(A) LTD is significantly attenuated by pretreatment with the protein synthesis inhibitor cycloheximide (CHX, 60 μM, gray bar) in slices from WT animals (control: 74.3 ± 1.4%, n = 5 animals, 10 slices; CHX: 85.2 ± 2.8%, n = 4 animals, 7 slices; *p = 0.014). (B) CHX treatment has no effect on LTD in slices from Tsc2^+/− mice (control: 86.3 ± 3.1%, n = 6 animals, 12 slices; CHX: 85.3 ± 3.2%, n = 4 animals, 7 slices, p = 0.796). ANOVA: genotype *p = 0.041, treatment p = 0.089, genotype × treatment *p = 0.045. (C) Presynaptic LTD is not affected by genotype or CHX (see also Fig. S2). DHPG significantly increased PPF in slices from both WT and Tsc2^+/− mice (PPF with a 50 ms inter-stimulus interval: WT baseline: 1.37 ± 0.02, WT DHPG: 1.59 ± 0.06, n = 5 animals, 9 slices, *p = 0.003; Tsc2^+/− baseline: 1.39 ± 0.02, Tsc2^+/− DHPG: 1.64 ± 0.03, n = 5 animals, 9 slices, *p = 0.001) and this effect was not blocked by CHX (WT DHPG + CHX: 1.58 ± 0.06, n = 7 animals, 11 slices, p = 0.89; Tsc2^+/− DHPG + CHX: 1.64 ± 0.04, n = 6 animals, 7 slices, p = 0.94). (D) Metabolic labeling of hippocampal slices reveals a significant reduction of basal protein synthesis in Tsc2^+/− mice (WT: 100.0 ± 3.1%, Tsc2^+/−: 88.2 ± 3.3%, n = 13 animals; *p = 0.043). Differences in protein synthesis are exemplified by representative autoradiograph and total protein stain of the same membrane. (E) Immunoblotting experiments show that Arc expression is significantly reduced in Tsc2^+/− hippocampal slices (WT: 100.0 ± 4.7%, Tsc2^+/−: 76.6 ± 6.4%, n = 12 animals; *p = 0.005). (F) Arc translation was measured by metabolic labeling of hippocampal slices, followed by immunoprecipitation of Arc. Comparison of the ratios of ³⁵S-incorporated-to-total Arc reveals a significant reduction in Arc translation in the Tsc2^+/− hippocampus (WT: 100.0 ± 11.5%, Tsc2^+/−: 74.7 ± 6.8%, n = 19 animals; *p = 0.049). (G) Pretreatment of slices with the mTORC1 inhibitor rapamycin (RAP, 20 nM, gray bar) significantly enhances DHPG-induced LTD in slices from Tsc2^+/− mice (DMSO: 85.7 ± 2.1%, n = 8 animals, 17 slices; RAP: 72.9 ± 1.8%, n = 7 animals, 18 slices; *p = 0.002). (H) The rescue by rapamycin of DHPG-induced LTD in Tsc2^+/− mice is prevented by the protein synthesis inhibitor cycloheximide (DMSO: 87.1 ± 4.7%, n = 6 animals, 10 slices; RAP: 88.1 ± 2.4%, n = 7 animals, 9 slices; p = 0.796). ANOVA: rapamycin treatment *p = 0.043, cycloheximide treatment *p = 0.004, rapamycin × cycloheximide *p = 0.018. (I) Metabolic labeling experiments show that rapamycin (20 nM) normalizes protein synthesis in the Tsc2^+/− hippocampus to WT levels (WT DMSO: 100.0 ± 2.5%, WT RAP: 106.5 ± 3.6%, Tsc2^+/− DMSO: 88.8 ± 2.6%, Tsc2^+/− RAP: 100.4 ± 3.9%; ANOVA: genotype *p = 0.008, treatment *p = 0.006, genotype × treatment p = 0.430; t-test: WT vs. Tsc2^+/− DMSO *p = 0.003; WT vs. Tsc2^+/− RAP p = 0.344; Tsc2^+/− DMSO vs. RAP *p = 0.037; n = 22 animals). Error bars represent SEM.

(A) The data suggest that optimal synaptic function requires a narrow and tightly regulated level of synaptic protein synthesis and that deviations in either direction can impair function^3,43. TSC and FXS fall on different ends of this spectrum and respond to opposite alterations of mGluR5 signaling. These results raise the possibility that introducing both mutations to a mouse may normalize aspects of neural function. (B) Genetic rescue strategy. Heterozygous Tsc2 male mice (Tsc2^+/−) were bred with heterozygous Fmr1 females (Fmr1 x⁺/x⁻) to obtain male offspring of four genotypes: wild type (Tsc2^+/+, Fmr1^+/y), Fmr1 KO (Tsc2^+/+, Fmr1^−/y), Tsc2 Het (Tsc2^+/−, Fmr1^+/y), and Cross (Tsc2^+/−, Fmr1^−/y). (C) DHPG-induced LTD is significantly decreased in slices from Tsc2^+/− mice (*p = 0.002) and significantly increased in slices from Fmr1^−/y mice (*p = 0.017), as compared to WT slices. DHPG-LTD in slices from Tsc2^+/− × Fmr1^−/y mice is comparable in magnitude to WT slices (p = 0.558). (WT: 78.9 ± 2.1%, n = 7 animals, 17 slices; Fmr1: 71.2 ± 2.7%, n = 7 animals, 21 slices; Tsc2: 89.5 ± 2.6%, n = 7 animals, 15 slices; Cross: 77.4 ± 1.8%, n = 9 animals, 19). (D) Summary of LTD data. Bar graphs represent percent decrease from baseline in fEPSP (average of last 5 minutes of recording ± SEM); *p < 0.05, **p < 0.01. (E) Both mutations cause a deficit in context discrimination that is rescued in the double mutant. WT mice (Familiar: 42.9 ± 4.6%, n = 11; Novel: 27.8 ± 3.4%, n = 12; *p = 0.024), Fmr1^−/y mice (Familiar: 49.0 ± 5.6%, n = 11; Novel: 43.5 ± 6.7%, n = 12; p = 0.483), Tsc2^+/−(Familiar: 42.1 ± 6.8%, n = 12; Novel: 35.6 ± 6.0%, n = 12; p = 0.395) and Tsc2^+/− x Fmr1^−/y mice (Familiar: 50.5 ± 5.2%, n = 11; Novel: 29.8 ± 5.2%, n = 11; *p = 0.011). Error bars represent SEM.

(A) DHPG induces significantly less LTD in slices from Tsc2^+/− mice vs. littermate WT mice (WT: 74.3 ± 1.4%, n = 5 animals, 10 slices; Tsc2^+/−: 86.3 ± 3.1%, n = 6 animals, 12 slices; *p = 0.004). In this and all subsequent electrophysiology figures, representative field potential traces (average of 10 sweeps) were taken at times indicated by numerals and scale bars equal 0.5 mV, 5 ms, unless stated otherwise. Error bars represent SEM. (B) Synaptically-induced mGluR-LTD, elicited by delivering pairs of pulses (50 ms interstimulus interval) at 1 Hz for 20 minutes (PP-LFS, 1200 pulses) in the presence of the NMDA receptor antagonist D-(−)-2-Amino-5-phosphonopentanoic acid (D-AP5, 50 μM), is also deficient in slices from Tsc2^+/− mice (WT: 65.1 ± 2.1%, n = 3 animals, 9 slices; Tsc2^+/−: 85.0 ± 2.5%, n = 4 animals, 11 slices; *p = 0.003). (C) The magnitude of NMDA receptor-dependent LTD evoked by low frequency stimulation (LFS, 900 pulses at 1 Hz) does not differ between genotypes (WT: 79.8 ± 1.6%, n = 4 animals, 6 slices; Tsc2^+/−: 79.4 ± 1.9%, n = 6 animals, 6 slices; p = 0.610). (D) Hippocampal slices were stimulated with 50 μM DHPG for 5 min, and ERK1/2 activation (phosphorylation) assessed via immunoblot (normalized WT: 100.0 ± 6.1%, WT DHPG: 119.6 ± 5.5%, Tsc2^+/−: 97.5 ± 5.6%, Tsc2^+/− DHPG: 116.2 ± 3.9%; ANOVA: genotype p = 0.623, treatment *p = 0.0008, genotype × treatment p = 0.923; n = 9 animals). Results reveal that DHPG significantly increases ERK1/2 activation in both WT (*p = 0.040) and Tsc2^+/− (*p = 0.003). Error bars represent SEM.

(A) Model to account for effects of Tsc2^+/− and Fmr^1−/y mutations on mGluR5- and protein synthesis-dependent LTD. This model predicts that the impairment in Tsc2^+/− mice can be overcome either by inhibiting mTOR with rapamycin or by augmenting mGluR5 signaling with an mGluR5 PAM. (B) Consistent with the model, pretreatment of slices from Tsc2^+/− mice with CDPPB (10μM, gray bar) significantly enhances DHPG-induced LTD (DMSO: 86.4 ± 2.5%, n = 8 animals, 13 slices; CDPPB: 71.7 ± 3.9%, n = 7 animals, 12 slices; *p < 0.001). (C) CDPPB treatment fails to enhance DHPG-induced LTD in Tsc2^+/− mice when co-applied with the protein synthesis inhibitor cycloheximide (DMSO: 89.0 ± 4.4% n = 8 animals, 10 slices; CDPPB: 83.9 ± 2.1%, n = 7 animals, 9 slices; p = 0.64). ANOVA: CDPPB treatment *p = 0.008, CHX treatment p = 0.087, CDPPB × CHX *p = 0.034. (D) CDPPB (10 μM) restores protein synthesis in the Tsc2^+/− hippocampus to WT levels (WT DMSO: 100.0 ± 3.2%, WT CDPPB: 97.2 ± 1.9%, Tsc2^+/− DMSO: 86.1 ± 2.7%, Tsc2^+/− CDPPB: 94.9 ± 3.0%; ANOVA: genotype *p = 0.006, treatment p = 0.275, genotype × treatment *p = 0.041; t-test: WT vs. Tsc2^+/− DMSO *p = 0.012; WT vs. Tsc2^+/− CDPPB p = 0.538; Tsc2^+/− DMSO vs. CDPPB *p = 0.049; n = 17 animals). (E) CDPPB exposure significantly increases Arc translation in the Tsc2^+/− hippocampus (WT DMSO 100.0 ± 28.2%, WT CDPPB 121.0 ± 21.2%, Tsc2^+/− DMSO 59.2 ± 7.0%, Tsc2^+/− CDPPB 129.4 ± 20.3%; ANOVA genotype p = 0.554, treatment *p = 0.009, genotype × treatment p = 0.114; t-test: Tsc2^+/− DMSO vs. CDPPB *p = 0.026; n = 6 animals). Error bars represent SEM. (F) Experimental design of context discrimination task. (G) WT mice display intact memory by freezing more in the familiar context than the novel context (Black bars; Familiar: 50 ± 7.7%, n = 12; Novel: 34.1 ± 3.2%, n = 14; *p = 0.003). A single injection of CDPPB (10 mg/kg, i.p.) 30 minutes prior to training has no effect on WT context discrimination (Familiar: 42.3 ± 3.7%, n = 12; Novel: 26.4 ± 3.6%, n = 12; *p = 0.005). Control Tsc2^+/− mice display an impairment in context discrimination (Blue bars; Familiar: 40.9 ± 5.3%, n = 11; Novel: 39.3 ± 5.2%, n = 14; p = 0.501), but this deficit is corrected by a single injection of CDPPB (Familiar: 44.5 ± 4.3%, n = 11; Novel: 31.6 ± 3%, n = 12; *p = 0.034). Error bars represent SEM.