with an intraperitoneal injection of LiCl (the unconditioned stimulus [US[), which produces visceral malaise, results in its future avoidance. The strength of this associative memory is mainly dependent on two parameters, that is, the strength of the US and the interstimuli interval (ISI). In rats, COA has been observed only with ISIs of ≤15 min and LiCl (0.15 M) doses of 2.0% of bodyweight, when tested 48 h after acquisition (long-term memory [LTM]). However, we previously reported a robust aversion in rats trained with ISIs up to 60 min when tested 4 h after acquisition (short-term memory [STM]). Since memories get reactivated during retrieval, in the current study we hypothesized that testing for STM would reactivate this COA trace, strengthening its LTM. For this, we compared the LTM of rats trained with long ISIs or low doses of LiCl initially tested for STM with that of rats tested for LTM only. Interestingly, rats conditioned under parameters sufficient to produce STM, but not LTM, showed a reliable LTM when first tested for STM. These observations suggest that under suboptimal training conditions, such as long ISIs or low US intensities, a CS-US association is established but requires reactivation in the short-term in order to persist in the long-term.

Figure 1.

CS–US association at low US intensities and reactivation-induced long-term memory in COA. (A) Experimental protocol. (B) Preference Index (P.I.) of LTM test, no significant reduction was observed with 0.5 or 1.0% BW LiCl. (C) P.I. of STM test, a significant reduction was observed with all doses tested. (D) P.I. of LTM_Reactivated test (same groups previously tested for STM shown in C), a significant reduction was observed at all doses of LiCl tested. One-way ANOVA, followed by Dunnett's post-hoc test for all groups. (***) P < 0.001. (E) Linear regression analysis of all doses and conditions tested, only LTM test groups showed a significant LiCl dose-dependent reduction on P.I. and the slope of the curve was significantly different to STM and LTM_Reactivated groups. Group size in parentheses.

Figure 2.

CS–US association at long ISIs and reactivation-induced long-term memory in COA. (A) Experimental protocol. (B) Preference Index (P.I.) of LTM test, no significant reduction was observed after 30 min. (C) P.I. of STM test, a significant reduction was observed on all ISIs tested. (D) P.I. of LTM_Reactivated test (same groups previously tested for STM shown in C), a significant reduction was observed at all ISIs tested. One-way ANOVA, followed by Dunnett's post-hoc test for all groups. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (E) Linear regression analysis of all ISI and all conditions tested, only LTM test groups showed a significant ISI length-dependent reduction on P.I. and the slope of the curve was significantly different to STM and LTM_Reactivated groups. Group size in parentheses.

Figure 3.

Memory reactivation or medicine-like effect? (A) Experimental protocol; only CS+ was presented during the STM test. (B) STM test of LiCl 0.5% groups. Water intake was similar between saline and LiCl i.p. injected groups (t-test, P > 0.05). (C) STM test of long ISI groups. ISI 60-min rats drank significantly less than its respective saline control (t-test, P < 0.001). (D,E) P.I. during the LTM test. Only the ISI group reached a statistical significance (t-test, P < 0.01).

Figure 4.

US intensity and time to first test correlation with conditioned response. (A) P.I. for all times tested for 0.5% LiCl, there is a significant reduction at 4 and 8 h but not at 12 h. (B) P.I. of LTM_Reactivated test of groups shown in A, there is a significant reduction at all times, and a time-dependent gradient. (C) Linear regression showing a significant time-dependent increase of P.I. at first test (r² = 0.431, P < 0.001) but not at second test (r² = 0.1274, P > 0.05). (D) P.I. for 1.0% LiCl groups, there is a significant reduction at all times tested. (E) P.I. of LTM_Reactivated groups shown in D, there is a significant reduction at 4 and 8 h but not at 12 h. (F) Linear regression showing a significant time-dependent increase of P.I. at first test (r² = 0.1815, P < 0.05) but not at second test (r² = 0.1163, P > 0.05). (G) P.I. for 2.0% LiCl groups, there is a significant reduction at all times tested. (H) P.I. of LTM_Reactivated groups shown in G, there is a significant reduction at all times tested. (I) Linear regression showing no effect over time for either test (r² = 0.1297 and 0.0394, respectively; P > 0.05 for both). One-way ANOVA, followed by Dunnett's post-hoc test for all groups. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001.

Figure 5.

ISI length and time to first test correlation with conditioned response. (A) P.I. for 5-min ISI, there is a significant reduction at all times tested. (B) P.I. of LTM_Reactivated groups shown in A; there is a significant reduction at all times tested. (C) Linear regression showing no effect over time on either first or second test (r² = 0.1152 and r² = 0.03940, respectively; P > 0.05 for both). (D) P.I. for 15-min ISI, there is a significant reduction of P.I. at 4 and 8 but not at 12 h. (E) P.I. of LTM_Reactivated groups shown in D, there is a significant reduction at all times tested. (F) Linear regression showing a time effect during the first test (r² = 0.1544, P < 0.05) but not in the second test (r² = 0.0565, P > 0.05). (G) P.I. for 30-min ISI, there is a significant reduction at all times tested. (H) P.I. of LTM_Reactivated groups shown in G, there is a significant reduction at all times tested. (I) Linear regression showing no effect over time on either first or second test (r² = 0.01317 and 0.03998, respectively; P > 0.05 for both). (J) P.I. for 60-min ISI, there is a significant reduction at all times tested. (K) P.I. of LTM_Reactivated groups shown in J, there is a significant reduction at all times tested. (L) Linear regression showing no effect of time on P.I. on either first or second test (r² = 0.0990, P > 0.05 and r² = 0.0292, P > 0.05, respectively).