Fig. 1. A representative Western blot of the phosphorylation-independent epitopes of NF-L, NF-M, and NF-H proteins in 9-day-old control and KA-treated rats 30 min, 1, 3, and 6 h after SE (A). One control group (P9) was used for the treated rats at all time points. B shows a representative Western blot of the same epitopes as in A of control and KA-treated rats 1, 3, and 7 days after SE. Note that the corresponding age-matched controls were used for each time point and that the signal intensity of the NF protein triplet was increased during the development in control rats. (C) Semiquantitative analyses of the signal in Western blots for NF-L, NF-M, and NF-H are given as percentage of the corresponding age-matched controls. Data are given as means ± SEM (n = 8). Abbreviations: C indicates control; KA, kainic acid. The significance of differences between controls and KA-treated rats (*P < 0.05).

Fig. 2. (A) A representative Western blot of the phosphorylation-dependent epitopes of NF-H [NF-H(P)], and NF-M [NF-M(P)], and the nonphosphorylated NF-H [NF-H(NP)] epitope in 9-day-old control, in KA-treated rats 30 min, and 1, 3, and 6 h after the injection. (B) A representative Western blot of NF-H(P), NF-M(P), and NF-H(NP) in control and KA-treated rats 1, 3, and 7 days after the injection. Note that the corresponding age-matched controls were used for each time point, and the signal intensity in particular that of NF-H(P) and NF-H(NP) clearly increased during the follow-up time. C shows the semiquantitative analysis of the Western blots for NF-H(P), NF-M(P), and NF-H(NP) given as percentage of the corresponding age-matched controls. Data are given as means ± SEM (n = 8). Abbreviations: C indicates control; KA, kainic acid. The significance of differences between controls and KA-treated rats (*P < 0.05).

Fig. 3. A representative Western blot of the phosphorylation-independent epitopes of NF-L, NF-M, and NF-H proteins in adult control rats, and in KA-treated rats 6 h and 1 day after SE (A). B shows a representative Western blot of the phosphorylation-dependent epitopes of NF-H [NF-H(P)] and NF-M [NF-M(P)], and the nonphosphorylated NF-H [NF-H(NP)] epitope in adult control rats, and in KA-treated rats 6 h and 1 day after KA injection. In C, the semiquantitative analysis of the Western blots for NF-H, NF-M, NF-L, NF-H(P), NF-M(P), and NF-H(NP) in adult rats is given as percentage of the controls. Data are given as means ± SEM (n = 8–12). Abbreviations: C indicates control; 1d, 1 day. The significance of differences between controls and KA-treated rats (*P < 0.05).

Fig. 4. Immunocytochemical staining of NF proteins in control (A, C, E) and KA-treated (B, D, F) rats. A and B show an example of the NF-M staining in a control and KA-treated rat, respectively, 1 h after the injection. Note the moderate MF-M immunoreactivity in the MF region, but immunonegativity of cell bodies in CA1, CA3, and granule cells in both a control and KA-treated rat. C and D show an example of NF-H immunostaining in a control and KA-treated rat, respectively, 6 h after the injection. Note the moderate NF-H immunoreactivity in CA3 cell bodies, but again an absent immunoreactivity in cell bodies of CA1 and granule cells in both the control and KA-treated rat. A representative immunostaining with the SMI 31 antibody, which detects the phosphorylated epitopes of MF-M and NF-H is shown in a control (E) and KA-treated (F) rat 6 h after the injection. Scale bar in A–F: 200 μm. Abbreviation: DG indicates dentate gyrus.

Fig. 5. Representative images of the Timm's staining in control (A and C) and KA-treated (B and D) rats 2 weeks (A and B) and 4 weeks (C and D) after the KA-induced SE. The inserts in A–D show the DG region including the stratum granulare (sg) and the inner molecular layer (iml) with the higher magnification. Note the lack of staining in the inner molecular layer both in the control and KA-treated rats. Scale bar in A–D = 200 μm. Scale bar in the inserts is 100 μm. Abbreviations: DG indicates dentate gyrus; sg, stratum granulare; iml, inner molecular layer.

Fig. 6. Representative Fluoro-Jade B stainings 1 day (A) and 1 week (B), 2 (C), and 4 (D) weeks after the KA-induced SE. The insert in D shows the CA3 region of the hippocampus at the higher magnification. No FJB-stained fluorescent cells were detected at any time point after the KA-induced SE. E shows numerous FJB-stained neurons in the CA3a/b region of an adult rat 1 day after SE shown at higher magnification in F. Representative images of thionin staining of the hippocampus 2 weeks (G) and 4 weeks (H) after the KA treatment in developing rats show well-preserved cell layers in the hippocampus. Scale bar in A–E and G–H is 200 μm, and in F 50 μm. Abbreviation: DG indicates dentate gyrus.

NF-L, NF-M, NF-H, SMI 31, and SMI 32 protein immunoreactivity in different regions of the rat hippocampus in control and kainic acid-treated rats at the different time points after the treatment

Scoring is a mean of 10–12 slices from three to five rats in each group as described in Materials and methods. −, negative; −/+, weakly positive; +, positive; ++, moderately positive; +++, strongly positive. Abbreviations: Sp, stratum pyramidale; So, stratum oriens; Sr, stratum radiatum; Slm, stratum lacunosum moleculare; Sl, stratum lacunosum; Sg, stratum granulare; Mf, mossy fibers; Mi, inner molecular layer; Mm, medial molecular layer; Mo, outer molecular layer; C, control at P9; C7d, control 7 days after saline injection; K1h, 1 h after KA injection; K6h, 6 h after KA injection; K7d, 7 days after KA injection.