Overexpression of BDNF in CA3 pyramidal cells promotes mossy fiber pathfinding in the distal parts of the axons. (A) The expression viruses, including the CaMKII promoter were injected locally in the CA3 cell layer (green circle). Biocytin crystals were placed on the granule cell layer (pink circle) to visualize mossy fibers. (B) In nontreated control slices, mossy fibers visualized by the biocytin labeling (magenta) were fasciculated, and CA3 cells infected with mock viruses were identified by the co-expression of Venus (green). (C and D) K252a-treated slices with mock (C) or BDNF (D) expression are shown in high magnification images of the CA3c (2: solid box in 1) or CA3a (3: dotted box in 1) area. D shows abnormal distribution of mossy fibers in the CA3c area; these fibers began to accumulate in the CA3b area, which contained pyramidal cells overexpressing BDNF (green), and fasciculated tightly in the SL of the CA3a area. (E and F) Mossy fiber distribution estimated by biocytin labeling intensity in the CA3c (E) and CA3a (F) regions. *P < 0.05 and **, ##P < 0.01; Tukey's test after analysis of variance (n = 9 slices obtained from 4 independent experiments). SR: stratum radiatum, SP: stratum pyramidale, and SO: stratum oriens.