Abstract
The neocortex region of the mammalian brain is essential for motor control, spatial learning, language, thought and reasoning. Proper development of the neocortex is critical for appropriate cognitive performance. Animal models have shown that developmental blueprint issues at embryonic stages may give rise to autism spectrum symptoms and motor control associated diseases like Fragile X-associated tremor ataxia syndrome (FXT AS). The neocortex is shaped by tightly regulated proliferation, migration and differentiation of neural stem and progenitor cells, collectively known as neural precursor cells (NPCs). Moreover, microglia, immune cells of the brain, phagocytize (engulf) these NPCs and are thought to regulate their numbers in the developing neocortex. However, these factors alone do not explain a complete program that produces a functional neocortex. Nonetheless, interactions between different cell types, their behavior and their population size are important factors determining the developmental fate. Alterations in these factors can give rise to different developmental schematics and can be the root cause between normal and disease states. Individuals with FXTAS have reduced motor control, reflexes and brain volume. It is our hypothesis that microglia-mediated phagocytosis of NPCs causes abnormalities in NPC numbers as observed in a disease animal model. We tested this hypothesis in the murine model of FXTAS (FMRl premutation model) that has a premutation condition in the FMRl gene. Using antibodies to microglia and NPCs and confocal microscopy imaging, microglia quality and quantity were compared between the neocortex of normal and premutation animals. Analyses were done at two time points: Embryonic day 15 (E15) where the neocortex has plenty ofNPCs but not many microglia, and at post-natal day 21 (P21) when NPCs are scarce but microglia are in abundance. When imaged, we observed what appeared to be microglia-mediated NPC phagocytosis in the neocortex of murine tissues. Comparisons of El5 neocortexes indicated that an average of 36 and 39 microglia cells were located within 200μm-wide radial bins from normal and premutation animals respectively which was found to be not statistically different. Additionally, no variation was found in the morphological activation state of the microglia in either group. When P21 neocortexes were examined, poor cell signal and high background was evident. Multiple experiments were performed using different antibodies to improve staining, but only one neocortical section from normal and premutation animals produced patches of positively immunostained regions. These regions were analyzed but the number of microglia was also found to be similar between groups with no differences in the activation states. Nonetheless, the results indicate that microglia-mediated phagocytosis of NPCs does not appear to cause the permutation phenotype. Analysis of more samples and broader developmental stages should provide further insights. In addition to potentially causing disease states such as FXT AS, microgliamediated NPC phagocytosis could also be influencing the size and folding pattern of the neocortex. It has been noted that rats have small smooth brains, ferrets also have small brains but have cortical folds, and finally monkeys have larger brains with folds. We tested whether there was a correlation between the number of microglia and size and complexity of the brain. We hypothesized that an increase in number or activation of microglia as compared to NPCs could give rise to smaller or less complex (fold-less) brains, whereas, less microglia per NPCs would lead to larger and more complex (folded) brains. Microglia and NPCs were labeled in rat, ferret and monkey neocortexes and to our surprise, immunostains showed that monkeys had the most microglia per NPC followed by ferrets and then rats. This does not support the hypothesis that microglia determine the overall fate of the neocortex size and shape. Due to time constraint, we could not determine the Activation states of microglia were not analyzed due to time constraints. In conclusion, it appears that microglia numbers alone were unable to explain the neocortex disease condition found in FXTAS mice nor the neocortex size or complexity (folding). Further studies are warranted to determine what role microglia-mediated NPC phagocytosis serves in the neocortex.