A novel approach for visualizing mossy fibers in cultured hippocampal slices. (A) A schematic diagram of locally introduced lentiviral vectors. Lentivirus, which induces the expression of enhanced GFP (EGFP) under the influence of a CaMKII promoter, was injected into 2 or 3 sites of the granule cell layer in hippocampal slices. (B) Representative images of the results. Granule cells specifically expressed EGFP. Green: EGFP immunoreactivity, Magenta: Nissl staining of the neurons. (C and D) Images of control slices (C) and slices treated with K252a (300 nM) (D) in the CA3c (C1, solid box in B1) and CA3a (C2, dotted box in B1) areas. Mossy fibers in control slices (C) were observed primarily in the SL, similar to what was observed in vivo, while those in the K252a-treated slices were abnormally distributed outside of the SL as shown in D. (E and F) Distribution of the mossy fibers treated by 100 and 300 nM of K252a as assessed by the measurement of EGFP intensity in the CA3c (E) and CA3a (F) areas. *P < 0.05 and ##, **P < 0.01; Tukey's test after analysis of variance (n = 7–9 slices obtained from 4 independent experiments). DG: dentate gyrus, SR: stratum radiatum, SP: stratum pyramidale, and SO: stratum oriens.