Astrocytes recycle endocytic pro-BDNF for regulated secretion. (A) Western blot analysis of BDNF-YFP (mix) using α-BDNF or α–pro-BDNF antibodies. (B) Immunocytochemistry in astrocytes untreated (n = 12) or incubated for 10 min with BDNF-YFP (mix; n = 18) followed by acid strip. Bar, 10 μm. (C) Ultrastructural characterization of astrocytes exposed to BDNF-YFP gold for 10 min. Arrowheads point to gold particles contained in vesicular organelles. Bar, 500 nm. (D) Representative TIRF images of astrocytes incubated with BDNF-YFP for 5 min. The top sequence depicts exocytic fusion (arrowheads) in a selected astrocytic area (white rectangle) before and after perfusion with glutamate. The bottom sequence shows fusion of a single vesicle. Fluorescence intensity is measured in a circular mask centered over the vesicle and in a concentric annulus on the circle. Bars, 2 μm. (E) Time distribution of fusion events (flashes) after glutamate application. The inset shows the total number of flashes per astrocyte before (n = 17) and after (n = 13) glutamate. (F) ELISA quantification of BDNF secretion from astrocytes before (n = 18) and after (n = 22) glutamate application for 5 min. Astrocytes previously exposed to BDNF (mix) for 10 min were stimulated with glutamate in the absence (n = 14) or presence (n = 4) of TeNT. (G) Secretion of BDNF induced by AMPA or t-ACPD in the absence (n = 23) or presence (n = 17) of the respective antagonists CNQX or AIDA and by 50 Hz (n = 6). Data are means ± SEM (error bars). *, P ≤ 0.05.