Deficient mGluR1/5-regulated signaling to translation elongation suppresses mGluR-LTD in Tsc2+/− mice. a–c The levels of phosphorylated (P) and total (T) ERK (a), AKT (b), and eEF2 (c) in CA1-enriched hippocampal slices from wild-type and Tsc2+/− mouse littermates as determined by western blots under basal or DHPG-treatment condition (100 µM; 5 min). *p < 0.05; **p < 0.01; ***p < 0.001; 2 way ANOVA, post hoc Sidak’s test, n = 11–19 mice/genotype, 4–5 slices per drug condition/mouse. Asterisks below brackets indicate an effect of DHPG on phosphoprotein levels within genotype. Asterisks above brackets indicate effects between genotypes. d Schematic depicting that global slowing of ribosome movement through eEF2 phosphorylation or low dose cycloheximide (CHX) may promote protein synthesis of lowly translated mRNAs while inhibiting highly translated mRNAs. e Empirical cumulative distribution function (ECDF) plot of hippocampal TE for the Up and Down RNAs (left) and RPFs (middle) in DHPG/basal compared to all genes. Right: ECDF plot of hippocampal TE for FMRP binding targets compared to non-target mRNAs. FMRP binding targets are stratified into three groups based on binding stringency. Wilcoxon rank-sum test with correction by Bonferroni method. p values are indicated in the plot legend. f Left: Time course demonstrating that brief DHPG application (100 µM; 5 min) induces long-term synaptic depression (LTD) of field (f) EPSP slopes in WT hippocampal slices that is unaffected by low doses of cycloheximide (45–60 min pretreatment, 75 nM; n = 12 and 15 slices; 6–7 mice/condition). Right: In Tsc2+/− mice pretreatment with low-dose cycloheximide enhances LTD magnitude (n = 10 and 15 slices; 7 mice/condition). Plotted are group averages of fEPSP slope (mean ± SEM) normalized to pre-DHPG baseline as a function of time. Inset: Example fEPSP from baseline and during LTD (55–60 min post DHPG). Scale = 0.5 mV/10 ms. g LTD magnitude, measured at 55–60 min post-DHPG application in vehicle and cycloheximide in each genotype. There is a significant interaction between cycloheximide and Tsc2 genotype (F(1,47) = 5.197, p < 0.05; LMM). With post hoc pairwise comparisons, there is a strong trend for cycloheximide to enhance LTD in Tsc2+/− mice (veh: 83 ± 2% of baseline; cycloheximide; 75 ± 5%; n = 15 and ten slices from four mice/treatment; F(1,23) = 5.445, p = 0.058; LMM) but not WT mice (veh: 74 ± 2% of baseline; cycloheximide; 77 ± 3%; n = 11 and 15 slices from 6 and 5 mice, respectively/treatment; F(1,24) = 3.819, p = 0.14; LMM). In vehicle, LTD is reduced in Tsc2+/− mice in comparison with WT, but not in low-dose cycloheximide. (*p < 0.05; **p < 0.01). h Schematic showing DHPG induction of eEF2 phosphorylation in hippocampal slices. In WT slices, DHPG leads to increased levels of phospho-eEF2 through signaling to eEF2K, which presumably slows ribosome transit and promotes expression of lowly translated mRNAs required for mGluR-LTD, such as FMRP binding targets. In TSC slices, levels of phospho-eEF2 are elevated basally and correlate with increased mRNA abundance of FMRP binding targets. DHPG treatment does not further increase eEF2 phosphorylation, and Tsc2+/− slices may have excessive ribosome transit during mGluR signaling that contributes to deficient mGluR-LTD.