Microglia-astrocyte crosstalk regulates synapse remodeling via Wnt signaling

Unilateral whisker lesioning at age P4 leads to removal of thalamocortical synapses in layer IV of the barrel cortex over the course of the next 6 days. Synapse removal is mediated by microglia and induced by CX3CL1-CX3CR1 signaling between neurons and microglia. Downstream of CX3CL1-CX3CR1 activation, microglia induce astrocyte morphological changes. To identify molecular crosstalk between astrocytes and microglia, we performed translating ribosome affinty purification followed by RNA sequencing (TRAP-Seq). Astrocyte ribosomes were labeled using the transgenic Aldh1l1EGFP-L10a/+ mice and microglial ribosomes were labeled using Cx3cr1CreER/+; Eef1a1LSL-EGFP-L10a/+ mice. 24 hours after unilateral whisker lesioning in P4 mice, mice were euthanized with CO2, and brain regions of interest were dissected and flash-frozen for TRAP. The deprived and non-deprived barrel cortices were micro-dissected and GFP-tagged ribosomes from the cell type of interest were immunoprecipitated with a GFP antibody. RNA sequencing was performed on both the GFP antibody-bound and unbound fractions.