Measuring Protein Synthesis in Hippocampal Slices

Hippocampal slices were prepared from age-matched male WT and Fmr1 KO rats in an interleaved fashion as previously described (17 (link), 26 (link), 42 (link)). Briefly, hippocampi were rapidly isolated, and 500-μm slices were prepared from the dorsal half using a Stoelting tissue slicer. Slices were recovered for 4 hours in 32.5°C ACSF (NaCl, 124 mM; KCl, 3 mM; NaH₂PO₄, 1.25 mM; NaHCO₃, 26 mM; dextrose, 10 mM; MgCl₂, 1 mM; and CaCl₂, 2 mM, saturated with 95% O₂ and 5% CO₂) and then incubated for 30 min with 25 μM ActD to block transcription. To measure protein synthesis, slices were then transferred to fresh ACSF containing ³⁵S-Met/Cys (10 μCi/ml; PerkinElmer) and incubated for 30 min. After labeling, slices were homogenized, and labeled proteins were isolated by trichloroacetic acid precipitation. Samples were read with a scintillation counter and subjected to a protein concentration assay (Bio-Rad). Final data were expressed as counts per minute per microgram of protein, normalized to the ³⁵S-Met/Cys ACSF used for incubation, and the average incorporation of all samples was analyzed in that experiment (eight samples per experiment: four WT and four Fmr1 KO). All aspects of the experiments were performed blinded to genotype.