2.12. Biotinylated dextran amine (BDA) tracing

After PTZ kindling, rats were deeply anesthetized with pentobarbital (60 mg/kg), and 5 μL of BDA (10 000 MW; Invitrogen) was stereotaxically injected into the dentate gyrus (DG) region of the hippocampus (coordinates from Bregma: anterior‐posterior: −3.6 mm; medial‐lateral: −2.5 mm; dorsal‐ventral: −3.5 mm) using a microsyringe.

Fourteen days after BDA injection, rats were anesthetized with pentobarbital and perfused transcardially with 4% paraformaldehyde in PBS. The brains were removed, fixed overnight in 4% paraformaldehyde, dehydrated in sucrose, frozen, and sectioned at 40 μm. The sections were processed to reveal BDA‐positive fibers by incubation with an avidin‐biotin complex (ABC; BDA‐10,000 Neuronal Tracer Kit, Invitrogen) solution (1.0 µg/mL) in PBS containing 0.3% Triton X‐100 at 4°C overnight. The sections were treated with 0.03% DAB and 0.004% hydrogen peroxide. The procedure was performed as previously described.⁴² Mossy fiber pathways, indicated by BDA‐positive fibers, were visualized under a microscope (Leica DM600B Microsystems Heidelberg GmbH) with different objectives.