Chapter 5 168 We demonstrated that hPSC-derived microglia-like cells reduce cellular debris and decreased density of synaptophysin positive puncta. These results align well with the established roles of microglia in brain development and homeostasis, including their key functions in debris clearance and synaptic pruning (Paolicelli & Ferretti, 2017). While these changes are consistent with microglial phagocytic behavior, further investigation is needed to determine whether they are driven by direct microglial engulfment or mediated by microglia secreted factors. We demonstrated that neuronal morphology is altered in the presence of microglia-like cells. Given the well-documented interactions between microglia and neuronal structures (Nimmerjahn et al., 2005) and the secretion of neurotrophic and anti-inflammatory factors by microglia (Elkabes et al., 1996; Hanisch & Kettenmann, 2007) the identification of morphological changes aligned well with our expectations. Specifically, we observed a decrease in axonal and dendritic segments. Considering the secretion of neurotrophic factors by microglia, a decrease in axonal and dendritic segments seems contradicting. We hypothesize that the effects of microglial-secreted factors may have been masked, since both microglia-containing and microglia-free cultures were maintained in media supplemented with neurotrophic factors such as BDNF. Subsequently, the decrease in segments might be caused by phagocytosis or neurotoxins secreted by microglia-like cells that cleared nuclear debris (Bonkowsky et al., 2010; Giulian et al., 1994). More research is required to determine whether neurotrophic factor supplementation is necessary in these co-cultures and whether the microglia-like cells directly engulf axonal and dendritic structures or secrete neurotoxins. The functional impact of microglia on neuronal networks was evident from the MEA experiments, which revealed significantly increased neuronal activity in the presence of microglia-like cells. Enhanced spike rates, alongside trends toward increased burst activity and network synchronicity, suggest that microglia influence not only individual neuronal behavior but also broader network dynamics. Interestingly, this increase in activity cooccurred with morphological changes, including a reduction in synaptophysin-positive puncta density—a proxy for synapse density. As synapses are the physical sites of action potential transmission between neurons, these findings may seem counterintuitive. However, less active synapses are more likely to be pruned (Schafer et al., 2012), suggesting that microglia may retain active synapses, thereby enhancing overall network activity.
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