Persistence of early-generated neurons in the rodent subplate: Assessment of cell death in neocortex during the early postnatal period

Abstract

In the rat, the deepest neocortical layer forms a conspicuous cell band known as layer VIb. Cells in layer VIb are among the first to differentiate, and it has been regarded as an homolog to the subplate of primates and carnivores. Cell death has been considered a universal feature of subplate cells. In order to assess the validity of this assertion, we examined the sequence of generation and the extent of cell death in layer VIb. This was achieved using injections of 3H-thymidine and two methods for the direct visualization of apoptotic figures. Single injections of 3H-thymidine were performed between E12 and E15 (E0 is the day of insemination), and brains were examined at different postnatal ages between P1 and P63. The number of heavily labeled cells were counted in layer VIb in six standard, equally spaced coronal sections in each brain. Single injections at E12 labels about 3% of the entire population of layer VIb cells, 17% at E13, 30% at E14, and < 1% at E15. Our results indicate that the absolute number of heavily labeled cells in layer VIb remains constant. The analysis of variance (one-way ANOVA) showed that the difference among the group means was not significant from P1 to P63 after injections at either E12, E13, or E14. In order to confirm these results, we evaluated the distribution of pyknotic (apoptotic) cell bodies in the neocortex. Apoptotic cells were visualized in Nissl preparations and by histochemical staining using an in situ apoptosis detection kit. The analysis was performed in rats from E18 to P15. Both methods gave comparable results. We found that the amount of cell death in layer VIb is neither particularly prominent nor significantly different from that which occurs in the remaining neocortical layers, apart from layer II and in the white matter of the corpus callosum. We conclude that neuronal death does not play any significant role in the rodent subplate.