Fig. 3

Recruitment of mTORC1 to late endocytic compartments. A Changes in the neuronal distribution of mTOR after NMDAR activation. Immunostaining of DIV18 hippocampal neurons for endogenous mTOR and LAMP1, with or without NMDAR stimulation (incubation in Mg²⁺ free ACSF for 15 min). Mg²⁺ removal induced the appearance of clusters of mTOR that co-distribute with LAMP1 positive structures in both soma and dendrites. Bar, 10 µm. B Neurons were incubated for 15 min in Mg²⁺-free ACSF, double labelled for mTOR and LAMP1 and imaged with a TIRFM/SIM microscope with a lateral resolution of ~ 100 nm. Structures enriched in mTOR were also positive for LAMP1 confirming that mTOR was recruited on LEs. Bar, 5 µm. C Quantification of mTOR recruitment to LEs in dendrites. mTOR fluorescence was measured in LEs (IN) and in the cytosol area surrounding LEs (OUT) in dendrites from 100X epifluorescence images stained for LAMP1, mTOR, and MAP2. Shown is the ratio (IN/OUT) (8–10 images per condition per experiment, n exp indicates the number of experiments). Removal of Mg²⁺ induced a rapid AP5-sensitive increase in mTOR recruitment to LEs. Similar results were obtained with Mg²⁺-free ACSF plus glycine or in cLTP. D Same as in (C) but after a 45 min starvation period in ACSF to reduce possible amino acid-dependency (5 experiments including 3 with AP5). E Addition of exogenous BDNF (50 ng/mL in ACSF) does not promote the recruitment of mTOR to LEs after 10 or 30 min (3 and 5 experiments respectively) compared to NMDAR activation (3 experiments for glycine and one for cLTP). (F) Contribution of endogenous BDNF to the NMDAR-dependent translocation of mTOR to LEs. Neurons were treated as in (C) in ACSF or in Mg²⁺-free ACSF plus glycine. The TrkB inhibitors ANA12 (25 µM) reduced the recruitment of mTOR to LEs (2 experiments)