Transfer of pro-BDNF from neurons to perineuronal astrocytes. (A) Schematic representation of the slice preparation. (B) Western blot analysis of BDNF (mix) or cleavage-resistant pro-BDNF (Mowla et al., 2001) using α-BDNF– or α–pro-BDNF–specific antibodies. (C) Field potential amplitudes (black circles) and BDNF levels (gray circles) upon basal (control) or TBS stimulations. After recording, slices were immunostained using α–pro-BDNF. Immunoreactivity is shown in two adjacent areas corresponding to areas A1 and A2 of A. (D) Immunohistochemistry using α-BDNF. Bars, 100 μm. (E) High resolution confocal images of A1 in a slice 20 min after TBS. Pro-BDNF immunoreactivity is shown at the site of astrocytic contact with a neuron (box and inset 1), the astrocytic cell body (box and inset 2), and processes (box and inset 3). Colocalization of pro-BDNF with GFAP immunoreactivity is shown and superimposed onto the 3D reconstruction of the GFAP signal. Arrowheads indicate pro-BDNF immunoreactive puncta distributed along the astrocytic processes. Bar, 20 μm. (F) Time course of pro-BDNF/GFAP colocalization (four slices and nine cells). (G) Pro-BDNF/GFAP colocalization in astrocytes of control (three slices and 12 cells) or TBS slices in the absence (six slices and 24 cells) or presence of anisomycin (five slices and 11 cells), TrkB-Fc (four slices and nine cells), and plasmin (five slices and 24 cells) 10 min after stimulation. NeuN, neuronal nuclei. Data are means ± SEM (error bars). *, P ≤ 0.05.