Eight naturally-delivered male pups from six pregnant ferrets, which were purchased from Japan SLC (Hamamatsu, Japan), were used in the experiments. The pups were reared with lactating dams (3–5 pups/mother) in stainless steel cages (80 cm × 50 cm × 35 cm) maintained at 21.5 ± 2.5 °C under 12-h artificial illumination in the Facility of Animal Breeding, Nakaizu Laboratory Japan SLC (Izu, Japan). All lactating dams were fed a pellet feed (High-Density Ferret Diet 5L14, PMI Feeds, Inc., St. Louis, MO, USA) and ad libitum tap water.
The thymidine analogs and VPA were administered on a schedule designed according to a previous study [10 (link)]. Eight male pups were intraperitoneally injected with 30 μg/g body weight of 5-ethynyl-2′-deoxyuridine (EdU) (Sigma-Aldrich, St. Louis, MO, USA) at PD 5 and 30 μg/g body weight of 5-bromo-2′-deoxyuridine (BrdU) (Sigma-Aldrich) at PD 7, respectively. Four pups were intraperitoneally administered 200 µg/g body weight of VPA on PDs 6 and 7. Four unexposed pups were used as controls. VPA was injected a second time at the same time as the BrdU injection. On PD 20, all animals were perfused with 4% paraformaldehyde (PFA) (Merck, Darmstadt, Germany)-phosphate buffered saline (PBS) (pH 7.4) under deep anesthesia with ~2% isoflurane gas. The cerebellum was removed and immersed in the same fixative.
The thymidine analogs and VPA were administered on a schedule designed according to a previous study [10 (link)]. Eight male pups were intraperitoneally injected with 30 μg/g body weight of 5-ethynyl-2′-deoxyuridine (EdU) (Sigma-Aldrich, St. Louis, MO, USA) at PD 5 and 30 μg/g body weight of 5-bromo-2′-deoxyuridine (BrdU) (Sigma-Aldrich) at PD 7, respectively. Four pups were intraperitoneally administered 200 µg/g body weight of VPA on PDs 6 and 7. Four unexposed pups were used as controls. VPA was injected a second time at the same time as the BrdU injection. On PD 20, all animals were perfused with 4% paraformaldehyde (PFA) (Merck, Darmstadt, Germany)-phosphate buffered saline (PBS) (pH 7.4) under deep anesthesia with ~2% isoflurane gas. The cerebellum was removed and immersed in the same fixative.
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