IGF-II activates both IGF-I and IGF-II receptors but with different affinity29
. To determine whether IGF-II-mediated memory enhancement recruits one or both of these receptors, we tested the effect of IGF-I and IGF-II receptor (R) selective inhibitors. Specific inhibitors of IGF-IIR (anti-IGF-IIR antibody) but not IGF-IR (JB1) co-injected with IGF-II completely abolished the memory enhancement compared to respective controls (). The inhibitors alone did not affect memory retention (). Similarly to the antisense experiments, compared to control IgG, a single bilateral hippocampal injection of anti-IGF-2R antibody immediately after training did not affect memory retention (), whereas double injections, immediately- and 8 hours after training, caused a complete amnesia 24h after training ().
The role of IGF-II receptors, de novo protein synthesis, and Arc in memory consolidation and IGF-II-mediated enhancement
We next asked whether IGF-II-mediated memory enhancement recruits new protein synthesis. Since memory consolidation per se
requires new protein synthesis in the hippocampus, blocking protein synthesis in IGF-II-injected rats after training would not be informative. However, because new protein synthesis is not required in the hippocampus for IA reconsolidation15
, we tested the effect of protein synthesis inhibition on retrieval-dependent IGF-II-mediated enhancement.
Bilateral hippocampal co-injection of IGF-II and the protein synthesis inhibitor anisomycin immediately after Test1, 24h after training, showed that anisomycin, compared to vehicle, completely disrupted the IGF-II-mediated memory enhancement tested 24h later () without changing the training-induced retention levels. Hence, memory enhancement, but not reconsolidation, requires hippocampal de novo protein synthesis.
To begin identifying which proteins are required for the memory enhancement, we investigated the role of C/EBPβ. Bilateral hippocampal injection of β-ODN 5h after retrieval (Test1) did not affect the IGF-II-mediated memory enhancement tested 48h after training (supplementary Fig. S9
). The timing of the ODN injections was based on previous kinetics studies showing maximal disruptive effect of β-ODN7,15
. To test whether a prolonged β-ODN treatment could affect the post-retrieval IGF-II-mediated memory enhancement we injected β-ODNs at both 1h before and 5h after reactivation. This treatment, compared to control SC-ODN, also failed to affect the IGF-II-evoked memory enhancement (supplementary Fig. S9
), suggesting that, although de novo
protein synthesis is critical for memory enhancement, C/EBPβ is not.
We therefore hypothesized that the protein synthesis-mediated enhancement may recruit synaptic rather than cell-wide, transcriptional mechanisms. One rapidly regulated translation known to occur at activated synapses and critical for long-term plasticity and memory is that of activity-regulated cytoskeletal-associated protein (Arc)30
. Bilateral hippocampal injection of Arc antisense (Arc-ODN), compared to relative SC-ODN, 1h before retrieval (Test1), completely blocked the post-retrieval IGF-II-mediated memory enhancement, without affecting the basal level of the memory 2 days after training ().
Thus, IGF-II-mediated enhancement requires IGF-II- but not IGF-I receptors. Furthermore, retrieval-dependent IGF-II-mediated enhancement requires de novo protein synthesis and Arc but not C/EBPβ, suggesting that it may use synaptic rather than cell-wide-regulatory mechanisms.
Memory consolidation requires the CREB-C/EBP-dependent gene cascade2
. In IA, both CREB phosphorylation in ser133 (pCREB) and the expression of C/EBPβ are significantly increased in the hippocampus for more than 20 hours after training20
. Here, we examined whether IGF-II mediated memory enhancement following training correlates with an enhanced hippocampal activation of the CREB-C/EBP pathway. Quantitative western blot analyses confirmed that training significantly increased both pCREB and C/EBPβ in the hippocampus 20 h later20
naïve-vehicle, ). Compared to vehicle, IGF-II treatment immediately after training resulted in only a tendency toward a further increase in both markers (). Thus, IGF-II-mediated memory enhancement does not correlate with significant enhancement in the activation of the CREB-C/EBP cascade, strengthening our hypothesis that IGF-II-regulated mechanisms may be synaptic rather than cell-wide. We therefore investigated the synaptic expression levels of GluR1 and GluR2 AMPA receptor subunits. Synaptic GluR1 AMPA receptors have been shown to rapidly increase following IA training and play a critical role in consolidation31,32
. Furthermore, AMPA receptor subunit trafficking is known to accompany long-term plasticity in LTP and long-term depression (LTD)33
. Quantitative western blot analyses of synaptoneurosomal extract (see supplementary Fig. S10
for the biochemical characterization), revealed that there was an increased expression of synaptic GluR1 30 minutes after training compared to naive, which, however, was not significant, probably due to the relatively low shock intensity used (). Importantly, synaptic GluR1 levels were significantly increased in trained rats treated with IGF-II compared to vehicle. This increase was completely abolished by anti-IGF-IIR antibody. On the other hand, GluR2 levels remained unchanged across groups.
Mechanisms of IGF-II-mediated memory enhancement. IGF-II promotes LTP
Previous studies reported that AMPA receptor trafficking and dendritic expression of GluR1 in neurons are regulated by GSK334
and, interestingly, IGF-II has been implicated in GSK3 regulation35
. As depicted in , the IGF-II-mediated significant increase of GluR1 was paralleled by a significant synaptic activation of GSK3β (measured by its dephosphorylation at ser936
), which was also completely abolished by anti-IGF-IIR antibody. Furthermore, while blocking hippocampal GSK3 function with pretraining injection of the inhibitor SB216763 completely disrupted IA memory (data not shown), the same treatment delivered immediately after retrieval (Test1) selectively eliminated the IGF-II-mediated enhancement tested 2d after training (Test2) without affecting memory reconsolidation ().
Thus, IGF-II-dependent memory enhancement requires the activation of GSK3β and correlates with increased synaptic expression of GluR1.