Accumulation of abnormal adult-generated hippocampal granule cells predicts seizure frequency and severity

Abstract

Accumulation of abnormally integrated, adult-born, hippocampal dentate granule cells (DGCs) is hypothesized to contribute to the development of temporal lobe epilepsy (TLE). DGCs have long been implicated in TLE, because they regulate excitatory signaling through the hippocampus and exhibit neuroplastic changes during epileptogenesis. Furthermore, DGCs are unusual in that they are continually generated throughout life, with aberrant integration of new cells underlying the majority of restructuring in the dentate during epileptogenesis. Although it is known that these abnormal networks promote abnormal neuronal firing and hyperexcitability, it has yet to be established whether they directly contribute to seizure generation. If abnormal DGCs do contribute, a reasonable prediction would be that the severity of epilepsy will be correlated with the number or load of abnormal DGCs. To test this prediction, we used a conditional, inducible transgenic mouse model to fate map adult-generated DGCs. Mossy cell loss, also implicated in epileptogenesis, was assessed as well. Transgenic mice rendered epileptic using the pilocarpine-status epilepticus model of epilepsy were monitored continuously by video/EEG for 4 weeks to determine seizure frequency and severity. Positive correlations were found between seizure frequency and (1) the percentage of hilar ectopic DGCs, (2) the amount of mossy fiber sprouting, and (3) the extent of mossy cell death. In addition, mossy fiber sprouting and mossy cell death were correlated with seizure severity. These studies provide correlative evidence in support of the hypothesis that abnormal DGCs contribute to the development of TLE and also support a role for mossy cell loss.

Figure 1.

EEG and hippocampal abnormalities in pilocarpine-treated animals. Cortical EEG recording showing a spontaneous seizure (A). Seizure frequencies in animals exhibiting pilocarpine-induced SE ranged from zero to three seizures per day (B). The average spontaneous seizure duration between animals ranged from 15 to 43 s per seizure (C), with higher seizure frequencies being positively correlated with longer seizure durations (E). The average behavioral seizure score for each animal ranged from 2.0 to 4.4 (D). Four weeks of 24 h video/EEG monitoring revealed the typical “seizure clustering” phenomenon associated with the pilocarpine model (F). No significant differences were found between dorsal and ventral hippocampus for the percentage of ectopic DGCs (G), the percentage of DGCs harboring basal dendrites (BD; I), the percentage increase of MFS (K), or hilar mossy cell density (M). Dorsal and ventral hippocampus were significantly correlated for the percentage of newborn DGCs with basal dendrites (J), MFS (L), and density of surviving mossy cells (N) but not for ectopic DGCs (H). Error bars are means ± SEM.

Figure 2.

Ectopically located newborn granule cells correlate with seizure frequency. Images are confocal maximum projections showing GFP-expressing newborn granule cells in the dorsal hippocampus of bitransgenic mice. Mice experienced 0.1 (A, C) and 3.0 (B, D) seizures per day. C and D are higher-resolution images of the boxed regions in A and B, respectively. The dentate hilus in each image is located between the dotted lines. Note the large number of ectopic granule cells in D and the absence of such cells in C. E, Schematics showing the sample regions in red for ectopic cell measures for dorsal and ventral hippocampus (the entire hilus). F, G, The percentage of newborn granule cells ectopically located in the dentate hilus was significantly correlated with seizure frequency but not seizure duration (regression line is in red). Scale bars: A, B, 250 μm; C, D, 20 μm.

Figure 3.

Basal dendrites and seizure frequency. Images are confocal maximum projections from dorsal hippocampus showing GFP-expressing newborn granule cells with (B, D) and without (A, C) basal dendrites. Images have been processed using a depth filter, which assigns different colors to processes located at different depths within the tissue. Mice had mean seizure frequencies of 0.1 (A, C) and 3.0 (B, D) seizures/d. C and D are high-magnification images of the boxed regions in A and B, respectively. The entire dentate was scored for dorsal hippocampus (E, region in red in the left), whereas ventral hippocampus was sampled in the upper and lower blades of both hemispheres (E, right). F, G, Scatter plots display a nonsignificant trend between the percentage of normotopic DGCs harboring basal dendrites and seizure frequency and duration (regression line is in red). Scale bars: A, B, 20 μm; C, D, 10 μm.

Figure 4.

MFS correlates with seizure frequency and duration. Images are confocal maximum projections showing ZnT3 and GFP immunostaining in dorsal hippocampus of mice that exhibited means of 0.1 (A–F) and 2.1 (G–L) seizures per day. Arrowheads (I) highlight sprouted granule cell mossy fiber axons within the dentate IML. M, Schematics showing the sample regions in red for MFS measures for dorsal and ventral hippocampus. Greater MFS density was significantly correlated with increased seizure frequency (N) and seizure duration (O) (regression line is in red). GCL, Granule cell layer; h, hilus. Scale bars: A–C, G–I, 250 μm; D–F, J–L, 50 μm.

Figure 5.

Mossy cells density negatively correlates with seizure frequency and duration. Images are confocal maximum projections of GluR2 immunoreactivity in ventral hippocampus of mice that exhibited means of 0.1 (A) and 3.0 (B) seizures per day. Arrows denote presumptive mossy cells, whereas arrowheads denote presumptive hilar ectopic granule cells. Note the almost complete loss of mossy cell labeling in B. C, Schematics showing the sample regions in red for mossy cell counts for dorsal and ventral hippocampus. D, E, Scatter plots show low hilar mossy cell density in mice exhibiting high seizure frequency and duration (regression line is in red). Green dots denote mossy cell densities of age-matched control mice that did not undergo SE (not included for statistical analysis of correlations). Scale bar, 25 μm.