Fig. 4. Fluorescence micrographs (recorded in the red channel) of the axonal growth cones of hippocampal neurons from membrane addition experiments. Neurons were labeled with BODIPY-ceramide for 30 minutes at room temperature and then chased for 2.5-3 hours at 37°C. Growth cones were challenged with control medium, IGF-1 or BDNF, after factor deprivation, for the time indicated in minutes. Whereas fluorescent vesicle clusters persist in controls (vehicle only; growth cone of the neuron shown in Fig. 2A) and in the presence of BDNF, red fluorescence rapidly dissipates upon challenge with 20 nM IGF-1 (growth cone of the neuron shown in Fig. 2B).