Novel Translational Control in Arc-dependent Long Term Potentiation Consolidation in Vivo^*

Electrophysiology and Intrahippocampal Infusion

Experiments were performed on 220 adult male Sprague-Dawley rats. The electrophysiological methods have been detailed elsewhere (27, 28). Briefly, rats were anesthetized with urethane, and electrodes were positioned for selective unilateral stimulation of the medial perforant fibers in the angular bundle and extracellular recording of evoked field potentials in the hilar region of the dentate gyrus (illustrated in Fig. 1A). Recordings were done with borosilicate glass micropipettes (tip size 1–5 μm) filled with 1 m NaCl (input impedance 1–1.5 megaohms). Drugs were infused with a second micropipette (tip size 10–15 μm) connected via a polyethylene (PE50) tube to a 5-μl Hamilton syringe (Reno, NV) and infusion pump. The two micropipettes were clamped together on a micromanipulator with a vertical tip separation of 700 μm. The tip of the infusion cannula was located in deep stratum lacunosum-moleculare of field cornu ammonis 1 (CA1), ∼300 μm from the nearest medial perforant path-granule synapses in the upper blade of the dorsal dentate gyrus. Test pulses were applied at 0.033 Hz throughout the experiment except during the period of HFS. Responses were allowed to stabilize, and 20 min of base-line recording was obtained. After base-line recording, drugs were locally infused at 0.06 μl/min, with volumes ranging from 0.3 to 2 μl depending on the drug. After completing the infusion, test responses were collected for a further 45 min followed by application of HFS. HFS was given in three sessions with 5 min between each. Each session consisted of four 400-Hz stimulus trains (8 pulses/burst) with a 10-s interval between each train. The total HFS duration was 10.5 min, and the total pulse number was 128 pulses. After HFS, evoked responses were collected for periods of 15 min, 2 h, or 4 h.

Drugs and Antibodies

2-Amino-5-phosphonopentanoic acid (AP5, 50 mm prepared in 1× PBS; Tocris), anisomycin (86.7 mm in 1 n NaOH diluted in saline; Sigma), actinomycin D (5 mg/ml in saline; Sigma), U0126 (100 μm in 1× PBS with 1% dimethyl sulfoxide (DMSO); Promega), rapamycin (100 μm in 1× PBS with 0.1% DMSO) and CGP57380 (2 mm in 1× PBS with 0.1% DMSO; Sigma).

Antibodies used for Western blotting were as follows (all were obtained from Cell Signaling Technology, Beverly, MA, unless otherwise indicated): Arc (1:400; C-7 Santa Cruz), phospho-eIF2α-Ser⁵¹ (1:1000), eIF2α (1:1000), eEF1A (1:1000; Upstate Biotechnology), phospho-eIF4E Ser²⁰⁹ (1:500), eIF4E (1:1000), phospho-eEF2 Thr⁵⁶(1:1000), eEF2 (1:1000), GAPDH (1:400; Sigma mouse monoclonal G8795), phospho-rpS6 Ser^240/244 (1:1000), rpS6 (1:1000), phospho-p70S6K Thr⁴²¹/Ser⁴²⁴ (1:1000) (Sigma), phospho-p70S6K1-Thr³⁸⁹) (1:1000), p70S6K1 antibody (1:1000), phospho-mTOR Ser²⁴⁴⁸ (1:1000), anti-mTOR (1:1000), 4E-BP2 (1:1000), phospho-MNK1 Thr^197/202 (1:500), MNK1 (1:1000), and eIF4G1 (1:1000) (from the Sonenberg laboratory).

SDS-PAGE and Western Blotting

At the end of electrophysiological recording rats were decapitated, and the dentate gyrus was rapidly dissected on ice and homogenized as previously described (26). Samples were boiled in sample buffer (Bio-Rad) and resolved on 10% or 8% SDS-PAGE minigels. Proteins were transferred to polyvinylidene difluoride membranes (Amersham Biosciences) which were then blocked, probed with antibodies, and developed using chemiluminescence reagents (ECL, Amersham Biosciences). The blots were scanned using Gel DOC EQ (Bio-Rad), and bands intensity were quantified using analytical software (Quantity one 1D analysis software; Bio-Rad). Blots treated with phospho-specific antibody were stripped with 100 mm 2-mercaptoethanol, 2% SDS, and 62.5 mm Tris-HCL, pH 6.7, at 50 °C for 30 min, washed, blocked, and reprobed with antibody recognizing total protein. Optical density values were expressed per unit of protein (GAPDH) applied to the gel lane. The phosphoproteins were normalized relative to the total protein on the same lane. Total proteins were normalized to GAPDH. Values from the treated dentate gyrus were expressed in percent of the values from contralateral control dentate gyrus. Significant differences between the treated and non-treated dentate gyrus were determined using Student's t test for dependent samples. Group comparison was done by analysis of variance. The p value for significance was 0.05.

Cap Analogue Pulldown Assay

Microdissected dentate gyri were homogenized on ice in homogenization buffer (50 mm Tris, pH 7.5, 150 mm KCl, 1 mm dithiothreitol, 1 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride), and protein concentration was determined using the Pierce BCA protein assay reagent (Thermo Scientific, Pierce). 250 μg of tissue lysates were incubated with 7-methyl-GTP-Sepharose 4B beads (GE Healthcare) at 4 °C for 12 h with moderate shaking to precipitate eIF4E and the associated proteins. The beads were pelleted, washed, and resuspended in 40 μl of 2× SDS-PAGE loading buffer and denatured at 100 °C for 5 min. Equivalent proportions of precipitated proteins in each group were separated on a 7–15% gradient gel and analyzed by immunoblotting. In some experiments an m⁷GDP-agarose resin was used as indicated. Precipitated material was washed 3 times and then eluted with 40 μl of 2× sample buffer followed by SDS-PAGE.

Immunohistochemical Staining

The brains were fixed and processed for staining as described (22). Briefly, sections were blocked and then incubated with primary antibodies recognizing phospho-rpS6-Ser^240/244 (1:1000) and Arc (1:400) overnight at 4 °C. After primary antibody incubation the sections were washed and incubated in respective biotinylated secondary antibodies (1: 200) (Vector Laboratories, Burlingame, CA) at room temperature for 2 h. The sections were washed and incubated in Vector ABC reagents (Vector Laboratories) for 1 h, and final color was developed using 3,3′-diaminobenzidine with nickel as the chromogen. Sections were mounted on slides, dehydrated through alcohols to xylene, and covered with dibutyl phthalate xylene.

In Situ Hybridization

RNA probes were prepared as previously described (23). Fixed floating sections were washed in PBS for 5 min, treated with pre-warmed proteinase K (10 μg/ml) for 5 min at 37 °C, and post-fixed (5 min with 4% paraformaldehyde/PBS) at room temperature. After post-fixation, sections were incubated 3 min in 0.1 m triethanolamine (TEA, pH 8.0) and treated with 0.25% acetic anhydride in 0.1 m TEA, pH 8.0, for 10 min, washed twice in 2× SSC (1× SSC = 0.15 m NaCl and 0.015 m sodium citrate), and blocked for 10 min in prehybridization buffer. Hybridization with an Arc riboprobe was carried out in a humidified chamber at 60 °C overnight. Sections were washed twice with 2× SSC at room temperature for 20 min, once with 50% formamide in 2× SSC at 65 °C, and twice in 2× SSC at 37 °C. Unbound probe was removed by incubation with 20 μg/ml RNase A at 37 °C for 30 min, and the RNase A was sequentially inactivated by incubating the sections in RNase A buffer for at 65 °C for 30 min. After blocking in 2% blocking reagent for 2 h at room temperature, AP-coupled anti-digoxigenin antibody (1:2000, Roche Applied Science) was applied overnight at 4 °C. Visualization was done with the chromogenic substrates nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Roche Applied Science) according to the supplier's instructions. Sections incubated with a control, sense probe for Arc did not show any staining.

LTP-specific Modulation of eIF4E and eIF2a Phosphorylation State

LTP at medial perforant path-granule cell synapses of the dentate gyrus requires Arc synthesis and is associated with rapid Arc transcription, transport of mRNA to dendrites, and enhanced protein expression (26, 29, 30). Local infusions with Arc antisense oligodeoxynucleotides revealed that LTP consolidation requires sustained Arc synthesis during a time-window that starts within 15 min of HFS and lasts between 2 and 4 h (26). The present study examines translation control mechanisms across the critical period of Arc-dependent LTP consolidation. Stable LTP of the field excitatory postsynaptic potential (fEPSP) slope was induced by HFS of the medial perforant pathway (Fig. 1). The total duration of the HFS paradigm was 10.5 min (3 sessions of 400-Hz stimulation with 5 min between sessions). Dentate gyrus tissue was collected at 15 min, 2 h, and 4 h after completing HFS, and Western blot analysis of homogenate samples was used to determine changes in the expression of phosphorylated translation factor. In all figures levels of phosphorylated translation factors are normalized to total levels of the respective translation factor proteins.

We first examined the possible changes in the phosphorylation state of initiation factors eIF4E and eIF2α. Cap-dependent translation starts with recognition of the m⁷GpppN cap structure on the 5′-end of the mRNA by eIF4E, resulting in recruitment of an initiation complex that includes the 40 S ribosomal subunit. This complex scans the mRNA to the start site, where 60 S subunit joining occurs, and the elongation step of protein synthesis commences. Phosphorylation of eIF4E on Ser²⁰⁹ is generally correlated with enhanced rates of translation, whereas dephosphorylation is associated with decreased translation (31). Phosphorylation of eIF4E promotes its dissociation from the cap, which could serve to speed ribosomal scanning or promote further rounds of initiation (32). As shown in Fig. 1D, HFS led to a rapid increase in Ser²⁰⁹ phosphorylation of eIF4E that declined during the course of LTP maintenance. Phospho-eIF4E levels in homogenate samples from HFS-treated dentate gyrus were significantly elevated 65.3 ± 20.7% above levels in the contralateral control dentate gyrus at 15 min post-HFS and 30 ± 1.1% above control at 2 h post-HFS but were not significantly elevated at 4 h.

eIF2α is a heterotrimeric GTP-binding protein required for recruitment of tRNA_i^Met to the 40 S subunit. GTP is hydrolyzed during translation initiation, and regeneration of active eIF2α-GTP requires the guanine-nucleotide exchange factor eIF2B. When phosphorylated on Ser⁵¹, eIF2α competitively inhibits eIF2B, thus blocking recycling of eIF2 and inhibiting translation (5, 8). As shown in Fig. 1E, HFS resulted in rapid dephosphorylation of eIF2α-Ser⁵¹, indicating rapid activation of initiation. Activation of eIF2α followed a decremental time course that paralleled the effects on phospho-eIF4E, although levels of phosph-eIF2α remained significantly decreased at 4 h post-HFS. We then asked whether these changes are specific to NMDAR-dependent LTP induction. Rats were briefly infused with the NMDAR antagonist AP5 (0.3 μl, 5 min, 50 mm, 15 nmol) or vehicle (PBS) 45 min before HFS. The tip of the infusion pipette was located in stratum lacunosum-moleculare of CA1, some 300 μm from the nearest medial perforant path synapses in the upper blade of the dorsal dentate gyrus. fEPSPs were reduced briefly after AP5 but resumed the stable base line by 25 min before HFS. AP5 had no effect on fEPSPs evoked by low frequency test stimulation over 2 h of recording (Fig. 1B) and no effect on initiation factor phosphorylation in tissue collected 15 min, 2 h, and 4 h after AP5 infusion (not shown). As expected, LTP induction was blocked in AP5-treated rats (Fig. 1C). Furthermore, AP5 abolished the HFS-induced changes in eIF4E and eIF2α phosphorylation state (Fig. 1, D–F). Thus, the time course of initiation factor modulation is LTP-specific and corresponds to the window of Arc synthesis. Regulation of total eIF4E and eIF2α expression was also observed, as detailed in a later section on translation factor expression (see Fig. 6).

ERK-dependent Regulation of Initiation Factor Activity and eIF4F Formation

Pharmacological and genetic approaches have demonstrated the role of MEK-ERK signaling in LTP maintenance and memory. MEK inhibitors such as U0126 block ERK phosphorylation and attenuate early and late phase LTP in the CA1 region and dentate gyrus (7, 25, 28, 33, 34). Here, we investigated the effects of MEK-ERK signaling on dynamic changes in translation factor activity during in vivo LTP. Rats were infused with U0126 (0.6 μl, 9.8 min, 100 μm, 0.06 nmol) or vehicle (DMSO-PBS) 45 min before HFS (Fig. 2A). Base-line synaptic transmission was not significantly affected by U0126 or vehicle infusion. HFS in the vehicle-infused group induced non-decremental enhancement of the fEPSP. As expected, HFS in the presence of U0126 elicited a decremental potentiation of the fEPSP slope that decayed within ∼15 min to a stable level that was not significantly different from base line. Strikingly, U0126 treatment blocked the HFS-induced increase in eIF4E phosphorylation and the decrease in eIF2α phosphorylation at both 15 min and 2 h post-HFS (Fig. 2B). The combination of U0126 and low frequency test stimulation (LFS) had no effect on initiation factor phosphorylation state (Fig. 2C).

In addition to regulation of eIF4E phosphorylation, ERK signaling might function in initiation complex (eIF4F) formation, as detected by binding of eIF4E to the scaffolding protein eIF4G. To assess this possibility an m⁷GDP cap analogue pulldown assay was performed in homogenate samples from HFS-treated and contralateral control dentate gyrus and the resulting precipitate was immunoblotted with antibodies recognizing total eIF4E, Ser²⁰⁹ phosphorylated eIF4E and eIF4G1. HFS resulted in significantly enhanced eIF4G association with cap-bound eIF4E as well as enhanced phosphorylation of eIF4E (Fig. 2, D and E). These effects were blocked by UO126, demonstrating ERK-dependent enhancement of initiation complex formation.

Given the complete block of eIF4E phosphorylation and eIF4F protein complex formation, we anticipated that U0126 would inhibit LTP-associated changes in Arc expression. As shown in Fig. 3, immunoblot analysis revealed robust enhancement of Arc protein expression at 15 min and 2 h post-HFS in vehicle treated-control that was abolished by local infusion of U0126 or AP5 before HFS. Treatment with the general protein synthesis inhibitor, anisomycin, similarly blocked LTP consolidation and Arc synthesis (Fig. 3C).

LTP Maintenance in the Dentate Gyrus Does Not Require Rapamycin-sensitive mTORC1 Signaling

Rapamycin-sensitive mTORC1 signaling is necessary for several forms of protein-synthesis-dependent synaptic plasticity, as studied extensively in the hippocampal CA1 region (17, 35–37). We, therefore, expected that rapamycin would impair LTP in the dentate gyrus. Previous work showed that brief intrahippocampal infusion of 60 nm rapamycin effectively blocks acquisition of inhibitory avoidance learning and mTORC1 activation in hippocampus (38). Using a similar protocol, we infused rapamycin 45 min before HFS. In contrast to expectation, HFS in rapamycin-treated rats evoked non-decremental LTP that was indistinguishable to that obtained in vehicle-infused controls over 4 h of post-HFS recording (Fig. 4A). A range of concentrations were tested, and the results shown are based on the maximal rapamycin concentration of 100 μm (2 μl, 32.5 min, 0.2 nmol). Systemic injection of rapamycin at high dosage (50 mg/kg intraperitoneally) 30 min before HFS similarly failed to inhibit LTP over 4 h of recording (n = 6; not shown).

Surprised by this finding, we examined mTORC1 activation using antibodies recognizing Ser²⁴⁴⁸-phosphorylated mTOR. Immunoblots showed rapid and sustained activation of mTORC1 at 15 min and 2 h post-HFS (Fig. 4, B and C). Rapamycin blocked this effect at both time points, thus verifying the efficacy of the drug in blocking mTORC1. Notably, however, rapamycin failed to inhibit the rapid modulation of eIF4E and eIF2α at 15 min post-HFS, although significant inhibition was detected at the 2-h time point. Immunoblotting for Arc further showed that rapamycin does not block the LTP-associated increase in Arc synthesis (Fig. 4, D and E), although this synthesis was abolished by anisomycin (Fig. 3C). Taken together this suggests that ERK, but not mTORC1 signaling, is required for rapid regulation of translational initiation Arc synthesis, and LTP maintenance.

These results suggest that the branch of mTORC1 signaling involved in the release of eIF4E from 4E-BP2, the main 4E-BP isoform in brain, is not necessary for LTP in the dentate gyrus. To address this issue an m⁷GDP cap analogue pulldown assay was performed in homogenate samples from HFS-treated and contralateral control dentate gyrus, and precipitates were immunoblotted with antibodies recognizing eIF4E and 4E-BP2. LTP was not associated with detectable reductions in the 4E-BP signal relative to eIF4E, indicating that 4E-BP2 is not being released from the complex. A quantitative analysis of the 4E-BP2 subunit/eIF4E ratio and representative blots based on similar results obtained at 15 min and 2 h post-HFS (6 rats at each time point) are shown in supplemental Fig. S1. Consistent with these observations, no signal could be detected using an antibody recognizing phosphorylated 4E-BP2 (results not shown). Analysis of eIF4G-eIF4E interaction further showed significant enhancement of initiation complex formation in rapamycin-infused rats equivalent to vehicle-infused controls (Fig. 4, F and G).

LTP Is Associated with mTORC1 Signaling to Ribosomal Protein S6 and Enhanced Translation Factor Expression

Next we examined regulation in the branch of mTORC1 signaling to p70 S6 kinase (p70S6K) and its substrate, rpS6. p70S6K is activated by mTORC1 through direct phosphorylation of Thr³⁸⁹ (39, 40). HFS resulted in enhanced phosphorylation of p70S6K-Thr³⁸⁹ at 15 min and 2 h post-HFS (Fig. 5, A and C). Rapamycin inhibited significant changes in p70S6K-Thr³⁸⁹ phosphorylation, with the strongest inhibition detected at 15 min post-HFS. p70S6K activation is thought to additionally require prior phosphorylation of an ERK-dependent site (Thr⁴²¹/Ser⁴²⁴) located in the module IV region of the enzyme (41, 42). We found that U0126, but not rapamycin, blocked phosphorylation of the ERK-site on p70S6K (Fig. 5, A and C). We then assayed rpS6 regulation using antibodies recognizing the Ser^240/244 site that is directly phosphorylated by p70S6K. A more than 3-fold increase in rpS6 phosphorylation was observed at 15 min, 2 h, and 4 h post-HFS (Fig. 5), and these effects were abolished by treatment with rapamycin and U0126. Taken together, we conclude that rapamycin effectively blocks HFS-evoked mTORC1 activation and downstream signaling to p70S6K and rpS6 while having no effect on LTP maintenance during 4 h of recording.

Immunohistochemical staining was used to assess the distribution of phospho-rpS6 in the dentate gyrus after induction of LTP (supplemental Fig. S2). Staining was observed in scattered granule somata of the non-stimulated, contralateral dentate gyrus. In the HFS-treated dentate gyrus, expression of phospho-rpS6 was dramatically enhanced in the granule cell layer and across the dendritic field of the molecular layer. Similar somatodendritic expression was observed at 15 min, 2 h, and 4 h post-HFS. Local infusion of AP5 blocked the enhanced expression of phospho-rpS6 in both the dorsal and ventral blades of the dentate gyrus.

mTORC1 and ERK-dependent de Novo Synthesis of Translation Factors

In situations demanding intensive protein synthesis, as during cell division, translational capacity may be maintained through the enhanced synthesis of ribosomal proteins and translation factors. Many of these proteins are encoded by TOP mRNAs and undergo enhanced translation through an mTORC1-dependent pathway (43, 44). LTP in the CA1 region in vitro is associated with mTORC1-dependent activation of p70S6K and synthesis of several TOP-containing mRNAs including rpS6, eEF2, and eEF1A (16, 18). Here, we examined changes in total translation factor expression (normalized to GAPDH levels) after LTP induction in the dentate gyrus in vivo (Fig. 6). At 15 min, expression of eIF4e, eIF2α, and rpS6 were significantly enhanced in the HFS-treated dentate gyrus relative to control, whereas expression of eEF2 and eEF1A were not significantly altered. At 2 h, no significant change in translation factor levels was observed. At 4 h, eIF4E expression was elevated, revealing a biphasic pattern of expression specific to this translation factor. AP5 infusion blocked all HFS-induced increases in translation factor expression at 15 min post-HFS as well as the enhanced expression of eIF4E at 4 h (Fig. 6A and 7B). Interestingly, however, HFS in the presence of the NMDAR antagonist led to enhancement of eIF4E expression at 2 h post-HFS, suggesting that NMDAR signaling counteracts an unknown mechanism for enhanced eIF4E expression at this time. U0126, rapamycin, and anisomycin all blocked the HFS-induced enhancement of eIF4E, eIF2α, and rpS6 expression as measured 15 min post-HFS (Fig. 6). The results show that mTORC1 and ERK signaling are both critically required for rapid de novo synthesis of translation factors during NMDAR-dependent LTP in vivo. Notably, however, inhibition of translation factor expression by rapamycin does not block LTP maintenance.

Dissociation of eEF2 Phosphorylation from LTP and Arc Expression

Although eEF2 phosphorylation is associated with reduction in global protein synthesis, translation of certain plasticity-associated mRNAs, including Arc and α-calmodulin kinase II, is maintained (20, 23, 25). In the present study, HFS resulted in rapid and sustained phosphorylation of eEF2-Thr⁵⁶. This sustained increase outlasts the critical period of Arc synthesis and contrasts with the slowly declining pattern of eIF4E and eIF2α activation.

Blockade of NMDAR-dependent LTP failed to inhibit the enhanced expression of phospho-eEF2 as observed 15 min and 2 h post-HFS (Fig. 7). At 15 min, phospho-eEF2 levels were equally enhanced in the AP5-treated and PBS control groups (p > 0.05). At 2 h, phospho-eEF2 levels in the AP5 group (75.18 ± 23.57% increase) were significantly elevated ∼2-fold above rats receiving PBS infusion and HFS (35.1 + 5.3% increase, p < 0.05). Remarkably, eEF2 phosphorylation at 4 h post-HFS was completely blocked by AP5 treatment. Thus, eEF2 phosphorylation during LTP consists of an early NMDAR-independent component and a late NMDAR-dependent component. Blockade of mTORC1 signaling with rapamycin blocks the hyperphosphorylation of eEF2 at 15 min and 2 h post-HFS but has no effect on Arc synthesis. Taken together, these results dissociate eEF2 phosphorylation from enhanced Arc synthesis during LTP.

ERK Couples Arc Transcription and Translation

The foregoing data demonstrates ERK-dependent eIF4E phosphorylation, initiation complex formation, and enhanced expression of Arc during LTP. However, the effects of the U0126 on Arc protein expression could also be due to inhibition of Arc transcription (45, 46). We considered that Arc translation might be selectively blocked by applying U0126 well after the induction of Arc mRNA (26). U0126 or vehicle solution were, therefore, infused 10 min after HFS, and homogenates samples were collected for immunoblot analysis of translation initiation factor phosphorylation and Arc protein expression at 30 min post-HFS. Post-HFS administration of U0126 inhibited LTP maintenance (Fig. 8A), changes in eIF4E and eIF2α phosphorylation state (Fig. 8, B and C), and enhancement of Arc protein expression in immunoblots (Fig. 8D). A series of perfusion-fixed brains was collected for in situ hybridization and immunohistochemical analysis of Arc mRNA and protein expression, respectively. As illustrated in Fig. 8E (top panel), vehicle-infused rats exhibited robust increases in Arc mRNA and protein expression in the granule cell layer and across the dendritic field of the molecular layer. Remarkably, U0126 abolished increases in Arc mRNA expression in parallel with Arc protein (Fig. 8E, middle panel). In an attempt to elicit Arc protein expression from pre-existing RNA, HFS was applied in the presence of the transcriptional inhibitor actinomycin D, but we were unable to detect Arc protein increases in the absence of de novo Arc mRNA (Fig. 8E, bottom panel). Taken together, this suggested that Arc protein expression derives from ERK-dependent transcription well into the maintenance phase of LTP.

To isolate ERK effects on translation, we blocked activation of mitogen-activated protein kinase-interacting kinase (MNK), the kinase that couples ERK to phosphorylation of eIF4E (5, 13, 47, 48). Local infusion of the MNK inhibitor CGP57380 strongly attenuated LTP maintenance in a manner similar to U0126 (Fig. 9A). Western blot analysis of tissue collected 15 min post-HFS showed that CGP57380 blocks phosphorylation of MNK1-Thr^197/202 and eIF4E and abolishes enhancement of Arc synthesis, whereas HFS-evoked phosphorylation of rpS6 was not affected by MNK inhibition (Fig. 9B). CGP57380 infusion in a low frequency test stimulation group had no significant effects on base-line fEPSPs or MNK1 phosphorylation state (Fig. 9B). Given these effects we further considered that ERK signaling to MNK might contribute to initiation complex formation itself. As shown in Fig. 9C, CPG57380 completely blocked enhanced loading of eIF4G onto eIF4E in the cap pulldown assay. Imunohistochemistry confirmed the block of Arc protein expression in dentate granule cells 15 min post-HFS. In contrast, in situ hybridization revealed intact HFS-evoked up-regulation of Arc mRNA in granule somata of CGP57380-infused rats (Fig. 9D). We conclude that MNK inhibition blocks eIF4F formation, eIF4E phosphorylation, and Arc synthesis without disrupting Arc RNA expression.