To further validate that microglia phagocytose RGC inputs, pHrodo-dextran, an anterograde tracer and pH-sensitive dye, was used to label RGC inputs (Figures S1A and S1B; Deriy et al., 2009 and Miksa et al., 2009). Because

pHrodo only fluoresces Cabozantinib clinical trial once it enters acidic compartments of lysosomes, any pHrodo-positive fluorescence within a microglia confirms phagocytosis of RGC inputs. Similar to previous experiments, pHrodo-positive RGC inputs were localized within microglia (Figures S1A and S1B). Furthermore, in addition to anterograde tracing with CTB and pHrodo, RGC input engulfment was also assessed within the P5 dLGN using a genetic approach, double transgenic mice expressing tdTomato under the control

of Chx10, a transcription factor expressed by RGCs (Chx10-cre/Rosa26-STOP-tdTomato) IWR-1 ic50 (Figures S1C–S1F). Similar to CTB experiments, we observed tdTomato-labeled RGC inputs within lysosomal compartments of microglia. Importantly, these experiments exclude the possibility that engulfment is due to injury secondary to ocular injections. Together, we demonstrate that microglia phagocytose RGC inputs during a peak period of synaptic pruning in the dLGN. To begin to address whether microglia-mediated engulfment of RGC inputs contributes to the normal process of synaptic pruning, we assessed the developmental regulation of microglia phagocytic capacity. We first characterized microglia activation state through development and observed a unique class of microglia in the early postnatal dLGN as compared to older ages (P30) (Figure S2). Microglia within the early postnatal dLGN had characteristic features of more “activated” cells traditionally Oxalosuccinic acid associated with disease including increased phagocytic capacity (assessed by morphology and CD68 immunoreactivity; Figures S2C and S2D). Interestingly, early postnatal microglia also had processes, a morphological characteristic of ‘resting’ microglia which are resident in

the healthy adult brain (Figure S2B; Lynch, 2009 and Ransohoff and Perry, 2009). To address whether engulfment of RGC inputs was developmentally regulated, we developed an in vivo phagocytosis assay (Figure 2A). Using high-resolution confocal microscopy followed by 3D reconstruction and surface rendering (Figure 2D), internalization of ipsilateral (CTB-647; blue) and contralateral (CTB-594; red) RGC inputs was quantified within the volume of each microglia (CX3CR1+/EGFP) throughout the dLGN. To control for variation in microglia volume, the following calculation was used: % Engulfment = Volume of internalized RGC inputs (μm3)/Volume of microglia (μm3). Consistent with microglial involvement in normal developmental synaptic pruning, engulfment of RGC inputs was developmentally regulated.