A critical role for IGF-II in memory consolidation and enhancement

Abstract

We report that, in the rat, administering insulin-like growth factor II (IGF-II, also known as IGF2) significantly enhances memory retention and prevents forgetting. Inhibitory avoidance learning leads to an increase in hippocampal expression of IGF-II, which requires the transcription factor CCAAT enhancer binding protein β and is essential for memory consolidation. Furthermore, injections of recombinant IGF-II into the hippocampus after either training or memory retrieval significantly enhance memory retention and prevent forgetting. To be effective, IGF-II needs to be administered within a sensitive period of memory consolidation. IGF-II-dependent memory enhancement requires IGF-II receptors, new protein synthesis, the function of activity-regulated cytoskeletal-associated protein and glycogen-synthase kinase 3 (GSK3). Moreover, it correlates with a significant activation of synaptic GSK3β and increased expression of GluR1 (also known as GRIA1) α-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid receptor subunits. In hippocampal slices, IGF-II promotes IGF-II receptor-dependent, persistent long-term potentiation after weak synaptic stimulation. Thus, IGF-II may represent a novel target for cognitive enhancement therapies.

Figure 1. C/EBPβ-dependent IGF-II expression significantly increases following training

a, Northern blot examples and densitometric analyses of IGF-II (cyclophilin-normalized). Data are expressed as mean %±s.e.m. of 0h− (one-way ANOVA comparing all groups F_8,59=2.46, P=0.0249, post-hoc t-test *P<0.05). b, Real-time PCR of hippocampal IGF-II and IGF-I mRNA (18S RNA-normalized). Data are expressed as mean fold change± s.e.m. of 20h−/0h− (Student’s t-test ***P< 0.0001). c, Western blot analyses of hippocampal IGF-II from 0h−, unpaired (Un) and trained (+) rats euthanized 20, 72 or 96h later (actin-normalized). Data are expressed as mean %± s.e.m. of 0h− (one-way ANOVA comparing 0h−, 20un, and 20h+ F_2,29=4.69, P=0.0172, Newman-Keuls post-hoc test, *P<0.05). d, Western blot analysis of hippocampal IGF-II from trained or Un rats injected (↓) with either SC-ODN or β-ODN 5h post-training and euthanized 24h post-training (actin-normalized). Data are expressed as mean %± s.e.m. of SC-ODN-Un (two-way ANOVA F_1,19=4.62, P=0.0447 for interaction, F_1,19=1.45, P=0.2434 for ODN-treatment, F_1,19=6.46, P=0.0199 for training-paradigm, Bonferroni post-hoc **P<0.01;*P<0.05).

Figure 2. Hippocampal IGF-II is required for memory consolidation

Schedules shown above figures. a, Mean latency± s.e.m. of rats given double hippocampal injections (↓) of SC-ODN or IGF-II-ODN (one-way ANOVA for treatment F_5,47= 2.54, P=0.043, post-hoc Student’s t-test * P<0.05 for 0h/8h and 24h/32h). b, Mean latency± s.e.m. of rats given double hippocampal injections (↓) of SC-ODN or IGF-II-ODN with either IGF-II or IGF-I (two-way ANOVA F_1,33=4.29, P=0.0468 for interaction, F_1,31=6.34, P=0.173 for ODN-treatment, F_1,31=11.38, P<0.0021 for IGF-treatment, Bonferroni post-hoc **P<0.01)

Figure 3. Hippocampal post-training IGF-II administration enhances memory and prevents forgetting

Schedules shown above figures. a, Mean latency± s.e.m. of trained rats given hippocampal injection (↓) of vehicle, IGF-II or IGF-I and tested 24h and 7-days later (two-way ANOVA F_2,38=0.44, P=0.6463 for interaction, F_2,38=26.7, P<0.0001 for treatment, F_1,38=4.24, P=0.0466 for test, Bonferroni post-hoc test **P<0.01, ***P<0.001). b, Mean latency± s.e.m. of trained rats given an hippocampal injection (↓) of vehicle or IGF-II (Student’s t-test *P=0.0261). c, Mean % freezing of trained rats injected with vehicle or IGF-II (Student’s t-test *P<0.0434). d, Mean latency± s.e.m. of trained rats given bilateral amygdala injection (↓) of vehicle or IGF-II.

Figure 4. Post-retrieval IGF-II administration enhances memory and the effect is temporally limited

Schedules shown above figures. a, Mean latency± s.e.m. of trained rats, tested 24h post-training and, immediately after, injected (↓) with IGF-II or IGF-I. Non-reactivated rats (NoR) were trained and injected (↓) without testing. Rats were tested 48h post-training (two-way ANOVA F_1,26=5.67, P=0.0249 for interaction, F_1,26=9.82, P=0.0042 for treatment, F_1,26=13.67, P=0.0001 for test, Bonferroni post-hoc **P<0.01, *P<0.05). b, Mean latency± s.e.m.of trained rats, tested 14d post-training and, immediately after, injected (↓) with vehicle or IGF-II; memory was tested 15d after training.

Figure 5. The role of IGF-II receptors, de novo protein synthesis, and Arc in memory consolidation and IGF-II-mediated enhancement

Schedules shown above figures. a, Mean latency± s.e.m. of trained rats injected (↓) with vehicle, IGF-II, IGF-II/Anti-IGF-2R, IGF-II/JB1, Anti-IGF-2R, or JB1 (one-way ANOVA F_5,40=3.82, P=0.0023, Newman-Keuls post-hoc test *P<0.05 **P<0.01). b, Mean latency± s.e.m. of trained rats given double injections of IgG or anti-IGF-2R antibody (Student’s t-test **P<0.0041). c, Mean latency± s.e.m. of rats trained, tested then injected (↓) with vehicle, IGF-II or IGF-II+anisomycin (two-way ANOVA F_2,34=5.25, P=0.0103 for interaction, F_2,34=4.68, P=0.0161 for treatment, F_1,34=13.7, P=0.0008 for test, Bonferroni post-hoc **P<0.01,***P<0.001). d, Mean latency± s.e.m. of rats trained, tested and injected (↓) with vehicle+SC-ODN, vehicle+Arc-ODN, IGF-II+SC-ODN, or IGF-II+Arc-ODN (two-way ANOVA F_1,18=7.8, P=0.0119 for interaction, F_1,18=17.3, P=0.0006 for ODN-treatment, F_1,18=12.3, P=0.0025 for veh/IGF-II-treatment, Bonferroni post-hoc ***P<0.001).

Figure 6. Mechanisms of IGF-II-mediated memory enhancement. IGF-II promotes LTP

a, Western blot analysis of hippocampal pCREB and C/EBPβ from naïve or trained rats injected (↓) with vehicle or IGF-II and euthanized 20h later (actin-normalized). Data are expressed as mean %± s.e.m. of naïve-Veh (one-way ANOVA, pCREB: F_2,20=4.3, P=0.0287, C/EBPβ: F_2,19=5.7, P=0.0117, Newman-Keuls post-hoc test, *P<0.05). b, Western blot analysis of hippocampal GluR1 and GluR2 from naïve or trained rats injected (↓) with vehicle, IGF-II, IGF-II+Anti-IGF-IIR antibody (actin-normalized). Data are expressed as mean%± s.e.m. of naïve-Veh (one way ANOVA F_3,19=4.24, P=0.0188, Newman-Keuls post hoc test * P<0.05). c, Western blot analysis of hippocampal pGSK3β and GSK3β from the same extracts as in (B) (actin normalized). Data are expressed as mean%± s.e.m. of naïve-veh (one-way ANOVA F_3,19=4.93, P=0.130, Newman-Keuls post hoc test * P<0.05,**P<0.01). d, Mean latency± s.e.m. of rats trained, tested and injected (↓) with vehicle, IGF-II, SB216763 (SB) or IGF-II+SB (two-way ANOVA F_3,56=4.44, P=0.0072 for interaction, F_3,56=5.07, P=0.0035 for treatment, F_1,56=9.12, P=0.0038 for test, Bonferroni post-hoc **P<0.01,***P<0.001) e, Time-courses of fEPSPs in area CA1 stratum radiatum are shown with sample traces obtained during the baseline period, 2 min after high frequency stimulation (HFS) and 100 min after HFS (gray traces: no HFS; black traces: HFS). Scale bars: 0.5 mV, 5 ms. Left panel: Weak HFS induced only transient potentiation that returned to baseline levels within 100 min. Middle panel: In the presence of IGF-II, the same protocol induced stable LTP (Student’s t-test P<0.05). Right panel: In slices pretreated with antibodies against the IGFII receptor, IGF-II failed to facilitate the induction of stable LTP.