Assuming that an excitatory stimulus will cause more granule cells to discharge leading to more IEG expression in mutants than in controls, we next evaluated expression of the immediate-early gene (IEG) in response Protein Tyrosine Kinase inhibitor to kainic acid (KA) injection. As expected, in the acute phase KA injection (20 mg/kg i.p.) evokes expression of both Zif268 and c-Fos in more granule cells in mutants than

in controls (Figures 5C– 5E), and regardless of genotype, no such expression occurs in the untreated condition. In the chronic phase, however, differences in IEG expression between mutants and controls is negligible (Zif268, 165.8 ± 58.6 for control [n = 8], 226.9 ± 79.1 for mutant [n = 7], t test, p = 0.54; c-Fos, 1766.5 ± selleck kinase inhibitor 557.7 for control, 2496.7 ± 973.3 for mutant, t test, p = 0.53). We also examined the intensity of KA-induced seizures in acute (Figure 5F) and chronic (Figure 5G) phases. With seizures scored every 5 min for 1 hr following i.p. injection of 20 mg/kg KA, the cumulative seizure score was significantly higher for mutant mice than for their control littermates during the acute phase (Figure 5F;

21.0 ± 2.3 for control, 27.8 ± 2.2 for mutant, t test, p < 0.05) but not during the chronic phase (Figure 5G; 18.2 ± 3.0 for control, 19.4 ± 2.5 for mutant, t test, p = 0.75). In mutants, the maximum seizure score was also significantly higher than PAK6 in controls during the acute (2.4 ± 0.2 for control, 3.2 ± 0.3 for mutant, t test, p < 0.05) but not the chronic phase (1.8 ± 0.3 for control,

2.3 ± 0.3 for mutant, t test, p = 0.38). Taken together, these results demonstrate granule cell hyperexcitability in response to mossy cell degeneration during acute, but not chronic phase. To see if granule cell axons sprout into denervated IML during chronic phase, as occurs when seizures induce hilar neuron loss (Jiao and Nadler, 2007; Kienzler et al., 2009), we used Timm staining (Figure 6A) and zinc transporter 3 (ZnT3) immunostaining (Figure 6B) to visualize mossy fibers 6 weeks after DT treatment (n = 6 mutants, n = 5 controls). Surprisingly, following extensive mossy cell loss, mutant mice show no detectable mossy fiber sprouting in the IML of either dorsal or ventral hippocampus. Semiquantitative analysis of Timm staining (Tauck and Nadler, 1985) shows no statistical difference between genotypes (0.17 ± 0.10 for controls, 0.13 ± 0.08 for mutants, Mann-Whitney U test, p = 0.80). Despite mossy cell loss confirmed in mutants by diminished band-like staining in IML (whether by Timm or anti-ZnT3) identified as mossy cell axons in controls (West and Andersen, 1980; Corbetta et al., 2009), moreover, Netrin G2 immunostaining (Nishimura-Akiyoshi et al., 2007) confirms the absence of detectable sprouting from perforant path axonal fibers (Figure S3A).