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. 2010 Feb 10;30(6):2051-9.
doi: 10.1523/JNEUROSCI.5655-09.2010.

The developmental stage of dentate granule cells dictates their contribution to seizure-induced plasticity

Michelle M Kron  1 , Helen Zhang, Jack M Parent
Affiliations

The developmental stage of dentate granule cells dictates their contribution to seizure-induced plasticity

Michelle M Kron et al. J Neurosci. .

Abstract

Dentate granule cell (DGC) neurogenesis persists throughout life in the hippocampal dentate gyrus. In rodent temporal lobe epilepsy models, status epilepticus (SE) stimulates neurogenesis, but many newborn DGCs integrate aberrantly and are hyperexcitable, whereas others may integrate normally and restore inhibition. The overall influence of altered neurogenesis on epileptogenesis is therefore unclear. To better understand the role DGC neurogenesis plays in seizure-induced plasticity, we injected retroviral (RV) reporters to label dividing DGC progenitors at specific times before or after SE, or used x-irradiation to suppress neurogenesis. RV injections 7 weeks before SE to mark DGCs that had matured by the time of SE labeled cells with normal placement and morphology 4 weeks after SE. RV injections 2 or 4 weeks before seizure induction to label cells still developing during SE revealed normally located DGCs exhibiting hilar basal dendrites and mossy fiber sprouting (MFS) when observed 4 weeks after SE. Cells labeled by injecting RV after SE displayed hilar basal dendrites and ectopic migration, but not sprouting, at 28 d after SE; when examined 10 weeks after SE, however, these cells showed robust MFS. Eliminating cohorts of newborn DGCs by focal brain irradiation at specific times before or after SE decreased MFS or hilar ectopic DGCs, supporting the RV labeling results. These findings indicate that developing DGCs exhibit maturation-dependent vulnerability to SE, indicating that abnormal DGC plasticity derives exclusively from aberrantly developing DGCs. Treatments that restore normal DGC development after epileptogenic insults may therefore ameliorate epileptogenic network dysfunction and associated morbidities.

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Figures

Figure 1.
Figure 1.
RV-GFP reporter labeling of DGCs generated at specific time points before or after SE. A, Timeline for RV labeling of newborn cells in the dentate gyrus born before or after SE. B–Q, Images of GFP-immunoreactive cells after injection of RV at the designated postnatal ages (P7, P28, P42, or P60) in rats receiving saline (B–I) or pilocarpine (J–Q) at P56. Higher magnification views of RV-GFP-labeled DGCs show granule cells with hilar basal dendrites (arrowheads) or hilar ectopic DGCs (arrows) only at specific RV labeling time points (P28, P42, or P60) with respect to SE at P56. ml, Molecular layer; gcl, granule cell layer; h, hilus. Scale bars: (in B) B–E, J–M, 50 μm; (in F) F–I, N–Q, 25 μm.
Figure 2.
Figure 2.
Quantification of the percentage of RV-GFP-labeled DGCs with hilar basal dendrites or ectopic migration to the hilus. A, Hilar basal dendrites were analyzed in GFP-positive cells in the DGC layer. The percentage of DGCs with hilar basal dendrites labeled with RV-GFP at P7 that were mature at the time of SE (P56) was not significantly increased compared with controls (p = 0.25), but cells labeled by RV injections at the three other time points before or after SE did show significantly greater proportions with hilar basal dendrites (*p < 0.005 for all groups vs controls; #p < 0.05 for P28, P42, and P60 vs P7 RV). B, A significantly increased number of hilar ectopic GFP-labeled DGCs were present compared with controls only when RV was injected after SE at P60. *p < 0.005 for the P60 group versus P60 control. Error bars indicate SEM.
Figure 3.
Figure 3.
Only DGCs still developing and not fully mature at SE display MFS. A, Representative image of DGCs labeled with RV-GFP at P7 that have axons extending normally into the hilus (h) (arrows) 4 weeks after experiencing SE on P56, with no GFP-labeled axons in the inner molecular layer (iml). B, P42 RV-GFP injection labeled a DGC 2 weeks of age at SE with GFP+ axon extending through the granule cell layer (gcl) into the iml (arrowheads). C, D, GFP/NFM double-labeled axon extending from the cell body toward the iml (arrows) 4 weeks after SE in an animal that received RV injection at P28. E, P28 RV-GFP injection into a saline-treated control rat labeled axons only in the hilus. F, P28 RV-GFP injection into an animal that subsequently experienced pilocarpine-induced SE on P56 labeled axonal sprouts in the iml (arrowheads) 4 weeks after SE. Scale bar: A, B, E, F, 75 μm; C, D, 40 μm.
Figure 4.
Figure 4.
Low-dose ionizing irradiation transiently suppresses DGC neurogenesis. A–C, Dcx immunostaining of immature neurons in the DG of rats irradiated (rad) beginning on P21 (P21/P23) shows decreased neurogenesis at 7 d (B, arrows) versus control (A), and recovery 28 d after rad (C). D, E, BrdU labeling shows cell proliferation also recovers 4 weeks after rad at P21/P23 (E) versus control (D). BrdU was given 2 h before killing on P49. F–J, Neurogenesis examined by Dcx immunolabeling is also transiently suppressed after rad beginning on P56 (P56/P58), with knockdown after 2 d (G, arrows) and 7 d (H, arrows), partial recovery at 14 d (I, arrows), and nearly total recovery to control levels (F) after 28 d (J). Scale bar, 100 μm.
Figure 5.
Figure 5.
Effect of irradiation before or after SE on MFS. A, Timeline for irradiation studies. The hatched areas show predicted timing of suppressed neurogenesis after 6 Gy x-irradiation (rad). B–F, Representative images of dentate gyrus Timm stain (arrows) in a naive control (B), a control receiving sham rad with SE (C), and rats receiving rad at specific time points before or after SE (D–F). Higher magnification views are shown in the insets. G, Quantitative analysis of pixel density revealed that 6 Gy fractionated irradiation administered 5 weeks before SE (P21) significantly decreased the amount of MFS (*p < 0.05). Irradiation either 2 weeks before or 1 week after SE did not have a significant effect on MFS at 4 weeks after SE. ml, Molecular layer; gcl, granule cell layer; h, hilus. Scale bars: B–F, 100 μm; insets, 25 μm. Error bars indicate SEM.
Figure 6.
Figure 6.
Effect of irradiation (rad) given after SE on hilar ectopic DGCs. A, A′, Prox1 immunostaining 28 d after SE (on P56) shows many hilar Prox1+ cells in a rat sham irradiated 4 d after SE. B, B′, Representative images from a rat that received 6 Gy rad beginning 4 d after SE (P60/P62) shows few Prox1-immunoreactive cells in the hilus (h). gcl, Granule cell layer. Scale bar, 50 μm.
Figure 7.
Figure 7.
Cells born after SE contribute to MFS 10 weeks after pilocarpine treatment. A, GFP immunolabeling of a control injected with RV-GFP 4 d after saline treatment and killed 10 weeks later shows axonal labeling in the hilus (h) but only GFP+ dendrites in the inner molecular layer (iml). B, C, At 10 weeks after SE, animals that received pilocarpine and then RV-GFP 4 d later display many GFP+ axonal processes in the iml (arrows) that appear identical with those seen in the granule cell layer (gcl) and hilus (arrowheads). D, E, Denser iml Timm staining is seen at 10 weeks after SE in a sham-irradiated control (D) than in a rat irradiated beginning 4 d after pilocarpine treatment (E). F, Densitometric analysis of the percentage area of inferior blade gcl and molecular layer that is Timm stain-positive. A dose of 6 Gy fractionated irradiation administered 4 and 6 d after SE significantly decreased the amount of Timm staining (*p < 0.005). Scale bars: (in A) A–C, 50 μm; (in D) D, E, 200 μm; insets, 50 μm. Error bars indicate SEM.
Figure 8.
Figure 8.
Diagrammatic model showing the influence of granule cell maturity on vulnerability to SE-induced structural plasticity. In this model, fully mature cells (born at P7; top row) are resistant to SE-induced remodeling. Some DGCs born at P28 (4 weeks of age at SE; second row), however, show abnormal hilar basal dendrites (HBDs) (b) and MFS (s). Those born on P42 (2 weeks of age at SE; third row) display extensive HBD formation 4 weeks after SE and to a lesser extent contribute to MFS seen 4 weeks after pilocarpine treatment. DGCs born 4 d after SE (P60 injections; bottom row) exhibit both HBDs and ectopic migration without MFS at 4 weeks after SE, but they develop MFS by 10 weeks. The model assumes that changes seen at 4 weeks remain stable for RV-GFP injections before SE (top 3 rows) as only animals administered RV-GFP after SE (bottom row) were examined at the 10 week time point in this study.
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