Seizure-associated, aberrant neurogenesis in adult rats characterized with retrovirus-mediated cell labeling

Abstract

Seizure activity within the hippocampal circuitry not only affects pre-existing structures, but also dramatically increases the number of newborn granule cells. A retroviral strategy was used to label dividing cells and their progeny in the adult dentate gyrus and to analyze the impact of epileptic activity on adult-generated cells labeled before or after seizures. We show that epileptic activity led to dramatic changes in the neuronal polarity, migration, and integration pattern of newborn granule cells, depending on the time of birth in relation to the epileptic insult. Aberrant neurons were stably integrated into the dentate circuitry, and the consequences on hippocampal neurogenesis were long lasting. The data presented characterized the consequences of seizure-associated plasticity on adult neurogenesis leading to long-term structural changes in the hippocampal circuitry that might represent a pivotal component of the epileptic disease process.

Figure 1.

Retroviral labeling of newborn cells in the adult hippocampus after seizure activity. A–G, Retroviral labeling of newborn granule cells in controls (A, F) and animals that had seizures (B–E, G) revealed altered polarity and ectopic localization of granule neurons born after SE. Whereas control cells 4 weeks after viral injection (A) extended an apical dendrite toward the ML, seizure-induced granule cells extended basal dendrites reaching deep into the hilus (B, C). Note that basal dendrites were covered with dendritic spines (D). Granule cells born after SE also ectopically migrated into the hilar/CA3 border (E), which was never observed in control animals. Under control conditions, once newborn granule cells survived the initial critical selection process, they became stably integrated into the circuitry for at least 1 year (F). Despite hilar dendritic growth, the same was true for aberrant granule cells, which were frequently observed 1 year after viral injection (G). H, The number of DCX-expressing immature neurons transiently increased in response to KA-induced seizures and returned to baseline 3 months after SE. Arrows indicate apical dendrites; arrowheads label basal dendrites. The pan-neuronal marker NeuN is red in all images. Small panels in A–C show the GFP signal alone (top) and a high-magnification view of the SGZ (bottom). Boxed areas in D, E, and G show the area that is shown in high power view in the insets in D, E, and G, respectively. Scale bars: A–C, F, G, 50 μm; D, E, 10 μm. SAL, Saline. Error bars in H represent SEM. *p < 0.05.

Figure 2.

Differential effects on granule cells born before or after onset of SE. A, Retroviral labeling of newborn granule cells 2 months after KA injection demonstrated that the detrimental effects of the initial SE on adult neurogenesis are long lasting as, even at this late time point after SE, a substantial fraction of granule cells extended spiny hilar basal dendrites (right panel shows GFP signal only). B, C, In contrast, the polarity of newborn granule cells was not significantly altered when dividing progenitors and their progeny were retrovirally labeled 1 week before SE (B), which was also true for the dendritic architecture of new granule cells labeled 4 weeks before KA injection (C). D, However, a substantial fraction of the latter cells showed extensive mossy fiber sprouting into the ML (right panel shows GFP signal only). Arrows indicate apical dendrites; arrowheads label basal dendrites. Blue arrows point toward axonal processes. The pan-neuronal marker NeuN is red in all images. Scale bars, 50 μm.

Figure 3.

Dendritic spine morphology of granule cells born after SE. A–E, Analyses of spine density and shape 4 weeks (A, B) and 3 months (C, D) after retrovirus injection in controls (A, C) and animals that had seizures (B, D) demonstrated that, despite no differences in spine density of apical dendrites, the number of mushroom spines was increased in animals that had seizures compared with controls 3 months after retrovirus injection (E). SAL, Saline. Error bars in E represent SEM. *p < 0.01.

Figure 4.

Synaptic integration of aberrant granule cells. A, B, Similar to apical dendritic spines of controls (A), spiny processes on basal dendrites extending from neurons born after SE (B) were often in close proximity (arrowheads) to the presynaptic protein synapsin (red). C–E, Electron microscopy of aberrant neurons (C) confirmed the formation of synapses on ectopic dendrites within the hilar region (D, E). F, Three-dimensional reconstruction of the ectopic dendritic spine (green, shown in D) contacting an axon (blue) filled with synaptic vesicles. The synapses shown in D–F were located in the area boxed in C. Scale bars: D, E, 1 μm.

Figure 5.

Activity-induced c-fos expression in aberrant granule cells extending basal dendrites. A, Three hours after a handling-induced seizure episode in epileptic rats (SE 5 weeks earlier), c-fos was dramatically upregulated in the dentate gyrus granule cell layer (bottom) compared with epileptic controls (top). B, C, Granule cells born after SE expressing NeuN (red) with aberrant dendrites (B) did not express c-fos (blue) under control conditions. Strong expression of c-fos is detectable in aberrant granule cells (C) 3 h after handling-induced seizure activity. Insets in C show a high-power view of the newborn granule cell (arrow) that extends a basal dendrite toward the hilus and expresses c-fos (top, blue; middle, gray). Scale bars, 50 μm.