Figure 1.

Exaggerated protein synthesis in the Fmr1 KO is ameliorated by mGluR5 antagonism and mimicked by mGluR activation in WT. A, Schematic illustrates experimental timeline: hippocampal slices were recovered in ACSF, incubated with 25 μm ActD for 30 min, then protein synthesis was measured with 10 μCi/ml ³⁵S-Met/Cys for 30 min. To measure the effect of after slice recovery time on protein synthesis, slices were incubated in ACSF for 0, 0.5, 1.5, 3.5, or 5.5 h before exposure to ActD and metabolic labeling. Quantification of multiple experiments showed that a 4 h postslice recovery time yields maximal protein synthesis, which is stable for at least another 2 h (ANOVA, p < 0.05; t test: 4 h vs 0.5 h, *p < 0.04; 4 h vs 1 h, *p < 0.03; 4 h vs 6 h, p = 0.89; n = 10). B, Protein synthesis was elevated in Fmr1 KO versus WT hippocampus (t test, *p < 0.02; n = 13). Differences in protein synthesis are exemplified by representative autoradiographs and total protein stain of the same membrane. C, Representative immunoblots and autoradiographs show IPs for α-CaMKII and GAPDH from WT and Fmr1 KO slices metabolically labeled with 50 μCi/ml ³⁵S-Met/Cys for 1 h. D, Quantification of multiple experiments reveals that the ratio of ³⁵S-incorporated total α-CaMKII is higher in Fmr1 KO slices than WT slices (t test, *p < 0.04; n = 6). In contrast, the ratio of ³⁵S-incorporated/total GAPDH is not elevated in Fmr1 KO versus WT slices (t test p = 0.31; n = 5). E, During the first 5 min of metabolic labeling, WT and Fmr1 KO slices were exposed to 50 μm MPEP or vehicle. Quantification of multiple experiments shows that MPEP treatment corrects protein synthesis in the Fmr1 KO back to WT levels (t test, *p < 0.03; n = 8). This treatment had no significant effect on WT protein synthesis (t test, p = 0.58; n = 8). F, WT and Fmr1 KO slices were preincubated ± 10 μm MPEP for 30 min, then metabolically labeled ± 10 μm MPEP for 30 min. Measurements taken from isolated CA1 regions show that MPEP corrects excessive protein synthesis in Fmr1 KO CA1 back to WT levels (t test, *p < 0.02; n = 8). This treatment had no effect on WT CA1 (t test p = 0.24; n = 8). G, WT and Fmr1 KO slices were stimulated ± 100 μm DHPG during the first 5 min of metabolic labeling. DHPG stimulation caused a robust increase in protein synthesis in WT (t test, *p < 0.0001), but not Fmr1 KO (t test, p = 0.62), hippocampus (n = 8). N represents number of animals per group, where 1–2 slices were analyzed per animal. Error bars represent SEM.