Fig. 1
Activation of mTORC1 in neurons by NMDA and BDNF receptors. A Epifluorescence images of P-pS6 detected by immunocytochemistry with anti-P-pS6 240/244 antibodies in cultured mouse hippocampal neurons (DIV 18–20) incubated in ACSF with Mg2+ for 45 min with BDNF (50 ng/mL), in the presence (Rapa) or the absence of rapamycin (10–25 neurons per large field images). Boxed region is shown at higher magnification with the P-pS6 signal and the corresponding MAP2 images. Shown in the graph is the P-pS6 mean fluorescence in the areas defined as MAP2 positive (BDNF: N = 50 fields from 5 independent experiments; Rapa: N = 20 fields from 2 experiments). Bar, 100 µm. B Impact of NMDAR activation on mTORC1 activity. Neurons were treated for 10 min under the indicated conditions and then in ACSF with Mg2+ for 20 min before processing for P-pS6 and MAP2 immunolabelling. Removal of Mg2+ and addition of glycine (N = 20 fields from 2 independent experiments) or glycine, bicuculline and strychnine (cLTP, N = 100 fields from 10 independent experiments including 3 with rapamycin and 3 with AP5) robustly activated mTORC1. The effect was suppressed by rapamycin and by AP5. C P-pS6 mean intensity for the entire neurons, somas or dendrites in control or cLTP conditions (N = 40 fields from 4 independent experiments used in (B). Areas tested are similar in both conditions. D Neurons were treated as in (B) (cLTP conditions) with or without inhibitors of BDNF receptor TrkB, 25–50 µM ANA12 or 200 nM cyclotraxin-B. Both inhibitors partially diminished the effect of NMDAR stimulation (6 independents experiments including 3 with ANA12 25 µM, 4 with ANA12 50 µM or CTX-B, (statistical comparison with ACSF with Mg2+ and AP5 are indicated in parentheses). E Neurons were starved in ACSF for 45 min, treated for 15 min in ACSF with Mg2+ supplemented with amino acids (AA) and processed 15 min later for immunofluorescence. (N = 10–30 large field images per condition from 2 independent experiments)