Since there is a brief window between phagocytosis and degradation of lysosomal contents, EM studies
may underestimate the synaptic content of microglial lysosomes. Together, these experiments suggest microglia can engulf presynaptic terminals of RGCs, though they do not rule out the engulfment of nonsynaptic Erastin supplier or postsynaptic structures, as has been seen in hippocampus ( Paolicelli et al., 2011). Microglia are exquisitely sensitive to injury and inflammation, and the above studies involved intraocular injections, which might cause microglia to target RGCs. To control for this possibility, a genetically encoded marker was used to label RGCs, eliminating the need for injections, and similar
microglial engulfment of RGC material was still seen. The idea that microglial engulfment is part of a normal developmental pathway is further supported by the fact that engulfment of RGC components in vivo roughly paralleled the timing of developmental remodeling. Previous work demonstrated that C3 is present at synapses during the early postnatal period and is required for normal developmental remodeling of retinogeniculate axons (Stevens et al., 2007). Given that microglia are the only known resident brain cells to express the C3 receptor, CR3, Schafer et al. (2012) hypothesized that AG14699 C3-CR3 interactions might recruit microglia to RGC axons as they remodel. To directly test the requirement for CR3 in remodeling, similar RGC-tracer experiments were performed in transgenic mice lacking functional CR3. Overlap between inputs from the two eyes was increased, Dipeptidyl peptidase and engulfment of RCG material by microglia was reduced, in CR3-deficient mice, effects that were mimicked by pharmacologically inhibiting microglial activity in WT animals. The increase in overlap in CR3-deficient mice was paralleled by an increase in synapse density in adults, as assessed by colocalization of VGlut2 (a marker for RGC presynaptic terminals)
and GluR1 (a marker of postsynaptic sites) by array tomography. These and other results show that in the absence of complement C3 or its microglial receptor, CR3, microglia contain less RGC-derived material than in WT, and inappropriate axon projections and synapses are present. Interestingly, CR3-deficient mice showed increase in both VGlut2-containing synapses and VGlut2 puncta not associated with synapses. Some of the nonsynaptic VGlut2 could represent retracting RGC axons that successfully underwent elimination in the absence of CR3-dependent mechanisms. This is likely, since significant pruning still occurs in CR3 knockouts. However, these recently pruned inputs should also be present in WT dLGNs. In fact, if pruning is impaired in CR3-deficient animals, this might be expected to lead to fewer recently pruned inputs than WT, not more.