BDNF as a mediator of OCN function in the brain. (A) Representation of the microfluidic device (top) composed of somatic and synaptic chambers connected by 500-µm-long microchannels. BDNF-mCherry-positive vesicles were imaged in a 100-µm-long distal region of axons for 30 s. Kymographs (bottom) extracted from the movies. Examples of traces of anterograde (green), retrograde (orange), and pausing (blue) vesicles are depicted on the kymograph. Bar, 10 µm. (B) Quantification of BDNF-mCherry transport in axons of hippocampal neurons cultured in microchambers treated with OCN (n = 108) or vehicle (n = 110) for 4 h (Mann-Whitney U test) compared with control condition. Anterograde and retrograde mean velocities of vesicles per axon, the number of anterograde and retrograde vesicles that are motile in a 100-µm length of axon during 30 s, the percentage of time spent by vesicles in pause, and the directional flux of vesicles within axons were measured. (C and D) Bdnf expression (quantitative PCR [qPCR]) in midbrain and hippocampi of 6-mo-old Gpr158−/− (n = 4–6) and WT (n = 6) littermates (Student’s t test; C) and in Gpr158−/− and WT hippocampal neurons (one-way ANOVA compared with vehicle-treated cells followed by Bonferroni’s post hoc test, n = 12) treated with either saline or OCN for 4 h (D). (E and F) BDNF accumulation (representative Western blot, E) and quantification of band intensities in hippocampi of 3-mo-old shRNA scramble- or shRNA Gpr158-injected (F). After a 10 d-long recovery, mice were injected with saline or OCN (10 ng) 16 h before collections (two-way repeated-measures ANOVA followed by Fisher’s least significantly different post hoc test, n = 4 per group). (G) Bdnf expression (qPCR) in hippocampi of 16-mo-old WT mice injected i.p. with either saline (n = 6) or OCN (n = 8; 500 ng/g BW) 2 h before collection (Student’s t test). (H) BDNF accumulation (representative Western blot, left) and quantification of band intensities (right) in hippocampi of older WT mice receiving plasma from older (n = 7) or young WT (n = 5) mice, from young Ocn−/− (n = 5) mice, or from young Ocn−/− mice supplemented with OCN (n = 5; 90 ng/g BW; one-way ANOVA compared with older WT mice receiving plasma from older WT mice, followed by Bonferroni’s post hoc test). α-Tubulin used as a loading control. Results are given as mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.