Developmental Dendritic Changes Of Newborn Granule Cells Following Pilocarpine-Induced Status Epilepticus

Background and ObjectivesThe mammalian dentate gyrus (DG) is a brain region where newborn granule cells are continuously added throughout life. Once being stimulated, neural stem cells in the subgranular zone differentiate and mature into granule cells, then integrate into the preexisting dentate network eventually. In rodents, newborn granule cells takes at least two months to display characteristic morphological and electrophysiological features as mature granule cells. Adult neurogenesis has been shown to contribute to learning, memory formation and mood regulation. Many physiological and pathological factors affect adult hippocampal neurogenesis. Status epilepticus transiently promotes hippocampal neurogeneis. Up-to-date, it is controversial about the role of enhanced neurogenesis in epileptogenesis. In this thesis, newborn granule cells are labeled by CAG-GFP retroviral vector. I investigated the developmental dendritic changes, including dendritic architecture, spine density and cellular activity of newborn granule cells after pilocarpine-induced status epilepticus.Materials and Methods Animals:Male Sprague-Dawley rats (140-170 g,6-7 weeks) were used. Methods: 1. Status epilepticus (SE) was induced by intraperitoneal injection of pilocarpine andintrahippocampal injection of retroviral vector.Animals suffered SE more than 2 hours with Ⅱ or more in Raince classificationwere used for futher experiments. Controlled rats received equivalent amount ofsaline injection. Five days after SE, CAG-GFP retroviral vector was injected intothe dentate gyrus at day 5. 2. Transcardial perfusion and brain section preparation Transcardiac perfusion with 4% paraformaldehyde was performed 5,28 or more than 70 days after retroviral vector injection. Upon removal, individual brains were post-fixed with 4%paraformaldehyde at 4℃ overnight. The tissue block containing the hippocampus was made and coronally cut into 100 μm or 40μm thick with a vibratome. 3. Immunohistochemistry GFP+cells were subjected to immunofluorescent staining. One hundred μm sections were stained for GFP (goat anti GFP) and the 40 μm hippocampal sections were chosen for Arc or NeuN stain. Alexa fluor 488 donkey anti-goat, Alexa fluor 568 donkey anti-rabbit and Alexa fluor 568 donkey anti-mouse secondary antibodies were used. 4. Dendritic morphological analyses GFP+cells in the suprapyramidal blade were selected for z-series stack scanning. After a 3D image was reconstructed for each cell, Sholl analysis was conducted in 2D image which was converted from the reconstructed 3D image by using which Image J software. The number of dendrites that crossed a series of concentric circles at 25 μm intervals from the center of the soma was counted and analyzed. 5. Quantification of dendritic spines For each suprapyramidal blade-resided GFP+ granule cell,3 dendritic segments located in middle molecular layer and another 3 dendritic segments located in outer molecular layer were randomly selected for z-series stack scanning. After a 3D image was created for each dendritic segment, the length of dendritic segments, total spines and mushroom-like spines were counted manually under Image J. Densities of total and mushroom-like spines were calculated.6. Quantification of Arc immunoreactivity Intensity of Arc immunoreactivity was measured in the GFP+ soma and 10 GFP-mature granule cells located in outer molecular layer under Image J. The ratio of Arc immunoreactivity intensity in the GFP+ cell to the averaged Arc immunoreactivity intensity in the 10 GFP- cells was used to indicate the activity of newborn granule cells.7. Statistical analysis Quantitative data are expressed as mean±standard error. Data were analyzed in GraphPad Prism Software. P≤0.05 is considered to be statistically significant.Results:1. Dentate gyrus neorogenesis exists in mouse treated with either saline or pilocarpine. Dividing cells were labeled with retroviral vectors that express green fluorescent protein (GFP). These GFP+cells include granule cells and a few glial cells. These newly generated dentate granule cells are located in the inner 1/3 of the granule cell layer and brightly labeled with GFP in their entirety including their cell bodies, full dendritic architectures, and mossy fibers.2. Sholl analysis showed that dendrites of 4-week-old newborn granule cells after SE were more complex than that in the controlled animals. However, this discrepancy was disappeared among 10-week-old newborn granule cells.3. The densities of total spines (Control:1.96±0.05 μm-1, n=5; SE:2.23±0.04 μm-1,n=4) and the mushroom-like spines (Control:0.06±0.002 μm-1, n=5; SE 0.07±0.002 μm-1, n=4) of SE group are significant higher in the middle molecular layer. Only mushroom-like spine density of dendrites in the outer molecular layer of SE group is significant higher than control group (Control: 0.10±0.007μm-1, n=6;SE:0.13±0.01μm-1, n=6).4. The fluorescence intensity of Arc immunoreactivity in 4-week-old GFP+ cells was denser than GFP- cells in both SEs and controls (Control:1.54±0.13, n=3; SE:1.45±0.17, n=7). However, the fluorescence intensity of Arc immunoreactivity in 10-week-old GFP+ cells was closer to GFP- cells in both SEs and controls (Control:1.18±0.10, n=6; SE:0.98±0.04, n=6).Conclusion:Compared to contemporary newborn granule cells under physiological condition, newborn granule cells following pilocarpine-induced status epilepticus show developmental changes in both dendritic morphology and cellular activity. Four-week-old newborn granule cells following pilocarpine-induced status epilepticus have a more complicated dendritic arborization than that of age-matched granule cells in control group. Also, their cellular activity is higher than preexisting mature granule cells, which indicates these cells are hyperexcitable at this time point. After maturation, their dendritic complexity and spine density are close to those of physiologically born ones. In addition, the cellular activity of these mature neurons in SE is lower than their predecessors. The data presented here suggest that the contribution made by newborn granule cells following status epilepticus to hyperexcitability in dentate gyrus is temporary and impermanent.