Fig. 3: Cyfip1 haploinsufficiency affects the ability if microglia to regulate number and apoptosis rates of immature neurons.
A Experimental setup, Mac-1-Sap was present in wild-type hippocampal progenitor cultures from 2 h after isolation until 3 days in vitro. Dose–response curves of microglial death after Mac-1-SAP administration show near-complete depletion of Iba1+ microglia was achieved within 48 h (n = 6). B Cultures where microglia were depleted (n = 4) showed a higher proportion of DCX+ cells than controls (n = 4). C Correspondingly, the fraction of DCX cells undergoing apoptosis, as marked by cleaved-caspase 3 staining, was significantly decreased in the absence of microglia. D The proportion of nestin+ cells was unaffected by microglia depletion. E Experimental setup, membrane inserts containing wild-type microglia prepared from P7–8 brain were added to cultures 2 h after progenitor isolation and were present throughout the remainder of the experiment. Due to the experimental conditions, no cell–cell contact between microglia and progenitors was possible, but secreted factors were free to diffuse. F The presence of microglia-containing inserts (n = 8) significantly decreased the proportion of DCX expressing cells compared to controls (n = 8). G Conversely, the added microglia significantly increased apoptosis in DCX+ cells. H No effect was seen in nestin expressing cells. I Experimental design, wild-type hippocampal progenitors were exposed to conditioned medium from either wild-type or Cyfip1+/− microglia. J In wild-type cultures, conditioned medium from wild-type microglia induced a significant reduction in the proportion of DCX+ cells, whereas medium from Cyfip1+/− microglia was unable to do this (n = 4 in all conditions). K An inverse pattern was seen in neuronal apoptosis where wild-type microglia medium increased apoptosis in DCX+ immature neurons but Cyfip1+/– medium did not. L Nestin+ cells were not significantly affected by the presence of microglial factors. M Experimental design, Cyfip1+/− hippocampal progenitors were exposed to conditioned medium from either wild-type or Cyfip1+/− microglia. N As seen in the wild-type cultures, a significant decrease in DCX+ cells was observed only after stimulation with medium from wild-type microglia (n = 4 in all conditions). O Similarly, medium from Cyfip1+/− microglia was unable to induce apoptosis in Cyfip1+/− progenitors. P Again, the nestin+ population was unaffected. All data are shown as mean ± SEM.
