Delayed wave of c-Fos expression in the dorsal hippocampus involved specifically in persistence of long-term memory storage

Abstract

Memory formation is a temporally graded process during which transcription and translation steps are required in the first hours after acquisition. Although persistence is a key characteristic of memory storage, its mechanisms are scarcely characterized. Here, we show that long-lasting but not short-lived inhibitory avoidance long-term memory is associated with a delayed expression of c-Fos in the hippocampus. Importantly, this late wave of c-Fos is necessary for maintenance of inhibitory avoidance long-term storage. Moreover, inhibition of transcription in the dorsal hippocampus 24 h after training hinders persistence but not formation of long-term storage. These findings indicate that a delayed phase of transcription is essential for maintenance of a hippocampus-dependent memory trace. Our results support the hypothesis that recurrent rounds of consolidation-like events take place late after learning in the dorsal hippocampus to maintain memories.

Fig. 1.

Hippocampal mRNA synthesis is required 24 h after training for LTM persistence but not for memory formation. (Upper) Schematic procedure. (Lower) Animals were infused into dorsal hippocampus with vehicle (Veh) or α-amanitin (0.5 μM; 1 μl/side) 9, 12, 18, 24, or 36 h after training. Data are expressed as mean ± SEM of training (TR, black bar) or test-session step-down latency, 2 d (white bars) or 7 d (gray bars) after training. **P < 0.01 vs. Veh; two-tailed Student’s t test, n = 10–12.

Fig. 2.

Memory processing is associated with two waves of c-Fos expression. (A) Time course of changes in hippocampal c-Fos levels after strong IA training. Bars indicate the percentage of change with respect to the naïve group for rats trained and killed immediately, 1, 3, 9, 12, 18, 24, or 30 h after training. Data are expressed as mean ± SEM. *P < 0.05; two-tailed Student’s t test; n = 5–8. (B) Strong (0.7 mA), but not weak (0.3 mA), IA training generates a persistent LTM. (Upper) Schematic procedure. (Lower) Data are expressed as mean ± SEM of TR (black bars) or test-session step-down latency at 2 or 7 d after weak (gray bars) or strong (white bars) IA training. ***P < 0.0001 vs. TR; two-tailed Student’s t test, n = 10. (C) Strong and weak training induced c-Fos expression 1 h after training (Left). In contrast, strong, but not weak, IA training results in increased c-Fos 24 h posttraining (Right). (Upper) Bars show normalized mean percentage levels of c-Fos with respect to the naïve group. Data are expressed as mean ± SEM of naïve (black bar), weak (gray bar), and strong (white bar) IA training. *P < 0.05; **P < 0.01; ***P < 0.001 vs. naïve in Newman–Keuls test after ANOVA; n = 6. (Lower) Representative Western blots showing c-Fos and actin levels. (D) Strong, but not weak, IA training is associated with increased c-Fos immunoreactivity in dorsal CA1 24 h after training.

Fig. 3.

Delayed expression of c-Fos is required for persistence but not memory formation. (A) Biotinylated ASO anatomical distribution and relative concentrations at different times after infusion. By 2 h, the ASO diffused throughout the dorsal hippocampus and remained there for at least 3 more h. After 24 h, the ASO was cleared out from the hippocampus. (B–E) Schematic of the procedure used. (B) Animals infused into dorsal hippocampus with MSO (white bars) or ASO (gray bars) 12 h after training (Upper). Twenty-four hours later, the dorsal hippocampus was dissected out and used for Western blot analysis (Lower). Bars show the normalized percentage levels with respect to naïve animals. *P < 0.05 in Newman–Keuls test after ANOVA; n = 6. (C) Animals were infused in the dorsal hippocampus with MSO (white bars) or ASO (gray bars) 4 h before training. Data are expressed as mean ± SEM of TR (black bars) or test session step-down latency 2 or 7 d after IA training. The dotted bar represents mean test-session step-down latency of ASO-infused animals that were retrained and tested 24 h later. *P < 0.05; **P < 0.01 vs. MSO group; Student’s t test; n = 10–12 per group. (D) Animals were infused in the dorsal hippocampus with MSO (white bars) or ASO (gray bars; 2 nmol/side) 8, 12, 18, or 24 h after training. Data are expressed as mean ± SEM of TR (black bars) or test session step-down latency 2 or 7 d after IA training. **P < 0.01 vs. MSO group; Student’s t test; n = 10–12 per group. (E) Intrahippocampal infusion of c-fos ASO 12 h posttraining impairs LTM persistence in a permanent way. Data are expressed as mean ± SEM of TR (black bars) or test-session step-down latency 7 and 14 d after IA training. *P < 0.05 vs. MSO group; Student’s t test; n = 10–12 per group.

Fig. 4.

Infusion of c-fos ASO 12 h after IA training does not affect locomotor activity, anxiety state, or exploratory behavior. (A) Number of rearings (Left) and crossings (Right) during a 5-min open-field (OF) session for animals that had received c-fos MSO (open bars) or c-fos ASO (gray bars; 2 nmol/μl; 1 μl/side) in dorsal CA1 12 h posttraining 7 d before the OF session. Data are expressed as mean ± SEM number of crossings or rearings (n = 8). (B) Total number of entries (Left), time spent in open arms (Center), and number of entries into the open arms (Right) during a 5-min plus maze session for rats that had received bilateral intra-CA1 infusion of c-fos MSO (white bars) or c-fos ASO (gray bars) 7 d before the OF session (n = 8).

Fig. 5.

Delayed posttraining infusion of NE promotes LTM persistence. (A) Schematic procedure. (Top) Animals infused into dorsal hippocampus with vehicle (Veh; light gray bar), MSO (white bar), or ASO (dark gray bar; 2 nmol/μl; 1 μl/side) 11 h after training. One hour later, they were also injected with Veh (light gray bar) or NE (white and dark gray bars; 0.6 μg/μl; 0.5 μl/side). Twenty-four hours after training, the dorsal hippocampus was dissected out and used for Western blot analysis (Bottom). Bars show the normalized percentage levels with respect to naïve (black bar) animals. *P < 0.05 in Newman–Keuls test after ANOVA (n = 6). (B) Animals infused in the dorsal hippocampus with vehicle (Veh; light gray bar), MSO (white bar), or ASO (dark gray bar) 11 h after training. One hour later, they were also injected with Veh (light gray bar) or NE (white and dark gray bars). Data are expressed as mean ± SEM of TR (black bars) or test-session step-down latency 2 (Left) or 7 d (Right) after training. ***P < 0.001 in Newman–Keuls test after ANOVA (n = 8–10).