Earlier, we generated a conditional transgenic mouse line in which diphtheria toxin receptor was selectively expressed in mossy cells using the Cre/loxP system. Within one week after diphtheria toxin injection, we observed 80% loss of mossy cells throughout the longitudinal axis. We found no obvious or sustained epilepsy-like discharges in the hippocampus as measured by in vivo local field potential recordings. Interestingly, no mossy fiber sprouting was detected by Timm staining. These results suggested that, in contrast to previous reports showing that lesions of the entire hilar region induce massive mossy fiber sprouting and epilepsy, selective in vivo elimination of mossy cells does not trigger behavioral epilepsy or mossy fiber sprouting. This year, we found that dentate granule cells in the DT-treated mutants became hyperexcitable to afferent stimulation in in vitro slice preparation, and during this hyperexcitable state deficits in contextual pattern separation were detected. We also evaluated the immediate-early gene (IEG) expression in response to kainic acid (KA) injection under the assumption that an excitatory stimulus would cause more granule cells to discharge and activate IEG expression in mutants compared to controls. KA injection evoked Zif268 expression in more granule cells in mutants than in controls. We also examined the KA-induced seizure intensity. The cumulative seizure score of mutants for the hour following KA injection was significantly higher than controls. Together, these results all suggested an increase in granule cell excitability following mossy cell ablation. In summary, we concluded that mossy cell loss in vivo renders the granule cells hyperexcitable. Contrary to the predicted epileptogenesis implicit in the dormant basket cell hypothesis, however, it was insufficient to trigger the mossy fiber sprouting and epileptic discharges. Perhaps, in addition to the loss of mossy cells, neurodegeneration of other limbic areas, such as entorhinal cortex, is necessary to induce medial temporal lobe epilepsy. These findings provide new insights into the mechanisms of epileptogenesis in the limbic cortex. This year, for the manuscript second and third revisions, we conducted some more additional experiments. First, we assessed the degree of mossy cell degeneration after DT treatment by Nissl staining, by Fluoro-Jade B staining, and by double immunostaining. We found that many mossy cell-like cells are already degenerated by post-DT 7 days;however, these cells, even degenerated, appear to be structurally present at least by 6 weeks after DT. Therefore, we decided to use the term mossy cell degeneration, instead of mossy cell deletion when we describe the process of cell death. Second, in response to the reviewers claim that the variability is high, we re-examined the cell counting of kainic acid-induced Zif268 and c-Fos positive cells 5 weeks after DT injection (chronic phase). Now the number of animals are n=8 for controls and n=7 for mutants. We found that the overall variability for both IEG IR-positive cells was not so high compared to the acute phase data. And our conclusion that dentate granule cells are transiently over-excited upon mossy cell degeneration is still valid, based on the IEG data. The manuscript is submitted again and under review.
| Jinde, Seiichiro; Zsiros, Veronika; Nakazawa, Kazu (2013) Hilar mossy cell circuitry controlling dentate granule cell excitability. Front Neural Circuits 7:14 |
| Jinde, Seiichiro; Zsiros, Veronika; Jiang, Zhihong et al. (2012) Hilar mossy cell degeneration causes transient dentate granule cell hyperexcitability and impaired pattern separation. Neuron 76:1189-200 |
