Microglia rapidly respond to changes in neural activity and inflammation to regulate synaptic connectivity. The extracellular signals, particularly neuron-derived molecules, that drive these microglial functions at synapses remain a key open question. Here we show that whisker lesioning, known to dampen cortical activity, induces microglia-mediated synapse elimination. This synapse elimination is dependent on signaling by CX3CR1, the receptor for microglial fractalkine (also known as CXCL1), but not complement receptor 3. Furthermore, mice deficient in CX3CL1 have profound defects in synapse elimination. Single-cell RNA sequencing revealed that Cx3cl1 is derived from cortical neurons, and ADAM10, a metalloprotease that cleaves CX3CL1 into a secreted form, is upregulated specifically in layer IV neurons and in microglia following whisker lesioning. Finally, inhibition of ADAM10 phenocopies Cx3cr1^−/− and Cx3cl1^−/− synapse elimination defects. Together, these results identify neuron-to-microglia signaling necessary for cortical synaptic remodeling and reveal that context-dependent immune mechanisms are utilized to remodel synapses in the mammalian brain.

Microglia, the brain resident macrophages, critically shape forebrain neuronal circuits. However, their precise function in the cerebellum is unknown. Here we show that human and mouse cerebellar microglia express a unique molecular program distinct from forebrain microglia. Cerebellar microglial identity was driven by the CSF-1R ligand CSF-1, independently of the alternate CSF-1R ligand, IL-34. Accordingly, CSF-1 depletion from Nestin⁺ cells led to severe depletion and transcriptional alterations of cerebellar microglia, while microglia in the forebrain remained intact. Strikingly, CSF-1 deficiency and alteration of cerebellar microglia were associated with reduced Purkinje cells, altered neuronal function, and defects in motor learning and social novelty interactions. These findings reveal a novel CSF-1–CSF-1R signaling-mediated mechanism that contributes to motor function and social behavior.

Significance: The main insight from this study is that the role of 5-hydroxymethylcytosine (5hmC) in postmitotic neurons is to sculpt the genome occupancy of the very abundant 5-methylcyctosine binding protein 2 (MeCP2). Accumulation of 5hmCG in transcribed genes replaces high-affinity 5mCG binding sites with low-affinity sites, decreasing MeCP2 occupancy over the transcription unit and removing its repressive effect. We refer to this role for 5hmCG as “functional demethylation” because its biochemical effect with respect to MeCP2 is equivalent to chemical demethylation: Loss of high-affinity sites for interaction in the genome. This concept reinforces the roles of 5hmC in demethylation in dividing cells by a mechanism that achieves the same goal without requiring cell division or DNA damage.